MONITORING

STEM CELL RESEARCH

A Report of

The President’s Council on Bioethics

Washington, D.C.

January 2004

www.bioethics.gov
CONTENTS

LETTER OF TRANSMITTAL TO THE PRESIDENT ix

MEMBERS OF THE PRESIDENT’S COUNCIL ON BIOETHICS xiii

COUNCIL STAFF AND CONSULTANTS xv

PREFACE xvii

1 INTRODUCTION 1

I. WHAT ARE STEM CELLS, AND WHY IS THERE CONTENTION

 ABOUT THEM? 2

II. BROADER ETHICAL ISSUES 5

III. TYPES OF STEM CELLS: AN INTRODUCTION 8

 A. EMBRYONIC STEM (ES) CELLS 8

 B. EMBRYONIC GERM (EG) CELLS 9

 C. ADULT (OR NON-EMBRYONIC) STEM CELLS 10

 D. CORD BLOOD STEM CELLS 11

IV. TERMINOLOGY 12

V. ABOUT THE REPORT 15

ENDNOTES 20

2 CURRENT FEDERAL LAW AND POLICY 21

I. A BRIEF HISTORY OF THE EMBRYO RESEARCH FUNDING

DEBATE 22

II. THE PRESENT POLICY 28

III. MORAL FOUNDATION OF THE POLICY 30

IV. THE SIGNIFICANCE OF FEDERAL FUNDING 37

V. IMPLEMENTATION OF THE PRESENT POLICY 41

VI. CONCLUSION 47

 ENDNOTES 49

v
vi CONTENTS

3 RECENT DEVELOPMENTS IN THE ETHICAL AND POLICY DEBATES 53

I. THE NATURE OF THE MORAL ARGUMENT 54

II. THE MORAL AIMS OF POLICY 58

 A. THE IMPORTANCE OF RELIEVING SUFFERING 58

 B. FREEDOM TO CONDUCT RESEARCH 61

 C. THE MORAL STANDING OF HUMAN EMBRYOS 62

III. THE CHARACTER OF THE POLICY 63

 A. ARBITRARY 63

 B. UNSUSTAINABLE 65

 C. INCONSISTENT 67

IV. THE MORAL STANDING OF HUMAN EMBRYOS 74

 A. CONTINUITY AND DISCONTINUITY 76

1. THE CASE FOR CONTINUITY. 76

2. THE CASE FOR MEANINGFUL DISCONTINUITY AND

 DEVELOPING MORAL STATUS. 78

 B. SPECIAL CASES AND EXCEPTIONS 84

1. IVF “SPARES.” 84

2. NATURAL EMBRYO LOSS. 88

3. PREDICTION OF NON-VIABILITY OF EMBRYOS. 89

4. CREATION OF NON-VIABLE EMBRYO-LIKE ARTIFACTS. 90

V. SOCIETAL SIGNIFICANCE AND PUBLIC RESPONSIBILITY 93

VI. CONCLUSION 97

 ENDNOTES 98

4 RECENT DEVELOPMENTS IN STEM CELL RESEARCH AND THERAPY 109

I. STEM CELLS AND THEIR DERIVATIVES 111

II. REPRODUCIBLE RESULTS USING STEM CELL PREPARATIONS

 AND THEIR DERIVATIVES 116

 A. INITIAL STEM CELL PREPARATIONS CAN

 CONTAIN MULTIPLE CELL TYPES 116

 B. GENETICALLY HOMOGENEOUS STEM CELLS

 THROUGH SINGLE CELL CLONING 117

 C. EXPANSION IN VITRO, PRESERVATION,

 AND STORAGE 118

 D. CHROMOSOME CHANGES 119
CONTENTS vii

 E. DEVELOPMENTAL HETEROGENEITY OF STEM CELL

 PREPARATIONS 119

 F. MICROBIAL CONTAMINATION 120

III. MAJOR EXAMPLES OF HUMAN STEM CELLS 121

 A. HUMAN ADULT STEM CELLS 122

1. HUMAN MESENCHYMAL STEM CELLS. 122

2. MULTIPOTENT ADULT PROGENITOR CELLS

 (MAPCS). 123

3. HUMAN NEURAL STEM CELLS. 124

4. ADULT STEM CELLS FROM OTHER SOURCES. 125

 B. HUMAN EMBRYONIC STEM CELLS 125

1. HUMAN EMBRYONIC STEM CELLS (ESCS). 126

2. EMBRYONIC GERM CELLS. 127

3. EMBRYONIC STEM CELLS FROM CLONED

 EMBRYOS (CLONED ESCS). 128

IV. BASIC RESEARCH USING HUMAN STEM CELLS 129

V. HUMAN STEM CELLS AND THE TREATMENT OF DISEASE 131

 A. WILL STEM CELL-BASED THERAPIES BE LIMITED BY

 IMMUNE REJECTION? 131

 B. CASE STUDY: STEM CELLS IN THE FUTURE TREATMENT OF

 TYPE-1 DIABETES? 133

1. THE DISEASE AND ITS CAUSES. 133

2. CURRENT THERAPY CHOICES AND

 OUTCOMES. 134

3. STEM CELL THERAPY FOR TYPE-1

 DIABETES? 135

 C. THERAPEUTIC APPLICATIONS OF MESENCHYMAL STEM CELLS

 (MSCS) 137

 D. EVALUATING THE DIFFERENT TYPES OF STEM CELLS 138

VI. PRIVATE SECTOR ACTIVITY 138

VII. PRELIMINARY CONCLUSIONS 139

 ENDNOTES 141

GLOSSARY OF TERMS 147

APPENDIX A. NOTES ON EARLY HUMAN DEVELOPMENT 157

STAFF OF THE PRESIDENT’S COUNCIL ON BIOETHICS

APPENDIX B. REMARKS BY THE PRESIDENT ON STEM CELL RESEARCH,

AUGUST 9, 2001 183

PRESIDENT GEORGE W. BUSH

APPENDIX C. BUSH ADMINISTRATION NIH GUIDELINES FOR EMBRYONIC

STEM CELL FUNDING 189
APPENDIX D. CLINTON ADMINISTRATION GUIDELINES FOR EMBRYONIC

STEM CELL FUNDING 191

COMMISSIONED PAPERS 198

APPENDIX E. LEGISLATORS AS LOBBYISTS: PROPOSED STATE REGULATION

OF EMBRYONIC STEM CELL RESEARCH, THERAPEUTIC

CLONING AND REPRODUCTIVE CLONING 199

LORI B. ANDREWS, J.D.

APPENDIX F. THE MEANING OF FEDERAL FUNDING 225

PETER BERKOWITZ, J.D., PH.D.

APPENDIX G. REPORT ON THE ETHICS OF STEM CELL RESEARCH 237

PAUL LAURITZEN, PH.D.

APPENDIX H. HUMAN EMBRYONIC GERM CELLS: JUNE 2001-JULY 2003,

THE PUBLISHED RECORD 273

JOHN GEARHART, PH.D.

APPENDIX I. CURRENT PROGRESS IN HUMAN EMBRYONIC STEM CELL

RESEARCH 279

TENNEILLE E. LUDWIG, PH.D., AND

JAMES A. THOMSON, PH.D.

APPENDIX J. MULTIPOTENT ADULT PROGENITOR CELLS: AN UPDATE 295

CATHERINE M. VERFAILLIE, M.D.

APPENDIX K. ADULT STEM CELLS 309

DAVID A. PRENTICE, PH.D.

APPENDIX L. STEM CELLS AND TISSUE REGENERATION: LESSONS FROM

RECIPIENTS OF SOLID ORGAN TRANSPLANTATION 347

SILVIU ITESCU, M.D.

APPENDIX M. POTENTIAL USE OF CELLULAR THERAPY FOR PATIENTS

WITH HEART DISEASE 365

SILVIU ITESCU, M.D.

APPENDIX N. THE BIOLOGY OF NUCLEAR CLONING AND THE POTENTIAL

OF EMBRYONIC STEM CELLS FOR TRANSPLANTATION

THERAPY 385

RUDOLF JAENISCH, M.D.

viii CONTENTS
LETTER OF TRANSMITTAL TO

THE PRESIDENT OF THE UNITED STATES

The President’s Council on Bioethics

1801 Pennsylvania Avenue N.W., Suite 700

Washington, D.C. 20006

January 14, 2004

The President

The White House

Washington, D.C.

Dear Mr. President:

I am pleased to present to you Monitoring Stem Cell

Research, a report of the President’s Council on Bioethics. Over

the past two years, in keeping with your stated intention, the

Council has been monitoring developments in stem cell

research, as it proceeds under the implementation of the

administration’s policy. We have consulted widely, heard

presentations, and commissioned review essays (included as

appendices in this volume) on all aspects of the topic—

scientific, ethical, and legal. Our desire has been both to

understand what is going on in the laboratory and to consider

for ourselves the various arguments made in the ongoing

debates about the ethics of stem cell research and the wisdom

of the current policy. Although both the policy and the research

are still in their infancy, the Council is now ready to give you

and the American people an update on this important area of

research.
Because this field and the current policy are so young, this

report can be no more than an “update.” It summarizes some

of the more interesting and significant developments since

August 2001, both in the basic science and medical

applications of stem cell research and in the related ethical,

legal, and policy discussions. It does not attempt to be a

definitive or comprehensive study of the whole topic. It

contains no proposed guidelines and regulations, nor indeed

any specific recommendations for public policy. Rather, it seeks

to shed light on where we are now—ethically, legally,

scientifically, and medically—in order that you, the Congress,

and the nation may be better informed as we all consider where

we should go in the future.

The report has four basic aims, three of them the subjects

of independent chapters devoted to their themes.

First, we have sought to clarify and explain the current

federal policy regarding stem cell research and to make clear

the legal, ethical, and prudential foundations on which the

policy rests: the desire to promote important biomedical

research without endorsing, funding, or creating incentives

for the future destruction of human embryos. We have also

sought to describe how that policy is being implemented,

especially by the National Institutes of Health. Many of these

matters have not been well understood or accurately

represented in public discussions since August 2001, and we

hope that the clarifications introduced in this report will enable

future discussions and debates to be better informed.

Second, we have tried to provide an overview of the ethical

and policy debates surrounding stem cell research in the past

two years. As you already know quite well, these are

immensely difficult and challenging matters, with the

obligations owed to nascent human life pitted against the

obligations to seek knowledge that might someday alleviate

much human suffering. Not surprisingly, arguments continue

on all aspects of the moral and political debate. We have sought

to present the arguments and counter-arguments, faithfully

x MONITORING STEM CELL RESEARCH
and accurately, so that all may learn what is at stake and where

the debate now stands.

Third, we have monitored recent scientific developments

in human stem cell research, embryonic and adult, basic

and applied. Our goal in the report is to enable (especially

non-scientific) readers to appreciate the reasons for the

excitement over stem cell research, the complexities of

working with stem cells, some early intriguing research and

therapeutic findings, and the difficult road that must be

traveled before we can reap therapeutic and other benefits

from this potentially highly fertile field of research.

The other three specific goals have been informed by a

fourth and overarching goal: to convey the moral and social

importance of the issue at hand and to demonstrate how

people of different backgrounds, ethical beliefs, and policy

preferences can reason together about it. We want everyone

to understand that biomedical research, being a human

activity, must always be regarded as a moral endeavor, to be

governed not only by the goals of gaining knowledge and

relieving suffering, but also by the obligation to safeguard the

inherent freedom and dignity of human life. Throughout the

Council’s deliberations and in this monitoring report, Council

members have tried to acknowledge the strengths and

importance of opinions and concerns held by people with

whom they disagree. We have aspired to be careful and fair in

our approach, precise in our use of language, accurate in

presenting data and arguments, and thoughtful in our laying

out of the various issues that remain before us. Above all, we

want all parties to these debates to understand that their

opponents, too, have something vital to defend, not only for

themselves but for all of us.

The policy debates over stem cell research that led you to

create this Council continue; they, and other debates on related

topics, are unlikely to go away any time soon. Our hope is that

our work will help to make those debates richer, fairer, and

better informed.

LETTER OF TRANSMITTAL TO THE PRESIDENT xi
Mr. President, allow me to join my Council colleagues and

our fine staff in thanking you for this opportunity to offer you

and the American people what we hope is a useful and

constructive review of where things stand, both in the

laboratory and in the public arena, with regard to this

promising and ethically challenging area of research.

Sincerely,

Leon R. Kass, M.D.

Chairman

xii MONITORING STEM CELL RESEARCH
MEMBERS OF

THE PRESIDENT’S COUNCIL ON BIOETHICS

LEON R. KASS, M.D., PH.D., Chairman.

Addie Clark Harding Professor, The College and the

Committee on Social Thought, University of Chicago. Hertog

Fellow, American Enterprise Institute.

ELIZABETH H. BLACKBURN, PH.D.

Professor, Department of Biochemistry and Biophysics,

University of California, San Francisco.

REBECCA S. DRESSER, J.D., M.S.

Daniel Noyes Kirby Professor of Law and Professor of Ethics

in Medicine, Washington University, St. Louis.

DANIEL W. FOSTER, M.D.

Donald W. Seldin Distinguished Chair in Internal Medicine,

Chairman of the Department of Internal Medicine, University

of Texas Southwestern Medical School.

FRANCIS FUKUYAMA, PH.D.

Bernard Schwartz Professor of International Political

Economy, Paul H. Nitze School of Advanced International

Studies, Johns Hopkins University.

MICHAEL S. GAZZANIGA, PH.D.

Dean of the Faculty, David T. McLaughlin Distinguished

Professor, Professor of Psychological and Brain Sciences,

Dartmouth College.

ROBERT P. GEORGE, J.D., D.PHIL.

McCormick Professor of Jurisprudence, Director of the James

Madison Program in American Ideals and Institutions,

Princeton University.

MARY ANN GLENDON, J.D., M. COMP. L.

Learned Hand Professor of Law, Harvard University.

xiii
MEMBERS OF

THE PRESIDENT’S COUNCIL ON BIOETHICS (CONTINUED)

ALFONSO GÓMEZ-LOBO, DR. PHIL.

Ryan Family Professor of Metaphysics and Moral Philosophy,

Georgetown University.

WILLIAM B. HURLBUT, M.D.

Consulting Professor in Human Biology, Stanford University.

CHARLES KRAUTHAMMER, M.D.

Syndicated Columnist.

WILLIAM F. MAY, PH.D.

Fellow, Institute for Practical Ethics and Public Life. Visiting

Professor, Department of Religious Studies, University of

Virginia.

PAUL MCHUGH, M.D.

University Distinguished Service Professor of Psychiatry,

Johns Hopkins School of Medicine. Professor, Department

of Mental Health, Bloomberg School of Public Heath, Johns

Hopkins University.

GILBERT C. MEILAENDER, PH.D.

Phyllis & Richard Duesenberg Professor of Christian Ethics,

Valparaiso University.

JANET D. ROWLEY, M.D.

Blum-Riese Distinguished Service Professor of Medicine,

Molecular Genetics and Cell Biology, and Human Genetics,

Pritzker School of Medicine, University of Chicago.

MICHAEL J. SANDEL, D.PHIL.

Anne T. and Robert M. Bass Professor of Government,

Harvard University.

JAMES Q. WILSON, PH.D.

James A. Collins Professor of Management and Public Policy

Emeritus, University of California, Los Angeles. Reagan

Professor of Public Policy, Pepperdine University.

xiv
COUNCIL STAFF AND CONSULTANTS

Dean Clancy

Executive Director

Peter Berkowitz

Senior Consultant

Eric Cohen

Senior Research Consultant

Judith E. Crawford

Administrative Director

Diane M. Gianelli

Director of Communications

Laura Harmon, Esq.

Senior Aide to the Chairman

Emily Jones

Executive Administrator

Yuval Levin

Deputy Executive Director

Michelle Powers

Law Clerk

Richard Roblin, Ph.D.

Scientific Director

Adam Schulman

Research Consultant

O. Carter Snead, Esq.

General Counsel

Catherine Thorp

Receptionist/Staff Assistant

Audrea R. Vann

Information Technology

Specialist

Rachel Flick Wildavsky

Director, Education Project

Lee L. Zwanziger

Director of Research

Allison Arensman

Intern

Dov Fox

Intern

Katie Goodman

Intern

Lauron Medley

Intern

Nelson Moussazadeh

Intern

Preface

Monitoring Stem Cell Research is a report of the President’s

Council on Bioethics, which was created by President George

W. Bush on November 28, 2001, by means of Executive Order

13237.

The Council’s purpose is to advise the President on

bioethical issues related to advances in biomedical science

and technology. In connection with its advisory role, the

mission of the Council includes the following functions:

• To undertake fundamental inquiry into the human

and moral significance of developments in

biomedical and behavioral science and technology.

• To explore specific ethical and policy questions

related to these developments.

• To provide a forum for a national discussion of

bioethical issues.

• To facilitate a greater understanding of bioethical

issues.

The President left the Council free to establish its own

priorities among the many issues encompassed within its

charter, and to determine its own modes of proceeding.

Stem cell research has been of interest to, and associated

in the public mind with, this Council since its creation. Taking

up the charge given to us by President Bush in his August 9,

2001, speech on stem cell research, the Council has from its

beginnings been monitoring developments in this fast-paced

and exciting field of research. The first formal discussions of

xvii
xviii MONITORING STEM CELL RESEARCH

the science and ethics of stem cell research took place at our

third meeting, in April of 2002, where presentations were made

by two prominent stem cell researchers (John Gearhart of

Johns Hopkins University and Catherine Verfaillie of the

University of Minnesota) and an ethicist (Gene Outka of Yale

University), at a time when we were still mostly engrossed in

our discussions of human cloning. Over the course of the

following year and a half, even as the Council was preparing

the reports Human Cloning and Human Dignity: An Ethical

Inquiry (July 2002) and Beyond Therapy: Biotechnology and

the Pursuit of Happiness (October 2003), it was gathering

information on stem cell research and devoting increasing

portions of its meeting agendas to this topic, which was

ultimately discussed at six meetings (specifically, in April and

July of 2002; and in June, July, September, and October of 2003).

The Council heard presentations from numerous experts in

the relevant scientific, ethical, social, advocacy, and

entrepreneurial arenas, and received public comment, oral and

written. The Members engaged in serious deliberation

throughout the process. All told, fourteen sessions, of ninety

minutes each, were devoted to the subject at public meetings.

Complete transcripts of all these sessions are available to the

public on the Council’s website at www.bioethics.gov.

The present monitoring report draws directly upon those

sessions and discussions, as well as on written material

prepared by Council members, staff, and consultants. As noted

in Chapter 1, it is in the spirit of an “update” and contains no

recommendations for policy.

We hope the report, with its overview chapters on the law,

ethics, and science of stem cell research, and its extensive

supporting material located in the appendices, will serve as a

source of clear, intelligible, and useful information for both

policymakers and the general public regarding the current

state of this important research and of the debates that

surround it.

In creating this Council, President Bush expressed his desire

to see us

consider all of the medical and ethical ramifications of

biomedical innovation. . . . This council will keep us
PREFACE xix

apprised of new developments and give our nation a

forum to continue to discuss and evaluate these

important issues. As we go forward, I hope we will

always be guided by both intellect and heart, by both

our capabilities and our conscience.

It has been our goal in the present report, as in all of our

work, to live up to these high hopes and noble aspirations.

LEON R. KASS, M.D.

Chairman
xx MONITORING STEM CELL RESEARCH
1

Introduction

This monitoring report has its origins in President George

W. Bush’s remarks to the nation on August 9, 2001. It was his

first major national policy address, and the topic was unusual:

federal funding of research on human stem cells.*

 In the speech,

the President announced that after several months of

deliberation he had decided to make federal funding available,

for the first time, for research involving certain lines of embryoderived stem cells. At the end of the speech the President

declared his intention to

name a President’s Council to monitor stem cell research,

to recommend appropriate guidelines and regulations,

and to consider all of the medical and ethical

ramifications of biomedical innovation. . . . This council

will keep us apprised of new developments and give our

nation a forum to continue to discuss and evaluate these

important issues.1

In keeping with the President’s intention, the Council has

been monitoring developments in stem cell research, as it

proceeds under the implementation of the administration’s

policy. Our desire has been both to understand what is going

on in the laboratory and to consider for ourselves the various

arguments made in the ongoing debates about the ethics of

*

 Throughout this report, excluding appendices, all references to embryos,

cells, or other biological materials are assumed to be of human origin unless otherwise stated.

1
2 MONITORING STEM CELL RESEARCH

stem cell research and the wisdom of the current policy.

Although both the policy and the research are still in their

infancy, the Council is now ready to give the President and

the public an update on this important and dynamic area of

research.

This report is very much an “update.” It summarizes some

of the more interesting and significant recent developments,

both in the basic science and medical applications of stem

cell research and in the related ethical, legal, and policy

discussions. It does not attempt to be a definitive or

comprehensive study of the whole topic. It contains no

proposed guidelines and regulations, nor indeed any specific

recommendations for policy change. Rather, it seeks to shed

light on where we are now—ethically, legally, scientifically,

and medically—in order that the President, the Congress, and

the nation may be better informed as they consider where we

should go in the future.

I. WHAT ARE STEM CELLS, AND WHY IS THERE

 CONTENTION ABOUT THEM?

The term “stem cells” refers to a diverse group of remarkable

multipotent cells. Themselves relatively undifferentiated and

unspecialized, they can and do give rise to the differentiated

and specialized cells of the body (for example, liver cells, kidney

cells, brain cells). All specialized cells arise originally from stem

cells, and ultimately from a small number of embryonic cells

that appear during the first few days of development.*

 As befits

their being and functioning as progenitor cells, all stem cells

share two characteristic properties: (1) the capacity for

unlimited or prolonged self-renewal (that is, the capability to

maintain a pool of similarly undifferentiated stem cells), and

(2) the potential to produce differentiated descendant cell

types. As stem cells within a developing human embryo

differentiate in vivo, their capacity to diversify generally

* These cells are grouped together as the “inner cell mass” of the embryo,

at the blastocyst stage of its development. Readers should consult the Glossary for definitions of technical terms and Appendix A for an illustrated

guide to the embryonic developments referred to in this report.
INTRODUCTION 3

becomes more limited and their ability to generate many

differentiated cell types generally becomes more restricted.

Stem cells first arise during embryonic development and

exist at all developmental stages and in many systems of the

body throughout life. The best described to date are the bloodforming (hematopoietic) stem cells of the bone marrow, the

progeny of which differentiate (throughout life) into the various

types of red, white, and other cells of the blood. It appears

that some stem cells travel through the circulatory system,

from their tissue of origin, to take up residence in other

locations within the body, from which they may be isolated.

Other stem cells may be obtained at birth, from blood contained

in the newborn’s umbilical cord. Once isolated and cultured

outside the body, stem cells are available for scientific

investigation. Unlike more differentiated cells, stem cells can

be propagated in vitro for many generations—perhaps an

unlimited number—of cell-doublings.

Stem cells are of interest for two major reasons, the one

scientific, the other medical. First, stem cells provide a

wonderful tool for the study of cellular and developmental

processes, both normal and abnormal. With them, scientists

hope to be able to figure out the molecular mechanisms of

differentiation through which cells become specialized and

organized into tissues and organs. They hope to understand

how these mechanisms work when they work well, and what

goes wrong when they work badly. Second, stem cells and

their derivatives may prove a valuable source of transplantable

cells and tissues for repair and regeneration. If these healing

powers could be harnessed, the medical benefits for

humankind would be immense, perhaps ushering in an era of

truly regenerative medicine. No wonder that scientists around

the world are actively pursuing research with stem cells.

Why, then, is there public contention about stem cell

research? Not because anyone questions the goals of such

research, but primarily because there are, for many people,

ethical issues connected to the means of obtaining some of

the cells. The main source of contention arises because some

especially useful stem cells can be derived from early-stage

human embryos, which must be destroyed in the process of

obtaining the cells. Arguments about the ethics of using human
4 MONITORING STEM CELL RESEARCH

embryos in research are not new. They date back to the mid1970s, beginning not long after in vitro fertilization (IVF) was

first successfully accomplished with human egg and sperm in

1969. A decade later, after IVF had entered clinical practice

for the treatment of infertility, arguments continued regarding

the fate and possible uses of the so-called “spare embryos,”

embryos produced in excess of reproductive needs and

subsequently frozen and stored in the assisted-reproduction

clinics. Although research using these embryos has never been

illegal in the United States (except in a few states), the federal

government has never funded it, and since 1995 Congress has

enacted annual legislation prohibiting the federal government

from using taxpayer dollars to support any research in which

human embryos are harmed or destroyed.

Although the arguments about embryo research had been

going on for twenty-five years, they took on new urgency in

1998, when the current stem cell controversy began. It was

precipitated by the separate publication, by two teams of

American researchers, of methods for culturing cell lines

derived, respectively, from: (1) cells taken from the inner cell

mass of very early embryos, and (2) the gonadal ridges of

aborted fetuses.2,3 (In this report, we shall generally refer to

the cell lines derived from these sources as, respectively,

embryonic stem cells [or “ES cells”] and embryonic germ cells

[or “EG cells”].) This work, conducted in university laboratories

in collaboration with and with financial support from Geron

Corporation, prompted great excitement and has already led

to much interesting research, here and abroad. It has also

sparked a moral and political debate about federal support

for such research: Is it morally permissible to withhold support

from research that holds such human promise? Is it morally

permissible to pursue or publicly support (even beneficial)

research that depends on the exploitation and destruction of

nascent human life?

Persons interested in the debate should note at the outset

that ES and EG cells are not themselves embryos; they are not

whole organisms, nor can they be made (directly) to become

whole organisms. Moreover, once a given line of ES or EG cells

has been derived and grown in laboratory culture, no further

embryos (or fetuses) need be used or destroyed in order to
INTRODUCTION 5

work with cells from that line. But it is not clear whether these

lines can persist indefinitely, and only very few lines,

representing only a few genetic backgrounds, have been made.

Thus there is continuing scientific interest in developing new

embryonic stem cell lines, and the existence of large numbers

of stored cryopreserved embryos in assisted-reproduction

clinics provides a potential source for such additional

derivations. Complicating the debate has been the study of

another group of stem cells, commonly called “adult stem

cells,” derived not from embryos but from the many different

tissues in the bodies of adults or children—sources exempt

from the moral debate about obtaining ES and EG cells. For

this reason, we often hear arguments about the relative

scientific merits and therapeutic potential of embryonic and

adult stem cells, arguments in which the moral positions of

the competing advocates might sometimes influence their

assessments of the scientific facts. Further complicating the

situation are the large commercial interests already invested

in stem cell research and the competition this creates in

research and development not only in the United States but

throughout the world. The seemingly small decision about the

funding of stem cell research may have very large implications.

II. BROADER ETHICAL ISSUES

While most of the public controversy has focused on the

issue of embryo use and destruction, other ethical and policy

issues have also attracted attention.*

 Although entangled with

the issue of embryos, the question of the significance and use

of federal funds is itself a contested issue: Should moral

considerations be used to decide what sort of research may or

may not be funded? What is the symbolic and moral-political

significance of providing national approval, in the form of active

support, for practices that many Americans regard as

abhorrent or objectionable? Conversely, what is the symbolic

and moral-political significance of refusing to support

*

 Introduced here, these issues and the discussions they have produced

will be reviewed in Chapter 3.
6 MONITORING STEM CELL RESEARCH

potentially life-saving scientific investigations that many

Americans regard as morally obligatory?

Even for those who favor embryo research, there are

questions about its proper limits and the means of establishing

and enforcing those limits through meaningful regulation.

Under the present arrangement, with the federal government

only recently in the picture, what is done with human embryos,

especially in the private sector, is entirely unregulated (save

in those states that have enacted special statutes dealing with

embryo or stem cell research). Is this a desirable arrangement?

Can some other system be devised, one that protects the

human goods we care about but that does not do more harm

than good? What are those human goods? What boundaries

can and should we try to establish, and how?

Although well-established therapies based on

transplantation of stem cell-derived tissues are still largely in

the future, concern has already been expressed (as it has been

about other aspects of health care in the United States) about

access to any realized benefits and about research priorities:

Will these benefits be equitably available, regardless of ability

to pay? How should the emergence of the new field of stem

cell research alter the allocation of our limited resources for

biomedical research? How, in a morally and politically

controverted area of research, should the balance be struck

between public and private sources of support? As with any

emerging discovery, how can we distinguish between genuine

promise and “hype,” and between the more urgent and the

less urgent medical needs calling out for assistance?4

There are also sensitive issues regarding premature claims

of cures for diseases that are not scientifically substantiated

and the potential exploitation of sick people and their families.

Some advocates of stem cell research have made bold claims

about the number of people who will be helped should the

research go forward, hoping to generate sympathy for

increased research funding among legislators and the public.

A few advocates have gone so far as to blame (in advance)

opponents of embryonic stem cell research for those who will

die unless the research goes forward today. At the same time,

other scientists have cautioned that the pace of progress will

be very slow, and that no cures can be guaranteed in advance.
INTRODUCTION 7

Which of these claims and counterclaims is closer to the truth

cannot be known ahead of time. Only once the proper scientific

studies are conducted will we discover the potential

therapeutic value of stem cells from any source. How, then, in

the meantime should we discuss these matters, offering

encouragement but without misleading or exploiting the fears

and hopes of the desperately ill?

Finally, questions are raised by some about the social

significance of accepting the use of nascent human life as a

resource for scientific investigation and the search for cures.

Such questions have been raised even by people who do not

regard an early human embryo as fully “one of us,” and who

are concerned not so much about the fate of individual embryos

as they are about the character and sensibilities of a society

that comes to normalize such practices.5

 What would our

society be like if it came to treat as acceptable or normal the

exploitation of what hitherto were regarded as the seeds of

the next generation? Conversely, exactly analogous questions

are raised by some about the social significance of refusing to

use these 150-to-200-cell early human embryos as a resource

for responsible scientific investigation and the search for cures.

What would a society be like if it refused, for moral scruples

about (merely) nascent life, to encourage every thoughtful and

scientifically sound effort to heal disease and relieve the

suffering of fully developed human beings among us?6

It is against the background of such moral-political

discussion and argument that the Council has taken up its

work of monitoring recent developments in stem cell research.

We are duly impressed with the difficulty of the subject and

the high stakes involved. All the more reason to enable the

debate to proceed on the basis of the best knowledge available,

both about science and medicine and about ethics, law, and

policy. Before proceeding to the results of our monitoring, we

complete this introduction with some additional comments on

the different types of stem cells, a few terminological

observations and clarifications, and an overview of the report

as a whole.
8 MONITORING STEM CELL RESEARCH

III. TYPES OF STEM CELLS: AN INTRODUCTION

Although we shall report later (in Chapter 4) on recent

developments in basic and clinical research using various

types of stem cells, we think the following introduction to the

“cast of characters” would be useful at the start.*

A. Embryonic Stem (ES) Cells

As noted above, ES cells are derived from the inner cell

mass of embryos at the blastocyst stage, roughly five to nine

days after fertilization—after the zygote has divided enough

times to result in about 200 cells, but before it has undergone

gastrulation and differentiation into the three primary germ

layers (see Appendix A).†

 The inner cell mass is the part of the

blastocyst-stage embryo whose cells normally go on to become

the body of the new individual. The outer cells of the blastocyststage embryo (the trophoblast cells) normally (that is, in vivo)

go on to become the fetal contribution to the placenta and

other structures that connect the developing individual to the

mother’s bloodstream and that otherwise support the

embryo’s further development. Collecting the cells of the inner

cell mass results in the destruction of the developing organism.

* The remarks about embryo-derived cells presented in the next two sections apply to human embryonic stem cells, as opposed to, say, mouse

embryonic stem cells (which will be referred to in several places because

they have provided the basis for much of what we now know about embryonic stem cells).

† In this report, we will not call the cells contained in the inner-cell-mass

“stem cells,” so long as they remain inside the intact embryo. We reserve

the term “stem cells” for those cells that are successfully cultured outside

the embryo, following artful derivation, and that demonstrate the characteristic capacities of “stemness”: a capacity for self-renewal and a capacity for differentiation. Inner-cell-mass cells may or may not be identical to

ES cells, though in an intact embryo the inner-cell-mass cells are still part

of a nascent organic whole. Indeed, it is important to remember that the

developmental fate of all cells inside the body is in part a function of their

location within the larger whole and of the influences of the local embryonic environments to which they are subject.
INTRODUCTION 9

The embryos from which human stem cells can be derived are

available (so far) only from in vitro fertilization (IVF): they have

been conceived by a combination of egg and sperm, occurring

outside the body.*

B. Embryonic Germ (EG) Cells

EG cells are stem cells that are isolated from the gonadal

ridge of a developing fetus. These are the cells that ultimately

give rise to sperm cells or egg cells, depending on the sex of

the fetus. The EG cells are collected from the bodies of five-tonine-week-old fetuses that have been donated after induced

abortions.† In federally funded research, collection of the EG

cells is governed by existing federal regulations for fetal-tissue

donation, designed (among other things) to ensure the

separation of the decision to terminate pregnancy from the

decision to donate the fetal tissue for research.7

Cell lines established from either of these two sources (ES

and EG cells, from embryos and fetal gonads, respectively)

have demonstrated two important properties: great ability to

multiply and form stable lines that can be characterized, and

great flexibility and plasticity. Their progeny can differentiate

in vitro into cells with characteristics of those normally derived

from all three embryonic germ layers (ectoderm, endoderm,

and mesoderm), which layers (in vivo) give rise in turn to all

the different types of cells in the body. Because they are so

flexible, it also seems likely that they could be used to produce

cell preparations that could then be transplanted (assuming

that the recipient’s immune response could be managed) to

repopulate a part of the body such as the pancreas or spinal

cord that has lost function due to disease or injury. As with

stem cells derived from the various tissues of the adult body,

*

 As of this writing, experiments in asexual methods of conceiving a human embryo, such as parthenogenesis or cloning, have not, to our knowledge, been successful beyond the very early stages of development. Embryos, fertilized in vivo, could also be procured for use in research by flushing them from the womb, but this procedure, though technically feasible,

has a very low yield and is rarely done.

† Abortion is legal throughout the United States, pursuant to a series of

federal Supreme Court decisions, the most important cases being Roe v.

Wade (1973) and Casey v. Planned Parenthood (1992).
10 MONITORING STEM CELL RESEARCH

ES cells and EG cells seem to hold out hope for an era of

regenerative medicine.

C. Adult (or Non-embryonic) Stem Cells

Adult stem cells are more differentiated than ES or EG cells,

but not yet fully differentiated. Like stem cells of embryonic

origin, they can give rise to lineages of cells that are more

specialized than themselves. The term “adult” is a bit of a

misnomer (“non-embryonic” would be more accurate): these

cells are found in various tissues in children as well as adults

(and in fetuses as well), and they have been isolated from

umbilical cord blood at the time of delivery. Despite its

inaccuracy regarding the origin of the cells, the term “adult”

helpfully emphasizes that the cells have been partially

differentiated. Although they can give rise to various cell types,

these non-embryonic stem cells are generally all within the

same broad type of tissue (for example, muscle stem cells,

adipose stem cells, neural stem cells). For this reason, it had

long been thought that they are less flexible than those derived

from embryos or fetal gonads. Yet this presumption has been

disputed in recent years by those who think that certain forms

of adult stem cells may be equally or nearly as plastic as nonadult stem cells. Indeed, possible exceptions to the

generalization that adult stem cells give rise only to cell types

found within their own broad type of tissue have recently been

reported (though most of these cells may well be shorter-lived

than ES cells, and, if so, potentially less useful in therapy).

This finding has ignited a debate about the relative merits of

embryonic stem cells and adult stem cells: which is more

valuable, both for research and (especially) for clinical

treatment?*

Research involving adult stem cells raises few difficult

ethical concerns, beyond the usual need to secure free and

*

 We shall review recent work with both kinds of cells in Chapter 4. Anticipating the implications of that discussion, we may safely say that not

enough is known to answer this empirical question. Work with both kinds

of cells seems promising. Some people argue that research with non-embryonic stem cells, being a morally unproblematic path, should be given

priority. Most researchers, meanwhile, support the advancement of work

with both kinds of cells simultaneously, to explore their potential.
INTRODUCTION 11

fully informed consent from donors and recipients, a favorable

benefit-to-risk ratio for all participants in attempts at therapy

and protection of privacy. Adult stem cells are less controversial

than embryonic ones, as we have noted, because the former

can be collected without lasting harm to the donor.

D. Cord Blood Stem Cells

Though clearly a type of non-embryonic stem cell, cord blood

stem cells deserve some special mention. Blood found in the

umbilical cord can be collected at birth and hematopoietic stem

cells (and other progenitor cells) isolated from it. It has been

proposed that individually banked cord blood cells may, at

some later time, offer a good match for a patient needing stem

cell-based treatments, whether the individual cord-blooddonor himself or a close relative, and in unrelated recipients

may require a less exact genetic match than adult bone

marrow.* †

*

 Several companies in the United States have sprung up to offer commercial storage services for cord blood in case the child or a closely genetically-matched sibling should later need the stem cells contained in the cord

blood for medical use. It is unclear whether individual banking of cord blood

will turn out to be valuable. It may turn out that, for the vast majority of

people, the cells are never needed, or that, when therapy is needed, the

stored cells are found to be unsuitable or incapable of meeting the need in

the time required. At the same time, Congress has recently allocated funds

to create a national non-commercial cord blood bank potentially available

to all patients. The authors of the legislation argue that a national bank

would have cord blood of many different types, increasing the odds that a

patient would find a match.

†

 The possibility of therapeutic use of cord blood stem cells has raised a

serious question unrelated to the ethics of stem cell research: whether

parents of a sick child may morally conceive another child, of genetic makeup appropriate for providing compatible cord blood cells, primarily to treat

the first child. In generating the second child, a prospective parent or parents might screen preimplantation embryos for genetic suitability to provide the cells (both compatible blood type and freedom from the genetic

disease affecting the older sibling). These and other ethical questions surrounding preimplantation genetic diagnosis (PGD) go beyond our present

subject and will not be considered further in this report.
12 MONITORING STEM CELL RESEARCH

IV. TERMINOLOGY

In considering complicated or contested public questions,

language matters—even more than it ordinarily does. Clear

thinking depends on clear ideas, and clear ideas can be

conveyed only through clear and precise speech. And fairness

in ethical evaluation and judgment depends on fair framing of

the ethical questions, which in turn requires fair and accurate

description of the relevant facts of the case at hand. Such

considerations are highly pertinent to our topic and to the

arguments it generates.

Confounding the discussions of stem cell research, there

are, to begin with, difficult technical concepts, referring to

complicated biological entities and phenomena, that can cause

confusion among all but the experts. Some of these concepts

we will clarify in Chapter 4, and others are defined in the

Glossary and, in some cases, illustrated in Appendix A on early

embryonic development. But the more important

terminological issues are those used to formulate the ethical

and policy issues about which people so vigorously disagree.

We pause to comment on three of them: “the embryo” (or “the

human embryo”), “spare embryos,” and “the moral status of

the embryo.”

Strictly speaking, there is no such thing as “the embryo,” if

by this is meant a distinctive being (or kind of being) that

deserves a common, reified name—like “dog” or “elephant.”

Rather, the term properly intends a certain stage of

development of an organism of a distinctive kind. Indeed, the

very term comes from a Greek root meaning “to grow”: an

embryo is, by its name and mode of being, an immature and

growing organism in an early phase of its development.*

 The

advent of in vitro fertilization, in which living human embryos

*

 In classical embryology, “embryo” is the name given—somewhat arbitrarily—to the developing human organism from the time of fertilization

until roughly eight weeks, the time that the first calcification of bone occurs. After that, the developing human organism is called a “fetus,” equally a reified name for a dynamic entity, an entity-in-the-process-of-becoming-more-fully-the-kind-of-organism-it-already-is.
INTRODUCTION 13

from their first moments are encountered as independent

entities outside the body of a mother, before human eyes and

in human hands, may also have contributed to this tendency

to reify “the embryo” in its early stages (though such reification

has likely always played a role in embryology). The ex vivo

existence of nascent human life is genuinely puzzling and may

invite terminology that can be distorting.*

If the term “the embryo” risks conveying the false notion

that embryos are distinct kinds of beings or things, the term

“spare embryo” risks making a difficult moral question seem

easier than it is. The term is frequently used to describe those

embryos, produced (each with reproductive intent, but in

excess of what is needed) in assisted-reproduction clinics, that

are not transferred to a woman in attempts to initiate a

pregnancy. No longer needed to produce a child, they are

usually frozen and stored for possible later use, should the

first efforts fail. But the “spareness” of a “spare embryo” is

not a property of a particular embryo itself; it bespeaks rather

our attitude toward it, now that it may no longer be needed to

serve the purpose for which it was initially brought into being.

Calling something “spare,” or only “extra,” invites the thought

that nothing much is lost should it disappear, because one

already has more than enough: one has “embryos to spare.” It

also abstracts from the distinct genetic individuality of each

embryo and invites the view that embryos are, like commercial

products, simply interchangeable—an outlook that may affect

the further judgment of any embryo’s moral standing. To be

sure, most of these unused embryos will die or be destroyed.

To be sure, if these unused embryos are otherwise destined

for destruction, a case can be made—and debated—that their

unavoidable loss should be redeemed by putting them to use

beforehand. But the moral question regarding their possible

*The Council is well aware of the fact that the debate about abortion shadows all these discussions about “embryos.” Yet in all of our work to date,

on cloning and stem cell research, we have called attention to the fact that

we face a rather different moral situation when we are dealing with embryos in the laboratory, in the absence of concerns for a pregnant woman’s

life and future. Accordingly, we explore the ethical issues of embryo research by addressing what we know (and how we know) about ex vivo

human embryos, separate from any issues that enter when the interests of

pregnant women are engaged.
14 MONITORING STEM CELL RESEARCH

use and destruction should not be decided—here, as

elsewhere—on terminological grounds, in this case, by the

naming of the embryo “spare.” Rather it should be decided on

the basis of a direct moral appraisal of the rights and goods

involved: on the basis of what we owe to suffering humanity

and the obligations we have to seek the means of its relief;

and on the basis of the nature of human embryos, what we

owe them as proper respect and regard, and whether and why

such respect or regard may be overridden.*

 For many people,

the moral question depends, in other words, on what some

bioethicists call—and we ourselves will sometimes call—“the

moral status of the embryo.” If embryos lacked all “moral

status,” there would be little moral argument about their use

and destruction.

Yet the notion “moral status” is problematic, even though

it is easy to understand why it has come into fashion. For many

people, the central ethical question regarding embryonic stem

cell research is whether an embryonic organism from which

cells may be removed to develop ES cells is fully “one of us,”

deserving the same kind of respect and protection as a

newborn baby, child, or adult. What they want to know is the

moral standing of these organisms—entities that owe their

existence, their extra-uterine situation, and their “spare-ness”

to deliberate human agency—at such early stages of

development. As we shall see, some people try to find

structural or functional markers—for example, the familiar

human form or the presence or absence of sensation—to decide

the moral worth of a human embryo. Others use an argument

from continuity of development to rebut any attempt to find a

morally significant boundary anywhere along the continuum

of growth and change. But, to judge from countless efforts to

provide a biologically based criterion for ascribing full human

worth, it seems certain that we shall never find an answer to

our moral question in biology alone, even as the answers we

give must take into account the truths of embryology. At least

until now, philosophical attempts to draw moral inferences

* Some Members of this Council (including Alfonso Gómez-Lobo and Robert George) hold that the moral question should be decided on the basis of

the prior consideration of the rightness or wrongness of intentionally destroying human beings for the sake of further goals, and then on whether

or not human embryos are human beings in the relevant sense.
INTRODUCTION 15

from the biological facts have not yielded conclusions that all

find necessary or sound.

Under these circumstances, some people believe that we

have no choice but to stipulate or ascribe some degree of moral

“status” to the entity, based either on how it strikes us and

the limited range of what we are able to know about it, or on

what we wish to do with it: we confer upon it some moral

status in regard to us, much as we confer one or another class

of immigration status upon people.8

 For this very reason, others

object to the term, fearing that it enables us to beg the question

of the intrinsic moral worth or dignity of the entity itself, seen

in its own terms and without regard to us. Different Members

of this Council hold different views of this terminological and

ontological matter, but we all recognize the moral freight

carried by attempts to speak about and ascribe “moral status”

to human embryos in their earliest stage of development.* We

encourage readers to be self-conscious about this and similar

terms, even as we proceed ourselves to make use of them.

V. ABOUT THE REPORT

Monitoring stem cell research can be a bit like watching

Niagara Falls. Not only do scientific reports pour forth daily,

as they do in many other areas of research, but a kind of mist

rises up for the torrent of news flashes and editorials, making

it difficult to separate knowledge from opinion and hope from

hype. The underlying biology—whether viewed at the level of

the gene, cell, tissue, organ, or organism—is dauntingly

complex, as is all cell biology. At any of these levels, in this

new and dynamic field, it is frequently difficult for even the

most knowledgeable scientist to be truly certain of “what really

causes what.” For example, how exactly do certain kinds of

stem cells have their apparently beneficial effects on heart

* It is, of course, possible to hold the view that the earliest human embryos

have no moral status or worth, because they are so small and undifferentiated or because they lack the ordinary human shape and form or the specifically human capacities for sensation or consciousness or the capacity

to develop on their own ex vivo. Some of these arguments are reviewed in

Chapter 3. Here it suffices to observe that at least one Member of this

Council (Michael Gazzaniga) holds this view.
16 MONITORING STEM CELL RESEARCH

disease when the cells are extracted from a cardiac patient’s

bone marrow or muscle, expanded in culture, and injected into

the patient’s heart? Or what is responsible for the positive

effects on a Parkinson Disease patient when cells from his own

brain are similarly extracted, treated, and re-injected? We do

not yet really know precisely what stem cell-based

preparations do when put into the body.

At the same time, all discussion in this area suffers from a

persistent background tension. The stakes are high, or seem

so, to many of the discussants, and there is much politicking

involved. As noted earlier, opponents of embryo research try

to tout the virtues of adult stem cells, because they regard

their use as a morally permissible alternative. Proponents, for

their part, often find it tempting to disparage or downplay all

adult stem cell studies and to emphasize instead what they

believe to be the superior potential of embryonic stem cells

for successful future therapeutic use. Navigating between

these tendencies in search of the full truth can be daunting,

and few people are altogether immune to the partial but

seductive calls from the scientific or moral side they prefer.

Yet without denying our individual differences on the ethical

and policy questions at issue, the Council has sought in this

monitoring report to present a fair-minded and thorough

overview, both of the ethical and policy debates and of the

scientific and medical results to date. To aid us in our task of

monitoring, we have commissioned six review articles and

heard several oral presentations on the state of research,

covering studies using embryonic and studies using adult stem

cells. We have commissioned a review article and heard a

presentation on the problem of immune rejection, a potential

major stumbling block to effective cell transplantation

therapies.

We have read papers, commissioned writings, heard

presentations, and debated among ourselves about the various

ethical and philosophical issues involved, from “the moral

status of the embryo,” to the existence of a moral imperative

to do research, to the meaning of federal funding of morally

controversial activities. We have read and heard public

testimony from both supporters and opponents of the current

policy on federal funding of ES cell research.
INTRODUCTION 17

We have considered arguments—presented by scientists

and patient-advocacy groups, and shared by some Members

of the Council—that the current policy is impeding potentially

life-saving research, for example, by offering researchers too

few useful ES cell lines to work with, by causing a chilling

effect on the whole field, or by allowing the field to be

dominated by private companies, less given (than are publiclyfunded academic scientists) to publishing and sharing the

results of their research. We have considered arguments—

presented by various critics and opponents of embryonic stem

cell research, and shared by some Members of the Council—

that the current policy has opened the path toward the

possibility of “embryo farming” or that it risks weakening our

respect for nascent life and our willingness to protect the

weakest lives among us. We have heard from ethicists and

scientific researchers, representatives of biopharmaceutical

companies and disease research foundations, and senior

government officials from such agencies as the National

Institutes of Health and the Food and Drug Administration.

We benefited from working papers prepared by the Council’s

staff and from existing reports on stem cell research, and in

particular reports by the National Bioethics Advisory

Commission (1999) and the National Academies (2001).9

Holding our own personal views in abeyance, we have tried

in the three chapters that follow to synthesize accurately and

fairly what we have heard and learned: about current law and

policy, about the state of the ethical debate, and about the

current state of scientific research.

Chapter 2, “Current Federal Law and Policy,” describes and

explains the current federal policy regarding stem cell research.

It locates that policy in relation to previous law and policy

touching this area of research and tries to make clear the

ethical, legal, and prudential foundations on which the policy

rests. It then describes the implementation of the policy and

other relevant considerations. Our goal in that chapter is to

describe and understand the present policy situation, in its

legal, political, scientific, and ethical colorations, and to present

accurately the various features of the current federal policy,

many of which are not generally well understood.
18 MONITORING STEM CELL RESEARCH

Chapter 3, “Recent Developments in the Ethical and Policy

Debates,” provides an overview of the ethical and policy

debates surrounding stem cell research in the past two years.

Special attention is, of course, given to arguments about what

may (or may not) be done with human embryos, and why. But

those arguments are also reviewed in relation to larger debates

about the other ethical and policy issues mentioned earlier.

Our goal in that chapter is to present the arguments and

counter-arguments, faithfully and accurately, rather than finally

to assess their validity.

Finally, in Chapter 4, “Recent Developments in Stem Cell

Research and Therapy,” we offer an overview of some recent

developments in the isolation and characterization of various

kinds of stem cell preparations and a partial account of some

significant research and clinical initiatives. In addition, by

means of a selected case study, we consider how stem cellbased therapies might some day work to cure devastating

human diseases, as well as the obstacles that need to be

overcome before that dream can become a reality. Our goal in

that chapter, as supplemented by several detailed

commissioned review articles contained in the appendices, is

to enable (especially non-scientific) readers to appreciate the

reasons for the excitement over stem cell research, the

complexities of working with these materials, some early

intriguing research and therapeutic findings, and the difficult

road that must be traveled before we can reap therapeutic

and other benefits from this potentially highly fertile field of

research.

After these three substantive chapters—on policy, ethics,

and science—we offer a Glossary and a series of appendices,

beginning (in Appendix A) with a brief primer on early human

embryonic development. That primer aspires to provide the

basic facts and concepts that any thoughtful and publicspirited person needs to know about human development and

especially about (early) human embryos if he or she is to

participate intelligently in the ethical and political

deliberations that are certain to continue in our society for

some time. There follow the texts of President Bush’s August

9, 2001, stem cell speech and the NIH guidelines (for both the

Clinton and Bush administrations) regarding the funding of
INTRODUCTION 19

embryonic stem cell research. Completing the appendices are

the texts of all the papers that the Council commissioned, as

revised by their authors in light of subsequent developments

or comments received. These papers appear in the authors’

own words, unedited by the Council.

In all that we offer in this monitoring report, we have aspired

to be careful and fair in our approach, precise in our use of

language, accurate in presenting data and arguments, and

thoughtful in our laying out of the various issues that remain

before us. It is up to our readers to judge whether or not we

have succeeded. The policy debates over stem cell research

that led to the creation of this Council continue; they, and other

debates on related topics, are unlikely to go away any time

soon. Our hope is that our work will help to make those debates

richer, fairer, and better informed.
20 MONITORING STEM CELL RESEARCH

ENDNOTES

1

 “Remarks by the President on Stem Cell Research,” Crawford, Texas, August 9,

2001. Text made available by the White House Press Office, August 9, 2001. (Also

available in full at Appendix B of this report.)

2

 Thomson, J., et al., “Embryonic stem cell lines derived from human blastocysts,”

Science 282: 1145-1147 (1998).

3

 Shamblott, M., et al., “Derivation of pluripotent stem cells from cultured human

primordial germ cells,” Proceedings of the National Academy of Science 95: 13726-

13731 (1998).

4

 See, among others, Dresser, R., “Embryonic Stem Cells: Expanding the Analysis,”

American Journal of Bioethics 2(1): 40-41 (2003); and the personal statements of

Council Members Rebecca Dresser and William May, appended to the Council’s

July 2002 report Human Cloning and Human Dignity: An Ethical Inquiry.

5

 See, among others, Kass, L., “The Meaning of Life – In the Laboratory,” The Public

Interest, Winter 2002; Cohen, E., “Of Embryos and Empire,” The New Atlantis 2: 3-

16 (2003); and “The Moral Case against Cloning-for-Biomedical-Research” presented

by some Members of the Council in the Council’s July 2002 report Human Cloning

and Human Dignity: An Ethical Inquiry, Chapter 6.

6

 See, for instance, “The Moral Case for Cloning-for-Biomedical-Research” presented

by some Members of the Council in the Council’s July 2002 report Human Cloning

and Human Dignity: An Ethical Inquiry, Chapter 6; and the personal statements of

Council Members Elizabeth Blackburn, Daniel Foster, Michael Gazzaniga, and Janet

Rowley appended to that report.

7

 45 C.F.R. § 46.204(h-i).

8

 See, for instance, Green, R., The Human Embryo Research Debates, New York:

Oxford University Press (2001). Also see the discussion of the Council in its October

17, 2003, meeting, particularly the comments of Council Member Alfonso GómezLobo. A transcript of that session is available on the Council’s website at

www.bioethics.gov.

9

 National Bioethics Advisory Commission (NBAC), Ethical Issues in Human Stem

Cell Research, Bethesda, MD: Government Printing Office (1999); National Research

Council/Institute of Medicine (NRC/IOM), Stem Cells and the Future of Regenerative

Medicine, Washington, D.C.: National Academy Press (2001).
2

Current Federal Law and Policy

Any overview of the state of human stem cell research under the current federal funding policy must begin with a thorough understanding of that policy. This is not as simple as it

may sound. From the moment of its first announcement, on

August 9, 2001, the policy has been misunderstood (and at

times misrepresented) by some among both its detractors and

its advocates. Its moral foundation, its political context, its

practical implications, and the most basic facts regarding the

policy’s implementation have all been subjects of heated dispute and profound confusion. Whether one agrees with the

policy or not, it is important to understand it as it was propounded, accurately and in its own terms, in the light also of

the historical and political contexts in which it was put forward.

This chapter attempts to place the policy in its proper

context; to articulate its moral, legal, and political

underpinnings (as put forward by its authors and advocates);

to offer an overview of its implementation thus far; and to begin

to describe its ramifications for researchers and for medicine.

By articulating the policy in its own terms, we intend neither

to endorse it nor to find fault with it.*

 Indeed, in the next chapter

*

 Some Members of the Council oppose the current policy, some Members

support it. Yet the descriptive account that we offer here aspires to be

seen as accurate and fair, regardless of where one personally stands on

the issue. Nearly all Members of this Council recognize, as we said in our

report Human Cloning and Human Dignity, that “all parties to this debate

21
22 MONITORING STEM CELL RESEARCH

we present an overview of arguments on all sides of the

question. Here we mean only to clarify, as far as we are able,

the original meaning and purpose of the policy, so as to be

better able to monitor its impact.

I. A BRIEF HISTORY OF THE EMBRYO RESEARCH

FUNDING DEBATE

The federal government makes significant public resources

available to biomedical researchers each year—over $20 billion

in fiscal year 2003 alone—in the form of research grants offered

largely through the National Institutes of Health (NIH). This

level of public expenditure reflects the great esteem in which

Americans hold the biomedical enterprise and the value we

place on the development of treatments and cures for those

who are suffering. But such support is not offered

indiscriminately. Researchers who accept federal funds must

abide by ethically based rules and regulations governing,

among other things, the use of human subjects in research.

And some policymakers and citizens have always insisted that

taxpayer dollars not be put toward specific sorts of research

that violate the moral convictions and sensibilities of some

portion of the American public. This has meant that

controversies surrounding the morality of some forms of

scientific research have at times given rise to disputes over

federal funding policy. Among the most prominent examples

has been the three-decade-long public and political debate

about whether taxpayer funds should be used to support

have something vital to defend, something vital not only to themselves but

also to their opponents in the debate, and indeed to all human beings. No

human being and no human society can afford to be callous to the needs of

suffering humanity, cavalier regarding the treatment of nascent human life,

or indifferent to the social effects of adopting in these matters one course

of action rather than another.” (Human Cloning and Human Dignity, p 121.)

Thus, whatever we think of the current funding policy, we recognize that

this is a genuine ethical dilemma and that reasonable people of good will

may come to different conclusions about where the best ethical or policy

position lies. We therefore also believe that not only results but also reasons matter, and that it behooves us to understand the principled or prudential reasons for the current policy (as well as for any alternative policy

that might be offered to replace it).
CURRENT FEDERAL LAW AND POLICY 23

research that involves creating or destroying human embryos

or making use of destroyed embryos and fetuses—practices

that touch directly on the much-disputed questions of the moral

status and proper treatment of nascent human life.

In the immediate aftermath of the Supreme Court’s 1973

Roe v. Wade decision legalizing abortion nationwide, some

Americans, including some Members of Congress, became

concerned about the potential use of aborted fetuses (or

embryos) in scientific research. In response to these concerns,

the Department of Health, Education and Welfare (DHEW, the

precursor to today’s Department of Health and Human

Services) initiated a moratorium on any potential DHEW

sponsorship or funding of research using human fetuses or

living embryos. In 1974, Congress codified the policy in law,

initiating what it termed a temporary moratorium on federal

funding for clinical research using “a living human fetus, before

or after the induced abortion of such fetus, unless such

research is done for the purpose of assuring the survival of

such fetus.”1

 Concurrently with that moratorium (and also

addressing concerns not directly related to embryo and fetal

research), Congress established a National Commission for

the Protection of Human Subjects of Biomedical and Behavioral

Research. Among its other tasks, Congress explicitly assigned

the Commission responsibility for offering guidelines for

human fetal and embryo research, so that standards for funding

might be established and the blanket moratorium might be

lifted. The statutory moratorium was lifted once the

Commission issued its report in 1975.2

In that report, the Commission called for the establishment

of a national Ethics Advisory Board within DHEW to propose

standards and research protocols for potential federal funding

of research using human embryos and to consider particular

applications for funding. In doing so, the Commission looked

ahead to the possible uses of in vitro embryos, since the first

successful in vitro fertilization (IVF) of human egg by human

sperm had been accomplished in 1969.*

 The Department

*

 In its discussion of “fetal” research, the commission defined the fetus as

the product of conception from the time of implantation onward, which

therefore included what we generally think of (and define in this report) as

embryos in utero. Its separate consideration of embryo research was therefore directed at in vitro embryos.
24 MONITORING STEM CELL RESEARCH

adopted the recommendation in 1975, established an Ethics

Advisory Board, and put in place regulations requiring that

the Board provide advice about the ethical acceptability of IVF

research proposals. The Board first took up the issue of research

on in vitro embryos in full in the late 1970s and issued its report

in 1979.3

By that time, human IVF techniques had been developed

(first in Britain) to the point of producing a live-born child (born

in 1978). These techniques, and their implications for human

embryo research, raised unique prospects and concerns that

were distinct from some of those involved in human fetal

research. As a consequence, starting in the late 1970s, funding

of embryo research and funding of fetal research came to be

treated as mostly distinct and separate issues. The Ethics

Advisory Board concluded that research involving embryos

and IVF techniques was “ethically defensible but still

legitimately controverted.” Provided that research did not take

place on embryos beyond fourteen days of development and

that all gamete donors were married couples, the Board

argued, such work was “acceptable from an ethical

standpoint,” but the Board decided that it “should not advise

the Department on the level of Federal support, if any,” such

work should receive.4

This left the decision in the hands of the DHEW, which

decided at that stage not to offer funding for human embryo

studies. The Ethics Advisory Board’s charter expired in 1980,

and no renewal or replacement was put forward, creating a

peculiar situation in which the regulations requiring the Ethics

Advisory Board to review proposals for funding remained in

effect, but the Board no longer existed to consider such

requests. Funding was therefore rendered impossible in

practice. Because the Ethics Advisory Board was never

replaced, a de facto ban on funding remained in place through

the 1980s.

In 1993, Congress enacted the NIH Revitalization Act, a

provision of which rescinded the requirement that research

protocols be approved by the non-existent Ethics Advisory

Board.5 This change opened the way in principle to the

possibility of NIH funding for human embryo research using

IVF embryos. The following year, the NIH convened a Human
CURRENT FEDERAL LAW AND POLICY 25

Embryo Research Panel to consider the issues surrounding

such research and to propose guidelines for potential funding

applications. The panel recommended that some areas of

human embryo research be deemed eligible for federal funding

within a framework of recognized ethical safeguards. It further

concluded that the creation of human embryos with the explicit

intention of using them only for research purposes should be

supported under some circumstances.6

 President Clinton

overruled the panel on the latter point, ordering that embryo

creation for research not be funded, but he accepted the panel’s

other recommendations and permitted the NIH to consider

applications for funding of research using embryos left over

from IVF procedures.7

Congress, however, did not endorse this course of action.

In 1995, before any funding proposal had ever been approved

by the NIH, Congress attached language to the 1996

Departments of Labor, Health and Human Services, and

Education, and Related Agencies Appropriations Act (the

budget bill that funds DHHS and the NIH) prohibiting the use

of any federal funds for research that destroys or seriously

endangers human embryos, or creates them for research

purposes.

This provision, known as the “Dickey Amendment” (after

its original author, former Representative Jay Dickey of

Arkansas), has been attached to the Health and Human

Services appropriations bill each year since 1996. Everything

about the subsequent debate over federal funding of

embryonic stem cell research must be understood in the

context of this legal restriction. The provision reads as follows:

None of the funds made available in this Act may be used

for—

(1) the creation of a human embryo or embryos for

research purposes; or

(2) research in which a human embryo or embryos are

destroyed, discarded, or knowingly subjected to risk of

injury or death greater than that allowed for research on

fetuses in utero under 45 CFR 46.204 and 46.207, and
26 MONITORING STEM CELL RESEARCH

subsection 498(b) of the Public Health Service Act (42

U.S.C. 289g(b)).*

(b) For purposes of this section, the term ‘human embryo

or embryos’ includes any organism, not protected as a

human subject under 45 CFR 46 as of the date of the

enactment of the governing appropriations act, that is

derived by fertilization, parthenogenesis, cloning, or any

other means from one or more human gametes or human

diploid cells.8

This law effectively prohibits the use of federal funds to

support any research that destroys human embryos or puts

them at serious risk of destruction. It does not, however,

prohibit the conduct of such research using private funding.

Thus, it addresses itself not to what may or may not be lawfully

done, but only to what may or may not be supported by

taxpayer dollars. At the federal level, research that involves

the destruction of embryos is neither prohibited nor supported

and encouraged.

The Dickey Amendment was originally enacted before the

isolation of human embryonic stem cells, first reported in 1998

by researchers at the University of Wisconsin, whose work

was supported only by private funds (largely from the Geron

Corporation and the University of Wisconsin Alumni Research

Foundation). The discovery of these cells and their unique and

potentially quite promising properties aroused great

excitement both within and beyond the scientific community.

It led some people to question the policy of withholding federal

funds from human embryo research. Most Members of

Congress, however, did not change their position, and the

Dickey Amendment has been reenacted every year since. For

many of its supporters, the amendment expresses their ethical

conviction that nascent human life ought to be protected

against exploitation and destruction for scientific research,

however promising that research might be, and that at the

very least such destruction should not be supported or

encouraged by taxpayer dollars.

*

 These legal citations refer to the federal regulations and federal statute

relating to research on living human fetuses.
CURRENT FEDERAL LAW AND POLICY 27

On its face, the Dickey Amendment would seem to close

the question of federal funding of human embryonic stem cell

research, since obtaining stem cells for such research relies

upon the destruction of human embryos. But in 1999, the

General Counsel of the Department of Health and Human

Services argued that the wording of the law might permit an

interpretation under which human embryonic stem cell

research could be funded. If embryos were first destroyed by

researchers supported by private funding, then subsequent

research employing the derived embryonic stem cells, now

propagated in tissue culture, might be considered eligible for

federal funding. Although such research would presuppose

and follow the prior destruction of human embryos, it would

not itself involve that destruction. Thus, the Department’s

lawyers suggested, the legal requirement not to fund research

“in which” embryos were destroyed would still technically be

obeyed.9

This has generally been taken to be a legally valid

interpretation of the specific language of the statute, and

indeed the subsequent policies of both the Clinton and Bush

administrations have relied upon it in different ways. But some

critics of the 1999 legal opinion argued that, though it might

stay within the letter of the law, the proposed approach would

contradict both the spirit of the law and the principle that

underlies it.10 It would use public funds to encourage and

reward the destruction of human embryos by promising

funding for research that immediately follows and results from

that destruction—thereby offering a financial incentive to

engage in such destruction in the future. By so doing, these

critics argued, it would at least implicitly state, in the name of

the American people, that research that destroys human

embryos ought to be encouraged in the cause of medical

advance. Supporters of the Clinton administration’s proposed

approach, however, argued that promoting such research—

especially given its therapeutic potential—was indeed an

appropriate government function, and that the policy proposed

by DHHS was neither illegal nor improper, given the text of

the statute and provided that the routine standards of research

ethics (including informed consent and a prohibition on

financial inducements) were met.11
28 MONITORING STEM CELL RESEARCH

The Clinton administration adopted this course of action

and drew up specific guidelines to enact it.*

 But the guidelines,

completed just before the end of the Clinton administration,

never had a chance to be put into practice, and no funding

was ever provided. Upon entering office in 2001, the Bush

administration decided to take another look at the options

regarding human embryonic stem cell research policy and

therefore put the new regulations on hold, pending review.

In conducting its review, the Bush administration stated

that it sought a way to allow some potentially valuable

research to proceed while upholding the spirit (and not just

the letter) of the Dickey Amendment, a spirit that the President

himself has advocated.12 The expressed hope was that the

government, while continuing to withhold taxpayer support

or encouragement for the destruction of human embryos, might

find a way to draw some moral good from stem cell lines that

had already been produced through such destruction—given

that this deed, even if immoral, could not now be undone. This

is the ethical-legal logic of the present stem cell funding policy:

it seeks those benefits of embryonic stem cell research that

might be attainable without encouraging or contributing to

any future destruction of human embryos.

II. THE PRESENT POLICY

The current policy on federal government funding of human

embryonic stem cell research, then, must be understood in

terms of the constraints of the Dickey Amendment and in terms

of the logic of the moral and political aims that underlie that

amendment.

At the time of the policy’s announcement, a number of

embryonic stem cell lines had already been derived and were

in various stages of growth and characterization. The embryos

from which they were derived had therefore already been

destroyed and could no longer develop further. As President

Bush put it, “the life and death decision had already been

made.”13

*

 These regulations, as published in the Federal Register, are provided in

Appendix D.
CURRENT FEDERAL LAW AND POLICY 29

The administration’s policy made it possible to use taxpayer

funding for research conducted on those preexisting lines, but

it refused in advance to support research on any lines created

after the date of the announcement. In addition, to be eligible

for funding, those preexisting lines would have had to have

been derived from excess embryos created solely for

reproductive purposes, made available with the informed

consent of the donors, and without any financial inducements

to the donors—standard research-ethics conditions that had

been attached to the previous administration’s short-lived

funding guidelines, as well as to earlier attempts to formulate

rules for federal funding of human embryo research. The policy

denies federal funding not only for research conducted on stem

cell lines derived from embryos destroyed after August 9, 2001

(or that fail to meet the above criteria), but also (as the proposed

Clinton-era policy would have) for the creation of any human

embryos for research purposes and for the cloning of human

embryos for any purpose.*

The moral, legal, and political grounds of this policy have

been hotly contested from the moment of its announcement.

Debates have continued regarding its aims, its character, its

implementation, and its underlying principles, as well as the

significance of federal funding in this area of research. For

example, many scientists, physicians, and patient advocacy

groups contend that the policy is too restrictive and thwarts

the growth of a crucial area of research. On the other side,

some opponents of embryo research believe the policy is too

liberal and legitimates and rewards (after the fact) the

destruction of nascent human life. Some ethicists argue that

there is a moral imperative to remove all restrictions upon

potentially life-saving research; other ethicists argue that there

is a moral imperative to protect the lives of human beings in

their earliest and most vulnerable stages. These and similar

arguments are reviewed in the next chapter. But before one

can enter into these debates, it is essential first to understand

the relevant elements of the policy itself as clearly and

distinctly as possible.

*

 The official NIH statement of this policy is provided in Appendix C.
30 MONITORING STEM CELL RESEARCH

III. MORAL FOUNDATION OF THE POLICY

In articulating its proposed funding policy in 1999 and 2000,

the Clinton administration expressed a firm determination that

funded research could use only those human embryos that

had been left over from IVF procedures aimed at reproduction

and that had been donated in accordance with the standards

of informed consent and in circumstances free of financial

inducements. Provided that these crucial conditions were met,

the administration argued that the potential benefits of stem

cell research were so great that publicly funded research

should go forward. In August of 2000, reflecting on the

guidelines put forward by his administration, President Clinton

remarked,

Human embryo research [as approved for funding by

the NIH guidelines] deals only with those embryos that

were, in effect, collected for in-vitro fertilization that

never will be used for that. So I think that the protections

are there; the most rigorous scientific standards have

been met. But if you just—just in the last couple of weeks

we’ve had story after story after story of the potential

of stem cell research to deal with these health

challenges. And I think we cannot walk away from the

potential to save lives and improve lives, to help people

literally to get up and walk, to do all kinds of things we

could never have imagined, as long as we meet rigorous

ethical standards.14

Given the promise of embryonic stem cell research, the

existence of many embryos frozen in IVF clinics and unlikely

ever to be transferred and brought to term, and the willingness

of some IVF patients to donate such embryos for research, the

Clinton administration reasoned that research using cell lines

derived from these embryos could ethically be supported by

federal funds. That position implies, of course, that the

destruction of embryos is not inherently or necessarily

unethical, or so disconcerting as to be denied any federal

support. The Clinton-era NIH Embryo Research Panel put
CURRENT FEDERAL LAW AND POLICY 31

succinctly one form of this view in stating that “the

preimplantation human embryo warrants serious moral

consideration as a developing form of human life, but it does

not have the same moral status as infants and children.”15 If

there is sufficient promise or reason to support research, the

claim of a human embryo to “serious moral consideration” (or,

as others, including some of us, have put it, to “special

respect”16) could be outweighed by other moral aims or

principles.

This (at least implicit) understanding of the moral status of

human embryos might be seen to have put the Clinton

administration at odds with the principle animating the

operative law on this subject (the Dickey Amendment). But

given its responsibility to carry out the laws as they are

enacted, the administration sought a way to advance research

within the limitations set by the statute. Its approach to the

funding of embryonic stem cell research, therefore, seems to

have sought an answer to this question: How can embryonic

stem cell research, conducted in accordance with standards of

informed consent and free donation, be maximally aided within

the limits of the law? The NIH guidelines published in 2000

represent the answer the Clinton administration found: funding

research on present and future embryonic stem cell lines, so

long as the embryo destruction itself is done with private

funds.

The Bush administration appears to have been motivated

by a somewhat different question, arising from what seems to

be a different view of the morality of research that destroys

human embryos. President Bush put the matter this way, in

discussing his newly announced policy in August of 2001:

Stem cell research is still at an early, uncertain stage,

but the hope it offers is amazing: infinitely adaptable

human cells to replace damaged or defective tissue and

treat a wide variety of diseases. Yet the ethics of medicine

are not infinitely adaptable. There is at least one bright

line: We do not end some lives for the medical benefit of

others. For me, this is a matter of conviction: a belief that
32 MONITORING STEM CELL RESEARCH

life, including early life, is biologically human, genetically

distinct and valuable.17 *

While expressing a desire to advance medical research, this

argument describes a line that such research should not cross,

and therefore past which funding should not be offered. That

line, in this context, is the destruction of a human embryo for

research purposes. The Bush administration thus appears to

share the view that underlies both the word and spirit of the

Dickey Amendment. In its approach to the stem cell issue it

has sought to answer a question that differs, subtly but

significantly, from that formulated by the previous

administration: How can embryonic stem cell research,

conducted in accordance with basic research ethics, be

maximally aided within the bounds of the principle that nascent

human life should not be destroyed for research?

In seeking to answer that question, the Bush administration

(like the Clinton administration) had to take account of the

existing situation and—as always in such instances—to mix

prudential demands and opportunities with an effort at

principled judgment. Given the existence of some human

embryonic stem cell lines, derived from human embryos that

had already been destroyed, the administration determined

that it might not simply have to choose between funding

research that relies on the ongoing destruction of embryos

(and therefore tacitly supporting and encouraging such

destruction by paying for the work that immediately follows

it) and funding no human embryonic stem cell research at all.

The decision regarding the funding of research on already-

*

 Using similar language, but speaking even more unambiguously, President Bush reiterated his ethical view of the destruction of human embryos

for medical research in a speech on human cloning legislation, saying, “I

believe all human cloning is wrong, and both forms of cloning ought to be

banned, for the following reasons. First, anything other than a total ban on

human cloning would be unethical. Research cloning would contradict the

most fundamental principle of medical ethics, that no human life should be

exploited or extinguished for the benefit of another. Yet a law permitting

research cloning, while forbidding the birth of a cloned child, would require the destruction of nascent human life.” (“Remarks by the President

on Human Cloning Legislation,” as made available by the White House

Press Office, April 10, 2002.)
CURRENT FEDERAL LAW AND POLICY 33

derived human embryonic stem cells came down to this

question: Can the government support some human embryonic

stem cell research without encouraging future embryo

destruction?

The present funding policy is therefore not an attempt to

answer the question of how the government might best

advance embryonic stem cell research while conforming to

the law on the subject. Rather, it is an attempt to answer the

question of how the government might avoid encouraging the

(presumptively) unethical act of embryo destruction and still

advance the worthy cause of medical research. Whether or

not one agrees with the premises defining the question, and

whether or not one accepts the logic of the answer, any

assessment of the policy must recognize this starting point.

From the very beginning, the policy has been described—

even by many of its supporters and defenders—as occupying

a kind of middle-ground position in the debate over the morality

of embryo research. It has been termed a “Solomonic

compromise.” But while it may be a prudential compromise

on the question of funding, it has been argued that the policy—

as articulated by its authors—does not seem to be intended

as a compromise on the question of the moral status of human

embryos or the moral standing of the act of embryo destruction.

In this sense, it appears to be not a political “splitting of the

difference” but an effort at a principled solution.18

To some extent, the effort reflects a traditional approach in

moral philosophy to an ancient and vexing question: Can one

benefit from the results of (what one believes to be) a past

immoral act without becoming complicit in that act?* The

moralists’ approach suggests that one may make use of such

benefits if (and only if) three crucial conditions are met: (1)

Non-cooperation: one does not cooperate or actively involve

oneself in the commission of the act; (2) Non-abetting: one

does nothing to abet or encourage the repetition of the act, for

*

 Readers should note that in reporting on this approach, as applicable to

President Bush’s stem cell decision, the Council is not itself declaring its

own views on whether the past act of embryo destruction was “immoral.”

(Some of us think it was, some of us think it wasn’t.) We are rather describing what we understand to be the moral logic of the decision as put forward.
34 MONITORING STEM CELL RESEARCH

instance by providing incentives or rewards to those who

would perform it in the future; and (3) Reaffirmation of the

principle: in accepting the benefit, one re-enunciates and

reaffirms the principle violated by the original deed in question.

As a plan for redeeming some good from embryo destruction

that has already taken place, while not encouraging embryo

destruction in the future, the administration’s policy appears

at least to seek to address each of these three conditions: (1)

No federal funds have been or, by this policy, would be used

in the destruction of human embryos for research. (2) By

restricting research funding exclusively to embryonic stem cell

lines derived before the policy went into effect, the policy

deliberately refuses to offer present or future financial or other

incentives to anyone who might subsequently destroy

additional embryos for research; this is the moral logic behind

a central feature of the policy, the cut-off date for funding

eligibility (though some argue that by failing to call for an end

to privately funded research the policy does not altogether

avoid complicity). And (3) the President, in his speech of

August 9, 2001, and since (as in the passage quoted above

and elsewhere), has reaffirmed the moral principle that

underlies his policy and the law on the subject: that nascent

human life should not be destroyed for research, even if good

might come of it. The policy as a whole draws attention to

that principle by drawing a sharp line beyond which funding

will not be made available.

Of course, since these terms from the parlance of moral

philosophy were not those explicitly employed by the policy’s

authors, they can go only so far in helping us to understand

the policy’s foundation. As in any public policy decision,

prudence is here mixed with principle, in the hope that the

two might reinforce (rather than undermine) each other, and a

variety of moral aims are brought together. The desire to afford

some aid to a potentially promising field of research moderates

what might otherwise have been an at least symbolically

stauncher stance against embryo destruction: no public

funding whatsoever, even for work on stem cell lines obtained

from embryos destroyed in the past. Moreover, the desire to

show regard for established principles and standards of ethical

research leads to an insistence that, to be approved, stem cell

lines must have been drawn from embryos produced for
CURRENT FEDERAL LAW AND POLICY 35

reproduction and obtained with consent and without financial

inducements. In these ways, the policy gives some due to

competing moral and prudential demands. But the policy’s

central feature—the announcement date separating eligible

from ineligible stem cell lines—holds firm to the principle that

public funds should not be used to encourage or support the

destruction of embryos in the future.

It is perhaps worth pointing out that one’s attitude

regarding the best federal funding policy is not simply

determined by one’s view regarding the moral standing of

human embryos, and that even persons who hold the same

view of the moral standing of human embryos may not all agree

on the best policy. For example, support for the current policy

does not necessarily require a belief that human embryos are

persons with full moral standing; and conversely, those who

believe that human embryos are persons do not necessarily

support the policy. One might believe, for instance, that an

embryo is a mystery, not clearly “one of us” but unambiguously

a life-in-process, and thus conclude that we should err on the

side of restraint (non-destruction) when moral certainty is

impossible. Or, one might believe that embryos are not simply

persons but are nonetheless either worthy of protection from

harm or at least worthy of more respect than ordinary human

tissues or animals, and that it would be wrong to begin a

massive public project of embryo research that offends the

deeply-held beliefs of many citizens. Meanwhile, an individual

who believes that human embryos have the same moral

standing as children or adults may be deeply unsatisfied with

the present policy, since merely denying federal

encouragement for future embryo destruction while taking no

action to prevent privately-funded stem cell research that

destroys embryos may be an insufficient response to the

ongoing destruction of nascent human life.

For some of its supporters, the policy goes as far as it seems

possible to go within the bounds of the spirit and aims of the

law—that the government should not encourage or support

the destruction of nascent human life for research. Yet at the

same time, it goes farther than the federal government has

gone before in the direction of actually funding research

involving human embryos, since no public funds had ever

before been spent on such research. To go further—say, by
36 MONITORING STEM CELL RESEARCH

funding research on the currently ineligible lines derived after

August 9, 2001—would not extend the logic of the policy or of

the law, but rather contradict them both: it would be a

difference not of degree but of principle. By implying that

research using embryos destroyed in the future might one day

be supported with public funding, such a policy shift would

at least implicitly encourage the very act (embryo destruction)

that the current policy aims not to encourage. Of course, such

a change might well be in order, but the case for it must address

itself to the moral argument and its principles, and not only to

the state of research and its progress or promise.

Rather than focus on this principled aspect of the policy,

the public debate has tended to concentrate on the precise

balance of benefits and harms resulting from the combination

of the administration’s policy and the state of the relevant

science. It has focused on whether there are “enough” cell

lines or on whether the science is advancing as quickly as it

could. And it has proceeded as though the administration’s

aim was simply to maximize progress in embryonic stem cell

research without transgressing the limits of the letter of the

law.

Had the decision been based on that aim alone, then claims

or evidence of slowed progress alone might, in themselves,

constitute an effective argument against it on its own terms

(on the ground that the law technically permits federal funding

of research on cells derived from embryos whose destruction

was underwritten by private funding). But if one accepts the

premise that the decision was grounded also in a discernible

(albeit highly controversial) moral aim, one cannot show that

the policy is wrong merely by pointing to the potential benefits

of stem cell research or the potential harm to science caused

by restrictions in federal funding. The present policy aims to

support stem cell research while insisting that federal funds

not be used to support or encourage the future destruction of

human embryos. To argue with that policy on its own terms,

therefore, one would need to argue with its view of the

significance of that aim. Concretely, this means arguing with

its ethical position regarding the destruction of nascent human

life and with its ethical-political position regarding the

significance of government funding of a contested activity.
CURRENT FEDERAL LAW AND POLICY 37

This latter point—regarding the meaning of government

funding—is much neglected in the current debates and

deserves further clarification. That will require delving into

the important distinction between government permission

(that is, an absence of prohibitions) of an activity and

government support for an activity. This ethical-political

distinction lies at the heart of the stem cell debate.

IV. THE SIGNIFICANCE OF FEDERAL FUNDING

The national debate over human embryonic stem cell

research often raises the most fundamental questions about

the moral status of human embryos and the legitimacy of

research that destroys such embryos. Yet, looking over this

debate, it is easy to forget that the question at issue is not

whether research using embryos should be allowed, but rather

whether it should be financed with the federal taxpayer’s

dollars.

The difference between prohibiting embryo research and

refraining from funding it has often been blurred by both sides

to the debate. Ignored in the battles over embryo research

itself, the ethical-political question regarding funding is rarely

taken up in full.

That question arises because modern governments do more

than legislate and enforce prohibitions and limits. In the age

of the welfare state, the government, besides being an enforcer of laws and a keeper of order, is also a major provider of

resources. Political questions today, therefore, reach beyond

what ought and ought not be allowed. They include questions

of what ought and ought not be encouraged, supported, and

made possible by taxpayer funding. The decision to fund an

activity is more than an offer of resources. It is also a declaration of official national support and endorsement, a positive

assertion that the activity in question is deemed by the nation

as a whole, through its government, to be good and worthy.

When something is done with public funding, it is done, so to

speak, in the name of the country, with its blessing and encouragement.

To offer such encouragement and support is therefore no

small matter. The federal government is not required to provide
38 MONITORING STEM CELL RESEARCH

such material support, even for activities protected by the

Constitution, let alone for those permitted but not guaranteed.19

The affording of most federal funding is entirely optional, and

the choice to make such an offer is therefore laden with moral

and political meaning, well beyond its material importance.

In the age of government funding, the political system is

sometimes called upon to decide not only the lowest standards

of conduct, but also the highest standards of legitimacy and

importance. When the nation decides an activity is worth its

public money, it declares that the activity is valued, desired,

and favored.

The United States has long held the scientific enterprise in

such high regard. Since the middle of the twentieth century,

the federal government, with the strong support of the

American people, has funded scientific research to the tune of

many hundreds of billions of dollars. The American taxpayer

is by far the greatest benefactor of science in the world, and

the American public greatly values the contributions of science

to human knowledge, human health, and human happiness.

And we Americans have overwhelmingly been boosters of

medical science and medical progress, deeming them worthy

of support for moral as well as material reasons.

But this enthusiasm for medical science is not without its

limits. As already noted, we attach restrictions to federally

funded research, for instance to protect human subjects. In

fact at times we even use funding to place restrictions on

research that might otherwise not be constrained. Indeed,

federal funding sometimes serves as a means by which private

research can be subjected to critical standards, since

institutions that receive federal funds are often inclined (and

given strong administrative incentives) to abide by the

prescribed ethical standards throughout all of their activities,

not only those directly receiving public dollars. Some

supporters of funding therefore argue that extending public

money to research is the most effective means of making

certain that nearly all researchers, public and private, adhere

to basic standards of ethics and safety. Public funding also

requires researchers to make their work available to the public

and for critical review by their peers, and it may encourage
CURRENT FEDERAL LAW AND POLICY 39

some degree of responsibility not necessarily encouraged by

commercial endeavors.*

In addition to conditions attached to government funding

of research, law sometimes erects specific limits on certain

practices that might be medically beneficial. For example, we

put limits on some practices that might offer life-saving

benefits, such as the buying and selling of organs for

transplantation, currently prohibited under the National Organ

Transplant Act. Also, as in the present case, many Americans

and their congressional representatives have moral reasons

for opposing certain lines of research or clinical practice, for

example those that involve the exploitation and destruction

of human fetuses and embryos.

The two sides of the embryo research debate tend to differ

sharply on the fundamental moral significance of the activity

in question. One side believes that what is involved is morally

abhorrent in the extreme, while the other believes embryo

research is noble or even morally obligatory and worthy of

praise and support. It would be very difficult for the

government to find a middle ground between these two

positions, since the two sides differ not only on what should

or should not be done, but also on the moral premises from

which the activity should be approached.

To this point, the federal government has pursued a policy

whereby it does not explicitly prohibit embryo research but

also does not officially condone it, encourage it, or support it

with public funds (though state governments have often taken

more active roles in both directions, as detailed in Appendix

E). This approach, again, combines prudential demands with

moral concerns. It has allowed the political system to avoid

banning embryo research against the wishes of those who

believe it serves an important purpose, while not compelling

those citizens who oppose it to fund it with their tax money.

This approach is also based, at least in part, on the conviction

that debates over the federal budget are not the place to take

* Indeed, some even argue that the terms and conditions set for federal

funding of research could be defined in such a way as not only to subject

private research to general standards but also to help influence the eventual distribution of the products of that research to all those in need, or to

serve other goods deemed publicly worthy.
40 MONITORING STEM CELL RESEARCH

up the anguished question of the moral status of human

embryos.

But the position is not only a compromise between those

who would have the government bless and those who would

have the government curse this activity. It is also a statement

of a certain principle: namely, that public sanction makes a

serious difference and ought not to be conferred lightly. While

embryo destruction may be something that some Americans

support and engage in, it is not something that America as a

nation has officially supported or engaged in.*

Of course, if the funding issue were merely a proxy for the

larger dispute over the moral status of human embryos, then

the present arrangement might appeal only to those who

would protect human embryos, and it would succeed only as

long as they were able to enact it. The argument might end

there, with a vote-count on the question of the moral status or

standing of human embryos. But some proponents of the

present law suggest that the particulars and contours of the

embryo research debate offer an additional rationale for that

arrangement. Here again, it is important to remember that the

issue in question is public funding, not permissibility. Opponents of embryo research have in most cases acquiesced (likely

owing to various prudential and moral factors) in narrowing

the debate at the federal level to the question of funding. They

do not argue for a wholesale prohibition of embryo research

by national legislation, even though many of them see such

work as an abomination and even a form of homicide. In return, proponents of the Dickey Amendment argue that it would

be appropriate for supporters of research to agree to do without federal funding in this particular field.

On the other hand, it might reasonably be argued that part

of living under majority rule is living with the consequences

of sometimes being in the minority. Were the Congress to

overturn the current policy of withholding public funds from

the destruction of embryos, opponents of funding for embryo

research would not be alone in being compelled to pay for

activities they abhor. We all see our government do things, in

our name, with which we disagree. Some of these might even

*

 The repeated reenactment of the Dickey Amendment by the Congress

may be taken as evidence of some support for this assertion.
CURRENT FEDERAL LAW AND POLICY 41

involve life and death questions of principle, for instance in

waging wars that some citizens deeply oppose. The existence

of strong moral opposition to some policy is not in itself a

decisive argument against proceeding with that policy.

These concerns give the question of funding its own crucial

ethical significance, even apart from the more fundamental

question of the legitimacy and propriety of the act being

funded. This matter of funding broadly understood, together

with the moral and prudential aims apparently motivating the

administration’s policy, as well as the legal context created

by the Dickey Amendment, are the essential prerequisites for

thinking about the underlying logic of the current policy. The

combination of these elements gives form not only to the

specific rules set forth in the administration’s funding policy,

but also to the implementation of that policy, to which subject

we now turn.

V. IMPLEMENTATION OF THE PRESENT POLICY

The complex and critical task of implementing the funding

policy falls largely to the National Institutes of Health, which

administers most federal funding of biomedical research. As

noted, the administration’s policy attempts to advance stem

cell research within the bounds already laid out regarding

further destruction of human embryos. Thus, while the funding

criteria of the policy set the bounds, the NIH, in its ongoing

work, is expected to advance the goal of maximally effective

funding and support within those bounds.

To this end, the NIH has worked to “jump-start” this field

of research through a series of coordinated activities.20 To plan

and oversee these activities, the NIH has established a Stem

Cell Task Force charged with determining the best uses for

public funds in the field and with putting in place the resources

required to make effective use of those funds.

The most basic material resources in question are the human

embryonic stem cell lines themselves. In August 2001,

President Bush announced that “more than sixty genetically

diverse stem cell lines” (or stem cell preparations) already

existed, and so would be eligible for funding under his policy.21
42 MONITORING STEM CELL RESEARCH

The NIH now believes the actual number to be somewhat

higher, so that seventy-eight lines (or preparations) are known

to be eligible for funding.*

 The lines are held by universities,

companies, and other entities throughout the world. According

to the National Institutes of Health’s latest report (September

2003), the following organizations have developed stem cell

derivations eligible for federal funding (that is, derived prior

to August 9, 2001, under the approved conditions):

 Number of

Name Derivations

BresaGen, Inc., Athens, Georgia 4

CyThera, Inc., San Diego, California 9

ES Cell International, Melbourne, Australia 6

Geron Corporation, Menlo Park, California 7

Göteborg University, Göteborg, Sweden 19

Karolinska Institute, Stockholm, Sweden 6

Maria Biotech Co. Ltd. – Maria Infertility

Hospital Medical Institute, Seoul, Korea 3

MizMedi Hospital –

Seoul National University, Seoul, Korea 1

National Centre for Biological Sciences/

Tata Institute of Fundamental Research,

Bangalore, India 3

Pochon CHA University, Seoul, Korea 2

Reliance Life Sciences, Mumbai, India 7

Technion University, Haifa, Israel 4

University of California, San Francisco, California 2

Wisconsin Alumni Research Foundation,

Madison, Wisconsin 5

Although all of these lines (or preparations) are deemed

eligible for funding according to the criteria of the

administration’s policy, not all are presently available for use

by researchers (nor is it clear that all of them will ever be

*

 These numbers took almost everyone by surprise. Prior to the President’s

announcement, the best estimates of the number of human embryonic stem

cell lines then existing worldwide ranged between 10 and 20. But eligibility is not the same thing as availability, as we will discuss.
CURRENT FEDERAL LAW AND POLICY 43

available for widespread use). Indeed, a point critical to

understanding the current situation is that as of the autumn

of 2003 only twelve lines are available for use,22 while most of

the other lines are not yet adequately characterized or

developed (some exist only as frozen stocks) and so have at

least not yet become available.*

 The process of establishing a

human embryonic stem cell line, turning the originally

extracted cells into stable cultured populations suitable for

distribution to researchers, involves an often lengthy process

of growth, characterization, quality control and assurance,

development, and distribution. In addition, the process of

making lines available to federally funded researchers involves

negotiating a contractual agreement (a “materials transfer

agreement”) with the companies or institutions owning the

cell lines, establishing guidelines for payment, intellectual

property rights over resulting techniques or treatments, and

other essential legal assurances between the provider and the

recipient.

The entire process—scientific and legal—has tended to take

at least a year for each cell line. Thus, determining which of

the 78 eligible lines are in sufficiently good condition, characterizing and developing those lines, and establishing the arrangements necessary to make them available has been a

demanding task. By September of 2003, slightly over two years

after the enactment of the funding policy, twelve of the eligible lines had become available to federally funded researchers.*

 The NIH has made available “infrastructure award” funds

(totaling just over $6 million to date) to a number of the institutions that possess eligible cell-lines, to enable them to more

quickly and effectively develop more lines to distribution quality. As a result, while the number of available lines (only one

in the summer of 2002 but risen to twelve in the autumn of

2003)* is expected to continue to grow with time, it is unclear

how many of the 78 lines will finally prove accessible and useful. According to the NIH, as of the autumn of 2003, the own-

*

 By the time of final publication of this document, in January 2004, the

number of available lines had risen to 15. This number is likely to rise further, and readers are advised to keep abreast of the current number and

availability of embryonic stem cell lines eligible for funding at the NIH

Stem Cell Registry website, stemcells.nih.gov.
44 MONITORING STEM CELL RESEARCH

ers of the available lines have distributed over 300 shipments

of lines to researchers. No information is presently available

on the number of individual researchers or institutions that

have received lines.23

Successful implementation of the current funding policy

depends not only on the availability of eligible lines, but also

on adequate allocation of financial resources to develop and

make use of those lines and to advance the field in general.

The funding policy, though it limits the targets of funding to

the eligible lines, does not directly delimit or restrict the

amount of money and other resources that the NIH may invest

in human embryonic stem cell research. The amount invested,

a decision left to NIH and the Congressional appropriations

process, is largely a function of the number of qualified

applicants for funding and of the NIH’s own priorities and

funding decisions. Of course, if more lines were eligible for

funding, it is quite possible that more funding would be

allocated, but the amount that can be allocated to work on

existing lines is not limited by the funding criteria. In fiscal

year 2002, the NIH devoted approximately $10.7 million to

human embryonic stem cell research. Based upon a September

2003 estimate, it will have spent approximately $17 million in

fiscal year 2003. It is expected that further increases will follow

as the field and the number of grant applications grow.

As of November 2003, NIH funds have been allocated to

support the following new and continuing awards for human

embryonic stem cell research: nine infrastructure awards to

assist stem cell providers to expand, test, and perform quality

assurance, and improve distribution of cell lines that comply

with the administration’s funding criteria (aimed at making

more of the eligible lines available); 28 investigator-initiated

awards for specific projects; 88 administrative supplements

(awarded to scientists already receiving funds for work on

other sorts of stem cells, either non-embryonic or non-human,

to enable them to begin to work with eligible human embryonic stem cell lines); two pilot and feasibility awards; three

awards to support exploratory human embryonic stem cell

centers; one institutional development award; four post-doctoral training fellowships; one career enhancement award; and

six awards to fund stem cell training (including short-term
CURRENT FEDERAL LAW AND POLICY 45

courses) to provide hands-on training to enable researchers

to learn the skills and techniques of culturing human embryonic stem cells.

The latter task, of training new researchers, the NIH regards

as one of its principal challenges in advancing the field, and,

along with available lines and available financial resources,

as a key measure of how the field is progressing. As NIH

Director Elias A. Zerhouni put it in his presentation before this

Council,

I don’t think the limiting factor is the cell lines. I really don’t.

I really think the limiting factor is human capital and trained

human capital that can quickly evaluate a wide range of

research avenues in stem cells.24

The NIH has therefore devoted funding to the training of

investigators and the cultivation of career development

pathways, including short-term courses in stem cell culture

techniques and (long-term) career enhancement awards in the

field. Some critics have contended, however, that the two

issues (funding restrictions and the scarcity of personnel) are

likely connected, and that limits on the cell lines eligible for

funding and the surrounding political controversy cause some

potential researchers to stay away from the field, contributing

to a shortage of investigators.25

These federal resources, then, have been directed toward

the advancement of human embryonic stem cell research

within the bounds of the determination to refrain from

supporting or funding new destruction of human embryos.

Scientists may receive federal funding—at any level

determined appropriate by the NIH—for any sort of meritorious

research, using as many of the approved lines as they are

eventually able to use. They can, of course, also receive federal

funding for using or deriving new animal embryonic stem cell

lines, to assess the potential of these cells for treatment of

animal models of human disease (though of course animal

models provide only limited information because they are not

in many cases exactly extrapolatable to the specific situations

that hold in human disease and development, and so cannot

replace human cell sources).
46 MONITORING STEM CELL RESEARCH

Researchers can, in addition, use federal funds for work

involving human embryonic germ cells, obtained from aborted

fetuses. They can carry out research projects using embryonic

germ cell lines already derived, following review and approval

of specific institutional assurances, informed consent

documents, scientific protocol abstracts, and Institutional

Review Board approvals by the NIH’s Human Pluripotent Stem

Cell Review Group (HPSCRG). They can also receive federal

funds for the derivation and study of new embryonic germ

cell lines following the same HPSCRG review and approval

process. In addition, of course, they can develop animal

embryonic germ cell lines to assess the potential of these cells

through animal models.

Also, researchers can receive federal funds for work

conducted on human adult (non-embryonic) stem cells. There

are no restrictions regarding what American scientists can do

with regard to adult stem cells using taxpayer funds, other

than those requiring them to honor the usual human subject

protections and clinical research requirements (if they are to

be transplanted into human patients). The NIH has devoted

substantial resources to the study of human adult stem cells,

allocating over $170 million to the field in fiscal year 2002, and

approximately $181.5 million in fiscal year 2003 (approximately

ten times the amount devoted to human embryonic stem cell

work).

Finally, researchers remain free to pursue work (including

the derivation of new lines of embryonic stem cells) in the

private sector, without government funding. Indeed, as

discussed above, embryonic stem cells were first isolated and

developed in the private sector, or in university laboratories

using private sector funds, and no work in the field was

publicly funded at all until 2001. Under present law, work

supported by private funds can proceed without restriction.

Under rules promulgated in the spring of 2002, such work does

not need to be conducted in a separate laboratory, but a clear

separation of the funds used to support this work from any

federally funded work of the laboratory is required. Of course,

because of the highly interlocking and complex nature of the

various aspects of operating a laboratory, such separation can

still prove extremely difficult to manage. It is not clear precisely
CURRENT FEDERAL LAW AND POLICY 47

how much privately funded work using human embryonic stem

cells has been undertaken in the past few years, but some

general figures are available. The most recent and thorough

survey available, based on figures from 2002,26 suggests that

approximately 10 companies in the United States were actively

engaged in embryonic stem cell work, employing several

hundred researchers and, cumulatively over the past several

years, spending over $70 million in the field, which is well

over twice what the NIH has so far spent.27 Those involved in

privately-funded research in the field, however, generally do

not see private funding as a substitute for federal funds, but

would much prefer that the field had the opportunity to benefit

from both. They also argue that restrictions on federal funds,

and the controversy surrounding the subject, act to dissuade

potential investors from entering the field, and thereby have a

“chilling effect” on private as well as publicly funded

research.28

Moreover, just as federal policy can affect privately

conducted research, so too a number of states have enacted

policies affecting stem cell research, ranging from all-out

prohibitions of such research to official statements of support

and positive encouragement.*

 The status of such research, and

the conditions to which it is subject, can vary dramatically

from state to state, independent of federal funding policy.

VI. CONCLUSION

The administration’s policy on the funding of embryonic

stem cell research rests on several moral and ethical-legal

principles, set upon the reality of existing law:

1. The law: The Dickey Amendment, which the President

is required to enforce.

2. The principle underlying the law: The conviction, voiced

by the administration, a majority of the Congress, and

some portion of the public, that federal taxpayer dollars

should not be used to encourage the exploitation or

* State policies regarding embryo research are detailed in Appendix E.
48 MONITORING STEM CELL RESEARCH

destruction of nascent human life, even if scientific and

medical benefits might come from such acts.

3. The principle underlying the desire to offer funding: That

efforts to heal the sick and the injured are of great

national importance and should be vigorously

supported, provided that they respect important moral

boundaries.

4. The significance of federal funding: That federal funding

constitutes a meaningful positive statement of national

approval and encouragement, which should be

awarded only with care, particularly in cases where

the activity in question arouses significant public moral

opposition.

The significance of the policy is best understood in light of

these key elements. Its soundness is most reasonably measured against them and against the policy’s implementation

by the National Institutes of Health.

Though the prudential and principled considerations raised

in this chapter governed the formulation of the policy, or at

least defined its articulation by its advocates and authors,

these are not the only terms by which federal funding policy

might be conceived or measured. In the next chapter we

present an overview of the ethical and policy debates that

have raged for the past two years around both the wisdom of

the present policy and the fundamental issues at stake in human embryonic stem cell research.
CURRENT FEDERAL LAW AND POLICY 49

ENDNOTES

1

 National Research Act, Pub. L. No. 93-348, § 213, 88 Stat. 342 (passed by the 93rd

Congress as H.R. 7724, July 12, 1974).

2

 National Commission for the Protection of Human Subjects of Biomedical and

Behavioral Research, Research on the Fetus: Report and Recommendations

(Washington, D.C.) 1975. Reprinted at 40 Fed. Reg. 33,526 (1975).

3

 “HEW Support of Human In Vitro Fertilization and Embryo Transfer: Report of the

Ethics Advisory Board,” 44 Fed. Reg. 35,033 (June 18, 1979) at 35,055-35,058.

4 Ibid.

5

 National Institutes of Health Revitalization Act of 1993, Pub. L. No. 103-43, § 121(c),

107 Stat. 122 (1993), repealing 45 C.F.R. § 46.204(d).

6 National Institutes of Health, Report of the Human Embryo Research Panel,

Bethesda, MD: NIH (1994).

7

 “Statement by the President,” as made available by the White House Press Office,

December 2, 1994.

8 The text of the Dickey Amendment can be found in each year’s Labor/HHS

Appropriations Bill. The original version, introduced by Representative Jay Dickey,

is in § 128 of Balanced Budget Downpayment Act, I, Pub. L. No. 104-99, 110 Stat. 26

(1996). For subsequent fiscal years, the rider is found in Title V, General Provisions,

of the Labor, HHS and Education Appropriations Acts in the following public laws:

FY 1997, Pub. L. No. 104-208; FY 1998, Pub. L. No. 105-78; FY 1999, Pub. L. No. 105-

277; FY 2000, Pub. L. No. 106-113; FY 2001, Pub. L. No. 106-554; and FY 2002, Pub. L.

No. 107-116. The most current version (identical in substance to the rest) is in

Consolidated Appropriations Resolution, 2003, Pub. L. No. 108-7, 117 Stat. 11 (2003).

9

 “Rendering legal opinion regarding federal funding for research involving human

pluripotent stem cells,” Memo from Harriet S. Rabb, General Counsel of the

Department of Health and Human Services to Harold Varmus, Director of the National

Institutes of Health, January 15, 1999. (Available through the National Archives.)

10 This case was made, for instance, in a letter authored by Rep. Jay Dickey and

signed by seventy other Members of Congress to DHHS Secretary Donna Shalala,

February 11, 1999.

11 DHHS Secretary Shalala argued this point in a letter responding to the

Congressional letter of opposition (see note 10, above), February 23, 1999.

12 President Bush has made a number of statements articulating the position that

nascent human life (including at the early embryonic stage) is deserving of protection

and ought not be violated. See especially: “Stem Cell Science and the Preservation

of Life,” The New York Times, August 12, 2001, p. D13; “Remarks by the President

on Human Cloning Legislation,” as made available by the White House Press Office,

April 10, 2002; “Remarks by the President at the Dedication of the Pope John Paul II

Cultural Center,” as made available by the White House Press Office, March 22,
50 MONITORING STEM CELL RESEARCH

2001; and “President Speaks at 30th Annual March for Life on the Mall,” as made

available by the White House Press Office, January 22, 2003.

13 “Remarks by the President on Stem Cell Research,” as made available by the

White House Press Office, August 9, 2001.

14 “Remarks by the President upon Departure for New Jersey,” as made available

by the White House Press Office, August 23, 2000.

15 National Institutes of Health, Report of the Human Embryo Research Panel,

Bethesda, MD: NIH, 1994.

16 See, for instance, the “Moral Case for Cloning-for-Biomedical-Research” presented

by some Members of the Council in the Council’s July 2002 report Human Cloning

and Human Dignity: An Ethical Inquiry, chapter 6.

17 Bush, G.W., “Stem Cell Science and the Preservation of Life,” The New York Times,

August 12, 2001, p. D13.

18 See, for instance, Council discussion at its September 3, 2003, meeting. A transcript

of that session is available on the Council’s website at www.bioethics.gov

19 This question has been addressed by the Supreme Court on a number of occasions,

in which the Court found that even activities protected as rights under the

Constitution are not thereby inherently worthy of financial support from the federal

government. See, for instance, Maher v. Roe 432 U.S. 464 (1977); Harris v. McRae 448

U.S. 297 (1980); and Rust v. Sullivan 500 U.S. 173 (1991). Also see Berkowitz, P. “The

Meaning of Federal Funding,” a paper commissioned by the Council and included

in Appendix F of this report.

20 The information provided in this section relies primarily on a presentation delivered

before the Council by NIH Director Elias Zerhouni on September 4, 2003, and on

data otherwise made available by the National Institutes of Health. The full transcript

of Director Zerhouni’s presentation may be found on the Council’s website at

www.bioethics.gov.

21 “Remarks by the President on Stem Cell Research,” as made available by the

White House Press Office, August 9, 2001.

22 As of the autumn of 2003, the following providers have eligible lines available for

distribution: BresaGen (2 available lines), ES Cell International, Australia (5 available

lines), MizMedi Hospital, South Korea (1 available line), Technion University, Israel

(2 available lines), University of California at San Francisco (1 available line),

Wisconsin Alumni Research Foundation (1 available line). A complete list of available

and eligible lines, updated as more lines become available, can be found at the NIH

Stem Cell Registry website at stemcells.nih.gov.

23 This information has been made available to the Council by the National Institutes

of Health.

24 Quoted from a presentation before the Council by NIH Director Elias Zerhouni,

September 4, 2003. The full transcript of Director Zerhouni’s presentation may be

found on the Council’s website at www.bioethics.gov.
CURRENT FEDERAL LAW AND POLICY 51

25 See, for instance, the presentation of Thomas Okarma, President and CEO of Geron

Corporation, before the Council on September 4, 2003. The full transcript of Okarma’s

presentation may be found on the Council’s website at www.bioethics.gov.

26 Lysaght, M.J., and Hazlehurst, A.L., “Private Sector Development of Stem Cell

Technology and Therapeutic Cloning,” Tissue Engineering 9(3): 555-561 (2003).

27 The dollar amount spent specifically on embryonic stem cell research in the private

sector is not apparent from Lysaght and Hazlehurst’s survey. The $70 million figure

is drawn from a presentation before the Council by Thomas Okarma, President and

CEO of Geron Corporation, the oldest and largest of the private companies involved

in embryonic stem cell research. Okarma told the Council, speaking only of Geron,

“We have spent over $70 million on this technology, most of it since 1999 after the

cells were derived. That’s a number against which the NIH disbursements pale by

both absolute and relative terms.” The full transcript of Okarma’s presentation may

be found on the Council’s website at www.bioethics.gov.

28 Ibid., and see also (for instance) Mitchell, S., “U.S. stem cell policy deters investors,”

The Washington Times, November 2, 2002 (original source: UPI).

3

Recent Developments in

the Ethical and Policy Debates

The announcement of the Bush administration’s human

embryonic stem cell research funding policy in the summer of

2001 certainly did not end the debates surrounding the issue.

The policy offered a particular target to which participants in

the debate could react, but the basic questions involved in

assessing how the federal government should approach embryonic stem cell research remained just as relevant, and just

as controversial, as they had been before. In this chapter, we

offer an overview of that still continuing debate as it has developed in the past two years. Without attempting to provide

anything like a full account of different positions and arguments, we hope, rather, to point to the major items under argument—to the issues that any interested citizen might wish

to ponder. First, we will outline the general form of the moral

argument as it has developed over time. Second, we will discuss specific questions and critiques regarding the current

policy, as those have emerged in public discussion. Third, we

will review the various positions on the moral standing of human embryos, seeking again to outline the chief fault lines in

that continuing debate. And finally, we will highlight some

critical ethical concerns that do not arise directly out of the

debate over the moral standing of human embryos but that

may be no less important to the larger question confronting

the country.

This array of subjects includes both some that are clearly

ethical questions and some that may fall closer to questions

53
54 MONITORING STEM CELL RESEARCH

of policy. We take up both together because we believe both

are essential to a full understanding of the issues involved

and because even the policy debates—as understood and

engaged in by participants on all sides—are clearly directed

to a set of underlying moral issues and informed by ethical

beliefs and opinions. Critiques (and defenses) of the present

policy, and proposals of alternatives, are almost without

exception based on moral grounds.

This chapter will, of course, revisit several of the themes

raised in the previous chapter. There, our aim was to present

some basic facts regarding the current policy, its context, and

its execution. Here, we take up arguments on all sides of the

issue—including those that dispute the understanding of the

policy put forward by its authors and those that raise other

issues or alternative courses of action.

While we will raise these arguments and counterarguments,

examining problems, questions, and concerns, our purpose is

not finally to assess the validity of the competing claims or to

arrive at a conclusion, but—in line with the Council’s charge

to monitor developments in this area—to present them more

or less as they have appeared in the public debates of the

past several years. This way of proceeding suffers from one

especially prominent drawback: it tends to present all

arguments as equal in importance or prevalence. We will seek

to avoid this whenever possible, by offering some sense of

which arguments have been most crucial for the public debate.

I. THE NATURE OF THE MORAL ARGUMENT

Before entering upon a detailed review of the debates, we

take a moment to consider the character of the moral reasoning

and argumentation we will confront. Different participants in

the stem cell debates tend to hold different views not only

regarding individual substantive judgments, but also

regarding the kind of moral question, in the most general terms,

we are facing in deciding about stem cell research policy. At

first glance, people seem to be disagreeing about whether a

balancing of competing interests and goods is called for, or

whether some one overriding moral duty ought to shape our
ETHICAL AND POLICY DEVELOPMENTS 55

judgment, though of course that dichotomy is not in every case

quite so stark.

In casual conversation, and sometimes in more theoretical

reasoning, moral questions are often analyzed in the language

of “weighing” and “balancing,” or else in terms of overriding

concerns, inviolable principles, or fundamental “rights.” These

two sets of terms and metaphors point to two distinct ways of

making difficult moral judgments. In some circumstances there

need be no irresolvable conflict between the two approaches.

Those who seek to respect fundamental rights and adhere to

inviolable principles need not ignore the complexities of moral

situations or the consequences of our actions when they form

their judgments. Likewise, those who seek to weigh or balance

competing goods may think of those goods as involving not

only benefits to be realized but also principles to be upheld.

There may also be circumstances, however, in which those

differences that do exist between these two approaches

constitute a fork in the road, constraining our decision. Thus,

to take an example from outside the domain of bioethics, the

principle that civilians should be safeguarded from direct,

intended attack in time of war may be understood to trump all

other competing goods (without denying the importance of

those goods), or it may be understood as one good to be

balanced against others. Which fork in the road of moral

reasoning one takes at such a point will have a decisive

influence on the character of the arguments employed and the

conclusions reached.

This has often also been the case in the public debate over

federal funding of human embryonic stem cell research. Many

observers have agreed that federal policy must seek a certain

balance between two competing goods or interests: the

progress of medical research and therapy, and respect for

nascent human life. Indeed, President Bush himself framed

the issue in these terms, saying a few weeks before his

decision was announced that his policy would “need to

balance value and respect for life with the promise of science

and the hope of saving life.”1

 But not all participants in the

debate have had the same idea of what such balance should

entail and, therefore, how weight should be assigned to the

competing demands.
56 MONITORING STEM CELL RESEARCH

For some, the degree of medical promise should profoundly

affect the degree of government support for embryonic stem

cell research. For them, the critical element (though of course

not the exclusive one) in establishing policy must be scientific

data about what might be achieved with human embryonic

stem cells.2 The greater the promise of the research, the more

support it should receive and the more it should outweigh

reasons offered for opposing the techniques involved.3

 On this

view, the concern given greatest prominence and weight in

reaching a judgment ought to be the very great good of medical

progress toward the relief of suffering. Most of the arguments

in favor of increased taxpayer funding of embryonic stem cell

research have begun from this premise—explicitly or

implicitly—and have proceeded by laying out the possible

medical benefits of the research or the possible harms (to

patients or to American science) of withholding support. We

shall review a number of these lines of argument in more detail

below. But it is worth noting at the start that most of them

tacitly assume that the policy decision at hand ought to be

based on a reasoned balancing of crucial concerns—all of

which matter but none of which simply overrides the others.

Others, however, have suggested that at least a substantial

portion of the opposition to the research rests upon the belief

that human embryos should not be violated and therefore—if

this claim is valid—that the threat to their life and worth cannot

be justified by the promise of research.4

 It follows, on this view,

that the federal government should do nothing to encourage

or support the future destruction of human embryos, regardless

of the promise of research. What remains then to be considered

is the extent to which the government might advance the

additional aim of progress in medical research within the

bounds of the principle.5 In this case, the moral reasoning is

understood to be decisively affected by an unbreachable

boundary, and only the extent of some particular provisions of

the policy are left to be settled by a weighing and balancing

of other priorities. Proponents of the various forms of this

position generally argue that the claim of human embryos to

our protection presents us with a fundamental duty, to be

overridden, if at all, only in extreme circumstances, rather than

with just one good to be balanced off against others.6
ETHICAL AND POLICY DEVELOPMENTS 57

In presenting the matter this way, adherents of this view

consciously appeal to the ethics governing research with

human subjects, which obliges those engaged in efforts to

advance knowledge and seek cures to keep from trespassing

upon human safety, freedom, and dignity.7 The decades-old

and nearly universal adherence of researchers to rules

protecting human subjects, these commentators suggest,

demonstrates that the needs of research are not always treated

as paramount and that the scientific community itself joins

the general public in recognizing instances in which

research—however important—must be limited for ethical

reasons.8

 Researchers do not weigh the interests of human

subjects against the importance of their work; rather they

respect a principled boundary—that human subjects are not

to be harmed or put at risk without their informed consent—

the importance of which trumps even the most promising

experiment. For some defenders of human embryos, the

prospect of embryo research raises precisely these concerns;

accordingly, they argue that this issue too should be decided

on the basis of a moral rule, not by a shiftable tally on a balance

sheet of benefits.9

But this assertion about the proper form of moral argument

depends on the truth of the claim that human embryos are

indeed human subjects of research. Therefore, one’s position

in the debate about the basic character of the moral issue may

depend, in many cases, on one’s understanding of the moral

standing of human embryos. As we shall see, the question of

the moral standing of embryos is by no means the only relevant

question. But in the actual public debate, as it has developed,

this question seems to have been most central and prominent

and probably most responsible for shaping the different basic

approaches pursued. It is this question that very often informs

the differing views regarding which aims or goods are more

weighty or which should not be compromised at all.

We turn next to arguments regarding that very issue: Which

moral aims or which concerns should be given priority in

shaping government policy toward human embryonic stem

cell research?
58 MONITORING STEM CELL RESEARCH

II. THE MORAL AIMS OF POLICY

A significant part of the public debate surrounding the

policy governing funding of embryonic stem cell research has

involved differing views of just which purposes or ideals should

most directly guide policymakers in this arena. The current

funding policy—while it appears to strike some balance

between protecting human embryos and advancing

biomedical research—seems to take as its overriding concern

the insistence that the federal government not encourage or

support the deliberate violation or destruction of human

embryos. But a number of commentators in recent years—and,

of course, particularly those who ascribe lesser moral status

to human embryos—have proposed alternative principles to

govern policy.

A. The Importance of Relieving Suffering

Many observers argue that the proper governing principle

should be the duty to relieve the pain and suffering of others—

the purpose that ultimately motivates the work of biomedical

science. This aim is broadly, perhaps universally, shared.

Indeed, the current policy, as outlined by its advocates, while

it seeks to protect nascent human life, explicitly seeks to

advance medical research as far as its authors believe is

morally permissible. Some commentators argue, however, that

the administration has chosen the wrong one of these aims as

the governing principle of its approach.

The cause of curing disease has a human face, the face of a

loved one or neighbor, bent under the suffering of an

incompletely understood or treated disease. As a result, the

aspiration to know is linked to a desire to relieve. How, wonder

many commentators, could anyone think of withholding

support for research that might yield therapies for devastating

diseases and conditions such as spinal cord injury, diabetes,

and Parkinson disease?10 Surely, they argue, the pain and

suffering of those in need should outweigh concerns for human

embryos frozen in a laboratory.11 Indeed, for many this is an
ETHICAL AND POLICY DEVELOPMENTS 59

especially critical issue in light of the likely ultimate fate of

these embryos. Will they be frozen indefinitely? If not, then

will they be thawed out and discarded and destroyed, or used

to potentially benefit mankind?

One form of this argument, heard increasingly over the past

few years, begins with data about disease prevalence and

suggests that anyone who obstructs public funding of research

that might someday help patients suffering from such diseases

must bear some of the responsibility for their future suffering

and (perhaps) death.12 Some opponents have countered that

any assertion that makes relief of human suffering the highest

moral principle to this extent might logically impugn any and

all deflection of resources into less ultimate concerns such as

recreation, beautification, or social ceremony. Others respond

more directly that an unwillingness to violate one’s moral

principles in order to help relieve the sick does not make one

responsible for their sickness.13

In most cases, however, the arguments for grounding

federal funding policy in the importance of biomedical research

do not blame opponents for the suffering of the sick. Rather,

they focus on the promise of bringing relief to those who most

need it. They point to the immense benefits already delivered

to us by modern medicine and argue that the federal

government should advance this cause in whatever ways it

reasonably can. Many advocates of this view agree that

nascent human life should receive respectful treatment, but

they argue that the claims of our duties to human embryos—

whether in general or in particular circumstances, like those

stored in fertility clinic freezers—cannot simply trump the

claims of promising medical research and our duties to

suffering humanity. The obligation to aid the sick, they contend,

and the fact that the research in question might relieve the

pain and terrible suffering of countless patients and their

families, should lead us to do all that can reasonably be done

to find treatments and cures, and to offer help. This does not

mean simply ignoring the significance of human embryos, or

taking lightly the decision to destroy them in research, but,

they suggest, it should mean taking seriously the moral calling

to help the suffering and deciding how to proceed based on

more than one sort of obligation.14
60 MONITORING STEM CELL RESEARCH

In response, one commentator has argued in broad terms

against the underlying assumption that the demands of

biomedical research should somehow be seen as “imperative.”

Such work, he contends, should not be seen as inherently and

always obligatory, and claims to support it may be overridden

even by a level of respect for nascent human life that does not

suppose that embryos possess full human moral standing. He

suggests that it is not at all obvious that individuals or the

government have a definite responsibility to support such

research or that such a responsibility would override other

moral duties.15

Others point out that the duty to find cures for disease

cannot be an unqualified or absolute imperative. Pointing to

the present rules governing the treatment of human subjects

in research, they argue that the case for embryonic stem cell

research cannot rest on an alleged and overriding imperative

to pursue that research. These rules prohibit certain sorts of

procedures on human subjects of research, and the same, they

suggest, should be required in stem cell research. Yet those

who argue that the importance of medical research and

treatment should override the aim of protecting human

embryos presumably would not propose to override

protections for human subjects in research on children or

adults. Rather, they approach the present matter differently

because they do not consider human embryos the

developmental, anthropological, or moral equivalent of children

or adults, or worthy of the same protection. The difference,

therefore, has to do not so much with a dispute over the

imperative importance of research, but rather (at least to a

significant extent) with the status of nascent human life, which

again turns out to be the fundamental point at issue.16

Nevertheless, the moral claims of medical research and

treatment are extremely powerful in the debate over human

embryonic stem cell policy, and they are acknowledged as

profoundly important even by those who do not finally take

them to be decisive. Most arguments in opposition to the

present funding policy and in support of expanded embryonicstem cell research are grounded in these claims.
ETHICAL AND POLICY DEVELOPMENTS 61

B. Freedom to Conduct Research

Other opponents of the present policy argue not from the

value of medical benefits but on the basis of freedom to conduct

research, which they believe is the principle by which federal

policy ought to be governed. They regard government

restraints on scientific research as inherently offensive and

generally unjustifiable.17 The cherished ideals of freedom of

thought, freedom of conscience, and—specifically in this

context—freedom of inquiry, trump concerns over the moral

status of human embryos, they contend.18

The most common claim advanced for protecting research

as a basic right (employed, among others, by the American

Bar Association) involves some form of an appeal to the First

Amendment’s protection of free speech, interpreted through

the years by the Supreme Court to encompass free expression

and perhaps freedom of inquiry and thought.19 Some argue

that research is a form of expression, particularly when it is

politically or socially controversial, and when restraints upon

it are imposed for moral reasons.20 “One could make the case

that research is expressive activity and that the search for

knowledge is intrinsically within the First Amendment’s

protection for freedom of thought,” says one ethicist.21

Others, however, contend that this claim, never tested in

the courts, seems far-fetched. Most currently controversial

biological research involves experimental manipulation of

living matter, rather than theoretical exploration or mere

observation of natural objects. It is therefore as much action

as expression, as much creation as inquiry. It is difficult to

see, they argue, how such activity (as opposed to the reporting

of the results of such activity) could be classified as a form of

expression. “Scientists may have the right to pursue

knowledge in any way they want cognitively, intellectually,”

argues one observer, “but when it comes to concrete action in

the lab, that becomes conduct and the First Amendment

protection for that is far, far weaker.”22

Moreover, argues at least one commentator, even if one did

stipulate that research activity is to be protected from

governmental proscription or restriction, it is far from clear
62 MONITORING STEM CELL RESEARCH

that failure to provide federal funding would constitute such a

restriction.23 Indeed, legislative and judicial precedents

suggest it would not. The government routinely refrains from

funding activities it otherwise permits or even guards as

constitutionally protected. A line of Supreme Court decisions

stretching from 1977 to 1991, dealing with abortion and

government funding, established the principle that the

Constitution does not require the government to fund even

those activities that the Constitution protects.24 Because the

only issue in the present debate is one of federal funding, the

protected status of scientific activity seems not to be a

determining factor.25

Finally, some critics of the case for a paramount right to

research point again to the fact that scientific research—

conducted both with private and (especially) with government

funding—is already subject to certain restrictions, particularly

with regard to protecting human subjects. The proposition that

embryo research should not be subject to the same restrictions

hinges on an argument about the standing of human embryos,

rather than about the unrestrictable standing of research as

such.26 Once more, an important part of the question turns on

the status of extra-uterine human embryos.

C. The Moral Standing of Human Embryos

However they approach the matter, then, many people

engaged in the debate over federal funding policy find they

must consider the fundamental question of the moral standing

of human embryos: What are early human embryos, and how

should we regard them morally? Approaches to the question

of federal funding of embryonic stem cell research that propose

some other guiding principle—relief of suffering, freedom of

research—seem almost by necessity to assume that human

embryos do not possess the same human moral standing as

persons already born.27 Conversely, if human embryos ought

rightly to be treated as inviolable—as some have argued—

then questions of balancing other goods or giving priority to

other principles are largely rendered moot. Thus, to many

observers, some of the central questions in this arena would

appear to be those that surround human embryos: How ought
ETHICAL AND POLICY DEVELOPMENTS 63

we to think about and act toward human embryos? Should all

ex vivo human embryos be treated the same, or are some,

because of their circumstances, origins, or prospects, to be

treated differently from others? Or are embryos of sufficiently

little moral significance that we should simply decide the

funding question solely on the basis of the merits and promise

of the proposed biomedical research?

We shall, in due course, review the recent arguments on

these critical questions. But before doing so, we examine a

series of other more specific objections to the present policy.

These arguments recognize that the current policy rests on

the conviction that federal funds should not support or

encourage the violation or destruction of human embryos.

Rather than disputing this premise, a number of

commentators—including both supporters and critics—have

assessed the policy on its own grounds and judged it against

its own claims and terms. As a result, several general

categories of criticism of the policy itself have emerged.

III. THE CHARACTER OF THE POLICY

In addition to debating which aims ought to guide federal

policy, many observers in the past two years have also

assessed the particulars of the present funding policy,

considering them in scientific, political, and moral terms.

Critics have generally found fault with the policy through one

or more of three general lines of argument: that it is arbitrary,

that it is unsustainable, and that it is inconsistent. Defenders

of the policy, meanwhile, have usually sought to counter these

critics on these terms and to rebut these assertions and

criticisms.

A. Arbitrary

One quite common line of argument criticizes the present

funding policy as essentially arbitrary, because it relies on

what is deemed a capricious cutoff date. Cells derived from

embryos destroyed on August 9, 2001 are eligible for federal

funding, but those obtained from embryos destroyed the next
64 MONITORING STEM CELL RESEARCH

day are not. The only difference is the date of embryo

destruction, argue some critics, and what moral difference

could that possibly make? If the policy of funding human

embryonic stem cell research serves a genuine good, these

commentators suggest, would it not be equally good

regardless of when the cell lines were derived? Would it not,

therefore, make sense to permit funding for all cell lines derived

on either side of the date, provided they are otherwise

eligible?28 If, on the contrary, federal funding of embryo

research is unacceptable, then it should simply not be allowed

at all, regardless of when cell lines were first derived.29 “It is

difficult to see,” writes one critic, “what ethical reasoning

would commend a policy that takes as its central distinction

the time chosen for political convenience to deliver a

presidential address.”30

In response, some supporters of the policy contend that

while the cut-off date (August 9, 2001) certainly has no inherent

moral significance, it acquires moral meaning by the simple

fact that it was the operative date of a newly announced policy

that turned on a crucial distinction between the “up-until-now”

and the “from-now-on,” between past (irrevocable) deeds and

future (preventable) ones. That date of announcement was the

line between past embryo destruction, which could no longer

be undone, and future embryo destruction, which could still

be influenced by the federal government’s funding rules. If

the policy sought to avoid encouraging or offering incentives

for any future destruction of human embryos, they argue, then

drawing a hard line between past and future would be

indispensable. That line could only reasonably be drawn at

the moment of the decision’s announcement*

 (or before it),

since drawing it at some point in the future would create a

powerful incentive to quicken the pace of work until the cutoff date arrived. The date, they say, is therefore not a morally

arbitrary marker in the context of the policy, but is rather a

crucial and unavoidable element of the policy’s logic.31

*

 Only “the now,” created by an act of speaking, defines the difference

between past and future (tenses and deeds).
ETHICAL AND POLICY DEVELOPMENTS 65

B. Unsustainable

A further, and more common, critique of the current policy

suggests that its approach cannot be expected to hold over

time and that it will fairly quickly prove unsustainable.

One form of this argument suggests that the policy has

created a situation in which scientists may make some

progress using existing stem cell lines but would then be

prohibited from capitalizing on what they have learned and

making further progress using stem cell lines not now eligible

for federal funds. As the editors of the Washington Post put it:

“Mr. Bush’s compromise policy will be a reasonable one only

as long as the existing lines are capable of supporting the

research scientists need to perform.”32 Indeed, some have

argued that by explicitly encouraging and speaking of the

potential medical value of human embryonic stem cell

research, the President himself created the circumstances that

will make the constraints of the policy unsustainable. “Bush’s

decision was at its core an endorsement of the promise of

human embryonic stem cells and their importance to the

fledgling field of regenerative medicine,” wrote one critic, and

if that is the core message of the decision, then the resulting

policy seems insufficient.33

Others, arguing on more practical grounds, claim that,

regardless of the reasonableness of the principle behind the

policy, its effects will prove unbearable for American scientists,

who will then force a reconsideration.34 The limits placed on

funding in the current policy, several observers have predicted,

will seriously hamper and hold back embryonic stem cell

research work in the United States, perhaps causing prominent

scientists to leave the country in search of greater support

abroad.35 If that were to occur, it is argued, the policy would

prove genuinely damaging to American science, and therefore

to the national interest, and would need to be changed.36

In response to this specific point, some defenders of the

policy have noted that for the time being there has not been

news of any notable migration of prominent stem cell

researchers to foreign countries, that federal funding is now
66 MONITORING STEM CELL RESEARCH

available with no ceiling on its amount, and that American

researchers can continue to work with private funds.37

But even apart from worries about a “brain-drain” in the

field, some have argued that both the lack of funding, and

particularly the complexity of the set of conditions under which

research using such funding may be conducted, is preventing

progress in research and discouraging even private funding

in the field,38 and that the lines made available simply will not

be enough,39 or indeed that they have already proved

insufficient.40 Some argue that the very low number of

applications submitted to the NIH for postdoctoral and training

projects using the approved stem cell lines provides striking

evidence for the chilling effect of the current “in limbo”

situation. Some have also suggested that safety issues

connected with the way the eligible lines have been derived

and developed may make them less suitable for use in human

trials and treatments, thus making other cell lines necessary

(although presentations before this Council by NIH Director

Elias Zerhouni and FDA Commissioner Mark McClellan have

contradicted some of these claims).41 Others, meanwhile, worry

that the eligible lines do not offer sufficient genetic diversity

to be adequate for research needs or for eventual therapies.42

As scientists make their case that important work is being

hampered, it is argued, the policy will prove unsustainable

politically and practically.43

A similar argument has also been made in nearly the

opposite terms. That is, some have said that if or when ongoing

embryonic stem cell research produces a spectacular

breakthrough in understanding or treating disease, the

pressure to alter the policy would prove unstoppable.44 This

way, whether the future brings announcements of great

progress, or whether it brings no news of advances, the result

will be pressure for a policy that funds research more broadly.

Those defenders of the policy who have addressed these

claims of unsustainability have pointed out that the present

policy does not limit the amount of federal funds available to

the kind of research that may be performed with the approved

cell lines. They also point out that much of the pioneering work

in mouse stem cell research was done using very few

(approximately five) cell lines45 (though of course the challenge
ETHICAL AND POLICY DEVELOPMENTS 67

of working with human cell lines is more complex and

daunting). But they have also generally pointed to the policy’s

stated grounding in principle—a principle that would not

change in light of scientific advances or delays. In August of

2001, for instance, Health and Human Services Secretary

Tommy Thompson told reporters that “neither unexpected

scientific breakthroughs nor unanticipated research problems

would cause Bush to reconsider” the approach drawn by the

policy, because it is based on “a high moral line that this

president is not going to cross.”46 As another observer has

put it: “The general question is, well, will these cell lines be

enough . . . and a non-complicity argument [like the one implied

by the policy] will only work if the answer to that question is

well, I guess they’ll have to be enough.”47 Or, put differently, if

the policy is founded primarily in a determination to prevent

government funds from encouraging the destruction of human

embryos, and only secondarily in a judgment about the value

of embryonic stem cell research, then advances in research

alone (or the absence of advances alone) would not be

sufficient to overturn it. If it is sound before such advances,

some argue, it would still be valid after48—though again, of

course, whether it is right to begin with is itself a point of

great contention.

C. Inconsistent

In responding to critiques like those just discussed,

defenders of the administration’s policy generally point to the

principles that define the approach of the policy, as partially

laid out in the previous chapter. But some criticism of the policy,

directing itself precisely to the claim of consistent adherence

to principle, has charged that the policy is morally

contradictory, or at least inconsistent, in its own terms.

One common form of the charge of inconsistency concerns

the distinction the policy tacitly draws between public and

private funding. The current policy addresses itself only to

federal funding of embryo research and is silent on the conduct

of such research in the private sector. But the source of funding,

this line of criticism suggests, could have no bearing on the

question of the moral status of human embryos or the propriety
68 MONITORING STEM CELL RESEARCH

of using them in research. If federal funding for research that

destroys human embryos is so troublesome, then why should

such research be allowed to proceed when privately funded?

Acting to restrict one but not the other may be prudent, but it

seems inconsistent.49 Indeed, by implicitly excluding such

research from federal guidelines, it may actually encourage

more reckless practices and may simply transfer the problem

of complicity to the private sphere, where it is even more

difficult to monitor and moderate the uses to which human

embryos are put. Though these critics understand that the

President cannot simply ban embryo research by himself, they

argue that he could attempt to convince the Congress to do

so, and he could have made such an attempt as an element of

his funding policy decision. But he did not—thus bringing into

question (for these critics) not only his commitment to the

principle articulated by the policy, but also his own view of

the moral standing of human embryos, which the policy itself

does not make simply apparent.50 Moreover, critics argue, some

prominent defenders of the policy, by making the fact of

ongoing private research an element of their defense, might

be said to contribute to these doubts about its grounding in

consistent principle.51

There may be some political or structural reasons for

drawing a distinction in federal policy between what is funded

and what is permitted. Questions of federalism and other legal

realities no doubt enter the picture, and indeed those who

oppose the destruction of human embryos are, in many cases,

actively seeking prohibitions on all embryo research in

individual states.* But, critics point out, they generally say little

on the larger question of permissibility at the federal level. By

making funding the center of concern, these critics argue, the

policy puts into question the importance of preventing embryo

destruction more generally, casting some uncertainty over the

relation of that policy to one or another position regarding the

moral standing of early human embryos.

A related criticism contends that the distinction drawn

between research practices in which human embryos are

*

 Lori Andrews detailed both these ongoing efforts and existing legislation

in the states in her presentation at the Council’s July 2003 meeting and in

the accompanying paper. (See Appendix E.)
ETHICAL AND POLICY DEVELOPMENTS 69

destroyed and research practices that use the products of

previous embryo destruction is itself inconsistent—a

distinction without a difference, drawn for political cover.52

“Pretending that the scientists who do stem cell research are

in no way complicit in the destruction of embryos is just wrong,

a smoke and mirrors game,” writes one critic. “It would be

much better to take the issue on directly by making the

argument that destroying embryos in this way is morally

justified—is, in effect, a just sacrifice to make.”53 Similar

objections have also been raised by critics on the other side of

the embryo question, who believe that embryo destruction is

morally unjustified and that the present policy does not

sufficiently distance the federal government from such

destruction. “The federal government, for the first time in

history, will support research that relies on the destruction of

some defenseless human beings for the possible benefit to

others,” one critic contended in the immediate wake of the

August 9, 2001 announcement. “However such a decision is

hedged about with qualifications, it allows our nation’s

research enterprise to cultivate a disrespect for human life.”54

A further critique of the policy on grounds of inconsistency

focuses more particularly on specific elements of the implicit

claim to non-complicity, discussed in the previous chapter. If,

as many of its advocates argue, the policy takes embryo

destruction to be essentially a morally unjustifiable act, and if

its provisions aim to make use of the irreversible consequences

of that act without in any way encouraging or abetting the act

itself, then, critics contend, it is curious that the policy would

insist on requiring (in addition) that the eligible stem cell lines

must have been obtained from embryos originally intended

for reproduction and used with donor consent and without

financial inducements. If embryo destruction is in principle a

wrong, and if the policy’s provisions seek only to keep the

federal government from complicity in that wrong, then why

should it matter how precisely the wrong was originally

committed? The presence of these conditions, it is argued,

suggests that the policy is not in fact based in a consistent

and principled adherence to the proposition that human

embryos should not be destroyed. Indeed, some have argued

that this character of the policy suggests that its authors,

including the President, may not hold the view that human
70 MONITORING STEM CELL RESEARCH

embryos deserve the same protections as human children or

adults. These critics suggest not so much that the current

policy is necessarily inconsistent, but that it may only be

consistent with a view of human embryos as possessed of

intermediate or indeterminate moral standing, rather than a

view that holds that human embryos ought simply not to be

destroyed for the benefit of others.55

In response to these last arguments, some have suggested

that the qualifying conditions included in the policy reflect a

secondary commitment to long-standing principles of humansubject and embryo research and a recognition of pre-existing

standards in such research, rather than a contradiction of the

fundamental commitment to avoiding any support for the

destruction of nascent human life. The government, they argue,

has multiple aims in this area, and these need not undercut

each other. In addition to discouraging the creation and use of

embryos for purposes other than producing children, one

commentator argues, the government also seeks to support

the requirement for informed consent to all procedures

involving human subjects and to discourage commercial

trafficking in human material.56 Another observer has

suggested that the additional conditions are an expression of

the fact that some standards for stem cell derivation did exist

before August 9, 2001, including those reflected in the Clinton

administration’s funding guidelines.57 Even in rejecting the

legitimacy of the act of embryo destruction and seeking to

discourage it in the future, it is still reasonable to recognize

the value of those earlier standards. The policy, it is argued,

while establishing a new standard, still takes account of

previous standards.58

In response to the more general complaints of

inconsistency, defenders of the policy, within and outside the

administration, have described the present policy as both

principled and prudent. The policy, as articulated by these

defenders, aims (at least minimally) to uphold and advance

the principled conviction that the federal government should

not offer support or incentives for the destruction of nascent

human life for research. At the same time, they say, it seeks,

as much as reasonably possible within the bounds of the

principle, to benefit from the results of embryo destruction

that has already occurred and can no longer be undone.59 As
ETHICAL AND POLICY DEVELOPMENTS 71

President Bush put it in announcing the policy, “This allows

us to explore the promise and potential of stem cell research

without crossing a fundamental moral line, by providing

taxpayer funding that would sanction or encourage further

destruction of human embryos that have at least the potential

for life.”60 The policy, argue some of its advocates, aims to put

into practice the moral principle of not destroying nascent

human life, even if it does not do so on every possible front, or

to the greatest possible extent.

Regarding the alleged inconsistency of withholding federal

funding but not calling for a ban on all private embryo research,

some have pointed out that the Dickey Amendment, under

which the President was acting, is itself a “don’t fund, don’t

ban” law. Moreover, they argue that neither statesmanship

nor prudence requires that the President do battle with what

is settled practice (the free use of embryos in private-sector

research) or push zealously against everything to which he is

morally opposed, especially in a pluralistic society that is

deeply divided on the moral issue. An analogous case may be

found in the administration’s approach to abortion, a practice

that the President says he opposes deeply on moral grounds:

the administration supports the legislative ban on federal

funding, but has not called for a constitutional amendment

that would ban abortion. As President Bush told the pro-life

“March for Life” participants in January 2002: “Abortion is an

issue that deeply divides our country. And we need to treat

those with whom we disagree with respect and civility. We

must overcome bitterness and rancor where we find it and

seek common ground where we can. But we will continue to

speak out on behalf of the most vulnerable members of our

society.”61 In a similar way, its defenders contend that the

current administration policy on stem cell research funding

keeps the public ethical conversation open, may be acceptable

to some individuals who hold that human embryos possess

an intermediate or unknowable moral standing, and, at the

same time, also advances the cause of those who contend that

human embryos should be protected from destruction

altogether.62

The present funding policy has also been defended from

the charge of inconsistency on rather different grounds, which,

for their advocates, carry different consequences for future
72 MONITORING STEM CELL RESEARCH

federal funding. This defense contends that, if the early embryo

is morally equivalent to a child, then the proper moral response

would be to ban all future embryo destruction and all stem

cell research (public and private) on embryos destroyed after

August 9, 2001, and not merely deny it federal funding.

Confronted with a practice that involved killing children so

that their organs could be used to save the lives of others,

advocates of this view contend, no one would simply deny it

federal funding while allowing the gruesome practice to

continue. But, they point out, there is no reason to attribute to

the current funding policy the assumption that the early

embryo is morally equivalent to a child. In fact, they contend,

the President has never said that early embryos are inviolable,

or are persons, or morally equivalent to children. While

President Bush explained his funding policy by arguing that

“it is unethical to end life in medical research,”63 he has not

sought to prohibit privately-funded embryonic stem cell

research. This leads some to conclude that implicit in the

President’s policy is either the view that the embryo has an

intermediate moral status (worthy of serious moral

consideration as a developing form of human life) or

uncertainty about its moral status. Those who interpret the

President’s policy in this way point out that, in his address to

the nation on stem cell research, he spoke in terms of a human

embryo’s “potential for life”:

Research on embryonic stem cells raises profound ethical

questions, because extracting the stem cell destroys the

embryo, and thus destroys its potential for life. Like a

snowflake, each of these embryos is unique, with the

unique genetic potential of an individual human being.64

This, they contend, does not constitute an argument for

treating human embryos as possessed of fully human moral

standing.65

If the present policy is seen to reflect either the

intermediate or uncertain view of the moral standing of human

embryos, these advocates argue, it is not inconsistent to

withhold federal funding, at least for now, when the medical

benefits of stem cell research are still speculative, while

permitting privately funded embryo research to proceed. They
ETHICAL AND POLICY DEVELOPMENTS 73

argue that restricting or limiting federal funding is a reasonable

way of registering either doubt about the moral status of the

embryo, or the moral unease felt by someone who takes

nascent human life seriously and does not want it used

wantonly, and who therefore wants scientists to prove the

promise of this research before permitting them to go further

with the support of federal funding. Indeed, they argue, this

interpretation is consistent with the President’s words in his

August 9th address:

[W]hile we’re all hopeful about the potential of this

research, no one can be certain that the science will live

up to the hope it has generated. . . . Embryonic stem cell

research offers both great promise and great peril. So I

have decided we must proceed with great care.”66

Viewing the administration’s policy as based on an

intermediate or uncertain view of the moral standing of human

embryos also makes plausible, in the view of these observers,

the fact that the President has neither called for a ban on

privately funded embryo research, nor called upon scientists

to desist from research on stem cell lines created after August

9, 2001. They contend that it also makes sense of the

requirements, discussed above, that stem cell lines created

before that date must, in order to qualify for federal funding,

be derived from excess embryos created for reproduction, with

donor consent, and without financial inducements.

Those who interpret the policy as reflecting an intermediate

or uncertain view of the moral status of human embryos argue

further that, if embryonic stem cell research vindicates its

promise, there would be no categorical reason to prevent a

reconsideration of federal funding in the light of medical

advances. A further implication is that people may reasonably

draw different conclusions about whether this principle

justifies federal funding of stem cell lines derived after August

9, 2001 provided they are derived from embryos left over from

IVF procedures, donated with consent, and without financial

inducement.67 Although the President has declared that he

regards destructive embryo research as unethical without

additional qualifications,68 and although HHS Secretary Tommy

Thompson has said that scientific advances would not cause
74 MONITORING STEM CELL RESEARCH

the President to reconsider his policy,69 those who interpret

the President’s policy as reflecting an intermediate or uncertain

view of the embryo argue that the moral logic of this principle

admits the possibility that significant medical breakthroughs

could justify a reconsideration.

IV. THE MORAL STANDING OF HUMAN EMBRYOS

Many elements (though, as we shall make clear, not all

elements) of the ongoing debate about federal funding of

human embryonic stem cell research seem, as we have

reviewed them, to turn on a basic disagreement about the

nature, character, and moral standing of human embryos.

Public debate over the moral standing and appropriate

treatment of human embryos has been quite contentious and

divisive in recent years. In part, this had to do with its almost

inevitable entanglement with the abortion debate, itself a deep

and thorny controversy in America. In part, too, this has been

connected to the fact that the question of the moral standing

of human embryos touches many other fundamental moral and

existential questions involving human origins, human dignity,

the moral significance of our biology, and its relation to

numerous traditional and widely shared moral teachings.70

Differences of opinion on the moral standing of human embryos

often suggest differences on these larger questions of overall

worldview.71 Nonetheless, the question of the moral standing

of human embryos itself has been taken by nearly all

commentators to be amenable to human reason and argument,

and a lively debate has raged despite (or perhaps precisely

because of) these widely diverging starting assumptions.

In the public arena, the question of the moral standing of

human embryos has often been summed up in the question,

“When does (a) human life begin?” This question suggests

something of the quandary, although the academic and

intellectual debate generally takes a somewhat more nuanced

question as its starting point. That question has as its unstated

premise the fact that under normal circumstances we regard

all born human beings (from newborns through adults) as

possessing equal moral worth and meriting equal legal

protection. It then reflects upon the ways in which human
ETHICAL AND POLICY DEVELOPMENTS 75

embryos are similar and different from live-born human

individuals, the moral significance of those similarities and

differences, and therefore whether embryos should or should

not be afforded protections.72

The first and most common recourse in seeking an answer

to such questions has been human biology, and particularly

human embryology. Nearly all participants in the dispute make

some reference to biological findings, whether to claim that

they teach us about an embryo’s essential continuity with and

similarity to human beings at other stages of life, or to argue

that they reveal profound and morally meaningful

discontinuities between embryos and live-born persons.

While we examine these differing contentions, it is crucial

to remember—as several commentators in recent years have

noted—that the biological findings, however relevant, are not

themselves necessarily decisive morally.73 They may serve

better to challenge moral positions founded on erroneous

assumptions than to ground some positive moral affirmation

or conclusion. For example, a recognition of biological

continuity might in some measure undermine the argument

that embryo destruction is permissible when certain biological

markers or states of development are absent. But it would not

by itself show indisputably that embryos are to be treated as

simply inviolable.74 Meanwhile, recognizing the biological

significance of some particular point, marker, characteristic,

or capacity would not, in itself, imply some decisive moral

significance. A description of early embryonic development

is necessary though not sufficient to an understanding of the

nature and worth of an early embryo.75 It is not sufficient

because any purely biological description requires some

interpretation of its anthropological and moral significance

before it can function as a guide to action.76

With these provisions in mind, we offer the following brief

review of developments in the debate over the moral standing

of human embryos in the past several years.*

*

 Readers may find it helpful to consult Notes on Early Human Development (Appendix A).
76 MONITORING STEM CELL RESEARCH

A. Continuity and Discontinuity

Many participants in the debate take the question of the

biological continuity or discontinuity between nascent and

later human life to be crucially significant. Some argue that

the fundamental organismal continuity from the moment of

fertilization until natural death means that no lines can be

drawn between embryos and adults. Others argue, on the

contrary, that some particular point of discontinuity (or the

sum of several such points) marks a morally significant

distinction between stages, which difference should guide our

treatment of human embryos.

1. The Case for Continuity.

Many of those who seek to defend human embryos base

their case on some form of the argument for biological

continuity and sameness through time. For example, they argue

that a human embryo is an organic whole, a living member of

the human species in the earliest stage of natural development,

and that, given the appropriate environment, it will, by selfdirected integral organic functioning, develop progressively

to the next more mature stage and become first a human fetus

and then a human infant. Every adult human being around us,

they argue, is the same individual who, at an earlier stage of

life, was a human embryo. We all were then, as we still are

now, distinct and complete human organisms, not mere parts

of other organisms.77

This view holds that only the very beginning of a new

(embryonic) life can serve as a reasonable boundary line in

according moral worth to a human organism, because it is the

moment marked out by nature for the first visible appearance

in the world of a new individual. Before fertilization, no new

individual exists. After it, sperm and egg cells are gone—

subsumed and transformed into a new, third entity capable of

its own internally self-directed development. By itself, no

sperm or egg has the potential to become an adult, but zygotes

by their very nature do.78

Many authors therefore regard the activation of the oocyte

(by the penetration of the sperm)79 or the completion of
ETHICAL AND POLICY DEVELOPMENTS 77

syngamy (the combining of paternally- and maternallycontributed haploid pro-nuclei to result in a unique diploid

nucleus of a developing zygote) as a meaningful marker of the

beginning of a new human life worthy of protection.80 After

this point, there is a new genome, in a new individual

organism, and there is a zygote (single-celled embryo) already

beginning its first cleavage and embarking on its continuous

developmental path toward birth.

All further stages and events in embryological development,

they argue, are discrete labels applied to an organism that is

persistently itself even as it continuously changes in its

dimensions, scope, degree of differentiation, and so on. We

can learn names for the various stages as if they were static

and discrete, but the living and developing embryo is

continuously dynamic.81 More to the point, in the view of these

commentators no discrete point in time or development would

seem to give any justification for assuming that the embryo in

question was one thing at one point and then suddenly became

something different (turning, for example, from non-human to

human or from non-person to person). None of the biological

events (or “points” in processes), they contend, is sufficient

to tell us what we are morally permitted or obligated to do to

human embryos, in the absence of one or another additional

premises that shape one’s view of these biological events. And

if one’s guiding premise is that all human persons possess

equal moral standing—regardless of their particular powers,

size, or appearance—then there are no grounds for denying

the earliest human embryo full moral standing as a person. 82

Some critics of this position argue that it makes too much

of mere genetic identity and (uncertain) potential or that it

does not make enough of present condition and the

significance of development itself.83 There is more to being

human, some observers argue, than possessing a human

genome or spontaneous cell division, and it matters that the

early human embryo is but a ball of cells, without sentience or

sensation and without human form.84 It matters, too, they

argue, that an ex vivo human embryo does not have the

potential to develop independently, without further technical

intervention.85 Indeed, some argue that a human embryo in

its earliest stages is essentially no different from any human
78 MONITORING STEM CELL RESEARCH

tissue culture in the laboratory,86 or that, because the ex vivo

embryo cannot develop if left to itself, it cannot be thought of

as truly continuous with more developed human organisms.

It may be, in the description of one observer, not much different

from a pile of building materials stored in a warehouse.87

Nonetheless, advocates of the argument from continuity

suggest that it is dangerous to begin to assign moral worth

on the basis of the presence or absence of particular capacities

and features, and that instead we must recognize each member

of our species from his or her earliest days as a human being

deserving of dignified treatment. They contend that a human

embryo already has the biological potential needed to enable

the exercise, at a later stage of development, of certain

functions. Sentience and sensation come in later in the process

of development, but their seeds are there right from the

beginning. And the fact that an embryo cannot develop outside

the body is not an argument for leaving it outside the body.

There is, they argue, no clear place to draw a line after the

earliest formation of the organism, and so there can be no stark

division between the moral standing of nascent human life

and that of more mature individuals.88

2. The Case for Meaningful Discontinuity and Developing Moral

Status.

Many other observers, however, argue that some

biologically and morally significant discontinuities do in fact

present themselves in the course of early human development.

These arguments generally do not simply hinge on biological

descriptions—which are, in the absence of analysis, largely

devoid of obvious moral significance—but instead begin from

some implicit or explicit claim regarding the importance of a

particular feature, capacity, form, or function (or the

progressive accumulation of these) in defining a developing

organism as meaningfully a member of the human race.*

 Not

simply grounded in biology, they appeal also to a moral or even

metaphysical claim about the meaning of humanity.89 They

*

 Also, several traditional religious views of the embryo (among some Jews

and some Muslims, for instance) have attributed humanity to the embryo

only after a particular point in its development, for example the 40th day.
ETHICAL AND POLICY DEVELOPMENTS 79

suggest that the developing human organism might become

(at once or progressively) deserving of protection as it becomes

able to feel pain, or to exhibit neural activity, or rudimentary

features of consciousness, or some elements of the human form,

or the capacity to function independently—or as it

progressively exhibits more and more of these or other criteria.

Until that time, many argue, a developing human deserves

some respect because of what it might become, but not

protection on par (or nearly so) with that afforded to fully

human subjects.90 They suggest that genetic identity and

organismic continuity are not sufficient; what matters is

present form and function, more than mere potential.91

Several particular putative discontinuities (and

combinations of them) have been proposed as candidates for

the division between early stages, when a human embryo may

be disaggregated for research, and later stages, when it

deserves some greater level of protection.

(a) Primitive streak: The most popular candidate for a

meaningful point of discontinuity is the appearance of the

primitive streak, the earliest visible “structure” that defines

the region of the embryo along which the vertebral column

will form. The primitive streak generally appears around

the 14th day after first cell division. It is taken to indicate

the anterior-posterior axis of an embryo (in vertebrates),

although recent studies suggest that polarity may be

established much earlier, and in fact may be indicated by

the point at which the sperm enters the egg.92

A principal reason for the importance placed on the

primitive streak has to do with the biology of twinning. Prior

to the appearance of the primitive streak, an embryo may

(rarely, and for unknown reasons) divide completely to form

identical twins. Some conclude that individuality must not

yet be established, since the embryo might yet become two

embryos.93 Since individuality is essential to human

personhood and capacity for moral status, since

individuality presumes a definitive single individual, and

since the singularity of the embryo is not irrevocably settled

prior to the appearance of the primitive streak, they argue

that the entity prior to the primitive streak stage lacks
80 MONITORING STEM CELL RESEARCH

definitive individuality and hence also moral status.94 Critics

of this line of reasoning point to the low statistical

probability of monozygotic twinning: one in 240 live births

(though rates clearly vary among populations in different

regions, and precise figures probably cannot be known

because some zygotes that split likely later fuse into one,

and some singleton births may conceal a twin who died

early in gestation). Critics also point to evidence that

twinning results, not from an intrinsic drive within the

embryo, but from a disruption of the fragile cell dynamics

of embryogenesis. Evidence for this, they suggest, may be

seen in the increased incidence of monozygotic twinning

(up to tenfold in blastocyst transfer) associated with IVF.

This suggests, in their view, that twinning is neither a proof

of the absence of an integrated individual organism with a

drive in the direction of development nor a demonstration

ex post facto of the absence of moral worth of the embryo

before twinning.95

Nonetheless, for this reason, and for others (discussed

below) having to do with the formation of the nervous

system, the primitive streak has often been taken to be a

highly significant marker of embryological development,

and many commentators suggest it as a reasonable

candidate for a meaningful point of discontinuity. For this

reason, many supporters of embryo research regularly

propose the 14th day of development as a logical stopping

point for permissible embryo research.96

(b) Nervous system: A second argument for discontinuity

focuses on the developing nervous system. Many observers

regard the nervous system as an especially important

marker of humanity, both because the human brain is critical

for all “higher” human activities, and because the nervous

system is the seat of sensation and, especially relevant to

this case, the sensation of pain. Proponents of this view

hold that before an embryo has developed the capacity for

feeling pain (or, in some forms of the argument, before

sentience), we cross no crucial moral boundary in subjecting

it to destructive research.97 For some, this is taken to mean

that the primitive streak, as the first marker of a future
ETHICAL AND POLICY DEVELOPMENTS 81

nervous system, is a crucial feature of developing life. For

others, only later points of neural development (where pain

might plausibly be experienced) are held to be decisive.98

Critics meanwhile contend that neural development as well

as development of other systems (such as the cardiovascular system) are the natural outcome of the genetic

program in action, and should be explained by reference to

the previous stage and as leading to the subsequent stage,

rather than marking a significant discontinuity.99 They

maintain that the human being is, from the start, an

inseparable psycho-physical unity, rather than a pure

rationality or consciousness that exists with no meaningful

ties to our bodies. From a scientific perspective, such critics

hold, there is no meaningful moment when one can

definitively designate the biological origins of a human

characteristic such as consciousness, because our mind

works in and through our body, and the roots of

consciousness lie deep in our development. The earliest

stages of human development serve as the indispensable

and enduring foundations for the powers of freedom and

self-awareness that reach their fullest expression in the

adult form.100 Some of those who believe that neural

development is crucial, however, argue that the fact of nonsentience and of an inability to experience pain possess

great moral significance, quite apart from the question of

probable potential.

(c) Human form: Some observers also argue that certain

rudimentary features of the human form must be apparent

before we can consider a human embryo deserving of

protection. In this view, the human form truly signals the

presence of a human life in the making and calls upon our

moral sentiments to treat the being in question as “one of

us.”101 Different versions of this argument appeal to different

particular elements (or combinations of elements) of the

human form as decisive, but all suggest that a “ball of cells”

is not recognizably human and therefore ought not to be

treated as simply one of us.102 Some critics of this view argue

that humans have different external forms or shapes

throughout their lives, and that an organism, particularly

in its early stages, should not be judged by its external
82 MONITORING STEM CELL RESEARCH

shape, but rather by its biological constitution, and

especially its genetic identity.103 But adherents of the

argument that human form matters contend that genetic

identity cannot simply be decisive of moral worth.

These various particular cases for discontinuity (and these

are not the only ones that have been propounded over the

years) are not mutually exclusive. Indeed, many of them point

to more than one particular element of early human

development as finally decisive of moral standing. But they

share the belief that moral status accrues only at some later

stage of the developing human organism. Their claim, in the

broadest terms, is that in its earliest stages a human embryo

is not yet simply a human being or a human person, and that

it need not be treated as though it were.104 Human

development, they contend, is an essential element in any

understanding of human life, and an organism at the earliest

stages of that process is not to be treated the same as one

much farther along.105 There are developmental differences,

and these differences matter, in ways to be determined by

human choice and understanding, as well as by a grasp of the

biological facts.106

Critics of this view contend that while it is certainly true

that human beings at different stages of development are not

to be treated the same (as children are not given the

responsibilities of adults), the crucial treatment here at issue

is destructive treatment. No human being, at any stage, they

argue, should simply be destroyed for research, and the “use”

of an embryo for research, no matter how valuable one deems

the research to be, could not amount to treatment of that

embryo as “deserving some degree of respect.” The degree of

respect granted in destroying the embryo would be zero, they

contend.107

Nonetheless, the case for developing moral status, as

articulated by a great number of participants in the policy

debates of the past several years, often results in an expression

of what has sometimes been termed the “special respect”

approach to human embryos: an embryo in its earliest stages

is not accorded the full moral standing of a human person, but

it is nonetheless regarded as deserving some degree of respect

and is treated as more than a mere object or collection of
ETHICAL AND POLICY DEVELOPMENTS 83

somatic cells in tissue culture. In practice, adherents of this

view tend to accept the use of early human embryos in

medically valuable research under some circumstances, but

they seek to apply some scrutiny to the reasons for which

embryos will be used, the circumstances under which those

embryos are obtained, and other relevant factors. Several

bodies advising the federal government on human embryo

research over the years have expressed this view, to varying

degrees. The Ethics Advisory Board to the Department of

Health, Education and Welfare concluded in 1979 that the early

human embryo deserves “profound respect” as a form of

developing human life (though not necessarily “the full legal

and moral rights attributed to persons”).108 The NIH Human

Embryo Research Panel agreed in 1994 that “the

preimplantation human embryo warrants serious moral

consideration as a developing form of human life,” though the

panel argued that this did not mean that research should be

prohibited.109 In 1999, the National Bioethics Advisory

Commission (NBAC) cited broad agreement in society that

“human embryos deserve respect as a form of human life,”110

but, like its predecessors, did not recognize the embryo as

having the full rights of a human person. The special respect

position has been held by its advocates to be consistent with

a range of possible particular policies on embryo research,

including fairly restrictive ones, and indeed could be held

consistent even with an outright restriction on the destruction

of human embryos.*111 To consider the embryo “inviolable” (and

therefore not a mere utility to be instrumentally used), it is not

necessary to presuppose its moral equality with a child, an

adult or even a later stage gestating fetus. There may be

increasing moral obligations (and natural sentiments)

associated with a deepening relationality that extend moral

duty without in any way implicitly eroding an imperative of

protection at earlier stages of developing life. Most of those

who have articulated the special respect position in the public

debate have, however, tended to then argue that some research

*

 This is partly due to the fact that “special respect,” and “intermediate

moral status,” are rather vague terms, and embrace a very wide range of

degrees of “specialness” and “intermediacy.”
84 MONITORING STEM CELL RESEARCH

on embryos should be permitted within certain boundaries,

even if they have not always agreed on the permissible extent

or the appropriate boundaries.

B. Special Cases and Exceptions

Some arguments in favor of permitting and funding embryo

research, grounded in the “special respect” approach, do not

in fact explicitly (or exclusively) rely on arguments about

discontinuity or a biologically grounded view of developing

moral status. Instead, or in addition, they rely upon questions

of embryo viability and potential, and they are aimed at

exploring unique circumstances to address and perhaps

resolve questions of the moral standing of certain particular

human embryos.

1. IVF “Spares.”

Much of the debate surrounding embryonic stem cell

research has focused on the use of cryogenically preserved

IVF embryos, left over from assisted reproduction procedures

and stored, perhaps indefinitely, in the freezers of IVF clinics.

One recent study suggests there are hundreds of thousands

of such embryos in the United States alone, though only a very

small percentage of them (less than 3 percent, approximately

3,000 or more) has ever been donated for research.112 Although

all were produced with reproductive intent and were stored

for further reproductive efforts should the first attempt fail,

most of these frozen embryos may never be claimed by the

original egg and sperm donors for use in assisted reproduction

procedures. They are almost certain to remain frozen and,

eventually, to die without developing further. Although there

have been some efforts to build interest in adopting such

embryos, such adoption, some commentators argue, is very

unlikely to become a large-scale phenomenon or to affect the

fate of most of these stored embryos. 113 Under the present

funding policy, if these frozen embryos were donated for

research and embryonic stem cells were derived from them,

research on the resulting cells would not be eligible for federal

funding. Many observers argue that it should be: Since these
ETHICAL AND POLICY DEVELOPMENTS 85

particular embryos are almost certainly destined to die without

ever developing, it would be appropriate to at least redeem

some possible good from their existence and unavoidable

demise.114

Some people have pushed the point further. Since the vast

majority of the (huge number of) cryopreserved embryos will

almost certainly not be adopted, and since many may not be

viable even if they were to be transferred to a woman’s uterus,

a few observers have argued that, practically speaking, the

frozen embryos are virtually all already lost.115 To be sure, they

are not already dead, but they are in a so-called “terminal

situation” from which no rescue is practically possible. In view

of this situation, one commentator proposes extending to these

embryos the principle that sometimes, he argues, permits the

killing of innocents. That is, killing may be morally permissible

in cases where the person will soon die for other unavoidable

reasons and where there is another person who can somehow

be rescued through or as a result of such a normally

impermissible act of killing (thus, as he puts it, “nothing more

is lost”). He admits that the case of cryopreserved embryos

stretches the application of the “nothing is lost” principle

beyond its previous uses, because the embryos in question

are alive and at risk of death only because of human choices

and designs specifically directed toward them. The principle

is also stretched because the lives that might someday be

saved through today’s embryo deaths are quite remote. The

potential lives saved are those of unspecified future persons

with diseases that might be treated by therapies that as yet

do not exist and may or may not exist in the future. However,

against the weight of all these ifs, which some find formidable,

there is the present fact that (like the embryos used to create

stem cell lines derived before August 9th of 2001) the

cryopreserved embryos are already here, with little or no

prospect of rescue—they are, in this observer’s description,

already lost.116

Presumably, if destruction of “spare embryos” for human

embryonic stem cell research were generally agreed to be

permissible through this “nothing is lost” principle, it could

be federally funded, subject to such routine secondary

considerations as the need for free and informed consent by

donors.
86 MONITORING STEM CELL RESEARCH

Yet this argument, not surprisingly, has met with opposition.

Some critics have claimed that it employs circular moral

reasoning. The embryos, they argue, are in a “terminal

situation” because of human choice and design; thus to then

decide that, since they are going to die anyway, they may as

well be put to good use is to ignore the moral implications of

the original decision to create and freeze them. Critics argue,

moreover, that when thinking about our responsibilities to

those who are soon to die, we would normally say that it makes

a considerable moral difference whether we simply accept

their dying or whether we positively embrace it as our aim.117

Yet some proponents of using IVF spares argue that the present

situation is best understood as a forced choice between two

regrettable alternatives for the final disposition of stored

embryos (whether donated for research or abandoned). One

choice may then be justified as the lesser evil. Even if one

deems the original decisions leading to the creation and

storage of these embryos questionable, the embryos exist, and

the earlier decisions cannot be undone.118

Some have also worried about the possibility of a “slippery

slope,” by which the uses of “spare” IVF embryos under this

justification might open the door to their wider use in

experiments in natural embryogenesis, toxicological studies

or chimerizations, or perhaps their development in an artificial

(or natural) endometrium,119 (though the reasonableness of

“slippery slope” arguments is often disputed).120 Other critics

point out that the “nothing is lost” principle is not permitted

to govern decisions regarding lethal experiments on the

terminally ill, on death-row inmates, or even on fetuses slated

for abortion.121

A further issue involves the question of whether accepting

the “nothing is lost” principle for already existing embryos

condones in principle the use of future excess embryos, or

whether the principle actually requires efforts to prevent the

creation or storage of “excess” embryos in the future.122 Further,

this application of the principle relates only to embryos

originally created for the purpose of reproduction but not

transferred to initiate a pregnancy. Should their use in research

be accepted, however, it is not clear that it would be possible

to differentiate between embryos created originally for

reproduction and extra embryos created with an eye to
ETHICAL AND POLICY DEVELOPMENTS 87

research uses, since the process of producing them would be

identical (though consideration of the extra risks involved for

the woman egg donor could act as a counter against any largescale embryo-creation-for-research).123

Other observers, however, begin from the presence of

cryogenically preserved embryos but extend further the

argument justifying their use in research. They argue that there

is good reason to use embryos for research, not only because

a situation some judge tragic already exists but because

donating embryos is an act of beneficence and using them is

a social obligation incurred by their gift.124 This approach would

have us start by recognizing that, in the current situation, there

is a set of embryos (in IVF clinics) none of which will ever

enter a uterus, or even a (still hypothetical) artificial uterus.125

These embryos, in one commentator’s term, are “unenabled”

and have no potential for full development. Since there is no

possible way for such embryos to develop, there is no “possible

person” to whom any “unenabled” embryo corresponds;

therefore using them in research involves no loss of possible

life.126

Critics of this approach argue that in effect it allows the

moral standing of any given embryo to be decided simply by

those responsible for it. Thus, whether a given embryo has

moral standing depends only on whether it has a practical

prospect of developing, as evidenced by whether it will be

transferred to a uterus. But whether it enters a uterus depends

on human choices, so the moral standing of a given embryo

depends on human choices. If we choose to withhold essential

enabling conditions for an embryo’s development, these critics

argue, then that embryo will lack not moral standing but

merely the opportunity to develop. Its intrinsic nature,

including its own potential to develop (given needed

conditions), has not changed.127

Nonetheless, in the view of a number of commentators, the

fact that so many frozen embryos already exist (even if only a

small percentage of them are donated for research) changes

the balance of duties and respect owed to any single ex vivo

embryo. Several observers have argued that the presence of

these frozen embryos, with little or no chance of attaining birth,

creates special circumstances in which the use of embryos in

research (whether destroying them in the course of research
88 MONITORING STEM CELL RESEARCH

or allowing them to die and subsequently using them)128 is

independent of any final judgment regarding the moral

standing or worth of the ordinary human embryo, as such.129

2. Natural Embryo Loss.

Some authors tie their moral arguments regarding the use

of embryos in research to the fact of the high rate of embryo

loss under natural conditions of sexual intercourse and

unassisted reproduction. They argue that directed destruction

of ex vivo embryos for the purpose of research would not result

in greater embryonic loss than occurs in this natural process

and would result in far greater benefits for humanity.130 The

rate of natural embryo loss after conception in unassisted

human reproduction (taking in losses both before and after

implantation), though not accurately known, is thought to be

high, some suggest as high as 80 percent.131 Moreover, the

fact of natural loss is now fairly well known, so that persons

who engage in unprotected intercourse, whether seeking to

reproduce or not, are knowingly bringing about the conception

of many embryos that will die. We generally do not regard

this embryo loss as unacceptably tragic, and we do not engage

in great efforts to avert it or to find ways to diminish it (beyond

research to prevent miscarriage, for instance). For this reason,

these commentators argue, using artificially created embryos

for purposes of research would not destroy a greater portion

of those embryos than ordinarily die in natural unassisted

reproduction.132

Moreover, they suggest, the high rate of natural embryo

loss should call into question the views of those who believe

that early-stage human embryos merit equal treatment with

human children and adults. If so many die in the natural course

of things, why do we not treat natural procreation as a great

fountain of tragedy and carnage? The natural rate of embryo

loss, and most people’s failure to mourn its consequences, they

suggest, should teach us something about the limited

significance of human embryos in the earliest stages. One

observer adds that the same logic should diminish our

concerns about creating extra human embryos for research,

as long as sufficient embryos are created for implantation to

keep the chances of survival for any given embryo as good as
ETHICAL AND POLICY DEVELOPMENTS 89

the chances in natural reproduction.133 Another argues further

that human embryonic stem cell research might actually raise

the probability of longer survival for all humans, including

embryonic ones, and is therefore a case of permissible taking

of life, even on the assumption that the embryos are persons.134

Opponents of this view, however, have argued that natural

deaths of embryos and the deaths caused by intentional efforts

to destroy ex vivo embryos for research are not morally

equivalent. There are many things that happen naturally that

we are not therefore justified in doing deliberately. Indeed,

the rate of natural loss of live-born human beings is 100

percent, but that does not justify their killing. And, they argue,

even if one were permitted to analogize the deaths of frozen

embryos in vitro to the embryonic wastage in vivo, in neither

case were the embryos lost destined or created for anything

other than their procreative end. In contrast, they argue, using

embryos for research bears no relation to their natural direction

or trajectory. Critics also argue that the character of our reaction

to the natural embryonic death does not justify our practice of

destructive embryo research. For they believe that a creature’s

moral worth is not dependent on the emotional reaction of

others to its death.135 The absence of moral sentiment does

not imply an absence of moral obligation (nor a right of adverse

intervention in a naturally developing human life). Moreover,

critics contend, it is not clear how many of these “natural

losses” were in fact failures of the fertilization process, so that

there was never a unified, integrated organism in the first

place, and thus never the loss of a human embryo. It is also

unclear, they argue, how much of the supposedly “natural”

loss rate is actually due to contingent and changeable factors,

such as environmental pollution, pesticides, or endometrial

problems, and so is not simply an unavoidable fact of

embryonic existence.136

3. Prediction of Non-Viability of Embryos.

Some people, hoping to get around the moral dilemma of

destroying even “unenabled” embryos, seek to identify a

subset of embryos that might reasonably be regarded in

advance as non-viable. One proposal has involved the

possibility that cloned human embryos, created by somatic
90 MONITORING STEM CELL RESEARCH

cell nuclear transfer, may prove to be non-viable or unable to

develop beyond a certain point (biological evidence that this

may be the case is presented by Rudolph Jaenisch in Appendix

N) or may even, by their nature and origins, simply not

constitute the equivalent of human embryos. If this turns out

to be true, it is further argued, it might be possible to use

them without arousing some of the ethical dilemmas that

accompany the use of otherwise potentially viable human

embryos.137 Others point to a possible sub-group of those

embryos currently frozen in storage as potentially non-viable.

Although those embryos are not yet dead and, if thawed, would

still exhibit some cellular function, some would be unlikely to

survive even were transfer attempted. Since IVF procedures

usually produce more embryos than can be transferred at one

time, goes the argument, the clinicians choose for transfer

those among the available embryos that look “the best” and

seem most likely to survive and develop—so that those that

are frozen are those deemed less likely to develop. Moreover,

by applying similar judgment to the unimplanted embryos,

we might identify those that would be least likely to survive

even under the most favorable circumstances. These embryos

might then reasonably be regarded as non-viable and therefore

available for research since their use will not disrupt a potential

life.138

There has not been much direct reaction to this view in the

ongoing ethical debates. Yet some observers have noted, in

other contexts, that the techniques used to identify which IVF

embryos are more or less likely to develop successfully—

estimates based usually on visual assessment of the

embryos—have never been proven effective or even tested to

ascertain their validity.139 Moreover, some argue, the true

viability of cloned human embryos or of cryogenically stored

embryos could not be determined in advance without

attempting to implant them.140

4. Creation of Non-Viable Embryo-Like Artifacts.

Others, seeking to bypass altogether the issue of viability,

propose the possibility of creating a biological entity that

cannot rightly be called a living organism, yet that has the

generic organic powers necessary to produce embryonic stem
ETHICAL AND POLICY DEVELOPMENTS 91

cells. They suggest that somatic cell nuclear transfer, or a

similar technique, could be used to create an entity that lacks,

by design, the qualities and capabilities essential to be

designated a human life in process. By intentional alteration

of the somatic cell nuclear components or the cytoplasm of

the oocyte into which they are transferred, researchers may

be able to construct an “artifact” that is biologically (and

morally) more akin to cells in tissue culture, but could still

provide a source of functional human embryonic stem cells.141

Proponents of this innovation aim to shift the ethical debate

from the question of whether or when a human embryo should

be considered a human being to the question of which

organized structures and potentials constitute the minimal

criteria for considering an entity to be a human embryo.142

Absent all the essential elements (including a full

complement of chromosomes, proper genetic sequence and

chromatin configuration, and cytoplasmic structures and

transcription factors), advocates of this proposal argue, there

can be no integrated whole, no organism, and hence no human

embryo. By technically constructing biological entities lacking

these essential elements yet bearing the partial organic

potential often found in failures of fertilization, they suggest

it may be possible to procure embryonic stem cells without

producing an organismal or embryonic entity that can

meaningfully be designated a being with moral standing.143

Proponents argue that there is a natural biological

precedent for such an entity lacking the qualities and

characteristics of an organism yet capable of generating cells

with the character of embryonic stem cells. Teratomas are germ

cell tumors that generate all three germ layers as well as more

advanced cells, tissues, and partial limb and organ primordia.

Yet these chaotic, disorganized, and nonfunctional masses lack

entirely the structure and dynamic character of an organism.

Likewise, failures of fertilization due to abnormal complements

or chromatin configurations (imprinting) of the chromosomes

may still proceed along partial trajectories of organic growth

without being meaningfully designated as organismal entities.

These natural examples of “partial generative potential”

(described by some as ‘pseudo-embryos’), together with other

observations of early embryonic process, have led to a diverse

array of suggestions for ways that embryonic stem cells may
92 MONITORING STEM CELL RESEARCH

be produced without raising the moral issues involved in the

creation and destruction of human embryos. These

suggestions include the use of aneuploidies, polyploidies,

viable cells from embryos in arrested development,

parthenotes, and chimeras of human nuclear material and

animal oocytes. Each presents its own particular technical

challenges and raises unique and unfamiliar moral

considerations. The scientific prospects for such projects

remain largely unexplored in humans, though some animal

work has shown promise, and proponents argue that they are

within the reach of current technology.144

The crucial principle in all such efforts, proponents argue,

is the preemptive nature of the intervention: undertaken at a

stage before the transition to organismal status. They contend

that just as we have learned that neither genes, nor cells, nor

even whole organs define the locus of human moral standing,

in this era of developmental biology we will come to recognize

that cells and tissues with “partial generative potential” may

be used for medical benefit without a violation of human life

or dignity. Moreover, they argue, the moral distinctions

essential to discern and define the categories of organism,

embryo, and human being will be critical to progress in

scientific research involving embryonic stem cells, chimeras,

and laboratory studies of fertilization and early embryogenesis.

These advances in developmental biology, they contend, will

depend on clarifying these categories and defining the moral

boundaries in a way that at once defends human dignity while

clearing the path for scientific progress.145

This proposal has drawn criticism on several fronts. First,

critics suggest, it would require significant research to ensure

that the procedure reliably produced the desired sort of “nonembryonic” entity yet also still yielded normal human

embryonic stem cells, and such research might itself be morally

problematic. Second, this proposal raises a series of further

scientific and ethical questions, including those regarding the

minimal degree of “partial generative potential” for an entity

to be considered an organism, and for an entity to be

considered a human embryo. They point further to the risk of

creating entities that are so ambiguous as to leave their moral

standing in serious doubt, at least for those people who believe

that the early stages of human life have at least some moral
ETHICAL AND POLICY DEVELOPMENTS 93

worth. Finally, proposals to use human oocytes raise moral

concern regarding the source of supply, in this case as in the

larger arena of in vitro fertilization and experimentation.146

Although this approach has never been tested in humans,

animal experiments suggest it has potential, and it has begun

to play a part in the debates over the moral standing of human

embryos and the permissibility of embryo research more

generally.

V. SOCIETAL SIGNIFICANCE AND

PUBLIC RESPONSIBILITY

While the bulk of the public debate surrounding embryonic

stem cell research has been directed to the question of the

moral standing of human embryos, some commentators have

raised a number of other crucial and serious concerns. They

have argued that the debate suffers from focusing too narrowly

on questions of the standing of human embryos, when other

issues—including the duties and responsibilities of those who

engage in embryonic stem cell research, the implications of

such uses of nascent life for our society (rather than just for

the embryos themselves), the significance of the public debate,

and a series of other issues—also bear heavily on the subject,

and may illuminate it in ways at least as significant.147

Some authors, including some who do not believe that

human embryos should simply be treated as inviolable

persons, have argued that the instrumentalization of human

embryos—the seeds of the next generation—might tend to

coarsen the sensibilities of our society toward future

generations, and toward human life in general, quite apart

from the effects on the embryos themselves.148 Others also

argue that by setting down the path laid out by human

embryonic stem cell research, we open the way for other, and

more troubling, techniques and developments. Since human

suffering and disease will never come to an end, they suggest,

the resort to extreme and potentially exploitative methods is

unlikely to find a logical stopping point. Today, they argue,

scientists want to use only the earliest embryos; but what
94 MONITORING STEM CELL RESEARCH

will happen when it turns out that later-stage embryos are

even more valuable in developing treatments for disease?149

Critics of this view have generally argued that it fails to

offer a sufficient ground for impeding the promise and potential

of medical treatments that might result from embryonic stem

cell work. These critics see medical research as a central moral

duty, and they argue that a society that prevents such research

undermines not only medical progress, but the moral progress

of the community as a whole. While instrumentalization and

moral coarsening are real worries, these critics argue, longterm fears of a “slippery slope” do not justify renouncing the

potential of today’s research. Future difficulties, they say, can

be faced if and when they arrive in earnest.150

Other observers have raised concerns related to the ethical

and policy debate itself, rather than to the specifics of one

technique or another, or of one funding policy or another. Some,

for instance, have argued that what is needed in the human

embryonic stem cell research debate is not only an exchange

of views about the substantive issues (though that is surely

crucial) but also some sense of the appropriate democratic

process for deliberation and for establishing appropriate public

policy on such a profoundly contentious matter. The embryonic

stem cell debate, it is argued, offers a valuable opportunity to

think through the ways in which the American polity debates

the most contentious moral issues.151 Some even suggest that

such matters should be removed from the political process

altogether, and left in the hands of a regulatory body

specifically charged with monitoring and decision-making

authority.152

Other observers worry that the promise of embryonic stem

cell research to bring swift or immediate cures to those who

are now ailing has been oversold. They point out that, two

decades ago, similar claims of rapid cures were made on behalf

of fetal tissue transplantation research, but have not as yet

been realized (though, of course, the danger of unfulfilled hope

always looms over medical research). Such talk, some

observers have argued, tends to raise false hopes, and thus

does a genuine disservice to the sick and disabled.153 Worse,

some regard it as cruel and immoral to exploit the hopes and

fears of the desperately ill and their families, especially when—

as several scientists in the field have remarked—it is not very
ETHICAL AND POLICY DEVELOPMENTS 95

likely that stem cell based remedies will be available for most

people now suffering from the potentially targeted diseases.

This moral concern is tied directly to the longstanding

bioethical principle of truth-telling, which obliges physicians

to inform their patients honestly about their condition and

prognosis and encourages researchers to be truthful in their

assessments of the potential for new treatments and cures,

whether or not what they have to say is what patients and

their loved ones want to hear. “It is misleading to suggest

that stem cells will bring cures,” one writer observes,

“particularly cures for patients now coping with the serious

diseases the research targets.”154

A related concern, raised by some of the same

commentators, involves what has been termed “the

disproportionate emphasis on stem cell research in

contemporary health politics.”155 The prominence of this debate

in American politics, they argue, may tend to distract us from

the fact that many Americans, and even more people

elsewhere, lack very basic healthcare and have no access to

those medical tools and techniques that already exist and that

raise no profound controversy.156 The concern for justice, and

for the proper setting of priorities, they argue, requires us to

see this line of research in its proper perspective. Because

federal resources for research are limited, decisions must be

made about which areas should receive high (and low) priority,

and decisions must be made about how much to devote to

research and how much to devote to programs that provide

proven health care to patients. These commentators suggest

that this view does not mean that stem cell research should

not be funded, but only that we must keep in mind that funds

are also needed for many other approaches to fighting suffering

and disease.157 Critics of this view, however, respond that an

excessive preoccupation with existing health care needs can

jeopardize new medical research and medical progress, and

that today’s “basic medicine” was once experimental

research.158

Meanwhile, others have argued that the debate has been

too narrow in another way. Any federal policy, they suggest,

must take note not only of the potential promise of embryonic

stem cell science, but also possible alternatives to such

research, alternatives less morally troubling to many
96 MONITORING STEM CELL RESEARCH

Americans. Many opponents of public funding for human

embryonic stem cell research, for instance, have argued that

more attention should be paid, and more resources devoted,

to adult stem cell research, which raises few of the moral

difficulties present in the embryonic stem cell debates159

(though, for some, it still does raise a number of ethical

difficulties).160 Such work, they contend, might even make

embryonic stem cell research (or, at the very least, publicly

funded embryonic stem cell research)161 unnecessary, if it

proves sufficiently useful.*

In response, however, others point out that adult stem cell

research already receives about ten times the amount of federal

funding apportioned to human embryonic stem cell research.

Critics also argue that embryonic stem cells may possess

unique advantages over adult stem cells, just as (in some

circumstances) the opposite may be the case, and that

therefore both avenues, as well as research using stem cells

derived from fetal tissue, should be pursued simultaneously.162

And they contend that opponents of embryonic stem cell

research have oversold the promise of adult stem cells so that

the public might come to see embryonic stem cell research as

unnecessary.163 In the next chapter, we examine some of the

scientific facts regarding adult and embryonic stem cells, but

in the context of the ethical and political debates, the

distinctions between them have been quite important and

prominent.

In these ways, the controversial possibility of scientific

alternatives, as well as concerns about the health of American

culture and democracy, the honesty of a political debate that

touches on the hopes and fears of many who are suffering,

and the bigger picture of health-care politics all impinge upon

the question of federal funding of human embryonic stem cell

research.

* In 1999, the National Bioethics Advisory Commission stated that, “In our

judgment, the derivation of stem cells from embryos remaining following

infertility treatments is justifiable only if no less morally problematic alternatives are available for advancing the research,” though the commission

did not conclude that adult stem cells were sufficiently shown to offer such

an alternative. (National Bioethics Advisory Commission, Ethical Issues in

Human Stem Cell Research, Washington, D.C.: Government Printing Office,

1999, p. 53.)
ETHICAL AND POLICY DEVELOPMENTS 97

VI. CONCLUSION

Participants in the public debate surrounding human

embryonic stem cell research and the administration’s funding

policy have addressed themselves to many complicated and

difficult ethical matters: the character of the moral question at

issue; the nature, object, ethical assumptions, and premises

of the policy itself; the vexing question of what is owed to the

early human embryo; and a number of other related concerns.

As we have seen, strong and powerfully argued views have

been presented on various sides of each of these questions.

For now, neither side to the debate seems close to fully

persuading the other of the truth it thinks it sees. But the rich

and growing ethical debates do suggest the possibility of

progress toward greater understanding of the issues, and

toward more informed public decisionmaking, as all parties

to the deliberation appreciate better just what is at stake, not

only for them or their opponents, but indeed for all of us.
98 MONITORING STEM CELL RESEARCH

ENDNOTES

1

 “Press Conference by President Bush and Italian Prime Minister Berlusconi,” as

made available by the White House Press Office, July 23, 2001.

2

 See, for instance, Weissman, I. L., “Stem Cells—Scientific, Medical and Political

Issues,” New England Journal of Medicine 346(20): 1576-1579 (2002).

3

 See, for instance, Green, R., “Determining Moral Status,” American Journal of

Bioethics 2(1): 28-29 (2002).

4

 See, for instance, Orr, R., “The Moral Status of the Embryonal Stem Cell: Inherent

or Imputed?” American Journal of Bioethics 2(1): 57-59 (2002).

5

 See, for instance, Doerflinger, R., “Ditching Religion and Reality,” American Journal

of Bioethics 2(1): 31 (2002).

6

 See, for instance, Latham, S., “Ethics and Politics,” American Journal of Bioethics

2(1): 46 (2002).

7

 See, for instance, Marquis, D., “Stem cell research: The Failure of Bioethics,” Free

Inquiry 22(1): Winter 2002.

8

 See, for instance, the presentation of Richard Doerflinger before the Council on

June 12, 2003. A transcript of that presentation is available on the Council website

at www.bioethics.gov.

9

 See, for instance, FitzGerald, K., “Questions Concerning the Current Stem Cell

Debate,” American Journal of Bioethics 2(1): 50-51 (2002).

10 See, for instance, Haseltine, W., “Regenerative Medicine: A Future Healing Art,”

Brookings Review, Winter 2003.

11 See, for instance, Stolberg, S., “Scientists Urge Bigger Supply of Stem Cells,” The

New York Times, September 11, 2001, p. A1.

12 See, for instance, Perry, D., “Patients’ Voices: The Powerful Sound in the Stem Cell

Debate,” Science 287: 5457, 1423; and the presentation of Irving Weissman before

the Council on February 13, 2002, available on the Council website at

www.bioethics.gov.

13 This point was taken up by the Council in its report Human Cloning and Human

Dignity: An Ethical Inquiry (July 2002), Chapter 6, pp. 167-168.

14 One useful description of this approach to the issue is Zoloth, L., “Jordan’s Banks,

A View from the First Years of Human Embryonic Stem Cell Research,” American

Journal of Bioethics 2(1): 7 (2002).

15 Callahan, D., What Price Better Health: The Hazards of the Research Imperative.

Los Angeles, CA.: University of California Press, 2003; also see Daniel Callahan’s

presentation before the Council on July 24, 2003, available on the Council’s website

at www.bioethics.gov.
ETHICAL AND POLICY DEVELOPMENTS 99

16 FitzGerald, K., op. cit., pp. 50-51.

17 For examples, see Weiss, R., “Legal Barriers to Human Cloning May Not Hold

Up,” The Washington Post, May 23, 2001.

18 See, for instance, Elias, P., “States, Feds May Clash over Cloning Rules,” Genomics

and Genetics Weekly, April 12, 2002, p. 24.

19 This, for instance, was the line of reasoning of the American Bar Association

when, in August of 2002, it issued a resolution supporting limited therapeutic

research using cloned human embryos. The report of the ABA’s Section of Individual

Rights and Responsibilities (available from the ABA) lays out the case.

20 See, for instance, Russo, E., “Policy Questions and Some Answers,” The Scientist,

April 15, 2003.

21 R. Alta Charo, of the University of Wisconsin Law School, quoted in Coyle, M.,

“The Clone Zone,” The National Law Journal, May 15, 2002.

22 Alan Meisel of the University of Pittsburgh School of Law, quoted in Coyle, M.,

“The Clone Zone,” The National Law Journal, May 15, 2002.

23 Berkowitz, P., “The Meaning of Federal Funding,” a paper commissioned by the

President’s Council on Bioethics and included as Appendix F of this report.

24 See: Maher v. Roe 432 U.S. 464 (1977); Harris v. McRae 448 U.S. 297 (1980); Rust v.

Sullivan 500 U.S. 173 (1991).

25 Berkowitz, P., “The Meaning of Federal Funding,” a paper commissioned by the

President’s Council on Bioethics, and included as Appendix F of this report. See

also Khushf, G., and Best, R., “Stem Cells and the Man on the Moon: Should We Go

There from Here?” American Journal of Bioethics 2(1): 38 (2002).

26 FitzGerald, K., op. cit., pp. 50-51.

27 Doerflinger, R., op. cit., p. 31.

28 Childress, J., “Federal Policy Toward Embryonic Stem cell research,” American

Journal of Bioethics 2(1): 34 (2002).

29 Kahn, J., “Missing the mark on stem cells,” Bioethics Examiner 5:3, Fall 2001, p. 1.

30 Murray, T., “Hard Cell,” The American Prospect, September 24, 2001.

31 See, for instance, Lefkowitz, J., “The Facts on Stem Cells,” The Washington Post,

October 30, 2003, p. A23; and Bush, G.W., “Stem Cell Science and the Preservation

of Life,” The New York Times, August 12, 2001, p. D13.

32 “How many lines,” The Washington Post, August 31, 2001, p. A22.

33 Daley, G., “Cloning and Stem Cells—Handicapping the Political and Scientific

Debates,” New England Journal of Medicine 349(3): 211-212 (2003).

34 Kennedy, D., “Stem Cells: Still Here, Still Waiting,” Science 300: 865 (2003).
100 MONITORING STEM CELL RESEARCH

35 See, for instance, Brickley, P., “Scientists Seek Passports to Freer Environments,”

The Scientist 15:36 (2001).

36 See, for instance, Weissman, I., “Stem Cells—Scientific, Medical and Political

Issues,” New England Journal of Medicine 346(20): 1578-1579 (2002).

37 See, for instance, Weise, E., “USA’s Stem Cell Scientists Fear a Research BrainDrain,” USA Today, May 12, 2003, p. 6D.

38 Clark, J., “Squandering Our Technological Future,” The New York Times, August

30, 2001, p. A19. See also the presentation of Thomas Okarma, President and CEO

of Geron Corporation, before the Council on September 4, 2003. The full transcript

of Okarma’s presentation may be found on the Council’s website at

www.bioethics.gov.

39 Robertson, J., “Crossing the Ethical Chasm: Embryo Status and Moral Complicity,”

American Journal of Bioethics 2(1): 33 (2002).

40 See, for instance, Pizzo, P., “Remove Obstacles to Stem cell research,” San Jose

Mercury-News, June 19, 2003; and Holden, C. and Vogel, G., “‘Show Us the Cells’

U.S. Researchers Say,” Science 297: 923 (2002).

41 The most notable discussion of safety concerns has been Dawson, L., et al., “Safety

Issues in Cell-Based Intervention Trials” Fertility and Sterility 80: 5, 1077-1085, 2003.

We must note, however, that the assessment of the subject presented in that article

relies upon the assumption that “all of [the approved] lines were derived using

mouse feeder layers.” (p. 1078). In his presentation before the Council on September

4, 2003, NIH Director Elias Zerhouni flatly contradicted this assertion, telling the

Council that “there are at least those, which is about 16 lines, I believe, that have

not been exposed to either mouse or human cell—human feeder cell lines.” (The

transcript of Zerhouni’s presentation is available on the Council’s website at

www.bioethics.gov.) Those eligible unexposed lines, however, are not presently

among the lines available to researchers, and it cannot be known in advance if or

when they might become available or whether they will prove useable. In addition,

the FDA has stated that even in the case of those lines that were developed with

mouse feeders, “FDA does not intend that the agency’s regulation of

xenotransplantation will preclude the use of these hES cell lines.” (Food and Drug

Administration, “Guidance for Industry - Source Animal, Product, Preclinical, and

Clinical Issues Concerning Use of Xenotransplantation Products in Humans,” April

3, 2003.) FDA Commissioner Mark McClellan also made that clear in his presentation

before the Council, available at the Council’s website.

42 Faden, et al., “Public Stem Cell Banks: Considerations of Justice in Stem Cell

Research and Therapy” Hastings Center Report 33:6 (2003).

43 See, for instance, Capron, A., “Stem Cell Politics: The New Shape to the Road

Ahead,” American Journal of Bioethics 2(1): 35-36 (2002).

44 Robertson, J., op. cit., pp. 33-34.

45 See, for instance, the testimony of HHS Secretary Tommy Thompson before the

Senate Health, Education, Labor and Pensions Committee, September 5, 2001; and

Lefkowitz, J., “The Facts on Stem Cells,” The Washington Post, October 30, 2003, p.

A23.
ETHICAL AND POLICY DEVELOPMENTS 101

46 Brownstein, R., “Bush Won’t Budge on Stem Cell Position, Health Secretary Says,”

Los Angeles Times, August 13, 2001, p. A9.

47 This comment was made by Council Member Gilbert Meilaender in Council

discussion on September 4, 2003. A transcript of that discussion is available on the

Council’s website at www.bioethics.gov.

48 See, for instance, FitzGerald, K., op. cit., pp. 50-51.

49 Khushf, G., and Best, R., op. cit., p. 38.

50 See, for instance, the discussion of the Council in its September 4, 2003 meeting,

particularly the argument elucidated by Council Member Michael Sandel. A

transcript of that session is available on the Council website at www.bioethics.gov.

51 See, for instance, Stolberg, S., op. cit.

52 See, for instance, Robertson, J., “Ethics and Policy in Embryonic Stem cell

research,” Kennedy Institute of Ethics Journal 9(2): 109-136 (1999); Spike, J. “Bush

and Stem cell research: An Ethically Confused Policy,” American Journal of Bioethics

2(1): 46 (2003); and Capron, A., op. cit., p. 36.

53 The statement was made by bioethicist Glen McGee, in reference to the Clintonera proposed NIH rules on funding, quoted in Spanogle, J., “Transforming Life,”

The Baylor Line, Winter 2000, p. 30.

54 The remark was made by Bishop Joseph A. Fiorenza, President of the U.S.

Conference of Catholic Bishops, in a news release (“Catholic Bishops Criticize Bush

Policy on Embryo Research,” made available by the United States Conference of

Catholic Bishops, August 9, 2001).

55 This argument arose in the course of Council discussion on September 4, 2003

(see particularly the comments of Council Members Michael Sandel and Charles

Krauthammer in that discussion). A transcript of that session is available on the

Council’s website at www.bioethics.gov.

56 Ibid., see particularly the comments of Council Members Mary Ann Glendon and

Gilbert Meilaender in that discussion.

57 For these guidelines, see Appendix D.

58 Ibid., see particularly the comments of Council Member William Hurlbut in that

discussion.

59 Some reasons to regard public funding of activities in a light different from mere

permission are proposed in Khushf, G., and Best, R., op. cit., p. 38.

60 “Remarks by the President on Stem Cell Research,” as made available by the

White House Press Office, August 9, 2001.

61 “President’s Phone Call to March for Life Participants,” as made available by

the White House Press Office, January 22, 2002.

62 See Council discussion on September 4, 2003, especially comments by Council

Members Charles Krauthammer, Francis Fukuyama, Gilbert Meilaender, and Leon
102 MONITORING STEM CELL RESEARCH

Kass. A transcript of that session is available on the Council’s website at

www.bioethics.gov.

63 Bush, G.W., “Stem Cell Science and the Preservation of Life,” The New York Times,

August 12, 2001, p. D13.

64 “Remarks by the President on Stem Cell Research,” as made available by the

White House Press Office, August 9, 2001.

65 See, for instance, Council discussion on September 4, 2003 (particularly the

comments of Council member Michael Sandel and invited guest Peter Berkowitz in

that discussion). A transcript of that session is available on the Council’s website

at www.bioethics.gov.

66 “Remarks by the President on Stem Cell Research,” as made available by the

White House Press Office, August 9, 2001.

67 See, for instance, Council discussion on September 4, 2003 (particularly the

comments of Council Members Charles Krauthammer and James Wilson in that

discussion). A transcript of that session is available on the Council’s website at

www.bioethics.gov.

68 For instance, in discussing research on cloned human embryos, President Bush

said, “Research cloning would contradict the most fundamental principle of medical

ethics, that no human life should be exploited or extinguished for the benefit of

another. Yet a law permitting research cloning, while forbidding the birth of a cloned

child, would require the destruction of nascent human life.” (“Remarks by the

President on Human Cloning Legislation,” as made available by the White House

Press Office, April 10, 2002); and in explaining his stem cell funding policy, the

President wrote: “While it is unethical to end life in medical research, it is ethical to

benefit from research where life and death decisions have already been made.”

(Bush, G.W., “Stem Cell Science and the Preservation of Life,” The New York Times,

August 12, 2001, p. D13.)

69 As noted previously, in August of 2001 Health and Human Services Secretary

Tommy Thompson told reporters that “neither unexpected scientific breakthroughs

nor unanticipated research problems would cause Bush to reconsider.” (See,

Brownstein, R., “Bush Won’t Budge on Stem Cell Position, Health Secretary Says,”

Los Angeles Times, August 13, 2001, p. A9).

70 One useful account of these issues is Cohen, E., “Of Embryos and Empire,” The

New Atlantis 2: 3-16 (2003).

71 See, for instance, London, A., “Embryos, Stem Cells, and the ‘Strategic’ Element

of Public Moral Reasoning,” American Journal of Bioethics 2(1): 56 (2002).

72 For examples of this way of proceeding—both in arguments supporting embryo

research, and arguments opposing it—see, among numerous other sources, the

Council’s July 2002 report, Human Cloning and Human Dignity: An Ethical Inquiry,

Chapter 6.

73 See, for instance, McCartney, J., “Embryonic Stem cell research and Respect for

Human Life: Philosophical and Legal Reflections,” Albany Law Review 65: 597-624

(2002).

74 See, for instance, Orr, R., op. cit., pp. 57-58.
ETHICAL AND POLICY DEVELOPMENTS 103

75 See, for instance, Steinberg, D., “Can Moral Worthiness Be Seen Using a

Microscope?” American Journal of Bioethics 2(1): 49 (2002).

76 See, for instance, Green, R., op. cit., p. 20.

77 See, for instance, Doerflinger, R., op. cit., pp. 31-33; Orr, R., op. cit., pp. 57-58; and

the personal statements of Council Members William Hurlbut and Robert George

and Alfonso Gómez-Lobo, in the Council’s July 2002 report Human Cloning and

Human Dignity: An Ethical Inquiry, among others.

78 See, for instance, Orr, R., op. cit., pp. 57-58.

79 See, for instance, the personal statement of Council Member William Hurlbut, in

the Council’s July 2002 report Human Cloning and Human Dignity: An Ethical Inquiry.

80 See, for instance, Sullivan, D., “The Conception View of Personhood: A Review,”

Ethics and Medicine 19(1): 11-33 (Spring 2003).

81 A form of this argument was presented by some Members of the Council in the

Council’s July, 2002 report Human Cloning and Human Dignity: An Ethical Inquiry,

chapter 6, and in several of the personal statements appended to that report.

82 See, for instance, Steinberg, D., op. cit., p. 49.

83 See, for instance, the transcript of Council discussion on October 17, 2003,

especially the comments of Council Member Elizabeth Blackburn. The full transcript

is available on the Council’s website at www.bioethics.gov. Also see Green, R., op.

cit., pp. 20-30.

84 See, for instance, Lanza, R., et al., “The Ethical Validity of Using Nuclear Transfer

in Human Transplantation,” Journal of the American Medical Association, 284(24):

3175-3179 (2000). Also, see the views expressed by some Members of this Council

in our July 2002 report Human Cloning and Human Dignity: An Ethical Inquiry,

chapter 6, and in the personal statements of Council Members Elizabeth Blackburn,

Michael Gazzaniga, and Janet Rowley.

85 See, for instance, the transcript of Council discussion on April 25, 2002, especially

the comments of Council Member Daniel Foster. The full transcript is available on

the Council’s website at www.bioethics.gov.

86 See, for instance, the position articulated in “Position Number Two” of the “Moral

Case for Cloning for Biomedical Research” presented by some Members of the

Council in the Council’s July, 2002 report Human Cloning and Human Dignity: An

Ethical Inquiry, Chapter 6; and the personal statement of Council Member Michael

Gazzaniga, appended to that report.

87 See, for instance, the comments of Council Member Michael Gazzaniga (and

response from other Members) during Council discussion on February 14, 2002.

The full transcript is available on the Council’s website at www.bioethics.gov.

88 See, for instance, Orr, R., op. cit., pp. 57-59; Callahan, S., “Zygotes and Blastocysts:

Human enough to protect,” Commonweal, June 14, 2002; the position articulated

by several Members of this Council in our July 2002 report Human Cloning and

Human Dignity: An Ethical Inquiry, Chapter 6; and the personal statements of Council
104 MONITORING STEM CELL RESEARCH

Members Robert George and Alfonso Gómez-Lobo, and William Hurlbut in that same

report.

89 See, for instance, Steinberg, D., op. cit., pp. 49-50; and Green, R., op. cit., p. 20.

90 See, for instance, Meyer, M. J., and Nelson, L. J., “Respecting What We Destroy:

Reflections on Human Embryo Research,” Hastings Center Report 31(1): 16-23 (2001).

91 See, for instance, Strong, C., “The Moral Status of Preembryos, Embryos, Fetuses,

and Infants,” The Journal of Medicine and Philosophy 22(5): 457-478 (1997).

92 Pearson, H., “Your Destiny, from Day One,” Nature 418: 14-15 (2002).

93 See, for instance, McCartney, J., op. cit., pp. 601-602; Brogaard, B., “The moral

status of the human embryo: the twinning argument,” Free Inquiry Winter 2002;

Meyer, M. J., op. cit., p. 18. The question of twinning—pro and con—was also taken

up by the Council in its July 2002 report, Human Cloning and Human Dignity: An

Ethical Inquiry, Chapter 6.

94 See, for instance, Green , R., op. cit., pp. 21-22; Lanza, R., op. cit., p. 3177.

95 See, for instance, Ashley, B., and Moraczewski, A., “Cloning, Aquinas, and the

Embryonic Person,” National Catholic Bioethics Quarterly 1(2): 189-201 (2001); Orr,

R., op. cit., p. 58; Marquis, D., op. cit.; and Lori Andrews quoted in Green, R., op. cit.,

p. 22. The question of twinning—pro and con—was also taken up by the Council in

its July 2002 report, Human Cloning and Human Dignity: An Ethical Inquiry, Chapter

6, and in the personal statement of Council Member William Hurlbut, attached to

that report.

96 See, for instance, National Bioethics Advisory Commission, Ethical Issues in Human

Stem cell research, Washington, D.C.: Government Printing Office, 1999, p. 7; and

the position articulated by several Members of this Council in our July 2002 report

Human Cloning and Human Dignity: An Ethical Inquiry, Chapter 6, among many

others.

97 See, for instance, Lanza, R., op. cit., p. 3177.

98 See, for instance, Strong, C., op. cit., p. 467.

99 See, for instance, the views expressed by some Members of this Council in our

July 2002 report Human Cloning and Human Dignity: An Ethical Inquiry, Chapter 6,

and in the personal statement of Council Members Robert George and Alfonso

Gómez-Lobo.

100 See, for instance, the personal statement of Council Member William Hurlbut, in

the Council’s July 2002 report Human Cloning and Human Dignity: An Ethical Inquiry.

101 See, for instance, Wilson, J., “On Abortion,” Commentary January 1994; and the

position articulated by several Members of this Council in our July 2002 report

Human Cloning and Human Dignity: An Ethical Inquiry, Chapter 6.

102 See, for instance, Green, R., op. cit., pp. 20-30; and the position articulated by

several Members of this Council in our July 2002 report Human Cloning and Human

Dignity: An Ethical Inquiry, Chapter 6.
ETHICAL AND POLICY DEVELOPMENTS 105

103 See, for instance, the views expressed by some Members of this Council in our

July 2002 report Human Cloning and Human Dignity: An Ethical Inquiry, Chapter 6,

and in the personal statement of Council Members Robert George and Alfonso

Gómez-Lobo.

104 See, for instance, National Institutes of Health, Report of the Human Embryo

Research Panel, Bethesda, MD.: NIH, 1994; and the position articulated by several

Members of this Council in our July 2002 report Human Cloning and Human Dignity:

An Ethical Inquiry, Chapter 6.

105 See, for instance, McGee, G., “The Idolatry of Absolutizing in the Stem Cell

Debate,” American Journal of Bioethics 2(1): 53-54 (2002).

106 See, for instance, Green, R., op. cit., p. 20.

107 See, for instance, the views expressed by some Members of this Council in our

July 2002 report Human Cloning and Human Dignity: An Ethical Inquiry, Chapter 6,

and in the personal statements of Council Members Gilbert Meilaender, William

Hurlbut, Robert George and Alfonso Gómez-Lobo.

108 “Report of the Ethics Advisory Board,” 44 Fed. Reg. 35,033-35,058 (June 18, 1979)

at 35,056.

109 NIH, op. cit., Report of the Human Embryo Research Panel, p. 2.

110 National Bioethics Advisory Commission (NBAC), Ethical Issues in Human Stem

Cell Research Bethesda, MD.: Vol. I, p. ii; cf. p. 2 (September 1999).

111 See, for instance, Lebacqz, K., “On the Elusive Nature of Respect,” in Holland, S.,

Lebacqz K., and Zoloth, L., eds., The Human Embryonic Stem Cell Debate, Cambridge,

MA.: MIT Press, 2001, pp. 149-162; and the personal statement of Council Member

Rebecca Dresser, appended to the Council’s July 2002 report Human Cloning and

Human Dignity: An Ethical Inquiry.

112 Hoffman, D.I., et al., “Cryopreserved Embryos in the United States and Their

Availability for Research,” Fertility and Sterility 79: 1063-1069 (2003).

113 See, for instance, Spike, J., op. cit., p. 45.

114 See, for instance, the testimony of Michael West before the Labor, HHS, and

Education Subcommittee of the Senate Appropriations Committee, December 4,

2001, among others.

115 Outka, G., “The Ethics of Stem cell research,” a paper presented to the Council

for its April 2002 meeting, and available on the Council website at

www.bioethics.gov. A slightly revised version appeared in the Kennedy Institute of

Ethics Journal, 12:2, pp. 175-213.

116 Ibid.

117 See, for instance, “The Stem Cell Sell,” Commonweal, August 17, 2001; Meilaender,

G., “Spare Embryos,” The Weekly Standard, August 26, 2002; and Council discussion

of Gene Outka’s paper, in its April 2002 meeting, a transcript of which is available

on the Council website at www.bioethics.gov.

118 See, for instance, McCartney, J, op. cit., p. 615.
106 MONITORING STEM CELL RESEARCH

119 Council Member William Hurlbut, in Council communication.

120 See, for instance, the comments of Council Member James Wilson during Council

discussion on April 25, 2002. The full transcript is available on the Council’s website

at www.bioethics.gov.

121 A federal law, 42 U.S.C. § 289, is directed specifically against any such lethal

experimentation on these populations.

122 This concern is raised in Outka, G., “The Ethics of Stem Cell Research,” a paper

presented to the Council for its April 2002 meeting, and available on the Council

website at www.bioethics.gov. Also see the Council discussion of that paper,

available on the Council website; and see Meilaender, G., op. cit.

123 See, for instance, the transcript of Council discussion on April 25, 2002, available

on the Council’s website at www.bioethics.gov.

124 Guenin, L. M., “Morals and Primordials,” Science 292: 1659-1660 (2001).

125 Spike, J., op. cit., p. 45.

126 Guenin, L., op. cit.

127 See, for instance, Orr, R., op. cit., p. 58.

128 Mahowald, M. and Mahowald A., “Embryonic Stem Cell Retrieval and a Possible

Ethical Bypass,” American Journal of Bioethics 2(1): 42-43 (2002).

129 See, for instance, Zoloth, L., op. cit., pp. 4-5.

130 See, for instance, Harris, J., “The ethical use of human embryonic stem cells in

research and therapy,” in Burley, J. and Harris, J., eds., A Companion to Genetics:

Philosophy and the Genetic Revolution, Oxford: Basil Blackwell, 2001; and Savulescu,

J., “The Embryonic Stem Cell Lottery and the Cannibalization of Human Beings,”

Bioethics 16(6): 508-529 (2002).

131 This subject was discussed in a Council session on January 16, 2003. When

asked, in the course of a presentation before the Council, about the rate of natural

embryo loss, Dr. John Opitz (Professor of Pediatrics, Human Genetics, and Obstetrics/

Gynecology, School of Medicine, University of Utah) responded: “Estimates range

all the way from 60 percent to 80 percent of the very earliest stages, cleavage stages,

for example, that are lost.” The transcript of this session is available on the Council

website at www.bioethics.gov.

132 Spike, J., op. cit., p. 45.

133 Harris, J., “Stem Cells, Sex & Procreation,” a paper presented to the American

Philosophical Society, March 29, 2003.

134 Savulescu, J., op. cit., pp. 508-529.

135 See, for instance, Orr, R., op. cit., pp. 57-59.

136 See, for instance, Council discussion in its January 2003 meeting, a transcript of

which is available on the Council website at www.bioethics.gov.
ETHICAL AND POLICY DEVELOPMENTS 107

137 See, for instance, the comments of Rudolph Jaenisch before the Council in its

July 2003 meeting, and Dr. Jaenisch’s accompanying paper (included in Appendix

N of this report). See also the personal statement of Council Member Paul McHugh,

appended to the Council’s July 2002 report Human Cloning and Human Dignity: An

Ethical Inquiry.

138 See, for instance, Zoloth, L., op. cit., pp. 4-5.

139 See, for instance, Mandavilli, A., “Fertility’s New Frontier Takes Shape in the

Test Tube,” Nature Medicine 9: 1095 (2003).

140 See, for instance, Council discussion with Rudolph Jaenisch at the Council’s

July 2003 meeting, and discussion taken up in the Council’s July 2002 report Human

Cloning and Human Dignity: An Ethical Inquiry, Chapter 6.

141 See, for instance, the personal statement of Council Member William Hurlbut, in

the Council’s July 2002 report Human Cloning and Human Dignity: An Ethical Inquiry;

the transcripts of Council discussions on June 20, 2002 and July 24, 2003, available

on the Council’s website at www.bioethics.gov; and Weiss, R., “Can Scientists

Bypass Stem Cells’ Moral Minefield?” The Washington Post, December 14, 1998, p.

A3.

142 See, for instance, the transcript of Council discussion on July 24, 2003, available

on the Council’s website at www.bioethics.gov.

143 See, for instance, the transcript of Council discussion on June 20, 2002, available

on the Council’s website at www.bioethics.gov.

144 See remarks by Professor Rudolph Jaenisch before the Council’s July 24, 2003

meeting. A transcript of that session is available on the Council’s website at

www.bioethics.gov.

145 See, for instance, the personal statement of Council Member William Hurlbut, in

the Council’s July 2002 report Human Cloning and Human Dignity: An Ethical Inquiry.

146 See, for instance, a number of arguments raised in Weiss, R., “Can Scientists

Bypass Stem Cells’ Moral Minefield?” The Washington Post, December 14, 1998, p.

A3; and the transcript of Council discussion on June 20, 2002, available on the

Council’s website at www.bioethics.gov.

147 Lauritzen, P., “Report on the Ethics of Stem cell research,” a commissioned paper

prepared at the request of the Council and included in Appendix G of this report.

See also the Council’s July 2002 report Human Cloning and Human Dignity: An

Ethical Inquiry, Chapter 6.

148 See, for instance, Kass, L., “The Meaning of Life - In the Laboratory,” The Public

Interest, Winter 2002. Also see the Council’s July 2002 report Human Cloning and

Human Dignity: An Ethical Inquiry, Chapter 6.

149 This case is articulated by several Members of the Council in the Council’s July

2002 report Human Cloning and Human Dignity: An Ethical Inquiry, Chapter 6. See

also, “Biotechnology: A House Divided,” The Public Interest, Winter 2003.
108 MONITORING STEM CELL RESEARCH

150 This case is articulated, for instance, by several Members of the Council in the

Council’s July 2002 report Human Cloning and Human Dignity: An Ethical Inquiry,

Chapter 6.

151 Strong, C., “Those Divisive Stem Cells: Dealing with Our Most Contentious

Issues,” American Journal of Bioethics 2(1): 39-40 (2002).

152 See, for instance, the Council’s July 2002 report Human Cloning and Human

Dignity: An Ethical Inquiry, Chapters 6 and 8, available on the Council’s website at

www.bioethics.gov.

153 See, for instance, Dresser, R., “Embryonic Stem Cells: Expanding the Analysis,”

American Journal of Bioethics 2(1): 40-41 (2002); and FitzGerald, K., op. cit., pp. 50-

51.

154 Dresser, R., op. cit., p. 41.

155 Ibid.

156 See also FitzGerald, K., op. cit., pp. 50-51.

157 Dresser, R., op. cit., p. 41.

158 See, for example, the discussion and presentations at the Council’s July 2003

meeting, available on the Council’s website at www.bioethics.gov.

159 See, for instance, Orr, R., op. cit., pp. 57-58; “A review of the National Institute of

Health’s Guidelines for Research Using Human Pluripotent Stem Cells,” Issues in

Law and Medicine 3(17): 293; and the testimony of Representative David Weldon

before the Technology and Space Subcommittee of the Senate Commerce, Science

and Transportation Committee, January 29, 2003.

160 Lauritzen, P., “Report on the Ethics of Stem cell research,” a commissioned paper

prepared at the request of the Council and included in Appendix G of this report.

161 See, for instance, Oldham, R., “Stem Cells: Private Sector Can Do It Better,” The

Wall Street Journal, August 28, 2001, p. A14.

162 See, for example, the discussion and presentations at the Council’s April 2002

meeting, available on the Council’s website at www.bioethics.gov; and Verfaillie,

C., “Multipotent Adult Progenitor Cells: An Update,” a commissioned paper prepared

at the request of the Council and included in Appendix J of this report.

163 See, for example, the discussion and presentations at the Council’s July 2003

meeting, available on the Council’s website at www.bioethics.gov.
4

Recent Developments in Stem Cell

Research and Therapy

Research using human and animal stem cells* is an extremely active area of current biomedical inquiry. It is contributing

new knowledge about the pathways of normal and abnormal

cell differentiation and organismal development. It is opening

vistas of new cell transplantation therapies for human diseases. Although the availability of a variety of human stem cells

is relatively recent—the isolation of human embryonic stem

cells was first reported only in 1998—much is happening in

both publicly funded and privately funded research centers

around the world. It is difficult for anyone to stay abreast of all

the results now rapidly accumulating.

To help us fulfill our mandate to “monitor stem cell

research,” the President’s Council on Bioethics asked several

experts to survey the recent published scientific literature and

to contribute articles on various areas of stem cell research to

this report (see articles by Drs. Gearhart,1

 Ludwig and

Thomson,2 Verfaillie,3 Prentice,4 Itescu,5,6 and Jaenisch7 in the

Appendices). These reviews and the present chapter

emphasize peer-reviewed, published work with human stem

cells through July 2003. Interested readers should also consult

the wide variety of other review articles that have appeared.8

This chapter should be read in conjunction with the commissioned review articles cited above. It draws on their find-

* In this chapter, technical terms that are defined in the Glossary are underlined when they are used for the first time.

109
110 MONITORING STEM CELL RESEARCH

ings, as well as on the Council’s own monitoring activities,

but it makes no attempt to summarize all the complexity of

stem cell research or the vast array of results. Rather we offer

here some general observations and specific examples that

might help non-scientist readers understand the overall state

of present human stem cell research, its therapeutic promise,

and some of the problems that need to be solved if the research is to yield sound knowledge and clinical benefit. To

that end, we highlight the importance of well-characterized,

stable preparations of stem cells for obtaining reproducible

experimental results, and we identify several problems that

must be solved before these requirements can be fully met.

This chapter then describes, by way of illustration and example, some of the better-characterized adult and embryonic stem

cells. It also indicates some of the specific investigations that

are being conducted with their aid. Finally, it considers how

human stem cells are being used to explore their potential for

treating disease, using experiments in animal models of Type1 diabetes as an example, and it points out some of the difficulties that must be overcome before stem cell-based remedies may be available to treat human diseases.

We confine our attention here to newly identified types of

human stem cells and their potential use in research and future

medical treatment. Accordingly, we do not consider those stem

cell types that are already well established in medical practice

and research. Specifically, we will not examine those

preparations of bone marrow cells that have been clinically

used for some years to treat various forms of anemia and

cancer.9 Neither will we deal with hematopoietic (bloodforming) stem cells that have been isolated and purified from

bone marrow and are now being intensively studied.10

Although these developments lie beyond the scope of this

report, the demonstrated usefulness of these cells for research

and therapy encourages many researchers to expect similar

benefits from the newer stem cells that we shall consider here.
RESEARCH AND THERAPY DEVELOPMENTS 111

I. STEM CELLS AND THEIR DERIVATIVES

The adult human body, and all its differentiated cells,

tissues, and organs, arise from a small group of cells contained

within the early embryo at the blastocyst stage of its

development. During in vivo embryonic development, these

cells, constituting the inner cell mass (ICM), will divide and

differentiate in concert with each other and with the whole of

which they are a part, eventually producing the specialized

and integrated tissues and organs of the body. But when

embryos are grown [using in vitro fertilization (IVF)] in a

laboratory setting, these ICM cells may be removed and

isolated, and under appropriate conditions some will

proliferate in vitro and become embryonic stem cell lines.

These embryonic stem cells are capable of becoming many

different types of differentiated cells if stimulated to do so in

vitro [see endnote 2 for references]. However, it is not yet clear

that the cells that survive the in vitro selection process to

become embryonic stem cells have all of the same biological

properties and potentials as the ICM cells of the blastocyst.7

In particular, it is not known for certain that human embryonic

stem cells in vitro can give rise to all the different cell types of

the adult body.*

As noted in the Introduction to this report, stem cells are a

diverse class of cells, which can now be isolated from a variety

of embryonic, fetal, and adult tissues. Stem cells share two

characteristic properties: (1) unlimited or prolonged selfrenewal (that is, the capacity to maintain a pool of stem cells

like themselves), and (2) potency for differentiation, the

potential to produce more differentiated cell types—usually

* It is also not known whether stem cells, either human or animal, when

cultured in vitro apart from the embryonic whole from which they were

originally derived, will function in all respects like cells do when they act

as parts of a developing organic whole.
112 MONITORING STEM CELL RESEARCH

more than one and, in some cases, many.* When stem cells

head down the pathway toward differentiation, they usually

proceed by first giving rise to a more specialized kind of stem

cell (sometimes called “precursor cells” or “progenitor cells”),

which can in turn either proliferate through self-renewal or

produce fully specialized or differentiated cells (see Figure 1).

Figure 1. Schematic Diagram of Some Stages in Cell Differentiation

At the top of the figure is an undifferentiated stem cell; in

the central box are more “specialized” stem cells (or “precursor

cells” or “progenitor cells”); at the bottom are various

differentiated cells that are derived from the specialized stem

* Some stem cells, however, give rise to only one type of specialized cell.

For example, one type of stem cell found in the epidermis (skin) apparently

gives rise only to keratinoctyes (cells that produce the protein keratin, found

in hair and nails).
RESEARCH AND THERAPY DEVELOPMENTS 113

cells. Dashed arrows indicate symmetrical (in the sense that

both the daughter cells are stem cells) cell divisions that

produce more stem cells (self-renewal). Solid arrows indicate

asymmetric cell divisions that produce more differentiated

daughter cells. (There may also be self-renewal with

asymmetric division—not shown here—in which one daughter

cell initiates a differentiation pathway while the other remains

a stem cell.) Differentiation signals can be supplied by both

soluble proteins and by specific, cell-surface binding sites.

Some of the specialized stem cells inside the dashed box, for

example, mesenchymal stem cells, can be isolated from tissues

after birth and correspond to adult stem cells. Scientists are

currently investigating whether, at least in some cases, the

process can be reversed, that is, whether specialized cells may,

on appropriate signals, dedifferentiate to become precursor

or even fully undifferentiated stem cells.

The terminology used to describe different stem cell types

can be confusing. As used in this chapter, stem cells are selfrenewing, cultured cells, grown and preserved in vitro, that

are capable—upon exposure to appropriate signals—of

differentiating themselves into (usually more than one)

specialized cell types. Stem cells may be classified either

according to their origins or according to their developmental

potential.

Stem cells may be obtained from various sources: from

embryos, from fetal tissues, from umbilical cord blood, and

from tissues of adults (or children). Thus, depending on their

origin, stem cell preparations may be called adult stem cells,*

embryonic stem cells, embryonic germ cells, or fetal stem cells.

Adult stem cells [see (4)] are cells derived from various tissues

or organs in humans or animals that have the two characteristic

properties of stem cells (self-renewal and potency for

differentiation). Embryonic stem cells (ESCs) [see (2)] are

derived from cells isolated from the inner cell mass of early

embryos. Embryonic germ cells (EGCs) [see (1)] are stem cells

* As already noted in Chapter 1, “adult stem cells” is something of a misnomer. The cells are not themselves “adult.” As non-embryonic stem cells,

they are, however, partially differentiated and many of them are multipotent.

(See discussion in the text that follows shortly.)
114 MONITORING STEM CELL RESEARCH

derived from the primordial germ cells of a fetus. Fetal stem

cells (not further discussed in this chapter, but included for

the sake of completeness) are derived from the developing

tissues and organs of fetuses; because they come (unlike EGCs)

from already differentiated tissues, they are (like adult stem

cells) “non-embryonic,” and may be expected to behave as

such.

Depending on their developmental potential, cells may be

called pluripotent, multipotent, or unipotent. Cells that can

produce all the cell types of the developing body, such as the

ICM cells of the blastocyst, are said to be pluripotent. The

somewhat more specialized stem cells, of the sort found in

the developed organs or tissues of the body, are said to be

multipotent if they produce more than one differentiated tissue

cell type, and unipotent if they produce only one differentiated

tissue cell type.

We introduce in this chapter an additional term: stem cell

preparation. A stem cell preparation is a population of stem

cells, prepared, grown, and preserved under certain

conditions. Because different laboratories (or even the same

one) can have different preparations of the same type of stem

cell, it is important to recognize the potential differences

between particular preparations of embryonic stem cells.*

 It

will sometimes be important to call attention to this fact, by

speaking of a “preparation of ES cells” (or a preparation of

adult stem cells) rather than of “ES cells,” pure and simple.

We will use the term “stem cell preparations” when we are

speaking of a diverse group of stem cell cultures, when we

are speaking of stem cell cultures that contain an admixture

of other types of cells, or when the developmental homogeneity

of the stem cells in the population has not been defined.

Adult and embryonic stem cell populations have also been

called “stem cell lines.” In the past, the term “cell line” denoted a cell population (usually of cancer cells containing abnormal chromosome numbers or structure, or both) that could

* Embryonic stem cell cultures prepared from different embryos of a single

inbred mouse strain are more likely to have closely similar biological properties than will ESC cultures from genetically different individual human

beings.
RESEARCH AND THERAPY DEVELOPMENTS 115

grow “indefinitely” in vitro. Embryonic and some adult stem

cell preparations are capable of prolonged growth beyond 50

population doublings in vitro while retaining their characteristic stem cell properties and initially with no change in the

chromosome numbers and structure. It is not yet known whether any preparation of human ES cells (generally believed to be

much longer-lived than adult stem cells) will continue to grow

“indefinitely,” without undergoing genetic changes.

Under the influence of various cell-differentiation signals,

embryonic stem cells differentiate into numerous distinct types

of more specialized cells. Some of these are specialized stem

cells that can also self-renew, while retaining their ability also

to differentiate into multiple cell types. Recent research has

led to the isolation of an increasing number of adult (non-embryonic) stem cells (dashed box area of Figure 1) from such

tissues as bone marrow (for example, hematopoietic and mesenchymal stem cells), brain (for example, neural stem cells)

and other tissues [see (4)]. Although these stem cell preparations differ from one another in their future fates, they tend to

be grouped together (especially in the public policy debates)

under the name “adult stem cells,” even though they may have

been obtained from children or even from umbilical cord blood

obtained at the time of childbirth.

Subsequent exposure to additional differentiation signals

can cause these specialized stem cells to differentiate further,

so that they finally give rise to the variety of differentiated

cells that make up the adult body (labeled A-D in Figure 1). At

each stage of the differentiation process, specific sets of genes

are expressed (or “turned on”) and other sets are repressed

(or “turned off”), to produce the specific proteins that give

each cell its distinctive properties. At each stage along the

way, proteins called transcription factors play key roles in

determining which sets of genes are expressed and repressed,

and therefore what sort of a cell the newly differentiated cell

will become.
116 MONITORING STEM CELL RESEARCH

II. REPRODUCIBLE RESULTS USING STEM CELL

 PREPARATIONS AND THEIR DERIVATIVES

A major goal of scientific research is the acquisition of

reliable knowledge based on experiments that yield

reproducible results. Reproducible results are possible only if

the materials used in experiments remain constant and stable.

To obtain reproducible results in experiments using stem cells,

it is essential to produce, preserve, characterize, and

continually re-characterize preparations of stem cells in ways

that increase the likelihood that the cells used to repeat

experiments will remain unchanged—a technically

challenging task. The tendency of stem cells in vitro to

differentiate spontaneously into more specialized cells makes

the task of obtaining homogeneous and stable stem cell

preparations especially challenging, and much basic research

is needed to learn how to control the fate of these cells. Failure

to control the cells may yield experimental results that are

difficult or impossible to reproduce. The following more specific

observations make clear the dimensions of this difficulty.

A. Initial Stem Cell Preparations Can Contain Multiple Cell

Types

Isolation of adult stem cells from source tissues such as

bone marrow, brain, or muscle initially yields a heterogeneous

cell preparation. The initial preparation contains the several

cell types found in the source tissue, and it may also include

red blood cells, white blood cells, and (possibly) circulating

stem cells, owing to the presence of blood flowing through

the tissue in question. Initial mixtures of cells may then be

treated in various ways to remove unwanted contaminating

cells, thereby increasing the proportion of stem cells in the

preparation. But seldom, if ever, does one produce an adult

stem cell preparation that is 100 percent stem cells, unless

the adult stem cell preparation has been “single-cell cloned”

in vitro (see below).
RESEARCH AND THERAPY DEVELOPMENTS 117

The way in which human embryonic stem cells have been

produced from ICM cells also raises a question about the

“species homogeneity” of the initial cell preparations. In the

past, human embryonic stem cells were isolated and

maintained by in vitro growth on top of irradiated (so that they

no longer divide) “feeder layers” of mouse cells. It is thought

that the feeder cells secrete factor(s) that enable the stem cells

to divide while maintaining a relatively undifferentiated state.

Although the mouse cells have been treated to prevent their

cell division, should any of them happen to survive, human

embryonic stem cells prepared in this way may contain some

viable mouse cells.*

 More recently, several groups have shown

that it is possible to grow ESCs on feeder layers of human

cells, including fibroblasts obtained from skin biopsies, or

without any feeder cell layer at all.11 One way to be certain

that human embryonic stem cell preparations do not contain

any mouse feeder cells is through “single cell cloning” (see

below).

B. Genetically Homogenous Stem Cells through Single Cell

Cloning

Some preparations of stem cells growing in vitro have been

“single cell cloned,” that is, grown as a population derived

from a single stem cell. By placing a cylinder over a single cell

located with a microscope, scientists are able to isolate within

the cylinder all the progeny produced by subsequent cell

divisions beginning from this single cell. The result is a stem

cell preparation in which all the cells are descended from the

original single cell. The cells within the cylinder are then

harvested and grown to greater numbers in vitro, and the

resulting stem cell preparation is said to be “single cell cloned.”

The stem cells within a “single cell cloned” population are, at

least to begin with, genetically homogeneous because they

are all derived from the same original cell. Some of the ESC

preparations produced prior to August 9, 2001 have been

“single cell cloned.”12

* The issue of possible mouse virus contamination is dealt with in Section

F, below.
118 MONITORING STEM CELL RESEARCH

C. Expansion in Vitro, Preservation, and Storage

Reproducible results require that preparations of stem cells,

even if genetically homogenous when first isolated, remain

stable over time and during preservation. This, too, is not a

simple matter with stem cells, despite the fact that the selfrenewal characteristic of human embryonic and adult stem

cells enables them—unlike differentiated cells from many human tissues—to be grown in large numbers in vitro while

maintaining their essential stem cell characteristics. After such

expansion, many, presumably identical, vials of the cells can

be frozen and preserved at very low temperatures. Frozen stem

cell preparations can later be thawed and grown again in vitro to produce larger numbers of cells.

As with all dividing cells, stem cells are subject to a very

small but definite chance of mutation during DNA replication;

thus, prolonged growth in vitro could introduce genetic heterogeneity into an originally homogeneous population. During

this process of repeated expansion and preservation, subtle

changes in the growth conditions or other variables may give

rise to “selective pressures” that can increase the heterogeneity in a stem cell preparation by favoring the multiplication

of advantaged cell variants in the population. It is not known

at present how many of the 78 human ESC preparations, designated as eligible for federal funding under the current policy, have developed genetic variants that may make them unsuitable for further research.

Whether several cycles of freezing and thawing change the

phenotypic characteristics of stem cell preparations needs

detailed study. However, the practical advantages of

preserving stem cell preparations by freezing are too large to

ignore. Such preservation makes it possible to repeat an

experiment many times with a very similar stem cell

preparation. It would also make it possible, should stem cell

based therapies be developed in the future, to treat multiple

patients with a common, well-characterized cell preparation

derived from a single initial stem cell sample.
RESEARCH AND THERAPY DEVELOPMENTS 119

D. Chromosome Changes

In addition to the possible loss of homogeneity in stem cell

preparations owing to variability in growth conditions or to

freezing and thawing, there is the possibility of variation being

introduced during the processes of growth and cell division.

Normal human stem cells (like all human somatic cells) have

46 chromosomes. During the copying of chromosomal DNA and

the separation of daughter chromosomes at cell division, rare

mistakes occur that lead to the formation of abnormal

chromosomes or maldistribution of normal ones. Cells with

abnormal chromosomes or chromosome numbers can progress

to malignancy, so retention of the normal human chromosome

number and structure is an essential characteristic of useful

human stem cell preparations. The most studied preparations

of human stem cells generally have normal human chromosome

numbers and structure.3,13,* Nevertheless, vigilance is needed,

for even a small number of chromosomally abnormal cells could

end up causing cancer in future clinical trials of stem cell based

therapies.

E. Developmental Heterogeneity of Stem Cell Preparations

The in vitro growth conditions and the presence of specific

chemicals or proteins, or both, in the culture medium can

influence the differentiation pathway taken by stem cells as

they start to differentiate. Thus, even initially homogeneous,

“single cell cloned” stem cell preparations may become

developmentally heterogeneous over time, with respect to the

* As of November 2003, reports were available about the chromosome patterns of only 21 out of the 78 ESC preparations designated as eligible for

federal funding; 11 of the 12 preparations currently available as of that

time had their chromosome patterns characterized, and they appear normal. However, a recent publication, presenting results from two different

laboratories, reports abnormalities in chromosome number and structure

in some samples of three different human ESC preparations. Two of these

ESC preparations are among the preparations currently available for federal funding. [Draper, J.S., et al., “Recurrent gain of chromosomes 17q and

12 in cultured human embryonic stem cells,” Nature Biotechnology December 7, 2003, advance online publication.]
120 MONITORING STEM CELL RESEARCH

percentage of cells in the preparation that are in one or another

differentiated state. For example, a stem cell preparation after

growth in vitro under specific conditions might contain 75

percent fully differentiated (insulin-producing) cells and 25

percent partially differentiated cells. The biological properties

of the fully differentiated cells and the partially differentiated

cells are likely to be different. If such a cell preparation is used

in research, or transplanted into an animal model of human

disease and a biological effect is observed, one must do

additional experiments to determine whether the effect was

due to the fully differentiated cells or to the partially

differentiated cells (or perhaps to both acting together) in the

now mixed preparation.

F. Microbial Contamination

Stem cell preparations originally isolated from humans and

expanded in vitro may also be variably contaminated with

human viruses, bacteria, fungi, and mycoplasma. ESC

preparations isolated using mouse feeder cell layers might also

be contaminated with mouse viruses. Specific tests need to

be performed on the source tissue and periodically on the

resulting stem cell preparations to rule out the presence of

these contaminants. Some of these contaminants can also

multiply when stem cells are grown in vitro, and their presence

can influence the results obtained when stem cell preparations

are used in subsequent experiments. The presence of such

contaminants can also potentially affect the reproducibility of

the results of experiments in which stem cell preparations are

studied in vivo in experimental animals.

In summary, there are numerous challenges to obtaining

and preserving the uniform and stable preparations of stem

cells necessary for reliable research and, eventually, for safe

and effective possible therapies. Researchers must address

multiple factors in order to maximize the probability of

obtaining reproducible results with human stem cell

preparations. Human stem cell preparations that are
RESEARCH AND THERAPY DEVELOPMENTS 121

• “single cell cloned,” with a normal chromosome

structure and number, and

• stored as multiple samples that are preserved at very

low temperature, and

• compared in experiments where cells from the same

lot of frozen material are used, and

• well-characterized as to the absence of cellular, viral,

bacterial, fungal, and mycoplasma contaminants, and

• tested to determine the proportion of stem cells and

various differentiated cells in the cell preparation used

in the experiments,

are most likely to yield experimental results that will be

reproducible. Preparations with these properties will be the

most useful both in basic research and in investigations of

possible clinical applications.

III. MAJOR EXAMPLES OF HUMAN STEM CELLS

In this section we discuss major examples of human stem

cells that meet many of the criteria listed above. Among human

adult stem cells, we focus on mesenchymal stem cells (MSCs),4

multipotent adult progenitor cells (MAPCs),3 and neural stem

cells, and among human embryonic stem cells, on ESC2 and

EGC1 cells. For information on the wide variety of other human

stem cell preparations isolated from adult tissues, see reference

(4) (Appendix K).

Further research on some of these other adult stem cell

preparations may demonstrate that they can also be “single

cell cloned,” expanded considerably by growth in vitro with

retention of normal chromosome structure and number, and

preserved by freezing and storage at low temperatures. At

that point, it would be very important to compare the

properties of these other adult stem cells, and the more

differentiated cells that can be derived from them, with the
122 MONITORING STEM CELL RESEARCH

already characterized human embryonic and adult stem cell

preparations.

A. Human Adult Stem Cells

1. Human Mesenchymal Stem Cells.

Bone marrow contains at least two major kinds of stem cells:

hematopoietic stem cells,10 which give rise to the red cells

and white cells of the blood, and mesenchymal stem cells,*

which can be reproducibly isolated and expanded in vitro and

that can differentiate in vitro into cells with properties of

cartilage, bone, adipose (fat), and muscle cells.14

The characteristics (morphology, expressed proteins, and

biological properties) of these cells have been somewhat

difficult to specify, because they appear to vary depending

upon the in vitro culture conditions and the specific cell

preparation.15 However, there is a recent report indicating that

MSCs, if isolated using three somewhat different methods, give

rise to stem cell preparations whose properties are very similar

to one another.16 Using dual antibody staining and

fluorescence-activated cell sorting, Gronthos and colleagues17

isolated human MSCs in almost pure form and expanded them

substantially in vitro. Thus, human MSC preparations isolated

in different laboratories by different methods may have similar

but not identical properties.

A molecular analysis of genes expressed in a single-cellderived colony of MSCs provided evidence for the activity of

genes also turned on in bone, cartilage, adipose, muscle,

hematopoiesis-supporting stromal, endothelial, and neuronal

cells.15 These results are surprising in that MSCs derived from

a single cell appear to be expressing genes associated with

multiple major cell lineages. It is possible that different cells

within the colony had already entered into distinct

differentiation pathways, resulting in a developmentally

*

 The terms “stromal stem cells,” “mesenchymal stem cells,” and “mesenchymal progenitor cells” have all been used by different authors to describe these cells.
RESEARCH AND THERAPY DEVELOPMENTS 123

heterogeneous population composed of several different cell

types.

Mesenchymal stem cells are important for research and

therapy for several reasons. First, because they can be

differentiated in vitro into multiple cell types, they make

possible detailed research on the molecular events underlying

differentiation into bone,18 cartilage, and fat cell lineages.

Second, they have recently been shown to support the in vitro

growth of human embryonic stem cells.19 Thus, they could

replace the mouse feeder cells used previously, obviating the

need to satisfy FDA requirements for xenotransplantation,

should the ESCs or their derivatives ever be used in human

clinical research or transplantation therapy. Third, clinical

studies are already underway in which MSCs are cotransplanted with autologous hematopoietic stem cells into

cancer patients to replace their blood cell-forming system,

destroyed by radiation or high dose chemotherapy.20 It is

believed that the MSCs will support the repopulation of the

bone marrow by the injected hematopoietic stem cells.

In addition, injecting allogeneic MSCs (MSCs from a

genetically different human donor) may also prove valuable in

modulating the immune system to make it more accepting of

foreign tissue grafts [see Itescu review, reference (5)]. Finally,

MSCs have the potential for cell-replacement therapies in

injuries involving bone, tendon, or cartilage and possibly other

diseases. They are, in fact, already being tested as

experimental therapies for osteogenesis imperfecta,21

metachromatic leukodystrophy, and Hurler syndrome.22 These

last two studies are of great interest, since allogeneic MSCs

were used and no serious adverse immune reactions were

noted.

2. Multipotent Adult Progenitor Cells (MAPCs).

Verfaillie and coworkers recently described the isolation of

MAPCs from rat, mouse, and human bone marrow [see (3) and

references cited therein]. Like MSCs, MAPCs can also be

differentiated in vitro into cells with the properties of cartilage,

bone, adipose, and muscle cells. In addition, there is evidence

for the in vitro differentiation of human MAPCs into functional,

hepatocyte-like cells,23 a potential that has not so far been
124 MONITORING STEM CELL RESEARCH

shown for MSCs. There is increasing interest in MAPCs, both

as potential precursors of multiple differentiated tissues and,

ultimately, for possible autologous transplantation therapy.

The relationship between human MSCs and the human

MAPCs described by Verfaillie and coworkers [see (3)] needs

to be clarified by further research. Both kinds of cells are

isolated from bone marrow aspirates as cells that adhere to

plastic. Each can be differentiated in vitro into cells with

cartilage, bone, and fat cell properties. They express several

of the same cell antigens, but are reported to differ in a few

others.3

 MAPCs have to be maintained at specific, low cell

densities when grown in vitro, otherwise they tend to

differentiate into MSCs.3

 It remains important that the isolation

and properties of MAPCs be reproduced in additional

laboratories.

3. Human Neural Stem Cells.

The nervous system is made up of three major types of cells,

neurons or nerve cells proper, and two kinds of supporting or

glial cells (oligodendrocyte, astrocyte). Stem cells capable of

differentiating into one or more of these neural cell lineages

can be isolated from brain tissue (particularly the olfactory

bulb and lining of the ventricles)24,25 and grown in vitro. In the

presence of purified growth-factor proteins, the population of

cells can be expanded by growth in vitro as round clumps of

cells called neurospheres. However, many neurospheres grown

in culture are developmentally heterogeneous in that they

contain more than one neural cell type, and the number of

self-renewing cells is frequently low (less than five percent).26

Although neural stem cells are still insufficiently

understood, they are already proving valuable in basic research

on neural development. The ability to grow reproducible neural

stem cells in vitro has facilitated identification of important

neural stem cell growth factors and their cellular receptors.

For example, human neural stem cells from the developing

human brain cortex, expanded in culture in the presence of

leukemia inhibitory factor (LIF), allowed growth of a selfrenewing neural stem cell preparation for up to 110 population

doublings. Withdrawal of LIF led to decreased expression of
RESEARCH AND THERAPY DEVELOPMENTS 125

about 200 genes,27 which were specifically identified through

use of “gene chips” manufactured by Affymetrix. These genes

are presumably involved in promoting or preserving the stem

cell’s capacity for self-renewal in the undifferentiated state.

The number and specificity of the molecular changes

characterized in these experiments powerfully illustrate the

usefulness of neural and other stem cell preparations in basic

biomedical research.

Human neural stem cells are also being injected into animals

to test their effects on animal models of human neurological

disease. To track the fate of the introduced human cells, they

must first be modified or “marked” in ways that permit their

specific detection.*

 Marked human neural stem cells are easily

tracked after they are injected into experimental animals,

making it possible to determine whether they survive and

migrate following injection. Studies of this type have provided

evidence that human neural cells can migrate extensively in

the brain after injection.28 In addition, such cells can be injected

into animal models of human diseases such as intracerebral

hemorrhage and Parkinson Disease (PD) to study their effect

on the progression of the disease.29 Although human neural

stem cells may not yet be as well characterized as MSCs or

ESCs, they are being actively studied with the hope that they

can be used in future treatments for devastating neurological

diseases such as Alzheimer Disease and PD.

4. Adult Stem Cells from Other Sources.

Prentice [see (4)] has summarized a large amount of recent

information on preparations of stem cells isolated from

amniotic fluid, peripheral blood, umbilical cord blood, umbilical

cord, brain tissue, muscle, liver, pancreas, cornea, salivary

gland, skin, tendon, heart, cartilage, thymus, dental pulp, and

adipose tissue. Studies of many of the stem cell preparations

from these sources are just getting started, and further work

is needed to determine their biological properties and their

relatedness to other stem cell types. In some cases, the long-

*

 Stem cell preparations are frequently transduced in vitro with foreign

genes that, when expressed, produce readily visualized proteins, such as

Green Fluorescent Protein (GFP).
126 MONITORING STEM CELL RESEARCH

term expandability in vitro of these stem cells has not been

demonstrated. Yet, the demonstration that they can be isolated

from such tissue compartments in animals should spur the

search for similar human stem cell types.

As Prentice also reports,4 many attempts have already been

made using various preparations of adult stem cells to

influence or alter the course of diseases in animal models.

Despite the fact that the stem cell preparations used are not

well characterized, and reproducible results have yet to be

obtained, preliminary findings are sometimes encouraging. It

is of course not yet clear whether the injected cells are

functioning as stem cells, fusing with existing host cells, or

stimulating the influx of the host’s own stem cells into the

target tissue.*

 But, if reproduced, these preliminary findings

may point the way to future therapies, even in the absence of

precise knowledge of the mechanism(s) of cellular action.

B. Human Embryonic Stem Cells

1. Human Embryonic Stem Cells (ESCs).

Human embryonic stem cells have been isolated from the

inner cell masses of blastocyst-stage human embryos in

multiple laboratories around the world.† There is great interest

in understanding the properties of these cells because they

hold out the promise of being able to be differentiated into a

large number of different cell types for possible cell therapies,

as contrasted with the more limited number of cell types

available by differentiation of specific adult stem cell

preparations. As of July 2003, 12 ESC preparations (up from 2

such preparations a year earlier) out of a total of 78 “eligible”

preparations of human ESCs were available for shipment to

recipients of U.S. federal research grants.‡

 The review by

* In a recent review article on adult stem cell plasticity, Raff [see (8)] discusses the phenomenon of spontaneous cell fusion masquerading as cell

plasticity.

†

 According to published reports, laboratories in Australia, Britain, China,

India, Iran, Israel, Japan, Korea, Singapore, Sweden, and the United States

have isolated ESC preparations.

‡ For current information on available and eligible ESC preparations see

http://stemcells.nih.gov/registry/index.asp.
RESEARCH AND THERAPY DEVELOPMENTS 127

Ludwig and Thomson2 lists more than 40 peer-reviewed human

ESC primary research papers that have been published since

the initial publication in 1998.

Although isolated from different blastocyst-stage human

embryos in laboratories in different parts of the world, ESCs

have a number of properties in common. These include the

presence of common cell surface antigens (recognized by

binding of specific antibodies), expression of the enzymes

alkaline phosphatase and telomerase, and production of a

common gene-regulating transcription factor known as Oct4. At least 12 different preparations of ESCs have been

expanded by growth in vitro, frozen and stored at low

temperature, and at least partially characterized.13 Some of

these ESC preparations have been “single-cell cloned.”

Human ESCs have been differentiated in vitro into neural

(neurons, astrocytes, and oligodendrocytes), cardiac

(synchronously contracting cardiomyocytes), endothelial

(blood vessels), hematopoietic (multiple blood cell lineages),

hepatocyte (liver cell), and trophoblast (placenta) lineages.2

In the case of neural and cardiac lineages, similar results have

been obtained in different laboratories using different

preparations of ESCs, thus fulfilling the “reproducible results”

criterion described above. For other lineages, the results

described have not yet been reproduced in another laboratory.

2. Embryonic Germ Cells.

Human embryonic germ cells are isolated from the

primordial germ tissues of aborted fetuses. Gearhart1 has

summarized the results of recent research with human and

mouse EG cells. One study focused on regulation of imprinted

genes in EG cells: it showed “that general dysregulation of

imprinted genes will not be a barrier to their (EG cell) use in

transplantation studies.”30* In addition, Kerr and coworkers31

showed that cells derived from human EG cells, when

introduced into the cerebrospinal fluid of rats, became

* Previous work had shown that variation in imprinted gene expression

was observed in cloned mice, and that it might be partly responsible for

their subtle genetic defects. So it was reassuring that the pattern of imprinted gene expression appeared to be normal in EG cells.
128 MONITORING STEM CELL RESEARCH

extensively distributed over the length of the spinal cord and

expressed markers of various nerve cell types. Rats paralyzed

by virus-induced nerve-cell loss recovered partial motor

function after transplantation with the human cells. The

authors suggested that this could be due to the secretion of

transforming growth factor-α and brain-derived growth factor

by the transplanted cells and subsequent enhancement of rat

neuron survival and function.

Until recently, work with human EG cells came primarily

from one laboratory. Recently the isolation and properties of

human EG cells have been independently confirmed.32 Because

human EG cells share many (but not all) properties with ESCs,

these cells offer another important avenue of inquiry.

3. Embryonic Stem Cells from Cloned Embryos (Cloned ESCs).

Although it has yet to be accomplished in practice, somatic

cell nuclear transfer (SCNT) could create cloned human

embryos from which embryonic stem cells could be isolated

that would be genetically virtually identical to the person who

donated the nucleus for SCNT: hence cloned ESCs [see (7)]. In

theory, using such cloned embryonic stem cells from individual

patients might provide a way around possible immune

rejection (see below), though in practice this could require

individual cloned embryos for each prospective patient—a

daunting task. And clinical uses might require a separate FDA

approval for every single cloned stem cell line or its derivatives.

The ability to produce cloned mouse stem cells and

genetically modify them in vitro has made possible an

experiment demonstrating the potential of cloned human

embryonic stem cells in the possible future treatment of human

genetic diseases. Rideout et al.33 used a mutant mouse strain

that was deficient in immune system function. They produced

a cloned mouse embryonic stem cell line carrying the mutation,

and then specifically repaired that gene mutation in vitro. The

repaired cloned stem cell preparation was then differentiated

in vitro into bone marrow precursor cells. When these precursor

cells were injected back into the genetically mutant mice, they

produced partial restoration of immune system function.

Production of cloned human embryonic stem cell

preparations remains technically very difficult and ethically
RESEARCH AND THERAPY DEVELOPMENTS 129

controversial. Recently however, Chen and coworkers34 have

reported that fusion of human fibroblasts with enucleated

rabbit oocytes in vitro leads to the development of embryolike structures from which cell preparations with properties

similar to human embryonic stem cells can be isolated. This

work needs to be confirmed by repetition in other laboratories.

In addition, further work is needed to decisively settle the

question of whether rabbit (or human egg donor) mitochondrial

DNA and rabbit (or human egg donor) mitochondrial proteins

persist in the embryonic stem cell preparations. Persistence

of these foreign mitochondrial proteins in these human ESClike preparations could possibly increase the probability of

immune rejection of the cloned cells, thus limiting their clinical

application, although the immune reaction might not be as

severe as that to foreign proteins produced under the direction

of chromosomal genes. The presence of foreign or aberrant

mitochondria also carries the risk of transmitting mitochondrial

disease (caused by defects in mitochondrial DNA) that could

be detrimental to the cells and to the recipient into whom they

might eventually be transplanted.

IV. BASIC RESEARCH USING HUMAN STEM CELLS

Human stem cells are proving useful in basic research in

several ways. They are useful in unraveling the complex molecular pathways governing human differentiation. For example, because ESCs can be stimulated in vitro to produce more

differentiated cells, this transition can be studied in greater

detail and under better-controlled conditions than it can be in

vivo. In the best circumstances, these differentiated cells can

be grown as largely homogeneous cell populations, and their

gene expression profiles can be compared in detail.

Also, stem cell preparations can be used to produce

populations of specialized cells that are not easily obtained in

other ways. In one case, for example, this approach has

provided large quantities of human trophoblast-like cells that

have not been previously available.35 In addition, cultures of

differentiated cells derived from stem cells could be used to

test new drugs and chemical compounds for toxicity and
130 MONITORING STEM CELL RESEARCH

mutagenicity.36 As experience with these differentiated

derivatives of human ESCs grows, it may become possible to

reduce or eliminate the use of live animals in such testing

protocols.

In the near future, the differentiated state of various human

cell types will be characterized not just by a few biological

markers, but by the pattern and levels of expression of

hundreds or thousands of genes. Integration of this knowledge

with the catalog of all human genes produced during the

Human Genome Project will gradually give us knowledge of

which genes are key regulators of human development and

which genes are central to maintaining the stem cell state.37

Increased understanding of the molecular pathways of human

cell differentiation should eventually lead to the ability to direct

in vitro differentiation along pathways that yield cells useful

in medical treatment. In addition, when the normal range of

gene expression patterns is known, researchers can then

determine which genes are expressed abnormally in various

diseases, thus increasing our understanding of and ability to

treat these diseases.

A group of stem cell researchers has recently outlined a set

of important research questions that, once answered, will

greatly enhance our understanding of human embryonic stem

cells and their potential fates and possible uses.38 They include

the following:

• What is the most effective way to isolate and grow

ESCs?

• How is the self-renewal of ESCs regulated?

• Are all ESC lines the same?

• How can ESCs be genetically altered?

• What controls the processes of ESC differentiation?

• What new tools are needed to measure ESC

differentiation in vitro and in vivo?
RESEARCH AND THERAPY DEVELOPMENTS 131

V. HUMAN STEM CELLS AND THE

TREATMENT OF DISEASE

A major goal of stem cell research is to provide healthy

differentiated cells that, once transplanted, could repair or

replace a patient’s diseased or destroyed tissues. In pursuit

of this goal, one likely approach would start by isolating stem

cells that could be expanded substantially in vitro. A large

number of the cultivated stem cells could then be stored in

the frozen state, extensively tested for safety and efficacy as

outlined above, and used as reproducible starting material from

which to prepare differentiated cell preparations that will

express the needed beneficial properties when they are

transplanted into patients with specific diseases or

deficiencies.

To make more concrete both the potential of this approach

and the obstacles it faces, we will summarize, as a case study

example, some current information on the properties of cells

derived from human stem cell populations that have been used

in an animal model of Type-1 diabetes. But before doing so,

we discuss an obstacle to any successful program of stem cellbased transplantation therapy: the problem of immune

rejection of the transplanted cells.

A. Will Stem Cell-Based Therapies Be Limited by Immune

Rejection?

Much of the impetus for human stem cell research comes

from the hope that stem cells (or, more likely, differentiated

cells derived from them) will one day prove useful in cell transplantation therapies for a variety of human diseases. Such cell

transplantation would augment the current practice of whole

organ transplantation. To the extent that the healing process

works with in vitro derived cells, the need for organ donors

and long waiting lists for organ donation might be reduced or

even eliminated.

Will the recipient (patient) accept or reject the transplanted

human cells? In principle, the problem might seem avoidable
132 MONITORING STEM CELL RESEARCH

altogether: adult stem cells could be obtained from each

individual patient needing treatment. They could then be

grown or modified to produce the desired (autologous and

hence rejection-proof) transplantable cells. But the logistical

difficulties in processing separate and unique materials for

each patient suggest that this approach may not be practical.

The cost and time required to produce sufficient numbers of

well-characterized cells suitable for therapy suggest that it

will be cells derived from one or another unique stem cell line

that will be used to treat many (genetically different) individual

patients (allogeneic cell transplantation).

When allogeneic organ or tissue transplantation is currently

done using, for example, bone marrow, kidney, or heart,

powerful immunosuppressive drugs—carrying undesirable

side effects—must be used to prevent immunological rejection

of the transplanted tissue.5 Without such immunosuppression,

the patient’s T-lymphocytes and natural killer (NK) cells

recognize surface molecules on the transplanted cells as

“foreign” and attack and destroy the cells. Also, in whole organ

transplantation, donor T-lymphocytes and NK cells, entering

the recipient with the transplanted organ, can also destroy

the tissues of the transplant recipient (called “graft versus

host” disease).

Are the differentiated derivatives of human stem cells as

likely to incite immune rejection, when transplanted, as are

solid organs? Do their surfaces carry those protein antigens

that will be recognized as “foreign”? Experiments have been

done to examine human ESC and MSC preparations growing

in vitro for the expression of surface molecules known to play

important roles in the immune rejection process. Drukker and

coworkers39 showed that embryonic stem cells in vitro express

very low levels of the immunologically crucial major

histocompatibility complex class I (MHC-I) proteins on their

cell surface. The presence of MHC-I proteins increased

moderately when the ESCs became differentiated, whether in

vitro or in vivo. A more pronounced increase in MHC-I antigen

expression was observed when the ESCs were exposed to

gamma-interferon, a protein produced in the body during

immune reactions. Thus, under some circumstances, human
RESEARCH AND THERAPY DEVELOPMENTS 133

ESC-derived cells can express cell surface molecules that could

lead to immune rejection upon allogeneic transplantation.

Similarly, Majumdar and colleagues showed that human

mesenchymal stem cells in vitro express multiple proteins on

their cell surfaces that would enable them to bind to, and

interact with, T-lymphocytes. They also observed that gammainterferon increased expression of both human leukocyte

antigen (HLA) class I and class II molecules on the surface of

these MSCs.40 These results indicate that it will probably not

be possible to predict, solely on the basis of in vitro

experiments, the likelihood that transplanted allogeneic MSCs

would trigger immune rejection processes in vivo.

Many further studies in this area are badly needed. At this

time there is insufficient information to determine which, if

any, of the approaches to get around the rejection problem

will eventually prove successful.

B. Case Study: Stem Cells in the Future Treatment of Type1 Diabetes?

1. The Disease and Its Causes.

The human body converts the sugar glucose into cell energy for heart and brain functioning, and indeed, for all bodily

and mental activities. Glucose is derived from dietary carbohydrates, is stored as glycogen in the liver, and is released

again when needed into the bloodstream. A protein hormone

called insulin, produced by the beta cells in the islets of the

pancreas, facilitates the entrance of glucose from the bloodstream into the cells, where it is then metabolized. Insulin is

critical for regulating the body’s use of glucose and the glucose concentration in the circulating blood.

The body’s failure to produce sufficient amounts of insulin

results in diabetes, an extremely common metabolic disease

affecting over 10 million Americans, often with widespread

and devastating consequences. In some five to ten percent of

cases, known as Type-1 diabetes (or “juvenile diabetes”), the

disease is caused by “autoimmunity,” a process in which the
134 MONITORING STEM CELL RESEARCH

body’s immune system attacks “self.”* T-lymphocytes attack

the patient’s own insulin-producing beta cells in the pancreas.

Eventually, this results in destruction of ninety percent or so

of the beta cells, resulting in the diabetic state.

With a deficiency or absence of insulin, the blood glucose

becomes elevated and may lead to diabetic coma, a fatal

condition if untreated. Chronic diabetes, both Type-1 and the

much more common Type-2 diabetes (which is not

autoimmune, but largely genetic), causes late complications

in the retina, kidneys, nerves, and blood vessels. It is the

leading cause of blindness, kidney failure, and amputations

in the U.S. and a major cause of strokes and heart attacks.

Type-1 diabetes is a devastating, lifelong condition that

currently affects an estimated 550,000-1,100,000 Americans,41

including many children. It imposes a significant burden on

the U.S. healthcare system and the economy as a whole, over

and above the disabilities and impairments borne by individual

sufferers. Recent estimates suggest that treatment of all forms

of diabetes costs Americans a total of $132 billion per year.42

At 5-10 percent of all diabetes cases, the costs of Type-1

diabetes can be estimated as $6.5-$13 billion per year.

2. Current Therapy Choices and Outcomes.

The current treatment of Type-1 diabetes consists of insulin

injections, given several times a day in response to repeatedly

measured blood glucose levels. Although this treatment is lifeprolonging, the procedures are painful and burdensome, and

in many cases they do not adequately control blood glucose

concentrations. Whole pancreas transplants can essentially

cure Type-1 diabetes, but fewer than 2,000 donor pancreases

become available for transplantation in the U.S. each year, and

they are primarily used to treat patients who also need a kidney

transplant. Like all recipients of donated organs, pancreas

transplant recipients must continuously take powerful drugs

* Normally the immune system protects against infectious and toxic agents

and surveys for cancer cells with the intent of destroying them but does

not attack one’s own tissues. There are many other autoimmune diseases,

such as some forms of thyroiditis and lupus erythematosis.
RESEARCH AND THERAPY DEVELOPMENTS 135

to suppress the immunological rejection of the transplanted

pancreas.

In addition to treatment with whole pancreas

transplantation, small numbers of Type-1 diabetes patients

have been treated by transplantation of donor pancreatic islets

into the liver of the patient coupled with a less intensive

immunosuppressive treatment (the Edmonton protocol).43

Expanded clinical trials of this procedure are currently

underway. Scientists are also evaluating methods of slowing

the original autoimmune destruction of pancreatic beta cells

that produces the disease in the first place.

Whole pancreas and islet cell transplants ameliorate Type1 diabetes, but there is nowhere near enough of these materials

to treat all in need. To overcome this shortage, people hope

that human stem cells can be induced—at will and in bulk—

to differentiate in vitro into functional pancreatic beta cells,

available for transplantation. Of course, it would still be crucial

to prevent immunological destruction of the newly

transplanted stem cell-derived beta cells.

3. Stem Cell Therapy for Type-1 Diabetes?

Initial experiments in mice suggested that insulinproducing cells could be obtained from mouse embryonic stem

cells following in vitro differentiation.44 Can this approach be

extended to human stem cells? A number of attempts have

been made, with promising initial findings, yet they are not

easily evaluated, partly because the criteria for characterizing

the cells are not standardized. In a recent paper, Lechner and

Habener provided a list of six criteria to define the

characteristics of pancreas-derived “beta-like” cells that could

be potentially useful in treatment of Type-1 diabetes.45

We have used those criteria to facilitate assessment of the

current state of progress toward development of functional

“beta-like” cells that might eventually be tested in Type-1

diabetes patients. Table 1 summarizes and compares the

properties of human cell preparations recently produced in

research seeking this objective by Abraham et al.,46 Zulewski

et al.,47 Assady et al.,48 Zhao et al.,49 and Zalzman et al.,50 and

tested in mouse models of human diabetes.
136 MONITORING STEM CELL RESEARCH

Table 1: Comparison of Insulin-Producing Cells

Derived from Human Stem Cells

Beta-cell-specific markers: PDX-1: (a.k.a IPF-1), a regulatory gene

important for beta-cell function; Glucokinase (GK), an enzyme that

detects high levels of glucose and modulates insulin release; GLUT-2, a

protein associated with glucose-responsive insulin secretion. CK-19 is

a marker for pancreatic duct cells. Insulin production criteria: synthesis

of messenger RNA for insulin or preproinsulin; tests for the presence of

insulin protein; and ultrastructural studies (electron microscopy) to

determine the presence of typical insulin secretory granules. In addition,

the glucose-responsiveness of insulin production and release, an

essential characteristic of normal beta-cell function, was assessed in a

number of the studies described above. Both mouse models of Type-1

diabetes used mice that had a condition known as Severe Combined

Immunodeficiency (SCID) and were treated with streptozotocin (STZ),

a drug that induces selective destruction of the insulin-producing cells.

The mice in the Zalzman study were also born with a form of mouse

diabetes, and are called Non-Obese Diabetic (NOD) mice.

References

Cell Source:

Clonally

Isolated /

Marked?

Beta-cell

markers

Ultrastructural

Examination to

Ensure

Endogenous

Insulin

Production

Glucose-responsive

Insulin

Secretion?

In Vivo Studies Tumorigenicity?

Abraham et

al, 2002 (46);

Zulewski et

al, 2001 (47)

Clonally

isolated adult

stem cells

(derived from

adult

pancreatic

islets)

PDX-1 (+)

CK-19 (+)

Insulin

mRNA(+);

Insulin protein

(+); No ultrastructural

examination

Not

assessed None Not assessed

Assady et al,

2001(48)

Clonally

isolated

embryonic

stem cells

PDX-1 (-);

GK (+);

GLUT-2 (+)

Insulin mRNA

(+)

Insulin protein

(+); No

ultrastructural

examination;

possible insulin

uptake from

serum

No None Not assessed

Zhao et al,

2002 (49)

Uncloned

cadaver islets

(cultured in

vitro)

CK-19 (+)

Preproinsulin

mRNA (+);

Insulin protein

(+);electron

microscopyinsulin secretory

granuoles (+)

Yes

High blood

glucose

concentrations

reversed in

STZ/SCID mice

Not assessed

Zalzman et

al, 2003 (50)

Cloned fetal

liver cells:

immortalized

with human

telomerase

and

transduced

with rat PDX-1

Human and

rat PDX-1 (+);

GK (-); GLUT2 (-)

Insulin mRNA

(+); Insulin

protein (+); No

ultra- structural

examination

Yes

High blood

glucose

concentrations

reversed in

STZ/NOD-SCID

mice; high blood

glucose returned

upon graft

removal

No tumors at 3

months after

transplantation
RESEARCH AND THERAPY DEVELOPMENTS 137

As the results described in Table 1 indicate, cells derived

from some human stem cells transplanted into specific strains

of mice mimicking major aspects of Type-1 human diabetes51

were able to reverse high blood glucose concentrations.

Although these results are encouraging, the transplant

rejection question remains unanswered because the likely

immune rejection of the transplanted human cells was

prevented in these experiments by using special strains of

immunodeficient mice that lack the capacity to recognize and

attack foreign cells.

No tumors were observed in the transplanted mice, but the

experiments were terminated after about three months, an

insufficient time for much tumor development to occur. Because

many Type-1 diabetes patients are children and because a

largely effective therapy (insulin injection) is currently

available, the introduction of islet cell transplant therapy will

need a high degree of certainty that the introduced cells or

their derivatives will not become malignant over the course of

the patient’s life. Stringent tests of the cancer-causing potential

of candidate cell preparations will be required, including multiyear studies in animals that live longer than mice or rats. Longterm follow-up of children and adult patients who had received

bone marrow transplants many years ago has revealed an

increased risk of severe neurologic complications52 and a

variety of types of cancer.53

C. Therapeutic Applications of Mesenchymal Stem Cells

(MSCs)

Before stem cell based therapies are used to treat human

diseases, they will have to gain approval through the Food

and Drug Administration (FDA) regulatory process. The first

step in this process is filing an Investigational New Drug (IND)

application. As of July 2003, four IND applications have been

filed for clinical applications of mesenchymal stem cells. The

disease indications include: (1) providing MSC support for

peripheral blood stem cell transplantation in cancer treatment,

(2) providing MSC support for cord blood transplantation in

cancer treatment, (3) using MSCs to stimulate regeneration of

cardiac tissue after acute myocardial infarction (heart attack),

and (4) using MSCs to stimulate regeneration of cardiac tissue
138 MONITORING STEM CELL RESEARCH

in cases of congestive heart failure. The first two applications

are currently in Phase II of the regulatory process, with pivotal

Phase III trials scheduled to begin in 2004.54

D. Evaluating the Different Types of Stem Cells

A major unresolved issue at present involves the therapeutic potential of human adult stem cells compared with embryonic stem cells. The answer may well be different for different

diseases and for patients of different ages. For example, in

treating an elderly patient with Parkinson’s Disease, the use

of adult stem cells may be appropriate even if these cells may

have a more limited number of cell divisions remaining. On

the other hand, treating a child with Type I Diabetes, one may

want to use embryonic stem cells because of their potentially

greater longevity, or other factors. The only valid way to resolve these questions is by instituting rigorous therapeutic

trials which test the efficacy of the different types of stem cells

in treating a variety of different diseases to determine their

comparative efficacy. Clearly, such trials would be a long-term

endeavor, since it would take years to obtain answers to these

very critical questions.

VI. PRIVATE SECTOR ACTIVITY

In the United States, much of the basic research on animal

stem cells and human adult stem cells has been publicly

funded. Yet before 2001, research in the U.S., using human ESCs

could only be done in the private sector (the locus also of much

research on animal and human adult stem cells). The current

state of knowledge about human ESCs (and also about human

MSCs) reflects pioneering and on-going stem cell research

funded by the private sector in the U.S.54,55 For example, the

work that led to the 1998 reports of the first isolation of both

ESCs and EGCs, was funded by Geron Corporation. Embryonic

and adult stem cell research is today vigorously pursued by

many companies and supported by several private

philanthropic foundations,56 and the results of some of this

research have been published in peer-reviewed journals.57
RESEARCH AND THERAPY DEVELOPMENTS 139

Private sector organizations have pursued and been awarded

patents on the stem cells themselves and methods for

producing and using them to treat disease. As noted above,

at least one company (Osiris Therapeutics) has protocols under

review at the FDA for clinical trials with MSCs. It seems likely

that private sector companies will continue to play large roles

in the future development of stem cell based therapies.

VII. PRELIMINARY CONCLUSIONS

While it might be argued that it is too soon to attempt to

draw any conclusions about the state of a field that is changing

as rapidly as stem cell research, we draw the following

preliminary conclusions regarding the current state of the field.

Human stem cells can be reproducibly isolated from a

variety of embryonic, fetal, and adult tissue sources. Some

human stem cell preparations (for example, human ESCs,

EGCs, MSCs, and MAPCs) can be reproducibly expanded to

substantially larger cell numbers in vitro, the cells can be stored

frozen and recovered, and they can be characterized and

compared by a variety of techniques. These cells are receiving

a large share of the attention regarding possible future (nonhematopoietic) stem cell transplantation therapies.

Preparations of ESCs, EGCs, MSCs, and MAPCs can be

induced to differentiate in vitro into a variety of cells with

properties similar to those found in differentiated tissues.

Research using these human stem cell preparations holds

promise for: (a) increased understanding of the basic molecular

process underlying cell differentiation, (b) increased

understanding of the early stages of genetic diseases (and

possibly cancer), and (c) future cell transplantation therapies

for human diseases.

The case study of developing stem cell-based therapies for

Type-1 diabetes illustrates that, although insulin-producing

cells have been derived from human stem cell preparations,

we could still have a long way to go before stem cell-based

therapies can be developed and made available for this

disease. This appears to be true irrespective of whether one

starts from human embryonic stem cells or from human adult
140 MONITORING STEM CELL RESEARCH

stem cells. The transplant rejection problem remains a major

obstacle, but only one among many.

Human mesenchymal stem cells are currently being

evaluated in pre-clinical studies and clinical trials for several

specific human diseases.

Much basic and applied research remains to be done if

human stem cells are to achieve their promise in regenerative

medicine.58 This research is expensive and technically

challenging, and requires scientists willing to take a long

perspective in order to discover, through painstaking research,

which combinations of techniques could turn out to be

successful. Strong financial support, public and private, will

be indispensable to achieving success.
RESEARCH AND THERAPY DEVELOPMENTS 141

ENDNOTES

1

 Gearhart, J., “Human Embyronic Germ Cells: June 2001-July 2003. The Published

Record,” Paper prepared for the President’s Council on Bioethics, July 2003.

[Appendix H]

2

 Ludwig, T. E. and Thomson, J. A., “Current Progress in Human Embryonic Stem

Cell Research,” Paper prepared for the President’s Council on Bioethics, July 2003.

[Appendix I]

3

 Verfaillie, C., “Multipotent Adult Progenitor Cells: An Update,” Paper prepared for

the President’s Council on Bioethics, July 2003. [Appendix J]

4

 Prentice, D., “Adult Stem Cells,” Paper prepared for the President’s Council on

Bioethics, July 2003. [Appendix K]

5

 Itescu, S., “Stem Cells and Tissue Regeneration: Lessons from Recipients of Solid

Organ Transplantation,” Paper prepared for the President’s Council on Bioethics,

June 2003. [Appendix L]

6

 Itescu, S., “Potential Use of Cellular Therapy For Patients With Heart Disease,”

Paper prepared for the President’s Council on Bioethics, August 2003. [Appendix

M]

7

 Jaenisch, R., “The Biology of Nuclear Cloning and the Potential of Embryonic Stem

Cells for Transplantation Therapy,” Paper prepared for the President’s Council on

Bioethics, July 2003. [Appendix N]

8

 See, among others, Bianco, P., et al., “Bone marrow stromal cells: nature, biology

and potential applications,” Stem Cells 19: 180-192 (2001); Martinez-Serrano, A., et

al., “Human neural stem and progenitor cells: in vitro and in vivo properties, and

potential for gene therapy and cell replacement in the CNS,” Current Gene Therapy

1: 279-299 (2001); Nir, S., et al., “Human embryonic stem cells for cardiovascular

repair,” Cardiovascular Research 58: 313-323 (2003); Raff, M., “Adult stem cell

plasticity: fact or artifact?” Annual Review of Cell and Developmental Biology 19: 1-

22 (2003).

9

 Storb, R., “Allogeneic hematopoietic stem cell transplantation – Yesterday, today

and tomorrow,” Experimental Hematology 31: 1-10 (2003).

10 Kondo, M., et al., “Biology of Hematopoietic Stem Cells and Progenitors:

Implications for Clinical Application,” Annual Review of Immunology 21: 759-806

(2003) and references cited therein.

11 Xu, C., et al., “Feeder-free growth of undifferentiated human embryonic stem cells,”

Nature Biotechnology 19: 971-974 (2001); Richards, M., et al., “Human feeders support

prolonged undifferentiated growth of human inner cell masses and embryonic stem

cells,” Nature Biotechnology 20: 933-936 (2002); Amit, M., et al., “Human Feeder

Layers for Human Embryonic Stem Cells,” Biology of Reproduction 68: 2150-2156

(2003); Richards, M., et al., “Comparative Evaluation of Various Human Feeders for

Prolonged Undifferentiated Growth of Human Embryonic Stem Cells,” Stem Cells

21: 546-556 (2003).
142 MONITORING STEM CELL RESEARCH

12 Amit, M., et al., “Clonally derived Human Embryonic Stem Cell Lines Maintain

Pluripotency and Proliferative Potential for Prolonged Periods of Culture,”

Developmental Biology 227: 271-278 (2000); Amit, M. and Itskovitz-Eldor, J.,

“Derivation and spontaneous differentiation of human embryonic stem cells,” Journal

of Anatomy 200: 225-232 (2002).

13 Carpenter, M. K., et al., “Characterization and Differentiation of Human Embryonic

Stem Cells,” Cloning and Stem Cells 5: 79-88 (2003).

14 Pittenger, M. F. et al., “Multilineage potential of adult human mesenchymal stem

cells,” Science 284: 143-147 (1999); Pittenger, M., et al., “Adult mesenchymal stem

cells: Potential for muscle and tendon regeneration and use in gene therapy,” Journal

of Musculoskeletal and Neuronal Interactions 2: 309-320 (2002).

15 Tremain, N., et al., “MicroSAGE Analysis of 2,353 Expressed Genes in a SingleCell Derived Colony of Undifferentiated Human Mesenchymal Stem Cells Reveals

mRNAs of Multiple Cell Lineages,” Stem Cells 19: 408-418 (2001).

16 Lodie, T. A., et al., “Systematic analysis of reportedly distinct populations of

multipotent bone marrow-derived stem cells reveals a lack of distinction,” Tissue

Engineering 8: 739-751 (2002).

17 Gronthos, S., et al., “Molecular and cellular characterization of highly purified

stromal stem cells derived from bone marrow,” Journal of Cell Science 116: 1827-

1835 (2003).

18 Qi, H., et al., “Identification of genes responsible for osteoblast differentiation

from human mesodermal progenitor cells,” Proceedings of the National Academy of

Sciences of the United States of America 100: 3305-3310 (2003).

19 Cheng, L., et al., “Human adult marrow cells support prolonged expansion of

human embryonic stem cells in culture,” Stem Cells 21: 131-142 (2003).

20 Koc, O. N., et al., “Rapid hematopoietic recovery after coinfusion of autologousblood stem cells and culture-expanded marrow mesenchymal cells in advanced

breast cancer patients receiving high-dose chemotherapy,” Journal of Clinical

Oncology 18: 307-316 (2000).

21 Horwitz, E. M., et al., “Isolated allogeneic bone marrow-derived mesenchymal

cells engraft and stimulate growth in children with osteogenesis imperfecta:

Implications for cell therapy of bone,” Proceedings of the National Academy of

Sciences of the United States of America 99: 8932-8937 (2002).

22 Koc, O. N., et al., “Allogeneic mesenchymal stem cell infusion for treatment of

metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH),” Bone Marrow

Transplantation 30: 215-222 (2002).

23 Schwartz, R. E., et al., “Multipotent adult progenitor cells from bone marrow

differentiate into functional hepatocyte-like cells,” Journal of Clinical Investigation

109: 1291-1302 (2002).

24 Pagano, S. F., et al., “Isolation and characterization of neural stem cells from the

adult human olfactory bulb,” Stem Cells 18: 295-300 (2000).
RESEARCH AND THERAPY DEVELOPMENTS 143

25 Liu, Z., and Martin, L. J., “Olfactory bulb core is a rich source of neural progenitor

and stem cells in adult rodent and human,” Journal of Comparative Neurology 459:

368-391 (2003).

26 Pevny, L., and Rao, M. S., “The stem-cell menagerie,” Trends in Neurosciences 26:

351-359 (2003).

27 Wright, L. S., et al., “Gene expression in human neural stem cells: effects of

leukemia inhibitory factor,” Journal of Neurochemistry 86: 179-195 (2003).

28 See, for example, Englund, U., et al., “Transplantation of human neural progenitor

cells into the neonatal rat brain: extensive migration and differentiation with longdistance axonal projections,” Experimental Neurology 173: 1-21 (2002); Chu, K., et

al., “Human neural stem cells can migrate, differentiate, and integrate after

intravenous transplantation in adult rats with transient forebrain ischemia,”

Neuroscience Letters 343: 129-133 (2003).

29 See, for example, Jeong, S., et al., “Human neural cell transplantation promotes

functional recovery in rats with experimental intracerebral hemorrhage,” Stroke

34: 2258-2263 (2003); Liker, M., et al., “Human neural stem cell transplantation in

the MPTP-lesioned mouse,” Brain Research 971: 168-177 (2003).

30 Onyango, P., et al., “Monoallelic expression and methylation of imprinted genes in

human and mouse embryonic germ cell lineages,” Proceedings of the National

Academy of Sciences of the United States of America 99: 10599-10604 (2002).

31 Kerr, D. A., et al., “Human Embryonic Germ Cell Derivatives Facilitate Motor

Recovery of Rats with Diffuse Motor Neuron Injury,” The Journal of Neuroscience

23: 5131-5140 (2003).

32 Turnpenny, L., et al., “Derivation of Human Embryonic Germ Cells: An Alternative

Source of Pluripotent Stem Cells,” Stem Cells 21: 598-609 (2003).

33 Rideout, W., et al., “Correction of a genetic defect by nuclear transplantation and

combined cell and gene therapy,” Cell 109: 17-27 (2002); Tsai, R. Y. L., et al., “Plasticity,

niches and the use of stem cells,” Developmental Cell 2: 707-712 (2002); For political

and legislative aspects of the debate relative to these articles, see Daly, G., “Cloning

and Stem Cells—Handicapping the Political and Scientific Debates,” New England

Journal of Medicine 349: 211-212 (2003).

34 Chen, Y., et al., “Embryonic stem cells generated by nuclear transfer of human

somatic nuclei into rabbit oocytes,” Cell Research 13: 251-264 (2003).

35 Xu, R. H., et al., “BMP4 initiates human embryonic cell differentiation to

trophoblast,” Nature Biotechnology 20: 1261-1264 (2002).

36 Rohwedel, J., et al., “Embryonic stem cells as an in vitro model for mutagenicity,

cytotoxicity, and embryotoxicity studies: present state and future prospects,”

Toxicology In Vitro 15: 741-753 (2001).

37 Sato, N., et al., “Molecular signature of human embryonic stem cells and its

comparison with the mouse,” Developmental Biology 260: 404-413 (2003); RamalhoSantos, M., et al., “‘Stemness’: Transcriptional Profiling of Embryonic and Adult

Stem Cells,” Science 298: 597-600 (2002); Ivanova, N. B., et al., “A Stem Cell Molecular

Signature,” Science 298: 601-604 (2002).
144 MONITORING STEM CELL RESEARCH

38 Brivanlou, A. H., et al., “Stem cells. Setting standards for human embryonic stem

cells,” Science 300: 913-916 (2003).

39 Drukker, M., et al., “Characterization of the expression of MHC proteins in human

embryonic stem cells,” Proceedings of the National Academy of Sciences of the United

States of America 99: 9864-9869 (2002).

40 Majumdar, M. K., et al., “Characterization and functionality of cell surface molecules

on human mesenchymal stem cells,” Journal of Biomedical Science 10: 228-241 (2003).

41 American Diabetes Association, “Facts and Figures,” http://diabetes.org/main/

info/facts/facts.jsp (accessed June 23, 2003).

42 Hogan, P., et al., “Economic Costs of Diabetes in the US in 2002,” Diabetes Care

26: 917-932 (2003).

43 Ryan, E. A., et al., “Clinical outcomes and insulin secretion after islet

transplantation with the Edmonton protocol,” Diabetes 50: 710-719 (2001).

44 Soria, B., et al., “Insulin-secreting cells derived from embryonic stem cells normalize

glycemia in streptozotocin-induced diabetic mice,” Diabetes 49: 157-162 (2000);

Lumelsky, N., et al., “Differentiation of embryonic stem cells to insulin-secreting

structures similar to pancreatic islets,” Science 292: 1389-1394 (2001); Hori, Y., et

al., “Growth inhibitors promote differentiation of insulin-producing tissue from

embryonic stem cells,” Proceedings of the National Academy of Sciences of the United

States of America 99: 16105-16110 (2002).

45 Lechner, A. and Habener, J. F., “Stem/progenitor cells derived from adult tissues:

potential for the treatment of diabetes mellitus,” American Journal of Physiology -

Endocrinology and Metabolism 284: E259-266 (2003). The criteria that these authors

outlined were as follows:

• The stem or progenitor cell should be clonally isolated or marked;

“enrichment” of a certain cell type alone is not sufficient.

• In vitro differentiation to a fully functional beta cell should be

unequivocally established. Insulin expression per se does not make

a particular cell a beta cell. The expression of other markers of beta

cells (e.g. Pdx1/Ipf1, GLUT2, and glucokinase) or other endocrine islet

cells should be demonstrated.

• Ultrastructural studies should confirm the formation of mature

endocrine cells by identification of characteristic insulin secretory

granules.

• The in vitro function of endocrine cells, differentiated from stem cells,

should be reminiscent of the natural counterparts. For beta cells, this

would imply a significant glucose-responsive insulin secretion,

adequate responses to incretin hormones and secretagogues, and

the expected electrophysiological properties.

• In vivo studies in diabetic animals should demonstrate a reproducible

and durable effect of the stem/progenitor-derived tissue on the

attenuation of the diabetic phenotype. It should also be demonstrated

that removal of the stem cell-derived graft after a certain period of

time leads to reappearance of the diabetes.

• For future clinical use, the tumorigenicity of stem/progenitor tissue

should be determined.

• Additionally, immune responses toward the transplanted cells should

be examined.
RESEARCH AND THERAPY DEVELOPMENTS 145

46 Abraham, E. J., et al., “Insulinotropic hormone glucagons-like peptide-1

differentiation of human pancreatic islet-derived progenitor cells into insulinproducing cells,” Endocrinology 143: 3152-3161 (2002).

47 Zulewski, H., et al., “Multipotential Nestin-Positive Stem Cells Isolated From Adult

Pancreatic Islets Differentiate Ex Vivo Into Pancreatic Endocrine, Exocrine and

Hepatic Phenotypes,” Diabetes 50: 521-533 (2001).

48 Assady, S., et al., “Insulin production by human embryonic stem cells,” Diabetes

50: 1691-1697 (2001).

49 Zhao, M., et al., “Amelioration of streptozotocin-induced diabetes in mice using

human islet cells derived from long-term culture in vitro,” Transplantation 73: 1454-

1460 (2002).

50 Zalzman, M., et al., “Reversal of hyperglycemia in mice using human expandable

insulin-producing cells differentiated from fetal liver cells,” Proceedings of the

National Academy of Sciences of the United States of America 100: 7253-7258 (2003).

51 For a useful summary of the advantages and limitations of rodent models of

diabetes see: Atkinson, M. A. and Leiter, E. H., “The NOD mouse model of type 1

diabetes: As good as it gets?” Nature Medicine 5: 601-604 (1999).

52 Faraci, M., et al., “Severe neurologic complications after hematopoietic stem cell

transplantation in children,” Neurology 59: 1895-1904 (2002).

53 Baker, K. S., et al., “New Malignancies After Blood or Marrow Stem-Cell

Transplantation in Children and Adults: Incidence and Risk Factors,” Journal of

Clinical Investigation 21: 1352-1358 (2003).

54 Pursley, W. H., Presentation at the September 4, 2003, meeting of the President’s

Council on Bioethics, Washington, D.C., available at www.bioethics.gov.

55 Okarma, T., Presentation at the September 4, 2003, meeting of the President’s

Council on Bioethics, Washington, D.C., available at www.bioethics.gov.

56 See presentations from the Juvenile Diabetes Research Foundation International

and the Michael J. Fox Foundation at the September 4, 2003, meeting of the

President’s Council on Bioethics, Washington, D.C., available at www.bioethics.gov.

57 See, for example, Carpenter, M. K., et al., “Characterization and Differentiation of

Human Embryonic Stem Cells,” Cloning and Stem Cells 5: 79-88 (2003), and Pittenger,

M. F. et al., “Multilineage potential of adult human mesenchymal stem cells,” Science

284: 143-147 (1999), and Pittenger, M. F., et al., “Adult mesenchymal stem cells:

Potential for muscle and tendon regeneration and use in gene therapy,” Journal of

Musculoskeletal and Neuronal Interactions 2: 309-320 (2002).

58 Daley, G. Q., et al., “Realistic Prospects for Stem Cell Therapeutics,” Hematology

American Society for Hematology Education Program: 398-418 (2003).

GLOSSARY*

Adipose tissue: A type of connective tissue that stores fat.

Adult stem cell: An undifferentiated cell found in a differentiated

tissue that can renew itself and (with certain limitations) differentiate

to yield all the specialized cell types of the tissue from which it

originated. (NIH)

Allogeneic cell transplantation: Transplantation of cells from one

individual to another of the same species.

Amniotic fluid: Fluid that fills the innermost membrane, the amnion,

that envelopes the developing embryo or fetus.

Amnion: Innermost of the extra-embryonic membranes enveloping

the embryo in utero and containing the amniotic fluid. (SMD)

Aneuploid: Having an abnormal number of chromosomes. (SMD)

Angiogenesis: Development of new blood vessels. (SMD)

Antigen: A substance that, when introduced into the body, stimulates the production of protein molecules called antibodies that can

bind specifically to the substance.

Astrocyte: A type of nerve cell that has supportive and metabolic

functions rather than signal conduction.

Autologous: In transplantation, referring to a graft in which the

donor and recipient areas are in the same individual. (SMD)

*

 Definitions marked “(CR)” are from the Council’s report on human cloning (Human

Cloning and Human Dignity: An Ethical Inquiry, Washington, D.C.: Government

Printing Office, 2002). Definitions marked “(NIH)” are from the National Institutes

of Health on-line stem cell glossary at http://stemcells.nih.gov (accessed September

5, 2003). Definitions marked “(NRC)” are from the National Research Council report,

Stem Cell Research and the Future of Regenerative Medicine (Washington, D.C.:

National Research Council, 2001). Definitions marked “(SMD)” are from Stedman’s

Medical Dictionary.

147
148 MONITORING STEM CELL RESEARCH

Autosome: Any chromosome other than a sex chromosome, that is,

any chromosome other than an X or a Y. (SMD)

Bacteria: Any of numerous unicellular microorganisms, existing either as free living organisms or as parasites, and having a broad

range of biochemical, often pathogenic properties.

Blastocyst: (a) Name used for an organism at the blastocyst stage

of development. (CR) (b) A preimplantation embryo of about 150 to

200 cells. The blastocyst consists of a sphere made up of an outer

layer of cells (the trophectoderm), a fluid-filled cavity (the blastocoel),

and a cluster of cells on the interior (the inner cell mass). (NIH)

Blastocyst stage: An early stage in the development of embryos,

when (in mammals) the embryo is a spherical body comprising an

inner cell mass that will become the fetus surrounded by an outer

ring of cells that will become part of the placenta. (CR)

Bone marrow: The soft, fatty, vascular tissue that fills most bone

cavities and is the source of red blood cells and many white blood

cells.

Cardiomyoctes: Heart muscle cells.

Cartilage: A type of connective tissue that is firm but resilient. It is

found in joints and also as supportive structure, for example in the

ears.

Cell culture: Growth of cells in vitro on an artificial medium for

experimental research. (NIH)

Cerebrospinal fluid: A blood serum-like fluid that bathes parts of

the brain and the interior cavity of the spinal cord.

Chromosomes: Structures inside the nucleus of a cell, made up of

long pieces of DNA coated with specialized cell proteins, that are

duplicated at each mitotic cell division. Chromosomes thus transmit

the genes of the organism from one generation to the next. (CR)

Clone: A line of cells that is genetically identical to the originating

cell; in this case, a stem cell. (NIH)

Cord blood: Blood in the umbilical cord and placenta.

Cornea: Transparent tissue at the front of the eye.
GLOSSARY 149

Cryopreserved embryos: Embryos, generally those produced by in

vitro fertilization exceeding the number that can be transferred for

uterine implantation, that have been frozen.

Culture medium: The broth that covers cells in a culture dish, which

contains nutrients to feed the cells as well as other growth factors

that may be added to direct desired changes in the cells. (NIH)

Dental pulp: The soft part inside a tooth, containing blood vessels

and nerves.

Diploid: Refers to the full complement of chromosomes in a somatic

cell, distinct for each species (forty-six in human beings). (CR)

Diploid human cell: A cell having forty-six chromosomes. (CR)

Ectoderm: Upper, outermost layer of a group of cells derived from

the inner cell mass of the blastocyst; it gives rise to skin nerves and

brain. (NIH)

Edmonton protocol: A procedure (developed in Canada) for

transplanting pancreatic islet cells to the liver of a patient with Type

I diabetes.

Embryo: (a) In humans, the developing organism from the time of

fertilization until the end of the eighth week of gestation, when it

becomes known as a fetus. (NIH) (b) The developing organism from

the time of fertilization until significant differentiation has occurred,

when the organism becomes known as a fetus. An organism in the

early stages of development. (CR)

Embryonic germ cells: Cells found in a specific part of the embryo/

fetus called the gonadal ridge that normally develop into mature

gametes. (NIH)

Embryonic stem cells: Primitive (undifferentiated) cells from the

embryo that have the potential to become wide variety of specialized

cell types. (NIH)

Embryonic stem cell line: Embryonic stem cells, which have been

cultured under in vitro conditions that allow proliferation without

differentiation for months to years. (NIH)
150 MONITORING STEM CELL RESEARCH

Endoderm: Lower layer of a group of cells derived from the inner

cell mass of the blastocyst; it gives rise to lungs and digestive organs.

(NIH)

Endometrium: The mucous membranes lining the uterus.

Endothelial: relating to a flat layer of cells lining the heart, for

example, or blood vessels. (SMD)

Epidermal growth factor: A cell messenger protein that has effects

including stimulation of epidermal development, in newborn animals

it hastens eyelid-opening and tooth-eruption (SMD)

Ex vivo: Outside the body, frequently the equivalent of “in vitro.”

Fate (of cell progeny): The normal outcome of differentiation of a

cell’s progeny.

Feeder layer: Cells used in co-culture to maintain pluripotent stem

cells. Cells usually consist of mouse embryonic fibroblasts. (NIH)

Fertilization: The process whereby male and female gametes unite.

(NIH)

Fetus: A developing human from usually two months after

conception to birth. (NIH)

Fibroblast: A stellate (star-shaped) or spindle-shaped cell with

cytoplasmic processes present in connective tissue, capable of

forming collagen fibers. (SMD)

Gamete: A reproductive cell (egg or sperm). (CR)

Gamma-interferon: A type of small protein with antiviral activity,

made by T lymphocytes.

Gastrulation: The process whereby the cells of the blastocyst are

translocated to establish three germ layers. Also sometimes used

to mark the end of the blastocyst stage and the beginning of the

next stage of embryonic development. (Based on SMD)

Gene: A functional unit of heredity that is a segment of DNA located

in a specific site on a chromosome. A gene directs the formation of

an enzyme or other protein. (NIH)
GLOSSARY 151

Genome: The total gene complement of a set of chromosomes. (SMD)

Germ cells (or primordial germ cells): A gamete, that is, a sperm

or egg, OR a primordial cell that can mature into a sperm or egg.

(NRC)

Germ layers: The three initial tissue layers arising in the embryo—

endoderm, mesoderm, and ectoderm—from which all other somatic

tissue-types develop. (NRC)

Gonad: An organ that produces sex cells (testes or ovaries). (SMD)

Gonadal ridges: Embryonic structures arising in humans at about

five weeks, eventually developing into gonads (either testes or

ovaries).

Green Fluorescent Protein: A protein naturally occurring in some

animals including jelly fish that spontaneously fluoresces. It can be

used as a noninvasive marker in living cells by attaching it to different

proteins and then letting it fluoresce so as to track the cell.

Haploid human cell: A cell such as an egg or sperm that contains

only twenty-three chromosomes. (CR)

Hematopoietic stem cell: A stem cell from which all red and white

blood cells develop. (NIH)

Hepatocyte: Liver cell.

Histocompatible: The immunological characteristic of cells or tissue

that causes them to be tolerated by another cell or tissue; that allows

some tissues to be grafted effectively to others. (NRC)

Hurler syndrome: A heritable condition involving deficiency of an

enzyme (alpha-L-iduronidase), leading to abnormal accumulations

of materials inside cells, then resulting in abnormal development of

cartilage and bone and other systems. (SMD)

ICM cells: Cells from the inner cell mass, a population of cells inside

the blastula that give rise to the body of the new organism rather

than to the chorion or other supporting structures.

Immunodeficient: Unable to develop a normal immune response

to, for example, a foreign substance.
152 MONITORING STEM CELL RESEARCH

Immunosuppressive drugs: Drugs that prevent or interfere with

the development of an immunologic response. After a transplant,

immunosuppressive drugs are usually necessary in order to prevent

the recipient from rejecting the transplant.

Implantation: The attachement of the blastocyst to the uterine

lining, and its subsequent embedding there. (Based on SMD)

In vitro fertilization (IVF): The union of an egg and sperm, where

the event takes place outside the body and in an artificial

environment (the literal meaning of “in vitro” is “in glass”; for

example, in a test tube). (CR)

Inner cell mass: The cluster of cells inside the blastocyst. These

cells give rise to the embryonic disk of the later embryo and,

ultimately, the fetus. (NIH)

Karyotype: The chromosome characteristics (number, shape, etc)

of an individual cell or cell line, usually presented as a systematized

array in pairs. (SMD)

Leukemia inhibitory factor: A cell messenger protein originally

noted for inhibition of mouse M1 myeloid leukemia cells that also

has effects including inhibiting differentiation to maintain stem cells.

Lineage: The descendants of a common ancestor.

Long-term self-renewal: The ability of stem cells to renew

themselves by dividing into the same non-specialized cell type over

long periods (many months to years) depending on the specific type

of stem cell. (NIH)

Lymphocyte: A motile cell formed in tissues such as the lymph

nodes, that functions in the development of immunity.

Meiosis: A special process of cell division comprising two nuclear

divisions in rapid succession that result in four cells (that will become

gametes) with the haploid number of chromosomes. (Based on SMD)

Mesenchymal stem cells: Cells from the immature embryonic

connective tissue. A number of cell types come from mesenchymal

stem cells, including chondrocytes, which produce cartilage. (NIH)

Mesoderm: Middle layer of a group of cells derived from the inner

cell mass of the blastocyst; it gives rise to.bone, muscle, and
GLOSSARY 153

connective tissue. (NIH)

Metachromatic leukodystrophy: A heritable metabolic disorder,

usually of infancy, characterized by myelin loss and other

abnormalities of the white matter of the nervous system, leading to

progressive paralysis and mental retardation or dementia. (SMD)

Mitochondria: Small, energy-producing organelles inside cells.

Mitochondrial DNA: Genetic material inside the mitochondria.

Essentially all the mitochondria of an individual come from the

cytoplasm of the egg, so all mitochondrial DNA is inherited through

the maternal line.

Mitochondrial proteins: Proteins that are part of the mitochondria.

Mitosis: Cell division, resulting in two cells that each have the diploid

number of chromosomes and are just like the original cell.

Morphology: Configuration or structure, shape.

Mutagenicity: Tendency to promote mutations, that is, genetic

alterations.

Multipotent: As applied to stem cells, the ability to differentiate

into at least two, more differentiated descendant cells.

Multipotent adult progenitor cells (MAPC): Cells isolated from bone

marrow that can be differentiated into cells with characteristics of

cartilage, fat, and bone.

Mycoplasma: A general category of microorganisms that shares

some characteristics of bacteria.

Natural killer cell: A cell type of the immune system that destroys

tumor cells and cells infected with some types of organisms.

Olfactory bulb: A part of the brain involved in detecting and discriminating among different smells.

Oligodendrocyte: A type of neuroglia, that is, a particular type of

cell that is part of the nervous system with supportive and metabolic

functions rather than signal conduction, this type forms the myelin

sheath around nerve fibers.
154 MONITORING STEM CELL RESEARCH

Oocytes: Egg cells.

Osteogenesis imperfecta: A large and miscellaneous group of

conditions of abnormal fragility and plasticity of bone, with recurring

fractures on trivial trauma. (SMD)

Pancreas: An organ of the digestive system that secretes the

hormones insulin and glucagon, as well as digestive enyzymes.

Pancreatic beta cells: Cells of the pancreas (located in pancreatic

islets, or islets of Langerhans) that produce insulin.

Parkinson disease: A neurological syndrome usually resulting from

deficiency of the neurotransmitter dopamine . . . ; characterized by

rhythmical muscular tremors . . . (SMD)

Phenotypic characteristics: The genetically and environmentally

determined physical characteristics of an organism.

Placenta: The oval or discoid spongy structure in the uterus from

which the fetus derives it nourishment and oxygen. (NRC)

Pluripotent: having great developmental plasticity, as a pluripotent

stem cell. Cells that can produce all the cell types of the developing

body, such as the ICM cells of the blastocyst, are said to be pluripotent.

Polarity: The property of having two opposite poles, sides or ends

(for example, humans have left-right polarity, also front-back polarity

and head-tailward polarity).

Population doublings: The number of times cells growing in vitro

have increased the total number of cells by a factor of 2 compared

to the initial number of cells.

Primitive streak: A band of cells appearing in the embryo at the

start of the third week of development, that marks the axis along

which the spinal chord develops.

Primordial germ cell: A gamete, that is, a sperm or egg, OR a

primordial cell that can mature into a sperm or egg. (NRC)

Salivary gland: One of several pairs of glands in the mouth that

secrete saliva.
GLOSSARY 155

Skin biopsy: Process of removing tissue, in this case skin, from living

patients for diagnostic examination, or the tissue specimen obtained

by that process. (SMD)

“Single-cell cloned”: A procedure pertaining to cells in vitro in which

the descendants of a single cell are physically isolated from other

cells growing in a dish, and then expanded into a larger population.

Somatic cell nuclear transfer (SCNT): A method of cloning: transfer

of the nucleus from a donor somatic cell into an enucleated egg to

produce a cloned embryo.

Somite: One of the longitudinal series of segments into which the

body of many animals (including vertebrates) is divided. (MerriamWebster on line)

Stem cells: Stem cells are undifferentiated multipotent precursor

cells that are capable both of perpetuating themselves as stem cells

and of undergoing differentiation into one or more specialized types

of cells. (CR)

Stromal: Relating to the stroma of an organ or other structure, that

is, its framework, usually of connective tissue, rather than its specific

substance. (SMD)

Syngamy: The coming together of the egg and sperm at fertilization.

Thymus: An organ of the developing immune system, active mainly

in childhood.

T-lymphocyte: A cell type of the immune system that matures in

the thymus and is responsible for cell-mediated immunity.

Type-1 diabetes: A form of insulin dependent diabetes, usually

becoming evident in childhood, resulting from an autoimmune

reaction that destroys the pancreatic beta cells, so that the body

cannot produce its own insulin. In those cases where the condition

is not apparent until adulthood, it is called latent autoimmune

diabetes of adulthood (LADA).

Transcription factors: Specialized proteins that bind to specific sites

on DNA and turn on or turn off the expression of different sets of

genes.
156 MONITORING STEM CELL RESEARCH

Trophoblast: The extraembryonic tissue responsible for

implantation, developing into the placenta, and controlling the

exchange of oxygen and metabolites between mother and embryo.

(NIH)

Twinning: Development of monozygotic twins, that is, when a very

early embryo separates into two pieces, each of which continues

development, so that two embryos actually come from one zygote.

Uterine: Pertaining to the uterus.

Virus: A submicroscopic pathogen composed essentially of a core

of DNA or RNA enclosed by a protein coat, able to replicate only

within a living cell.

Xenotransplantion: A transplant of tissue from an animal of one

species to an animal of another species.

Zygote: The diploid cell that results from the fertilization of an egg

cell by a sperm cell. (CR)
Appendix A.

Notes on Early Human Development

The term “embryo” refers to an organism in the early stages of

its development. In humans, the term is traditionally reserved for

the first two months of development. After that point, the term

“embryo” is replaced by the term “fetus,” which then applies until

birth. Some authors further reserve the term “embryo” for the

organism only after it has implanted and established its placental

connection to the pregnant woman. Similarly many also reserve the

term “pregnancy” for the state of the woman only after implantation.

At the beginning of the individual’s development, the entity is a

single cell. After two months, it has limbs, distinct fingers and toes,

internal development, and countless cells. So the term “embryo”

applies to an individual throughout a vast range of developmental

change. This document is a description of early human development,

with emphasis on those events or structures that have figured most

prominently in recent discussions of research using human embryos

or their parts, especially for stem cell research.1

Development has fascinated centuries of observers, as they

pursued deeper understanding of the stability of species

characteristics at least from one generation to the next, as well as

the uniqueness of each offspring. Uniqueness is especially marked

in sexually reproducing organisms, that is, organisms where the

genetic make-up of the offspring comes from a combination of

maternal and paternal DNA, because a new genome is formed in

each instance of conception. The stability reflects inheritance

connecting one generation with the past and future members of its

line.

Organisms and the processes of their development have evolved.

As a result, the development of any organism has a species-specific

pattern, but also shares many of the same developmental processes

with other species related from its evolutionary origins. Many of the

processes discussed here are common not just to all humans, or to

all mammals, but to all vertebrates. In some cases they are shared

even with invertebrates as well.

The process whereby a new individual of the species comes into

being has been at the center of too many deep inquiries to list here,

let alone discuss in the depth they deserve. But even in this short

document it is important to note one question that is related to the

connection of one generation to the next and previous generations.

157
158 MONITORING STEM CELL RESEARCH

That is, how are we to understand the apparent directedness of

development, following a complex network of pathways from a single

cell to a multi-system, free-living, and even conscious being? This

process occurs in a reliable pattern time after time, but also is

sufficiently resilient to perturbations that developing entities can

recover from significant disturbances. For example, at early stages

of development an embryo may divide (or be cut) completely in half,

and then each half recovers to form an entire offspring, resulting in

identical twins.

Different notions of purposive directedness, functional

explanation, and even vital forces have been invoked to explain

development. One of the insights, from the relation of development

to evolution, is that the development of an individual reflects the

fact that it is descended from individuals that reproduced

successfully and, like its forebears whose DNA it inherited, its

development reflects their past survival with their particular

characteristics. This legacy of ancestral success at survival is

manifested in the new organism’s apparent directedness toward

development along lines that enhance its own survival. Even very

early embryos follow patterns of differentiation in the progeny of

different cells. These patterns, in embryology, are called the fate of

the progeny of a cell. The fate of the progeny of the newest single

cell embryo is maximally broad—if it survives it will give rise to

every type of cell of the species. But as the embryo becomes

multicellular, its cells specialize and, in the absence of artificial

perturbation, their progeny have increasingly specialized fates as

well.

The evolved events and processes of development include some

that reflect distant relations, such as the yolk sac that is conserved

in placental mammals, including human beings. Other events or

processes exhibit the evolution of more specific characteristics. In

animals such as human beings, the specialized and complex

membraneous structures that form the connection between the

individual body of the pregnant woman and the developing

individual body of the offspring begin to arise in the first week.

Human embryos implant in the uterine wall starting at about the

sixth day after conception, so of course they must arrive in the uterus

with membranes capable of participating in that bond. They do not

have a fully formed placenta at such an early stage, nor is the uterine

wall unilaterally ready, but rather the contact of embryo and

endometrium initiates complementary development finally resulting

in the fully developed placenta. One way to look at it is that the

early embryo’s very structure points to the future, showing its overall

developmental fate to be connected to the maternal body. Another

perspective is that this process reflects the past survival of many
APPENDIX A. 159

generations. In both senses, no moment of development can be

understood in isolation from the context of the organism’s reflection

of its predecessors in evolution, and its directed differentiation

toward its future functioning.

I. GERM CELLS

For the beginning of an embryo, one can look both at the newly

fertilized egg, and also further back, to embryos of the previous

generation. The beginning of an individual is, of course, the union

of egg and sperm, specifically the union of DNA in the nucleus of

each, so as to form a new complete genome. But the egg and sperm

in turn develop from primordial germ cells that were themselves

developed when the parents of the new individual were embryos.

This description starts at that point (Figure 1).

Figure 1: Developmental cycle, here of a frog. Note the continuity of germ plasm.

[Figure 2.1, page 26, in Gilbert, S. Developmental Biology. 6th Edition. Sunderland,

Mass.: Sinauer Associates Inc., 2000. Figure reproduced with permission of Sinauer

Associates.]

The primordial germ cells are the cells that will give rise to either

ova or sperm. They are large cells with some distinctive

characteristics that make it possible to track them in development.

Note that in Figure 1, they are highlighted throughout the life cycle

of the animal. Primordial germ cells appear in embryonic

development prior to the formation of the gonads (ovaries in female,

or testes in a male). In humans and other mammals, the primordial
160 MONITORING STEM CELL RESEARCH

germ cells actually develop first in the yolk sac. In either sex, the

primordial germ cells migrate in through the developing gut of the

embryo and then populate the new gonads of whichever type. In

humans, the primordial germ cells first appear by the end of the

fourth week of development, and begin their migration to the gonads.

The primordial germ cells share certain characteristics with

embryonic stem cells, including self-renewal and pluripotency.

Primordial germ cells have been recovered from fetuses that were

aborted (for reasons unrelated to research) and cell lines have been

established from them, the progeny of which showed characteristics

of multiple different types of cells.2

After the primordial germ cells populate the gonads, some

continue to divide by mitosis, producing more like themselves. The

primordial germ cells are diploid, meaning that they have all the

normal chromosomes of the organism in pairs. In humans, this means

that they have 22 pairs of autosomes, and one pair of sex

chromosomes, or 46 total. Mitosis is the name of the process whereby

the cell replicates its DNA and then divides equally to result in two

cells, each cell including an entire complement of DNA just like the

first cell before the division (in humans, that is the 46 total

chromosomes mentioned) (Figure 2).

But if a cell is to become an ovum or sperm ready to combine

with a gamete of the complementary type to produce a new organism

(at first a zygote) containing the normal number of chromosomes, it

must undergo a special type of cell division whereby each gamete

acquires only half the diploid number. Each mature ovum or sperm

must include only 23 single (not paired) chromosomes. Mature ova

or sperm cells are haploid, indicating that their 23 chromosomes in

their nuclei are unpaired (and after they combine, then the resulting

single cell the zygote is again diploid). The process whereby the

diploid primordial germ cells develop into haploid gametes is called

meiosis (Figure 3). Mitosis is part of the life cycle of any cell, but

meiosis or meiotic division occurs only in the development of haploid

ova and sperm from diploid primordial germ cells. The process itself

appears as though the cell nucleus is undergoing two rounds of

mitosis, but omits the step of replicating DNA on the second cycle.

In the “first round,” the differentiating primordial germ cell replicates

its DNA, and then in the “second round” it divides again (without

another replication). In the second division, the pairs of chromosomes

separate, leaving each of the new cells with just one copy of each of

the 22 (in humans) autosomes and just one sex chromosome.
APPENDIX A. 161

Figure 2: Schematic summary of the principal stages in mitotic cell division,

simplified to show the movement of just two pairs of chromosomes. [Figure 3-4,

page 61, in Carlson, B.M. Patten’s Foundations of Embryology. 4th Edition. New York:

McGraw-Hill, 1981. Figure(s) reproduced with permission of the McGraw-Hill

Companies.]

This process does not always occur flawlessly. Errors, such as

failure of the chromosomes to separate properly, sometimes produce

new cells that have the wrong number of chromosomes, a condition

called aneuploidy (that is, not the true number). One cell may have

an extra copy of one of the chromosomes while the other cell is

missing a copy. Such a condition can be detected in the lab by
162 MONITORING STEM CELL RESEARCH

Figure 3: Schematic summary of the major stages of meiosis in a generalized germ

cell, simplified to showing movement of two pairs of chromosomes at the start.

[Carlson, 4th ed., Figure 3-6, p. 64.]

collecting some cells when they are about to go through mitosis so

their chromosomes can be stained and be spread out so their number

and appearance can be examined. A normal set of chromosomes

produces a characteristic picture (22 recognizable pairs and a pair

of sex chromosomes) called the normal karyotype. If a cell is

aneuploid, it will produce an abnormal karyotype picture. If the

aneuploid cell becomes an egg or sperm and is then involved in a

conception, the embryo is also aneuploid. Aneuploidies are not

uncommon events in germ cell development, but aneuploid survival
APPENDIX A. 163

is uncommon; nearly all aneuploidies are fatal very early in

development.

II. FERTILIZATION AND CLEAVAGE

Like the word “embryo,” the word “conception” refers to a series

of events or processes, not an instantaneous occurrence. Human

development begins after the union of egg and sperm cells during a

process known as fertilization. Fertilization itself comprises a

sequence of events that begins with the contact of a sperm cell

with an egg cell and ends with the fusion of their two pronuclei

(each containing 23 chromosomes) to form a new diploid cell, called

a zygote. Fertilization normally occurs in the ampulla of the uterine

tube 12-24 hours after ovulation (Figure 4).

Before that, however, sperm must travel through the vagina and

the cervix, through the uterus, and then up the uterine tube. Smooth

muscle contractions in the uterine tubes as well as ciliary activity

(waving of hair-like structures) of the tube’s lining both are important

in the transport of sperm up, and of the ovum into and then down,

the uterine tube. Many more sperm, on the order of tens, or even

hundreds, of millions, are ejaculated than reach the ovum. Those

sperm that do come into the vicinity of the ovum must get through

the material covering the ovum (the corona radiata and the zona

pellucida) and finally contact and bind to the ovum’s membrane, by

means of specialized structures in the head of the sperm cell. When

a sperm does get into the ovum, then the ovum membrane changes

so that other sperm cannot enter. Meanwhile, the sperm cell in the

egg is also undergoing changes and its specialized structures fall

away. The haploid nuclei of both the sperm and the egg are now

called male and female pronuclei. Both swell, as their densely packed

DNA loosens up prior to replication, and they also migrate toward

the center of the ovum. Then their nuclear membranes disintegrate

and the paternally and maternally contributed chromosomes pair

up, an event called syngamy. In this integration, the diploid
164 MONITORING STEM CELL RESEARCH

Figure 4A-E: Steps in the process of fertilization. The sequence of events begins

with contact between a sperm and a secondary oocyte (a mature egg) in the ampulla

of the uterine tube, and ends with formation of a zygote. [A-E: Fig. 1-1, page 3, in

Moore, Essentials of Human Embryology, 1988, with permission from Elsevier.]

Figure 4F: (see following page) Shows fertilization, syngamy [from

www.visembryo.com by Mouseworks, Inc.]
APPENDIX A. 165

chromosome number is restored,

and a new complete genome comes

into being. The result of syngamy

is an entity with an individual

genome. Further, if all goes well, it

is an entity that is capable of

developing into a fully formed

individual of the species. The

fertilized egg is now called a

zygote. It is at this point already

entering the first stage of its first

mitotic division, and beginning

cleavage (Figure 5).

Figure 4F: Fertilization, Syngamy

Figure 5: Embryo after cleavage.

[www.visembryo.com by Mouseworks, Inc.]

Like other vertebrates, humans have polarity in three dimensions

(head-tail, or back-front, and left-right). Establishing polarity is one

Conception in the Lab

In Vitro Fertilization (IVF), literally

“fertilization in glass” is the

procedure of combining eggs and

sperm outside the body in a dish. The

zygotes that are the results of

successful conception, if any, are

grown in culture for a few days and

then transferred to the uterus of the

mother. It may be used when the

prospective mother has damaged

uterine tubes. Louise Brown, the first

baby from IVF was born July 25,

1978, in the UK. IVF was put into

practice in the U.S. starting in 1981

and there have since been over

114,000 U.S. IVF births.

Intracytoplasmic Sperm Injection

(ICSI) is a variation on IVF. Instead

of just allowing sperm and eggs to

come into contact in a dish, a

technician physically places a sperm

cell inside the egg cell through the

egg membrane. ICSI is used, among

other reasons, when the prospective

father has some condition affecting

fertility, such as a low sperm count.

Usually more eggs are collected for

fertilization than would be

transferred at one time, both to

increase likelihood of some

successful conceptions, and because

the process of collecting eggs

involves hormonal treatments that

can be uncomfortable and risky for

the woman. Any early embryos that

are not transferred right away are

usually stored frozen for later

transfer. But many of these are not

transferred. In the U.S. as of June

2002 there were approximately

400,000 embryos in storage.
166 MONITORING STEM CELL RESEARCH

of the most basic manifestations of emerging specialization. But the

egg is roughly spherical, and it is not readily apparent how polarity

is established. Although it had been shown long ago that the point

of sperm entry determines the plane of first cleavage (and thus

subsequent ones) in amphibian eggs, mammals were believed until

recently to remain spherically symmetrical until later in development.

Recent data on mammalian zygotes, however, suggests that the

point of sperm entry may similarly determine the cleavage plane.3

Even the first two cells resulting from the first cleavage may have

different propensities, which persist through the next divisions as

the progeny of one cell tend to become the body of the offspring

and progeny of the other cell become the embryo’s contribution to

the placenta and other supporting structures. The word “fate,”

however, might be too strong, because the cells of such very early

embryos are resilient to perturbations—if one cell is removed, the

remaining ones can compensate.

III. IMPLANTATION

After fertilization, the zygote proceeds immediately to the first

cleavage and subsequent cell divisions follow rapidly. The zygote

is a very large cell, but the first waves of rapid cell division occur

without increase in cell volume. The result is a closely bound mass

of cells each of more typical cell size. At this stage the cells are

called blastomeres, (“parts of the blast,” “blast” coming from the

Greek for “bud” or “germ”) and the organism as a whole is called a

morula (from the Latin for mulberry, descriptive of its appearance)

from the time it has 16 blastomeres to the next stage. The morula is

still encased in the zona pellucida. As it is undergoing this very

rapid cell division, the organism is also migrating down the uterine

tube toward the uterus. After it arrives in the uterus, at about day

five after the initiation of fertilization, the zona pellucida breaks up;

the process is called “hatching” and is a necessary prelude to

implantation.

Many zygotes do not survive this long. Estimates vary widely of

the rate of natural embryo loss prior to implantation or after

implantation but still early in gestation. One study of healthy women

trying to conceive found 22 percent of pregnancies (identified by

sensitive hormone measures) were lost prior to becoming detectable

clinically. Even after implantation, there is a substantial rate of loss,

still not known precisely but estimated at 25 to 40 percent.4

When the morula enters the uterus, fluid starts to accumulate

between its blastomeres. The fluid-filled spaces run together,

forming a relatively large fluid-filled cavity. At the point when the
APPENDIX A. 167

cavity becomes recognizable, the organism is called a blastocyst

(Figure 6). The outer cells of the blastocyst, especially those around

the blastocyst cavity, assume a flattened shape. The flattened cells

of the exterior blastocyst are the trophoblast. They become the

embryo’s contribution to the placenta and other supporting

structures. On one side of the blastocyst is a group of cells that

project inside into the blastocyst cavity; this is the inner cell mass,

or embryoblast, and its progeny form the body of the new offspring.

FORMATION OF THE BLASTOCYST

Figure 6A-C: Three stages of the mammalial (blastocyst) of the pig, drawn from

sections to show the formation of the inner cell mass.

[Carlson, 4th Ed., Fig. 4-10, page 124.]

Figure 6D: Early blastocyst (see following page).
168 MONITORING STEM CELL RESEARCH

The cells of the inner cell mass can give rise to progeny

differentiating into all the types of cells in the adult body, so they

are called pluripotent. They have not usually been described as

totipotent because, the inner cell mass having already differentiated

from trophoblast, the cells of the inner cell mass were believed to

be no longer able to give rise to the cells of the trophoblast. Recent

work, however, describes culture conditions under which human

embryonic stem cells can differentiate to trophoblast cells.5

Although the new offspring itself develops only from the inner call

mass, the trophoblast is not just passive padding. Its progeny are

the essential and specialized connection between the embryonic

and maternal systems. Embryonic stem cells can be isolated from

the inner cell mass (see Chapter 4).

IV. TROPHOBLAST TO PLACENTA

After the embyo covering degenerates, the blastocyst, now in

the uterus, enlarges and its trophoblast attaches to the endometrium

(the uterine lining) at about six days after fertilization. This begins

the process of implantation, during which the blastocyst becomes

integrated with the endometrium through specialized membranes.

The embryo is now beginning its second week of development. The

process of implantation takes three to four days, but is generally

completed by day twelve. The trophoblast area that binds to the

endometrium first differentiates into an inner layer of cells and an

exterior layer in which the membranes dividing the cells degenerate

and the cells fuse. As the blastocyst become more deeply embedded

in the endometrium, the layered area expands until finally the whole

trophoblast surface has divided into one layer or the other.

Figure 6D: Early Blastocyst [www.visembryo.com by Mouseworks.Inc.]
APPENDIX A. 169

Meanwhile, a sort of primitive circulation develops, supporting the

embedded blastocyst while more complex structures continue to

develop. The inner cell mass then separates itself from the overlying

trophoblast. The resulting space is called the amniotic cavity and

the layer of cells that forms its roof is called the amnion (Figure 7).

Another membrane called the chorionic sac develops from the

trophoblast and nearby tissue. Finally, outgrowths of trophoblast

from the chorion project into the endometrium and are called primary

chorionic villi, later giving rise to the placenta. Although the

blastocyst has become completely embedded in the endometrium

and maternal blood bathes the chorionic villi, the maternal blood

does not enter the blastocyst. Later, as the fetal circulation develops,

the fetal and maternal blood systems still remain distinct and do

not mingle. Nutrients, oxygen, and wastes diffuse in the appropriate

direction across the placenta, but the two blood systems are

individual and do not combine.
170 MONITORING STEM CELL RESEARCH

Figure 7A: Sections of completely implanted blastocysts at the end of the second

week, illustrating how the secondary yolk sac forms. The presence of primary

chorionic villi on the wall of the chorionic sac is characteristic of blastocysts at the

end of the second week. A primitive uteroplacental circulation is now present.

[Reprinted from Moore, Essentials of Human Embryology, 1988, Fig. 2-2, p. 13, with

permission from Elsevier.]
APPENDIX A. 171

 

Figure 7B: Photo micrographs of implantation beginning and completed.

[www.visembryo.com by Mouseworks, Inc.]

V. TWINNING

The usual case for human beings is for one ovum to be released,

and if all goes well, fertilized and developed to term. Less commonly,

more than one ovum may be released and fertilized so that more

than one embryo develops. These embryos would be genetically

distinct, sharing the uterus during the same gestation period. They

will have a family resemblance but no more genetic commonality

than any other set of siblings, and they may be of the same or

different sexes. These are called dizygotic twins (because they came

from two zygotes). More rarely, a single zygote may, during its early

cleavages, separate completely into two groups of cells. As

discussed above, the two cells resulting from the first cleavage may

already have different probable fates, the progeny of one contributing

to the body and the other to the supporting structures. Both,

however, at this stage are still totipotent and can, if disrupted, go

on to generate a full individual organism. If this separation occurs,

then monozygotic twins may be born (Figure 8). Monozygotic twins,

two offspring coming from one zygote, have the same genome and

are always of the same sex. When the twinning occurs in the first

cleavages and there are not yet any extraembryonic membranes

(Figure 8A), the two develop separately as do dizygotic twins, with

separate amnions, chorions and eventually placentae. If an embryo

should divide into two later in its development, between about days

four and eight, the twins will share the same chorion and therefore

eventually the same placenta, but a separate amnion will form

around each (Figure 8B). Should an embryo divide later than this,

between about the ninth and thirteenth days, the resulting twins
172 MONITORING STEM CELL RESEARCH

will share the same amnion, chorion, and placenta. It is very rare for

embryos to divide still later than this, but occasionally they do divide

after the fourteenth day. These divisions may not be complete, and

then the twins remain conjoined and can only be surgically separated

after birth (Figure 8C). The twin birth rate in the U.S. has increased

markedly in recent years, and was 30.1 per 1,000 live births in 2001.6

The rate of multiple births (most multiple births are twins; triplets

and so on are more rare) is higher with assisted reproductive

technologies and with higher maternal age. Dizygotic twins clearly

can result in ART from transferring more than one embryo to the

prospective mother. In addition, some assisted reproductive

practices, like age of the embryo transferred, may be associated

with more likelihood of monozygotic twinning,7

 though in general

the causes of monozygotic twinning are not known.

Figure 8A-C: Modes of monozygotic twinning. [Carlson, 4th Ed., Fig. 1-12, page 23]
APPENDIX A. 173

VI. THE PRIMITIVE STREAK AND GASTRULATION

While implantation is occurring, the inner cell mass is also

undergoing changes. First, the inner cell mass separates into two

layers, the epiblast, which is next to the amniotic cavity, and the

hypoblast, which is next to what was the blastocyst cavity but is by

this stage called the primary yolk sac. The epiblast thus forms the

floor of the amniotic cavity (as the amnion forms the roof) and is

connected with the amnion around the edges. The hypoblast is

connected around its edges with the exocoelomic membrane or

primary yolk sac. Thus, the supporting structures, collectively called

the extraembryonic membranes, are outside of the body that is

starting to develop and that will eventually be born, but during

embryonic development the membranes are also continuous with

that body. By the end of the second week, the hypoblast has

developed a thickened area, called the prochordal plate, that is

located at what will be the cranial (head) end of the individual. In

fact, the prochordal plate shows where the mouth will develop.

As the third week of development begins, dividing cells pile up

in a line to form a thicker band in the epiblast. The line or band

starts nearly directly across from the prochordal plate, and extends

from the edge toward the center of the embryonic disc. The band is

called the primitive streak. In many policy discussions, the

appearance of the primitive streak is an important boundary. This

summary will continue just a little longer, in order to discuss briefly

the nature of the primitive streak.

The end of the primitive streak that is toward the middle of the

disc (nearer the prochordal plate marking the mouth) is the cranial

end, and this end thickens more as more cells divide. This especially

thick end is called the primitive knot (formerly called Henson’s node).

The end of the primitive streak near the edge is the caudal (or tailward) end. As a model, think of the primitive streak as a zipper: the

epiblast cells that made the thickness now start to migrate across

the surface and into the zipper of the primitive streak. As the cells

enter the primitive streak, they do a U-turn around the edge and

continue to migrate back the way they came but underneath the

surface, displacing the hypoblast cells. This movement results in

three layers, all of epiblast origin: what was the epiblast on top, the

cells that used to be part of the epiblast but are now underneath it,

and the cells that remain in between (Figure 9).

These three layers get new names, and they also get newly

specified fates for their progeny. In the same order as above, they

are the ectoderm, mesoderm, and endoderm. The completion (during
174 MONITORING STEM CELL RESEARCH

the third week after fertilization) of forming these three layers is

called gastrulation. The ectodermal layer gives rise to progeny fated

to become the skin, the nervous system, and sensory structures of

the eye, ear, and nose; mesoderm gives rise to the skeletal and

muscular systems, connective tissue and blood vessels, and

endoderm gives rise to epithelial parts (e.g., the linings) of the

digestive and respiratory systems.

Gastrulation is a crucial event in the development of the body

plan of the individual, and it is a stage of development common to

all vertebrates. Our understanding of the significance of establishing

the three germ layers has grown more complex and subtle over the

years. Once interpreted as three completely separate paths or

compartments of development, we now know that the progeny of

the three layers are not totally isolated in their fates. Cartilage, for

example, was once thought to be entirely of mesodermal origin, but

now we know that some cartilaginous structures of the head and

neck come from ectoderm. Even more recently, work with certain

adult stem cell populations in culture and under special conditions

has suggested plasticity of cell progeny from one germ layer to

develop characteristics of cells typically from another germ layer,

long after gastrulation has assigned the cells of different germ layers

their different fates. Gastrulation is not the first differentiating event:

cells begin to acquire fates for different parts of the developing

embryo before the inner cell mass separates into epiblast and

hypoblast, indeed some results suggest even before the blastocyst

develops an inner cell mass and trophoblast. Yet these findings in

no way detract from the significance of gastrulation. They rather

facilitate our understanding of gastrulation by placing it in the

context of the entire process of differentiation, beginning from the

very earliest stages.
APPENDIX A. 175

Figure 9A-C: Schematic drawings of the embryonic disc and its associated

extraembryonic membranes during the third week. A: the amniotic cavity has been

opened to show the primitive streak, a midline thickening of the epiblast. Part of

the yolk sac has been cut away to show the bilaminar embryonic disc (epiblast and

hypoblast). The transverse section (lower right of A) illustrates the proliferation

and migration of cells from the primitive streak to form embryonic mesoderm. B

and C: drawings illustrating early formation of the notochordal process from the

primitive knot of the primitive streak. In the longitudinal sections on the right side,

note that the notochordal process grows cranially in the median plane between the

embryonic ectoderm and endoderm. [Reprinted from Moore, Essentials of Human

Embryology, 1988, Fig. 3-1, page 17, with permission from Elsevier.]
176 MONITORING STEM CELL RESEARCH

Figure 9D: Photo micrograph of Primitive Streak

[www.visembryo.com by Mouseworks, Inc.]

VII. NEURULATION

Neurulation is the series of developmental events that result in

the beginnings of the central nervous system (Figure 10). From the

cranial end of the primitive streak, a long stiff structure develops in

the mesoderm, elongating still further in the cranial direction. This

becomes the notochord, which marks the head/tail axis of the

embryo. Later, the vertebral column develops around it. But at this

time, the notochord and its adjacent tissue exert influence called

primary induction on the ectoderm lying over them, such that the

ectoderm thickens and becomes the neural plate.

The neural plate then actually pushes up to form folds (called

the neural folds) along each side of the tissue over the notochord.

The neural folds then meet and fuse to enclose the neural tube,

beginning at the middle of the (future) tube, like a zipper closing

from the middle toward each end. This process is completed by the

end of the third week. Some cells along the crests of the folds migrate

through the embryo. They are called neural crest cells, and they

give rise to a variety of nerve cells including dorsal root (spinal) and

autonomic nervous system ganglia, and some other nervous system

and endocrine structures.
APPENDIX A. 177

Figure 10A-H: Schematic drawings of the human embryo during the third and

fourth weeks. Left side: Dorsal views of the developing embryo illustrating early

formation of the brain, intraembryonic coelom, and somites. Right side: Schematic

transverse sections illustrating formation of the neural crest, neural tube,

intraembryonic coelom, and somites. [Reprinted from Moore, Essentials of Human

Embryology, 1988, Fig. 3-3, page 20, with permission from Elsevier.]
178 MONITORING STEM CELL RESEARCH

Figure 10I: Neurulation and Notochordal Process

[www.visembryo.com by Mouseworks, Inc.]

The mesoderm still adjacent to the neural tube resolves into the

form of paired blocks on either side of the tube, which are called

somites. The first pair of somites appears at about the twentieth

day after fertilization, at the cranial end of the neural tube. More

pairs appear in the caudal direction, up until about the thirtieth day.

Mesodermal cells from the somites give rise to most of the skeleton

and skeletal muscle.

Blood cell and blood vessel formation actually start at the

beginning of the third week after fertilization, first in the supportive

structures of the yolk sac and chorion. Blood vessel formation begins

in the embryo body about two days later, although blood is not

formed in the embryo itself until the fifth week. The heart begins

as a wide blood vessel, which later folds up to develop the chambers

of the fully formed heart. But even as a tube, the membranes of its

cells have the electrical and contractile capacity to begin beating

in the third week, and thus to begin primitive circulatory function

with blood. During this time the primary chorionic villi elaborate

branches and form capillary networks and vessels connected with

the embryonic heart. Oxygen and nutrients diffuse from the

maternal blood to the embryonic blood through these capillaries,

while carbon dioxide, urea, and other metabolic wastes diffuse from

the embryonic blood into the maternal blood. Meanwhile, even

firmer connections form between embryonic supporting
APPENDIX A. 179

membranes and the endometrium, finally completing the

development of the placenta.

VIII. ORGANOGENESIS

The basic structures and relations of all the major organ systems

of the body emerge during the fourth through the eighth weeks of

embryonic development. First, the embryo folds in several ways so

that the flat linear structure distinguished by neural tube flanked

by somites become roughly C-shaped. The effect of this is to bring

the regions of the brain, gut, and other internal organs into their

familiar anatomical relations. During the fourth week the neural

pores, the ends of the neural tube “zipper,” close. First the one at

the cranial or head end, which is called the anterior or rostral pore,

closes, and later the caudal or tail-ward pore closes. Closure of the

neural pores completes the closure of what will become the central

nervous system. Also during the fourth week, limb buds become

visible, first buds for arms and later for legs. Further, two

accumulations of cells along the neural tube become

distinguishable: the alar plate and the basal plate. Cells of the alar

plate go on to become mostly sensory neurons, while basal plate

cells give rise mostly to motor neurons. Already while the neural

tube is closing, its walls along the cranial area are thickening to

form early brain structure. Cranial nerves, for example the nerves

for the eye and for the muscles of the face and jaw, also are

beginning to develop at this time. The embryonic brain develops

rapidly in both size and structure especially during the fifth week,

and the optic cup that will form the retina of the eye becomes visible

as well.

IX. CONCLUSION AND CONTINUATION

Embryonic development continues with the emergence and

differentiation of organs, the skeleton, limbs, and digits, and with

the development of the face and further differentiation and

integration throughout the body. The development discussed above

is summarized briefly in Table 1.8

 But development continues, and

is a continuous process, past the eight-week mark, when the

organism is no longer called an embryo and instead is called a fetus.

Although the basic elements of the body plan have been established

during embryogenesis, a great deal of development of that body

plan, refinement and integration, continues in the fetal stage, also

called phenogenesis (emergence of the normal appearance of the

body). Development continues after birth as well.
180 MONITORING STEM CELL RESEARCH

Table1: Summary of Developmental Timecourse

Stage Week after

fertilization

Days after

fertilization Event

Pregenesis:

development of

parents

4th week

develop-ment

of parents

24

Parents' primordial germ

cells (PGCs) begin their

migration to parents'

gonads

Blastogenesis 1st week,

embryo is

unilaminar

1 Fertilization

1.5-3 1st cleavages, move to

uterus

4 Free blastocyst in uterus

5-6 Hatching, start

implantation

2nd week,

embryo is

bilaminar

7-12 Fully implanted

13 Primary stem villi and

primitive streak appear

3rd week,

embryo is

trilaminar

16 Gastrulation begins,

notochord forms

18 Primitive pit, neural

plate, neural groove

20 First somites, primitive

heart tube

4th week

22 Neural folds fuse,

pulmonary primordium,

24

PGCs begin migration,

Cranial neuropore

closes, optic vesicles

and pit form

26 Caudal neuropore

closes, arm limb buds

28 Leg limb buds, more

brain, eye/ear devel

Organogenesis 5th - 8th weeks 29-56

Phenogenesis 9th - 38th

weeks
APPENDIX A. 181

ENDNOTES

1

 There are many fine embryology texts, and the reader is urged to consult one or

more for deeper, broader and more extended treatment of embryology. The following

references are samples only, not a comprehensive bibliography, selected in part for

accessibility to the general though committed reader, and in part for recent

publication. A few examples concentrating on human embryology would include

Larsen, W. J., Essentials of Human Embryology. New York: Churchill Livingstone,

1988; Sadler, T. W., Langman’s Medical Embryology 8th Edition, Philadelphia:

Lippincott Williams and Wilkens, 2002; or Sweeney, L. J., Basic Concepts in

Embryology: A Student’s Survival Guide, New York: McGraw-Hill, 1988. For a more

comparative approach consider Carlson, B. M., Patten’s Foundations of Embryology.

6th Edition, New York: McGraw-Hill, 1996; and for more comparison and inclusion of

related topics, see Gilbert, S. J., Developmental Biology, 6th Edition, Sunderland,

MA: Sinauer Associates Inc., 2000. In addition, there are many fine web-based

resources, which the reader is encouraged to visit, for example http://anatomy.med.

unsw.edu.au/cbl/embryo/Embryo.htm and http://www.visembryo.com/ and to

accompany Gilbert’s text, http://www.devbio.com/. These sites provide links to

further resources as well.

2

 Shamblott M.J., et al., “Derivation of pluripotent stem cells from cultured human

primordial germ cells” Proceedings of the National Academy of Sciences. 95(23):

13726-13731 (1998). [Erratum in: Proc Natl Acad Sci USA 96(3): 1162 (1999).]

3

 Pearson, H., “Your destiny, from day one.” Nature 418(6893): 14-15 (2002).

4

 Wilcox, A. J., et al., “Incidence of early loss of pregnancy,” New England Journal of

Medicine 319(4): 189-194 (1988). See also this review article: Norwitz, E. R., et al.,

“Implantation and the survival of early pregnancy,” New England Journal of Medicine

345(19): 1400-1408 (2001). Some estimates are indeed much higher (as high as 80

percent for embryo loss before and after implantation).

5

 Xu, R.H., et al., “BMP4 initiates human embryonic stem cell differentiation to

trophoblast” Nature Biotechnology 20(12): 1261-1264 (2002).

6

 National Center for Health Statistics, “Births: Final Data for 2001.” National Vital

Statistics Reports 51(2) (2002), available at http://www.cdc.gov/nchs/data/nvsr/

nvsr51/nvsr51_02.pdf.

7

 Milki, A.A. et al., “Incidence of monozygotic twinning with blastocyst transfer

compared to cleavage-stage transfer” Fertility and Sterility 79(3): 503-506 (2003).

8

 Table 1 follows closely the table of events shown in Larsen (1998), p. xi, and also

the table presented by John M. Opitz, MD, at the January 16, 2003, meeting of the

Council. Not all the events listed in Larsen’s table were included in the Table 1

above, however.

Appendix B.

Remarks by President George W. Bush

on Stem Cell Research

August 9, 2001

The Bush Ranch

Crawford, Texas

THE PRESIDENT: “Good evening. I appreciate you giving me a few

minutes of your time tonight so I can discuss with you a complex

and difficult issue, an issue that is one of the most profound of our

time.

The issue of research involving stem cells derived from

human embryos is increasingly the subject of a national debate and

dinner table discussions. The issue is confronted every day in

laboratories as scientists ponder the ethical ramifications of their

work. It is agonized over by parents and many couples as they try

to have children, or to save children already born.

The issue is debated within the church, with people of

different faiths, even many of the same faith coming to different

conclusions. Many people are finding that the more they know

about stem cell research, the less certain they are about the right

ethical and moral conclusions.

My administration must decide whether to allow federal

funds, your tax dollars, to be used for scientific research on stem

cells derived from human embryos. A large number of these embryos

already exist. They are the product of a process called in vitro

fertilization, which helps so many couples conceive children. When

doctors match sperm and egg to create life outside the womb, they

usually produce more embryos than are planted in the mother. Once

a couple successfully has children, or if they are unsuccessful, the

additional embryos remain frozen in laboratories.

Some will not survive during long storage; others are

destroyed. A number have been donated to science and used to

create privately funded stem cell lines. And a few have been

implanted in an adoptive mother and born, and are today healthy

children.

183
184 MONITORING STEM CELL RESEARCH

Based on preliminary work that has been privately funded,

scientists believe further research using stem cells offers great

promise that could help improve the lives of those who suffer from

many terrible diseases — from juvenile diabetes to Alzheimer’s, from

Parkinson’s to spinal cord injuries. And while scientists admit they

are not yet certain, they believe stem cells derived from embryos

have unique potential.

You should also know that stem cells can be derived from

sources other than embryos — from adult cells, from umbilical cords

that are discarded after babies are born, from human placenta. And

many scientists feel research on these type of stem cells is also

promising. Many patients suffering from a range of diseases are

already being helped with treatments developed from adult stem

cells.

However, most scientists, at least today, believe that

research on embryonic stem cells offer the most promise because

these cells have the potential to develop in all of the tissues in the

body.

Scientists further believe that rapid progress in this research

will come only with federal funds. Federal dollars help attract the

best and brightest scientists. They ensure new discoveries are

widely shared at the largest number of research facilities and that

the research is directed toward the greatest public good.

The United States has a long and proud record of leading

the world toward advances in science and medicine that improve

human life. And the United States has a long and proud record of

upholding the highest standards of ethics as we expand the limits

of science and knowledge. Research on embryonic stem cells raises

profound ethical questions, because extracting the stem cell destroys

the embryo, and thus destroys its potential for life. Like a snowflake,

each of these embryos is unique, with the unique genetic potential

of an individual human being.

As I thought through this issue, I kept returning to two

fundamental questions: First, are these frozen embryos human life,

and therefore, something precious to be protected? And second, if

they’re going to be destroyed anyway, shouldn’t they be used for a

greater good, for research that has the potential to save and improve

other lives?

I’ve asked those questions and others of scientists, scholars,

bioethicists, religious leaders, doctors, researchers, members of

Congress, my Cabinet, and my friends. I have read heartfelt letters

from many Americans. I have given this issue a great deal of thought,

prayer and considerable reflection. And I have found widespread

disagreement.
APPENDIX B. 185

On the first issue, are these embryos human life — well,

one researcher told me he believes this five-day-old cluster of cells

is not an embryo, not yet an individual, but a pre-embryo. He argued

that it has the potential for life, but it is not a life because it cannot

develop on its own.

An ethicist dismissed that as a callous attempt at

rationalization. Make no mistake, he told me, that cluster of cells is

the same way you and I, and all the rest of us, started our lives. One

goes with a heavy heart if we use these, he said, because we are

dealing with the seeds of the next generation.

And to the other crucial question, if these are going to be

destroyed anyway, why not use them for good purpose — I also

found different answers. Many argue these embryos are byproducts

of a process that helps create life, and we should allow couples to

donate them to science so they can be used for good purpose instead

of wasting their potential. Others will argue there’s no such thing

as excess life, and the fact that a living being is going to die does

not justify experimenting on it or exploiting it as a natural resource.

At its core, this issue forces us to confront fundamental

questions about the beginnings of life and the ends of science. It

lies at a difficult moral intersection, juxtaposing the need to protect

life in all its phases with the prospect of saving and improving life

in all its stages.

As the discoveries of modern science create tremendous

hope, they also lay vast ethical mine fields. As the genius of science

extends the horizons of what we can do, we increasingly confront

complex questions about what we should do. We have arrived at

that brave new world that seemed so distant in 1932, when Aldous

Huxley wrote about human beings created in test tubes in what he

called a “hatchery.”

In recent weeks, we learned that scientists have created

human embryos in test tubes solely to experiment on them. This is

deeply troubling, and a warning sign that should prompt all of us to

think through these issues very carefully.

Embryonic stem cell research is at the leading edge of a

series of moral hazards. The initial stem cell researcher was at first

reluctant to begin his research, fearing it might be used for human

cloning. Scientists have already cloned a sheep. Researchers are

telling us the next step could be to clone human beings to create

individual designer stem cells, essentially to grow another you, to

be available in case you need another heart or lung or liver.

I strongly oppose human cloning, as do most Americans.

We recoil at the idea of growing human beings for spare body parts,

or creating life for our convenience. And while we must devote

enormous energy to conquering disease, it is equally important that
186 MONITORING STEM CELL RESEARCH

we pay attention to the moral concerns raised by the new frontier

of human embryo stem cell research. Even the most noble ends do

not justify any means.

My position on these issues is shaped by deeply held beliefs.

I’m a strong supporter of science and technology, and believe they

have the potential for incredible good — to improve lives, to save

life, to conquer disease. Research offers hope that millions of our

loved ones may be cured of a disease and rid of their suffering. I

have friends whose children suffer from juvenile diabetes. Nancy

Reagan has written me about President Reagan’s struggle with

Alzheimer’s. My own family has confronted the tragedy of childhood

leukemia. And, like all Americans, I have great hope for cures.

I also believe human life is a sacred gift from our Creator. I

worry about a culture that devalues life, and believe as your

President I have an important obligation to foster and encourage

respect for life in America and throughout the world. And while

we’re all hopeful about the potential of this research, no one can be

certain that the science will live up to the hope it has generated.

Eight years ago, scientists believed fetal tissue research

offered great hope for cures and treatments — yet, the progress to

date has not lived up to its initial expectations. Embryonic stem cell

research offers both great promise and great peril. So I have decided

we must proceed with great care.

As a result of private research, more than 60 genetically

diverse stem cell lines already exist. They were created from embryos

that have already been destroyed, and they have the ability to

regenerate themselves indefinitely, creating ongoing opportunities

for research. I have concluded that we should allow federal funds to

be used for research on these existing stem cell lines, where the life

and death decision has already been made.

Leading scientists tell me research on these 60 lines has

great promise that could lead to breakthrough therapies and cures.

This allows us to explore the promise and potential of stem cell

research without crossing a fundamental moral line, by providing

taxpayer funding that would sanction or encourage further

destruction of human embryos that have at least the potential for

life.

I also believe that great scientific progress can be made

through aggressive federal funding of research on umbilical cord

placenta, adult and animal stem cells which do not involve the same

moral dilemma. This year, your government will spend $250 million

on this important research.

I will also name a President’s council to monitor stem cell

research, to recommend appropriate guidelines and regulations, and

to consider all of the medical and ethical ramifications of biomedical
APPENDIX B. 187

innovation. This council will consist of leading scientists, doctors,

ethicists, lawyers, theologians and others, and will be chaired by

Dr. Leon Kass, a leading biomedical ethicist from the University of

Chicago.

This council will keep us apprised of new developments and

give our nation a forum to continue to discuss and evaluate these

important issues. As we go forward, I hope we will always be guided

by both intellect and heart, by both our capabilities and our

conscience.

I have made this decision with great care, and I pray it is

the right one.

Thank you for listening. Good night, and God bless America.”

* * *

Appendix C.

Bush Administration NIH Guidelines for

Embryonic Stem Cell Funding

Taken from the website of the National Institutes of Health

on September 26, 2003.

The text follows.

NOTICE OF CRITERIA FOR FEDERAL FUNDING OF RESEARCH

ON EXISTING HUMAN EMBRYONIC STEM CELLS AND

ESTABLISHMENT OF NIH HUMAN EMBRYONIC STEM CELL

REGISTRY

Release Date: November 7, 2001

NOTICE: NOT-OD-02-005

Office of the Director, NIH

On August 9, 2001, at 9:00 p.m. EDT, the President announced his

decision to allow Federal funds to be used for research on existing

human embryonic stem cell lines as long as prior to his

announcement (1) the derivation process (which commences with

the removal of the inner cell mass from the blastocyst) had already

been initiated and (2) the embryo from which the stem cell line was

derived no longer had the possibility of development as a human

being.

In addition, the President established the following criteria that must

be met:

o The stem cells must have been derived from an embryo that was

created for reproductive purposes;

o The embryo was no longer needed for these purposes;

o Informed consent must have been obtained for the donation of

the embryo;

o No financial inducements were provided for donation of the

embryo.

189
190 MONITORING STEM CELL RESEARCH

In order to facilitate research using human embryonic stem cells,

the NIH is creating a Human Embryonic Stem Cell Registry that

will list the human embryonic stem cells that meet the eligibility

criteria. Specifically, the laboratories or companies that provide the

cells listed on the Registry will have submitted to the NIH a signed

assurance. Each provider must retain for submission to the NIH, if

necessary, written documentation to verify the statements in the

signed assurance.

The Registry will be accessible to investigators on the NIH Home

Page http://escr.nih.gov. Requests for Federal funding must cite a

human embryonic stem cell line that is listed on the NIH Registry.

Such requests will also need to meet existing scientific and technical

merit criteria and be recommended for funding by the relevant

National Advisory Council, as appropriate. Further guidance is

accessible at http://grants.nih.gov/ grants/guide/ notice-files/NOTOD-02-006.html.

Inquiries should be directed to the Deputy Director for Extramural

Research DDER@nih.gov.

* * *
Appendix D.

Clinton Administration NIH Guidelines for

Embryonic Stem Cell Funding

As printed in the Federal Register, August 25, 2000

(“National Institutes of Health Guidelines for Research

Using Human Pluripotent Stem Cells,” 65 Fed. Reg. 51,975,

Aug. 25, 2000)

The text of the final Guidelines follows.

National Institutes of Health Guidelines for Research Using Human

Pluripotent Stem Cells

I. Scope of Guidelines

 These Guidelines apply to the expenditure of National Institutes

of Health (NIH) funds for research using human pluripotent stem

cells derived from human embryos (technically known as human

embryonic stem cells) or human fetal tissue (technically known as

human embryonic germ cells). For purposes of these Guidelines,

“human pluripotent stem cells” are cells that are self-replicating,

are derived from human embryos or human fetal tissue, and are

known to develop into cells and tissues of the three primary germ

layers. Although human pluripotent stem cells may be derived from

embryos or fetal tissue, such stem cells are not themselves embryos.

NIH research funded under these Guidelines will involve human

pluripotent stem cells derived: (1) From human fetal tissue; or (2)

from human embryos that are the result of in vitro fertilization, are

in excess of clinical need, and have not reached the stage at which

the mesoderm is formed.

 In accordance with 42 Code of Federal Regulations (CFR) 52.4,

these Guidelines prescribe the documentation and assurances that

must accompany requests for NIH funding for research using human

pluripotent stem cells from: (1) Awardees who want to use existing

funds; (2) awardees requesting an administrative or competing

supplement; and (3) applicants or intramural researchers submitting

applications or proposals. NIH funds may be used to derive human

191
192 MONITORING STEM CELL RESEARCH

pluripotent stem cells from fetal tissue. NIH funds may not be used

to derive human pluripotent stem cells from human embryos. These

Guidelines also designate certain areas of human pluripotent stem

cell research as ineligible for NIH funding.

II. Guidelines for Research Using Human Pluripotent Stem Cells That

Is Eligible for NIH Funding

A. Utilization of Human Pluripotent Stem Cells Derived From Human

Embryos

1. Submission to NIH

 Intramural or extramural investigators who are intending to use

existing funds, are requesting an administrative supplement, or are

applying for new NIH funding for research using human pluripotent

stem cells derived from human embryos must submit to NIH the

following:

 a. An assurance signed by the responsible institutional official

that the pluripotent stem cells were derived from human embryos

in accordance with the conditions set forth in section II.A.2 of these

Guidelines and that the institution will maintain documentation in

support of the assurance;

 b. A sample informed consent document (with patient identifier

information removed) and a description of the informed consent

process that meet the criteria for informed consent set forth in section

II.A.2.e of these Guidelines;

 c. An abstract of the scientific protocol used to derive human

pluripotent stem cells from an embryo;

 d. Documentation of Institutional Review Board (IRB) approval of

the derivation protocol;

 e. An assurance that the stem cells to be used in the research

were or will be obtained through a donation or through a payment

that does not exceed the reasonable costs associated with the

transportation, processing, preservation, quality control and storage

of the stem cells;

 f. The title of the research proposal or specific subproject that

proposes the use of human pluripotent stem cells;

 g. An assurance that the proposed research using human

pluripotent stem cells is not a class of research that is ineligible for

NIH funding as set forth in section III of these Guidelines; and

 h. The Principal Investigator’s written consent to the disclosure

of all material submitted under Paragraph A.1 of this section, as

necessary to carry out the public review and other oversight

procedures set forth in section IV of these Guidelines.
APPENDIX D. 193

2. Conditions for the Utilization of Human Pluripotent Stem Cells

Derived From Human Embryos

 Studies utilizing pluripotent stem cells derived from human

embryos may be conducted using NIH funds only if the cells were

derived (without Federal funds) from human embryos that were

created for the purposes of fertility treatment and were in excess of

the clinical need of the individuals seeking such treatment.

 a. To ensure that the donation of human embryos in excess of the

clinical need is voluntary, no inducements, monetary or otherwise,

should have been offered for the donation of human embryos for

research purposes. Fertility clinics and/or their affiliated laboratories

should have implemented specific written policies and practices to

ensure that no such inducements are made available.

 b. There should have been a clear separation between the decision

to create embryos for fertility treatment and the decision to donate

human embryos in excess of clinical need for research purposes to

derive pluripotent stem cells. Decisions related to the creation of

embryos for fertility treatment should have been made free from

the influence of researchers or investigators proposing to derive or

utilize human pluripotent stem cells in research. To this end, the

attending physician responsible for the fertility treatment and the

researcher or investigator deriving and/or proposing to utilize human

pluripotent stem cells should not have been one and the same

person.

 c. To ensure that human embryos donated for research were in

excess of the clinical need of the individuals seeking fertility

treatment and to allow potential donors time between the creation

of the embryos for fertility treatment and the decision to donate for

research purposes, only frozen human embryos should have been

used to derive human pluripotent stem cells. In addition, individuals

undergoing fertility treatment should have been approached about

consent for donation of human embryos to derive pluripotent stem

cells only at the time of deciding the disposition of embryos in excess

of the clinical need.

 d. Donation of human embryos should have been made without

any restriction or direction regarding the individual(s) who may be

the recipients of transplantation of the cells derived from the human

pluripotent stem cells.

 e. Informed Consent

 Informed consent should have been obtained from individuals who

have sought fertility treatment and who elect to donate human

embryos in excess of clinical need for human pluripotent stem cell

research purposes. The informed consent process should have
194 MONITORING STEM CELL RESEARCH

included discussion of the following information with potential

donors, pertinent to making the decision whether or not to donate

their embryos for research purposes.

 Informed consent should have included:

 (i) A statement that the embryos will be used to derive human

pluripotent stem cells for research that may include human

transplantation research;

 (ii) A statement that the donation is made without any restriction

or direction regarding the individual(s) who may be the recipient(s)

of transplantation of the cells derived from the embryo;

 (iii) A statement as to whether or not information that could

identify the donors of the embryos, directly or through identifiers

linked to the donors, will be removed prior to the derivation or the

use of human pluripotent stem cells;

 (iv) A statement that derived cells and/or cell lines may be kept

for many years;

 (v) Disclosure of the possibility that the results of research on the

human pluripotent stem cells may have commercial potential, and

a statement that the donor will not receive financial or any other

benefits from any such future commercial development;

 (vi) A statement that the research is not intended to provide direct

medical benefit to the donor; and

 (vii) A statement that embryos donated will not be transferred to

a woman’s uterus and will not survive the human pluripotent stem

cell derivation process.

 f. Derivation protocols should have been approved by an IRB

established in accord with 45 CFR 46.107 and 46.108 or FDA

regulations at 21 CFR 56.107 and 56.108.

B. Utilization of Human Pluripotent Stem Cells Derived From Human

Fetal Tissue

1. Submission to NIH

 Intramural or extramural investigators who are intending to use

existing funds, are requesting an administrative supplement, or are

applying for new NIH funding for research using human pluripotent

stem cells derived from fetal tissue must submit to NIH the following:

 a. An assurance signed by the responsible institutional official

that the pluripotent stem cells were derived from human fetal tissue

in accordance with the conditions set forth in section II.A.2 of these

Guidelines and that the institution will maintain documentation in

support of the assurance;

 b. A sample informed consent document (with patient identifier

information removed) and a description of the informed consent
APPENDIX D. 195

process that meet the criteria for informed consent set forth in section

II.B.2.b of these Guidelines;

 c. An abstract of the scientific protocol used to derive human

pluripotent stem cells from fetal tissue;

 d. Documentation of IRB approval of the derivation protocol;

 e. An assurance that the stem cells to be used in the research

were or will be obtained through a donation or through a payment

that does not exceed the reasonable costs associated with the

transportation, processing, preservation, quality control and storage

of the stem cells;

 f. The title of the research proposal or specific subproject that

proposes the use of human pluripotent stem cells;

 g. An assurance that the proposed research using human

pluripotent stem cells is not a class of research that is ineligible for

NIH funding as set forth in section III of these Guidelines; and

 h. The Principal Investigator’s written consent to the disclosure

of all material submitted under Paragraph B.1 of this section, as

necessary to carry out the public review and other oversight

procedures set forth in section IV of these Guidelines.

2. Conditions for the Utilization of Human Pluripotent Stem Cells

Derived From Fetal Tissue.

 a. Unlike pluripotent stem cells derived from human embryos,

DHHS funds may be used to support research to derive pluripotent

stem cells from fetal tissue, as well as for research utilizing such

cells. Such research is governed by Federal statutory restrictions

regarding fetal tissue research at 42 U.S.C. 289g-2(a) and the Federal

regulations at 45 CFR 46.210. In addition, because cells derived from

fetal tissue at the early stages of investigation may, at a later date,

be used in human fetal tissue transplantation research, it is the policy

of NIH to require that all NIH-funded research involving the

derivation or utilization of pluripotent stem cells from human fetal

tissue also comply with the fetal tissue transplantation research

statute at 42 U.S.C. 289g-1.

 b. Informed Consent

 As a policy matter, NIH-funded research deriving or utilizing

human pluripotent stem cells from fetal tissue should comply with

the informed consent law applicable to fetal tissue transplantation

research (42 U.S.C. 289g-1) and the following conditions. The

informed consent process should have included discussion of the

following information with potential donors, pertinent to making

the decision whether to donate fetal tissue for research purposes.

 Informed consent should have included:

 (i) A statement that fetal tissue will be used to derive human

pluripotent stem cells for research that may include human
196 MONITORING STEM CELL RESEARCH

transplantation research;

 (ii) A statement that the donation is made without any restriction

or direction regarding the individual(s) who may be the recipient(s)

of transplantation of the cells derived from the fetal tissue;

 (iii) A statement as to whether or not information that could

identify the donors of the fetal tissue, directly or through identifiers

linked to the donors, will be removed prior to the derivation or the

use of human pluripotent stem cells;

 (iv) A statement that derived cells and/or cell lines may be kept

for many years;

 (v) Disclosure of the possibility that the results of research on the

human pluripotent stem cells may have commercial potential, and

a statement that the donor will not receive financial or any other

benefits from any such future commercial development; and

 (vi) A statement that the research is not intended to provide direct

medical benefit to the donor.

 c. Derivation protocols should have been approved by an IRB

established in accord with 45 CFR 46.107 and 46.108 or FDA

regulations at 21 CFR 56.107 and 56.108.

III. Areas of Research Involving Human Pluripotent Stem Cells That

Are Ineligible for NIH Funding

 Areas of research ineligible for NIH funding include:

 A. The derivation of pluripotent stem cells from human embryos;

 B. Research in which human pluripotent stem cells are utilized to

create or contribute to a human embryo;

 C. Research utilizing pluripotent stem cells that were derived from

human embryos created for research purposes, rather than for

fertility treatment;

 D. Research in which human pluripotent stem cells are derived

using somatic cell nuclear transfer, i.e., the transfer of a human

somatic cell nucleus into a human or animal egg;

 E. Research utilizing human pluripotent stem cells that were

derived using somatic cell nuclear transfer, i.e., the transfer of a

human somatic cell nucleus into a human or animal egg;

 F. Research in which human pluripotent stem cells are combined

with an animal embryo; and

 G. Research in which human pluripotent stem cells are used in

combination with somatic cell nuclear transfer for the purposes of

reproductive cloning of a human.
APPENDIX D. 197

IV. Oversight

 A. The NIH Human Pluripotent Stem Cell Review Group (HPSCRG)

will review documentation of compliance with the Guidelines for

funding requests that propose the use of human pluripotent stem

cells. This working group will hold public meetings when a funding

request proposes the use of a line of human pluripotent stem cells

that has not been previously reviewed and approved by the HPSCRG.

 B. In the case of new or competing continuation (renewal) or

competing supplement applications, all applications shall be

reviewed by HPSCRG and for scientific merit by a Scientific Review

Group. In the case of requests to use existing funds or applications

for an administrative supplement or in the case of intramural

proposals, Institute or Center staff should forward material to the

HPSCRG for review and determination of compliance with the

Guidelines prior to allowing the research to proceed.

 C. The NIH will compile a yearly report that will include the

number of applications and proposals reviewed and the titles of all

awarded applications, supplements or administrative approvals for

the use of existing funds, and intramural projects.

 D. Members of the HPSCRG will also serve as a resource for

recommendations to the NIH with regard to any revisions to the

NIH Guidelines for Research Using Human Pluripotent Stem Cells

and any need for human pluripotent stem cell policy conferences.

Dated: August 17, 2000.

Ruth L. Kirschstein,

Principal Deputy Director, NIH.

* * *
The following commissioned papers were prepared at the request

of the President’s Council on Bioethics; the Council has not itself

verified the accuracy of the information contained therein, nor does

it necessarily endorse any of the authors’ conclusions or opinions.

Appendix E - Appendix N

198

COMMISSIONED PAPERS
Appendix E.

Legislators as Lobbyists:

Proposed State Regulation of Embryonic Stem Cell

Research, Therapeutic Cloning and Reproductive

Cloning

LORI B. ANDREWS, J.D.

Distinguished Professor of Law, Chicago Kent College of

Law, Illinois Institute of Technology

The issue of research involving embryos and fetuses has

been a matter of serious social concern for the past three decades.

As each new scientific and medical development is introduced that

affects embryos and fetuses, new state laws are proposed to deal

with them. In the wake of the 1973 U.S. Supreme Court abortion

decision in Roe v. Wade,1 for example, 24 states passed laws

prohibiting research on human conceptuses.2 When in vitro

fertilization (IVF) was accomplished, at least one state passed a

law attempting to discourage the procedure.3 After Dolly the cloned

sheep was born, nine states adopted laws restricting human

reproductive cloning.4 When researchers began creating and using

human embryos for stem cell research, states began introducing

laws to deal with that technology as well. In 2002 and 2003, dozens

of bills were introduced in state legislatures on cloning and stem

cell research. In total, 38 states considered such bills;5 some states

1

 Roe v. Wade, 410 U.S. 113 (1973).

2

 See Lori B. Andrews, Medical Genetics: A Legal Frontier 70 (Chicago:

American Bar Foundation, 1987).

3

 See description of the Illinois law in Lori B. Andrews, The Clone Age:

Adventures in the New World of Reproductive Technology 24-28 (New York:

Henry Holt, 1999).

4

 See Section IA, infra.

5

 Alabama, Arizona, Arkansas, California, Colorado, Connecticut, Delaware,

Florida, Georgia, Illinois, Indiana, Iowa, Kansas, Kentucky, Louisiana,

Maryland, Massachusetts, Michigan, Mississippi, Missouri, Nebraska, New

199
200 MONITORING STEM CELL RESEARCH

considered multiple and conflicting bills. In all, 133 separate bills

were introduced on these subjects. (See overview in Chart II and

specific provisions in Chart III).

Although state legislatures have addressed regulation of

research on the unborn for 30 years, the more recent technologies

are being debated within a new context. Pro-life and pro-choice

advocates are aligning in unusual ways. Unlike the clear line of

demarcation between the groups on the issue of abortion, some

pro-choice advocates oppose all forms of human cloning6

 and some

pro-life advocates support so-called “therapeutic” cloning.7 It would

seem that these unique alliances might allow more fruitful policy

discussions. However, despite agreement across traditional

boundaries on the substance of particular bills, some of the new

alliances break down because the sponsors of the bills include

preambles that evince either pro-life or pro-biotechnology

sentiments that go far beyond the substance of the bills themselves.

This is a new development in the policy considerations related to

human conceptuses. The legislators themselves are acting like

lobbyists by including within their bills language espousing a

particular position that goes far beyond the bills’ actual legal

substance.8

 Some bills seem to be driven more by political posturing

than by an attempt to actually get legislation enacted to deal with

the issue at hand.

This paper surveys the state law landscape by analyzing 1)

existing laws with implications for reproductive cloning, therapeutic

cloning and embryo stem cell research, 2) proposed bills addressing

those technologies, and 3) potential constitutional challenges to the

laws.

I. The Existing Legal Framework

A. Existing Bans on Cloning

1. The Restrictions

Hampshire, New Jersey, New York, North Dakota, Ohio, Oklahoma, Oregon,

Pennsylvania, Rhode Island, South Carolina, Tennessee, Texas, Vermont,

Virginia, Washington, West Virginia, Wisconsin.

6

 See, for example, Nigel Cameron and Lori Andrews, “Cloning and the

Debate on Abortion,” Chicago Tribune, August 8, 2001, at 17.

7

 Orin Hatch, for example.

8

 See Section II, infra.
APPENDIX E. 201

There are eight states that have enacted explicit laws to

ban reproductive cloning,9

 and one state that prohibits only the use

of state funds for reproductive cloning.10 (See Chart I). Of these

eight states, four also ban “therapeutic” cloning.11 Some states also

ban shipping, transferring, or receiving for any purpose an embryo

produced by human cloning.12

Under the existing reproductive cloning bans, some states

ban the purchase or sale of an ovum, zygote, embryo, or fetus for

the purpose of cloning a human being.13 Other states, however,

have bans on payment for eggs, embryos, or human tissue that could

also cover some of the transactions involved in reproductive or

“therapeutic” cloning.14

California’s initial anti-cloning bill, adopted in 1998, created

an advisory committee that met frequently over a five year period,

heard extensive testimony from scientists and members of the public,

and issued a report.15

9

 Ark. Code § 20-16-1001 et seq. (2003) (formerly AR SB 185); Cal. Bus. &

Prof. §§16004, 16105 (2003) and Calif. Health & Safety §§ 24185-24187 (2003)

(formerly CA SB 1230); Iowa Code § 707B.1-.4 (2003) (formerly IA SB 2046,

became IA SB 2118); La. Rev. Stat. Ann. § 1299.36.1-.6 (2003); Mich. Comp.

Laws §§ 333.26401-06, 333.16274, 16275, 20197, 750.430a (2003); ND Cent.

16.4-1 to .4-4 (2003); Va. Code Ann. § 32.1-162.21 to .22 (2003). Two of

these state laws could be evaded if a non-human egg was used instead of

a human egg as the incubation for the somatic cell DNA. Ark. Code § 20-

16-1001(5) (2003); La. Rev. Stat. § 1299.36.1 (2003).

10 Mo. Rev. Stat. § 1.217 (2003).

11 Arkansas, Iowa, Michigan, and North Dakota.

12 Ark. Code § 20-16-1002 (A)(3); Iowa Code § 707B.4(1)(c); ND Cent. Code

§§ 12.1-39-01 to 02; Va. Code Ann. § 32.1-162.22(A) (for purposes of

implantation).

13 See, for example, Cal. Health & Safety Code §§ 24185(b); La. Rev. Stat. §

1299.36.2(B).

14 Lori B. Andrews, “State Regulation of Embryo Stem Cell Research,” in

National Bioethics Advisory Commission, Appendix II at pages A1 – A13.

15 “Cloning Californians?” Report of the California Advisory Committee on

Human Cloning, January 11, 2002, Sacramento, California, available at http:/

/www.sfgate.com/chronicle/cloningreport/.
202 MONITORING STEM CELL RESEARCH

2. Exceptions for Certain Research

In some states, the ban on reproductive cloning is

accompanied by language supporting other types of research or

medical practices. In Louisiana and Michigan, for example, the

cloning bans say that they do not prohibit scientific research on a

cell-based therapy.16 Similarly, some states allow the use of nuclear

transfer or other cloning techniques to produce molecules, DNA,

cells other than human embryos, tissues, organs, plants, or animals

other than humans.17 The Arkansas and Rhode Island laws also

specifically allow in vitro fertilization and fertility enhancing drugs,

so long as they are not used in the context of human cloning.18 The

Iowa law more broadly allows in vitro fertilization and the use of

fertility drugs.19

3. Penalties

The penalties for violation of existing cloning bans range

widely. In Louisiana and Michigan, for example, penalties can be

up to 10 years imprisonment and fines of $10 million for an entity

such as a clinic or corporation and $5 million for an individual.20 In

16 La. Rev. Stat. Ann. § 1299.36.2(c); Mich. Comp. Laws § 750.430a(2).

17 See, for example, Ark. Code § 20-16-1003(A); Iowa 707B.4(2); R.I. Gen

Laws § 23-16.4-1 (specifically allows animal cloning in another section,

R.I. Gen Laws. § 23-16.4-2(c)(1)(iii)). Virginia allows gene therapy, cloning

of non-human animals, and cloning molecules, DNA, cells or tissue. Va.

Code Ann. § 32.1-162.22(B).

18 Ark. Code § 20-16.1003(B); R.I. § 23-16.4-2(c)(2)(i). Rhode Island’s bill

goes overboard in its hype about scientific progress. Rhode Island’s

preamble to its reproductive cloning ban states, “recent medical and

technological advances have had tremendous benefit to patients, and

society as a whole, and biomedical research for the purpose of scientific

investigation of disease or cure of a disease or illness should be preserved

and protected and not be impeded by regulations involving the cloning of

an entire human being; and . . . molecular biology, involving human cells,

genes, tissues, and organs, has been used to meet medical needs globally

for twenty (20) years, and has proved a powerful tool in the search for

cures, leading to effective medicines to treat cystic fibrosis, diabetes, heart

attack, stroke, hemophilia, and HIV/AIDS.” R.I. Gen. Laws. § 23-16.4-1.

19 Iowa Code § 707B.4(2).

20 La. Rev. Stat. § 1299.36.3; Mich. Comp. Laws § 750.430(a)(3) (in Michigan

even individuals too, can be fined up to $10 million.) Rhode Island has a

penalty of $250,000 for individuals and $1 million for entities. R.I. Gen.

Laws § 23-16.4-3.
APPENDIX E. 203

contrast, in Arkansas the penalty is a mere $250,000, but there are

also felony criminal penalties.21 Moreover, in some states, cloning

can result in the permanent revocation of a doctor’s license22 and

the denial of any other type of license or permit from the state

regarding any trade, occupation or profession.23

B. Existing Bans on Embryo Research

Twelve states’ laws apply to in vitro embryos. In New

Hampshire, the regulation of research on embryos prior to

implantation is minimal.24 The research must take place before day

14 post-conception,25 and the subject embryo must not be implanted

in a woman.26 These stipulations could readily be met by researchers

wanting to use IVF embryos as a source of stem cells.

Nine states ban research on in vitro embryos altogether,27

and two states ban destructive embryo research.28 In Louisiana, an

in vitro fertilized ovum may not be farmed or cultured for research

purposes;29 the sole purpose for which an IVF embryo may be used

is for human in utero implantations.30 In the other states, embryo

research is banned as part of the broader ban on all research

involving live conceptuses. These laws ban embryo stem cell

21 Ark. Code § 20-16-1002 (B ) to (D).

22 See, for example, La. Rev. Stat. § 1299.36.4; See also, Iowa § 707B.4(5).

23 La. Rev. Stat. § 1299.36.4; Iowa § 707B.4(6).

24 N.H. Rev. Stat. Ann. § 168-B:15.

25 N.H. Rev. Stat. Ann. § 168-B:15 at I.

26 N.H. Rev. Stat. Ann. § 168-B:15 at II.

27 Fla. Stat. Ann. § 390.0111(6); La. Rev. Stat. Ann. § 9:121 et. seq.; Me.

Rev. Stat. Tit. 22 § 1593; Mass. Ann. Laws. Ch. 112 § 12J; Mich. Comp.

Laws. §§ 333.2685 to 2692; Minn. Stat. Ann. § 145.421 (applies only until

265 days after fertilization); N.D. Cent. Code §§ 14-02.2-01 to –02; 18 Pa.

Cons. Stat. Ann. § 3216; R.I. Gen. Laws. § 11-54-1.

28 S.D. Codified Laws § 34-14-16; Iowa Code § 707B.1-4.

29 La. Rev. Stat. Ann. § 9:122.

30 La. Rev. Stat. Ann. § 9:122.
204 MONITORING STEM CELL RESEARCH

research. The penalties are high — in some states, the punishment

includes imprisonment.31

In some instances, the woman or couple who donate an

embryo or fetus for research purposes also face liability. Laws in

Maine, Michigan, North Dakota, and Rhode Island prohibit the

transfer, distribution, or giving away of any live embryo for research

purposes.32 In Maine, a person who does so is subject to a fine of up

to $5,000 and up to five years’ imprisonment.33

C. Existing Laws Specifically Addressing Embryo

Stem Cell Research

A California law declares that research shall be permitted

on human embryonic or adult stem cells from any source, including

somatic cell nuclear transfer.34 Such research must be reviewed by

an approved institutional review board and such research may not

be undertaken without written informed consent of the embryo

donor. Interestingly, the law does not say consent of the “donors,”

plural. So it would appear that the female patient of infertility

services (and not the woman and her husband) is the sole source of

consent. California also enacted a law urging Congress to ban

reproductive cloning, while permitting therapeutic cloning and

embryo stem cell research.

35

31 The Maine law, which applies both to research on embryos and research

on fetuses, carries a maximum five year prison term. Me. Rev. Stat. tit. 22

§ 1593. The Massachusetts and Michigan laws also carry with them a

potential prison sentence of up to five years. Mass Ann. Laws 112 §

12J(a)(V); Mich. Comp. Laws § 333.2691.

32 Me. Rev. Stat. Ann. tit. 22 § 1593; Mich. Comp. Laws Ann. § 333.2690;

N.D. Cent. Code § 14-02.2-2(4); R.I. Gen. Laws § 11-54-1(f). In addition,

S.D. Codified Law § 34-14-7 bans the sale or transfer of an embryo for

nontherapeutic purposes.

33 Me. Rev. Stat. Ann. tit. 22 § 1593.

34 Cal. Health & Safety Code § 125115.

35 California Senate Joint Resolution 38.
APPENDIX E. 205

II. Proposed Bills36

A. Preambles to the Bills – Hype versus Doom

Legislators are currently considering bills that govern the

technologies of cloning or embryo stem cell research. But even when

the provisions of those bills are narrowly tailored to deal with those

specific technologies, lawmakers include extensive preambles that

include sweeping pro-life or pro-biotechnology language. None of

the bills include a pro-choice preamble.

A Kentucky bill attempts to fuel a biotechnology sector in

the state. Its preamble mentions the “great potential” of nuclear

transplantation to treat “diseases and disorders, including but not

limited to Lou Gehrig’s disease, Parkinson’s disease, Alzheimer’s

disease, spinal-cord injury, cancer cardiovascular diseases, diabetes,

rheumatoid arthritis, and many others.”37 Its provisions would

require anyone engaging in therapeutic cloning to register with the

health department and pay a $50 fee. The bill also implores public

colleges in Kentucky to protect “the interest of the Commonwealth

or the institution relating to all intellectual property and other rights

to any research, experiments, or other activity related to human

nuclear transplantation. . . .”38

On the other hand, some bills’ preambles are designed to

cast aspersions on the technologies. The preamble of a Virginia

Senate resolution contains a hodge-podge of historical facts (from

slavery in Virginia 1619 to 1865 to Advanced Cell Technologies

cloning of an embryo), Presidential statements (from President

Reagan through President George W. Bush), and concerns about

36 This research was undertaken in Lexis current session bill text and bill

text archive for 2002 and 2003, using the words “human cloning,” “stem

cell research,” “embryo,” “cloning,” “twinning,” “oocyte,” and “somatic

cell nuclear transfer” in order to locate bills.

37 KY HB 265.

38 KY HB 265.

39 “WHEREAS, although the “commodification” of human of human beings

existed in this Commonwealth and the United States from 1619 to 1865,

the concept of human beings as property has been rejected by Americans

in their constitution and in their deeply held belief in the value of human

life; and
206 MONITORING STEM CELL RESEARCH

WHEREAS, Ronald Reagan, the 40th President of the United States, stated

this country understood that the personhood of every American should be

protected “from the moment of conception until natural death”; and

WHEREAS, the United States Patent and Trade Office rejected human

commercialization in a ruling on April 7, 1987, which stated “A claim

directed to or including within its scope a human being will not be

considered to be patentable subject matter” under the federal patent law;

and

WHEREAS, on August 25, 2000, the National Institutes of Health published

guidelines relating to stem cell research and the funding thereof that called

for the denial of funding for research involving stem cells derived from

embryonic human beings created for research purposes and noted that

President Clinton, many members of Congress and the NIH Human Embryo

Research Panel and the National Bioethics Advisory Committee had all

endorsed the “distinction between embryos created for research purposes

and those created for reproductive purposes”; and

WHEREAS, the NIH guidelines also called for assurances that “there can

be no incentives for donation” of human embryos and “any inducement for

the donation of human embryos for research purposes” would be prohibited;

and

WHEREAS, George W. Bush, the 43rd President of the United States noted

on August 9, 2001, that he is “deeply troubled” by the creation of “human

embryos in test tubes solely to experiment on them,” and described this

act as a “warning sign” to “all of us” as Americans; and

WHEREAS, Senator William Frist, distinguished physician representing the

State of Tennessee in the United States Senate, has proposed as a first

principle of ethical research that “the creation of human embryos solely for

research should be strictly prohibited”; and

WHEREAS, recently a Massachusetts research company claimed that it

had cloned the first human embryo, that “ [T]his work sets the stage for

human therapeutic cloning as a potentially limitless source of immunecompatible cells,” and that this work provides “hope for people with spinal

injuries, heart disease and other ailments”; and

WHEREAS, the Jones Institute of Norfolk recently published research

involving stem cell research conducted through the creation of

approximately 110 embryos developed with the purchased sperm and eggs

from men and women; and

WHEREAS, this conduct established a trade in new human life that treats

such lives as merchandise for manipulation and destruction; and

WHEREAS, reportedly, the Jones Institute screened and evaluated the

fitness of new human life according to the absence of “cosmetic handicaps,

and other eugenic formulations”; and

WHEREAS, the General Assembly of Virginia has, by way of HJR 607 of

2001, condemned past practices within the Commonwealth involving

institutional involvement in eugenics and eugenic ideology; now, therefore,

be it

RESOLVED by the House of Delegates, the Senate concurring, That a joint

subcommittee be established to study the medical, ethical, and scientific

issues relating to stem cell research conducted within the Commonwealth.”

VA HJR 148. See also VA HJR 573.
APPENDIX E. 207

eugenics and commodification of humans.39 The resolution would

establish a legislative task force, but its agenda is directed in a onesided way. It is supposed to “examine the medical, ethical and

scientific policy implications of prohibiting the creation of embryos

in vitro for any purpose other than bringing them to birth, and the

criminalizing of the transfer of compensation, in cash or in-kind, to

induce any person to donate sperm or eggs for any purpose other

than procreation. The joint subcommittee shall also examine the

efficacy of research using adult stem cells rather than embryonic

stem cells.”40

Some bills assert scientific or medical facts that are not

necessarily accurate. For example, Louisiana’s resolution says,

“stem cells derived from adult or placental tissues may hold greater

promise than embryonic stem cells for human medicine, due to a

lesser danger of immune reactivity against a recipient.”41

B. Proposed Bills on Reproductive Cloning

1. The Restrictions

In 2002 and 2003, 27 states introduced bills that would ban

reproductive cloning.42 One bill details a specific enforcement

mechanism for the human cloning ban. A New Jersey bill provides

that the Commissioner of Health and Senior Services may issue a

cease and desist order, with a subsequent administrative hearing

within 20 days, with judicial review available.43

40 VA HJR 148. See also VA HJR 573.

41 LA House Current Resolution (HCR) 29.

42 AL HB 9; AL SB 314; AZ HB 2108; CA SB 133; CA AB 267; CA SB 1557;

CO HB 1073; CT SB 407; CT HB 5639; CT SB 410; DE SB 55; DE SB 329; DE

SB 344; FL HB 285; FL SB 1726; FL HB 805; FL SB 1164; IL HB 253; IN HB

1538; IN HB 1984; IN SB 151; IN SB 138; KS HB 2736; KY HB 138; KY HB 153;

KY HB 265; LA HB 472; LA HB 1810; MA HB 1280; MA HB 3125; MA HB

2048; MA HB 2052; MA SB 1917; MO HB 163; MO HB 209; MO HB 1449; MO

SB 191; NE LB 602; NE LB 1067; NH HB 1464; NJ AB 2040; NJ AB 2840; NJ

AB 1379; NJ SB 542; NY AB 1819; NY AB 3295; NY AB 4533; NY AB 6249;

NY SB 206; NY SB 7638; NY SB 612; NY SB 3013; OK HB 1130; OK HB 2011;

OK HB 2036; OK HB 2142; OK SB 1552; OR HB 2538; OR HB 2504; SC HB

3819; SC HB 4408; SC SB 820; TN HB 1075; TN SB 1515; TN HB 2675; TN SB

2295; TX HB 1175; TX SB 1034; TX SB 156; VT HB 326; WA HB 1461; WA SB

5466; WA HB 2173; WA SB 5571; WV HB 2832; WV SB 402; WV SB 514; WI

AB 104; WI AB 246; WI SB 45; WI SB 699; WI AB 736; WI SB 404.

43 NJ AB 2040.
208 MONITORING STEM CELL RESEARCH

Many of these proposed laws suffer from drafting infirmities.

For example, some of the states’ proposals create a loophole by only

prohibiting the reproduction of a “genetically identical” individual

through cloning.44 Since the current cloning technique uses a

donated egg to create a clone, the resulting individual will have

some additional mitochondrial DNA from the enucleated egg, so he

or she will not be genetically identical to the original individual.45

In contrast, other states’ bills prohibit the reproduction of a “virtually

genetically identical” individual through human cloning using a

human or non-human egg,46 which would apply even if a donated

egg, containing additional mitochondria were used.

Certain states’ bills would prohibit the transferring of a

human cell into a human egg cell.47 But, in January 1998, scientists

at the University of Wisconsin revealed that cow eggs could serve

as incubators for nucleic DNA of other mammalian species.48 Thus,

the proposed laws could be evaded by making a human clone via

somatic cell DNA transfer into an enucleated cow egg. Other states’

bills prohibit human reproductive cloning using a human or nonhuman egg.49

A New Jersey bill has the opposite problem. Rather than

being too narrow, it is too broad, using vague language in places

and even potentially banning the creation of children through sexual

intercourse.50 The focus of the bill is to ban human cloning and

germline genetic engineering. But it defines human cloning as the

replication of one or more human beings, through “sexual or asexual

reproduction.” When two people create a child sexually are they

replicating themselves? The key word, “replication,” is not defined.

44 See, for example, IN HB 1538 § 1; IN SB 151 § 1.a; MO HB 163 § 565.305;

TN HB 1075 § 1; TN SB 1515 § 1.

45 Unless, of course, one used an egg from the to-be-cloned person’s own

mother.

46 See, for example, CA SB 133 § 1; FL SB 1726 § 2.A; KY HB 153 § 1.A; NE

LB 602 § 2.2; OK HB 1130 § C.1; SC SB 820 § 2.1; SC HB 3819 §2.1; WI SB 45

§ 1.c; WI AB 104 § 1.c.

47 See, for example, DE SB 55 § 3002; IL HB 253 § 10.c; NH HB 1464 § 141-

J:2; NY SB 206 § 3230; NY SB 7638 § 3230; OR HB 2538 § 1.

48 See Robert Lee Hotz, “Cow Eggs Used as Incubator in Cloning Boon,”

L.A. Times, Jan. 19, 1998, at A1.

49 See, for example, CA SB 133 § 1.

50 NJ AB 2040 (1). See also MA HB 3125 § 2A.
APPENDIX E. 209

The bill prohibits the destruction of human or non-human

embryos “for the purpose of human cloning.”51 There is a certain

vagueness to that provision. If a researcher working on primate

cloning generates data that might be useful to cloning human beings

could he or she be prosecuted?

2. Public funds

Six states’ bills would prohibit the use of public funds and

facilities to participate in cloning or attempted cloning.52

3. Other Means to Discourage Cloning

Some states’ bills attempt to discourage reproductive

cloning by prohibiting either the sale or purchase53 or the shipment

or receival of an ovum, zygote, embryo, or fetus to clone a human.54

Some states’ laws attempt to discourage cloning through liability

provisions. A Florida bill would create vast liabilities as another

means to discourage cloning.55 Participants in human reproductive

cloning would be liable to “the individual, the individual’s spouse,

dependents, and blood relatives, and to any woman impregnated

with the individual, her spouse, and dependents, for damages for

all physical, emotional, economic, or other injuries suffered by such

persons at any time as a result of the use of human cloning to produce

the individual.” Even more dramatically, participants in human

reproductive cloning would be liable to the cloned individual and

his or her legal guardian for support until the age of majority (or

longer, if the clone has “any congenital defects or other disability”

related to the cloning).

51 NJ AB 2040 (2)(c).

52 IN HB 1538; IN HB 1984; IN SB 138; IN SB 151; LA HB 472; MS SB 2747;

MO HB 481; MO SB 191; MO HB 163; NY AB 4533; OK HB 1130.

53 See, for example, CA SB 133.

54 See, for example, CO HB 1073; FL SB 1726; KY HB 153; OK HB 1130; SC

HB 820; SC HB 3819; WI SB 45; WI AB 104.

55 FL SB 1726.
210 MONITORING STEM CELL RESEARCH

4. Exceptions

Eighteen states’ bills have an exception allowing cloning to

create DNA, tissues, and organs.56 Eleven states’ bills would not

restrict the use of in vitro fertilization or fertility enhancing drugs.57

C. Proposed Bills on Therapeutic Cloning

There are 26 states that have bills about therapeutic

cloning.58 Two states have at least one bill that would ban

therapeutic cloning and at least one that would allow therapeutic

cloning.59

1. Ban Therapeutic Cloning

Twenty-six states have bills that would prohibit all human

cloning including therapeutic cloning.60 Some states prohibit

therapeutic cloning through a general cloning ban that defines

56 AL HB 9; AZ HB 2108; CA SB 133; CA SB 1557; DE SB 55 § 3003; DE SB

329; DE SB 344; FL HB 285; FL HB 805; FL SB 1164; FL SB 1726 § 5; IL HB

253; KS HB 2736; KY HB 138; KY HB 153 § 3; LA HB 1810; MA HB 2048; MA

HB 3125; NE LB 1067; NH HB 1464 § 1441-J:4; NY AB 3295; NY AB 6249 § 2;

NY SB 206 § 3232; NY SB 7638 § 3232; NY SB 3013; OK HB 2142; SC HB 3819

§ E; SC SB 820; TN HB 1075 § e; TN SB 1515 § e; TX HB 1175; WA HB 1461;

WA HB 2173; WA SB 5466.

57 AZ HB 2108; IN HB 1538; IN HB 1984; IN SB 138; IN SB 151; KS HB 2737;

LA HB 1810; NH HB 1464 § 1441-J:4; NY AB 6249 § 2; NY SB 206 § 3232; NY

SB 7638 § 3232; NY SB 612; NY SB 3013; OK HB 2142; OR HB 2538 § 2; VA

HB 2366; WA HB 2173; WI AB 736; WI SB 404.

58 AL HB 9; AZ HB 2108; CA SB 133; CA SB 1557; CO HB 1073; CT SB 407;

CT HB 5639; DE SB 55; FL HB 805; FL SB 1164; FL SB 1726; IL HB 253; IN HB

1538; IN HB 1984; IN SB 151; IN SB 138; KS HB 2736; KY HB 138; KY HB 153;

LA HB 1810; MA HB 3125; MO HB 1449; NE LB 602; NH HB 1464; NJ AB

2040; NY AB 4533; NY AB 6249; NY SB 206; NY SB 3013; NY SB 7638; OK HB

1130; OK HB 2011; OK HB 2142; OK SB 1552; OR HB 2538; SC HB 3819; SC

SB 820; SC HB 4408; TN HB 1075; TN SB 1515; TX HB 1175; TX SB 156; WA

HB 2173; WA SB 5571; WV HB 2832; WV SB 402; WV SB 514.; WI SB 699; WI

AB 104; WI AB 736; WI SB 404; WI SB 45. (Therapeutic cloning, as currently

undertaken, would also fall within the ban on destructive embryo research

discussed infra.)

59 See, for example, Indiana and New York.

60 AL HB 9; AZ HB 2108; CA SB 133; CA SB 1557; CO HB 1073; CT SB 407;

CT HB 5639; DE SB 55; FL HB 805; FL SB 1164; FL SB 1726; IL HB 253; IN HB
APPENDIX E. 211

human cloning as the creation of any human organism through

somatic cell nuclear transfer.61 Others prohibit therapeutic cloning

by banning not only the creation of the cloned embryo, but also the

“derivation of any product from human cloning.”62

2. Allow Therapeutic Cloning

Two states have bills that specifically allow therapeutic

cloning.63 In addition, twelve other states allow for the use of stem

cells from any source, including those derived from somatic cell

nuclear transplantation.64

One state bill would encourage researchers to do stem cell

research using only human adult or placental tissues obtained with

informed consent, and would discourage research involving human

embryonic or fetal tissue.65 One state’s bill urges state universities

to refrain from embryonic stem cell research.66

D. Proposed Bans on Embryo Stem Cell Research

1. Ban Embryo Stem Cell Research

Five states have bills that would prohibit acts where a human

fetus or embryo is destroyed or subject to injury.67 New Jersey has

1538; IN SB 151; IN SB 138; KS HB 2736; KY HB 138; KY HB 153; LA HB 1810;

MA HB 3125; MO HB 1449; NE LB 602; NH HB 1464; NJ AB 2040; NY AB

4533; NY SB 206; NY SB 7638; OK HB 1130; OK HB 2011; OK HB 2142; OK SB

1552; OR HB 2538; SC HB 3819; SC SB 820; SC HB 4408; TN HB 1075; TN SB

1515; TX HB 1175; TX SB 156; WA HB 2173; WA SB 5571; WV HB 2832; WV

SB 402; WV SB 514; WI AB 104; WI SB 699; WI AB 736; WI SB 404; WI SB 45.

61 See, for example, FL SB 1726.

62 See, for example, NE LB 602.

63 IN HB 1984; NY AB 6249; NY SB 3013.

64 MA HB 1280; MA HB 2052; NJ AB 2840; NJ SB 1909; NY AB 1819; NY SB

612; TX SB 1034; VT HB 326; WA HB 1461; WA SB 5466.

65 LA HCR 29A.

66 MI HR 189.

67 KS HB 2737; MI HB 4507; NJ AB 2040; VA HB 2366; WI AB 736; WI SB

404.
212 MONITORING STEM CELL RESEARCH

these provisions in a cloning bill, while Wisconsin has them in an

artificial insemination/in vitro fertilization bill.68 The others (Kansas,

Michigan, and Virginia) have these provisions in stem cell research

bills.69 One state’s bill would make all contracts for payment for the

extraction or use of embryonic stem cells against public policy, void,

and unenforceable.70 In addition, a Michigan bill, in its preamble,

opposes the White House decision to allow certain embryonic stem

cell research.71 The substance of the bill is a provision urging the

state’s public universities to refrain from embryonic stem cell

research.72

2. Allow Embryo Stem Cell Research,

Including Therapeutic Cloning

Twelve states have bills that would allow embryo stem cell

research from any source.73 While the bills literally say “from any

source,” some also have a list saying this includes human embryonic

stem cells, human embryonic germ cells, and human adult stem

cells, from any source, including somatic cell nuclear

transplantation.74 In the states where proposed bills would permit

embryonic stem cell research, there are often provisions regarding

how the research should be conducted. In eleven states, for example,

physicians undertaking in vitro fertilization on patients must discuss

options for the disposition of frozen embryos, including donation to

68 NJ AB 2040; WI AB 736; WI SB 404.

69 KS HB 2737; MI HB 4507; VA HB 2366.

70 VA HB 1361.

71 MI HR 189.

72 MI HR 189.

73 CA SB 771; CA SB 1272; IL HB 3589; MD HB 482; MA HB 2052; MA HB

1280; NJ AB 2840; NJ SB 1909; NY AB 1819; NY SB 612; PA HB 2984; PA HB

422; PA HB 945 (Limited to those from fertility clinics); RI SB 266; TN HB

945; TN SB 1654; TX SB 1034; VT HB 326; WA HB 1461; WA SB 5466.

74 See, for example, IL HB 3589 § 10.2.

75 CA SB 771; CA SB 1272; IL HB 3589 (does not require Dr. to inform patient

of embryo disposition options, but requires written informed consent); MA

HB 2052; MA HB 1280; NJ AB 2840; NJ SB 1909; NY AB 1819; PA HB 2984;

PA HB 422; PA HB 945 (Does not require Dr. to inform patient of embryo

disposition options, but requires written informed consent); RI SB 266; TN

HB 945; TN SB 1654; TX SB 1034; VT HB 326; WA HB 1461; WA SB 5466.
APPENDIX E. 213

research, and written informed consent for such donation would

have to be obtained.75 In at least one state, the proposed research

must be assessed by a state department,76 and in eleven states the

research must be assessed by an institutional review board.77 A

Kentucky bill requires that individuals engaged in therapeutic

cloning must register their name, address, and phone number, which

information shall be public record.78 Under one proposed California

bill, the State Department of Health Services would have the

responsibility to develop guidelines regarding the derivation or use

of human embryonic stem cells.79

In Illinois, care was taken in the proposed bill favoring

embryonic stem cell research not to create barriers for the research

due to contract or tort liability. Consequently, the proposed Illinois

bill states that “procuring, furnishing, donating, processing,

distributing, or using embryonic or cadaveric fetal tissue for research

purposes pursuant to this Act is declared for the purposes of liability

in tort or contract to be the rendition of a service by every person,

firm, or corporation participating therein, whether or not

remuneration is paid, and is declared not to be a sale of any such

items and no warranties of any kind or description nor strict tort

liability shall be applicable thereto.”80

3. Address Embryo Donation

Twelve states’ bills would expressly allow a person to donate

human embryos for research.81 Twelve states’ bills would also

prohibit the sale of embryonic or cadaveric fetal tissue for research

purposes.82

76 PA HB 2984; PA HB 422.

77 CA SB 322; CA SB 771; CA SB 1272; IL HB 3589; MD HB 482; MA HB

1280; MA HB 2052; NJ AB 2840; NJ SB 1909; NY AB 1819; RI SB 266; TN HB

945; TN SB 1654; TX SB 1034; VT HB 326; VA HB 639.

78 KY HB 265.

79 CA SB 771.

80 IL HB 3589.

81 CA SB 771; CA SB 1272; IL HB 3589 § 15; MD HB 482 § 20-901.C; MA HB

1280 § 3; MA HB 2052; NJ AB 2840 § 2.b.3; NJ SB 1909 § 2.b.2; NY AB 1819

§ 2452.2; PA HB 2984 § 5.b; PA HB 422 § 5.b; PA HB 945; RI SB 266 § 23-77-

2; TN HB 945; TN SB 1654; TX SB 1034 § 168.003; VT HB 326 § 9341.B; WA

HB 1461§ 4; WA SB 5466 § 4.1.
214 MONITORING STEM CELL RESEARCH

4. Review Committees

Six states’ bills would establish a subcommittee or task

force to review and/or study issues related to stem cell research.83

Some of these states have a bill that only establishes a task force.84

Some states establish this task force, subcommittee, or review board

within a bill relating to stem cell research.85 Maryland has one of

each.86

It has often been true in the bioethical area that when

controversial new technologies are introduced, states propose

commissions to address the issue.87 Often, the creation of the

commission is a stalling tactic to avoid having to confront the issue

head on or to postpone having to limit a technology, or to facilitate a

moratorium on the technology (while the Commission evaluates the

technology). Sometimes, though, state commissions have admirably

overseen a technology’s development, application and evaluation.

Such was the case in the 1970’s and 1980’s with Maryland’s newborn

screening commission. So it is no surprise that of the bills proposing

a state task force or commission, the Maryland bill provides the best

guidance about what the commission would do. The Maryland task

force would:

82 CA SB 771; CA SB 1272; IL HB 3589 § 15a; IN HB 1538; IN SB 138; IN SB

151; MD HB 482 § 20-901; MA HB 1280; MA HB 2052; NJ AB 2840 § c.1; NJ

SB 1909 § c.1; NY AB 1819 § 2453; PA HB 2984 § 6; PA HB 422 § 6; PA HB

945; TN HB 945; TN SB 1654; VT HB 326 § 9342; VA HB 2366 (If used in

destructive research); WA HB 1461 § 5.2; WA SB 5466 § 5.2.

83 CA SB 322; CA SB 771 (State department to oversee ESC research); MD

HB 72 (Task force would cover bioscience in general, including ESC

research); MD HB 1171; MA HB 1280 § 220; MA HB 2052; NY AB 6249 §

4372; NY SB 3013; PA HB 422 § 4; PA HB 2984 § 4; VA HJR 148; VA HJR 573;

VA HB 639 (Task force would cover all human research in general, including

ESC).

84 See, for example, California, Maryland, and Virginia.

85 See, for example, Illinois, Maryland, Massachusetts, New Jersey, New

York, Pennsylvania, Rhode Island, Texas, and Vermont.

86 MD HB 72; MD HB 1171.

87 This was the case, for example, with newborn screening and surrogate

motherhood.
APPENDIX E. 215

“(1) Make recommendations for state policy

regarding stem cells derived from embryos created

solely for research purposes;

(2) evaluate whether there should be

written informed consent requirements for

prospective donors of embryonic stem cells derived

from embryos created during infertility treatment;

(3) study constitutional, ethical, and policy

issues with respect to whether the state should

regulate or prohibit commerce in embryos or stem

cells;

(4) (i) determine what stem cell research

is already being done in the state by private

companies and academic institutions; and

 (ii) evaluate how these companies and

institutions are regulating themselves when doing

stem cell research;

(5) determine the effect on Maryland

businesses and institutions of Federal restrictions

that limit federally funded research and development

to existing cell lines on stem cell research;

(6) analyze the roles of, and interrelationship

between, federal and state oversight of stem cell

research; and

(7) review any other matter relating to

stem cell research that the task force considers

necessary and proper.”88

5. Public Funding or Tax Credits for

Stem Cell Research

States have also proposed bills allowing for or prohibiting

the granting of state funds for stem cell research. California’s

proposed “Biomedical Research and Development Act of 2004” would

award grants or loans to public or private institutions conducting

research in stem cell biology. 89 To be eligible for funding, all research

projects must be reviewed and approved by an institutional review

board.

The states of Indiana, Michigan, Mississippi, and Nebraska

have gone the opposite direction and introduced bills specifically

88 MD HB 1171.

89 CA SB 778.
216 MONITORING STEM CELL RESEARCH

prohibiting the use of state money or tax credits for stem cell

research.90 The Indiana bill proposes an amendment to the state tax

code denying tax exemptions and credits for research involving

human cloning or stem cell research with embryonic tissue.

A Mississippi House Bill and a Mississippi Senate Bill

provide that no public funds shall be used to assist in or to provide

facilities for stem cell research that uses cells from human embryos

and human cloning.

Finally, although not establishing public funds for

embryonic stem cell research, six Florida bills seek to establish a

center for “Universal Research to Eradicate Disease” which will

facilitate funding opportunities for stem cell research.91

E. Proposed Bills on Health Care Providers’ Right of

Conscience

In addition to proposing bills on the prohibition of or

allowance of embryonic stem cell research and cloning, a number

of states have proposed bills protecting a health care provider, payer,

or institution’s right to refuse certain services based on moral or

ethical convictions. Kansas, Louisiana, and Rhode Island have all

introduced bills entitled “Health Care Providers Rights of

Conscience.” 92

The Kansas bill introduced on January 28, 2002, specifically

allows all persons to refuse to participate in the provision of, or pay

for, a health care service related to human cloning and embryonic

stem cell and fetal experimentation. The Louisiana bill and the

identical Rhode Island House and Senate bills allow all health care

providers, institutions and payers to decline to counsel, advise, pay

for, provide, perform, assist, or participate in providing or performing

health care services that violate their consciences, including, human

cloning and embryonic stem cell research.

F. Constitutionality of State Law

Not all regulations affecting research are constitutional.

Laws restricting research on conceptuses may be struck down as

90 IN HB 1001; MI SB 1485; MI HB 4454; MS HB 361; MS SB 2747.

91 FL SB 2212; FL SB 572; FL SB 2390; FL HB 107A; FL HB 845; FL SB 2142.

92 KS HB 2711; LA SB 850; RI SB 906; RI HB 5846.
APPENDIX E. 217

too vague or as violating the right to privacy to make reproductive

decisions. Such a challenge was successful in a federal district court

case, Lifchez v. Hartigan,93 which held that a ban on research on

conceptuses was unconstitutional because it was too vague in that

it failed to define the terms “experimentation” and “therapeutic.”94

The court pointed out that there are multiple meanings of the term

“experimentation.”95 It could mean pure research, with no direct

benefit to the subject. It could mean a procedure that is not

sufficiently tested so that the outcome is predictable, or a procedure

that departs from present-day practice. It could mean a procedure

performed by a practitioner or clinic for the first time. Or it could

mean routine treatment on a new patient. Since the statute did not

define the term, it was unconstitutionally vague. It violated

researchers’ and clinicians’ due process rights under the fifth

amendment since it forced them to guess whether their conduct

was unlawful.96

A similar result was reached by a federal appellate court

assessing the constitutionality of a Louisiana law prohibiting

nontherapeutic experimentation on fetuses in Margaret S. v.

Edwards.97 The appeals court declared the law unconstitutional

because the term “experimentation” was so vague it did not give

researchers adequate notice about what type of conduct was

banned.98 The court said that the term “experimentation” was

impermissibly vague99 since physicians do not and cannot

distinguish clearly between medical experimentation and medical

93 735 F. Supp. 1361 (N.D. Ill. 1990).

94 Id. at 1364. The court also held that the statute violated couples’ right

to privacy to make reproductive decisions to undertake preimplantation

genetic screening, or procreate with a donated embryo. Id. at 1377. With

embryo stem cell research, the progenitors’ reproductive freedom is not an

issue, however.

95 Id. at 1364-65.

96 Id. at 1364.

97 Margaret S. v. Edwards, 794 F.2d 994 (5th Cir. 1986).

98 Id. at 999.

99 Id.

100 Id. A concurring judge found this analysis to be contrived and opined

that the provision was not unconstitutionally vague. Id. at 1000 (Williams,
218 MONITORING STEM CELL RESEARCH

tests.100 The court noted that “even medical treatment can be

reasonably described as both a test and an experiment.”101 This is

the case, for example, “whenever the results of the treatment are

observed, recorded, and introduced into the data base that one or

more physicians use in seeking better therapeutic methods.”102

A third case struck down as vague the Utah statute that

provided that “live unborn children may not be used for

experimentation, but when advisable, in the best medical judgment

of the physician, may be tested for genetic defects.”103 The Tenth

Circuit held “[b]ecause there are several competing and equally

viable definitions, the term ‘experimentation’ does not place health

care providers on adequate notice of the legality of their conduct.”104

In a fourth case, Forbes v. Napolitano,105 a number of

plaintiffs106 challenged the constitutionality of an Arizona statute

that criminalized medical experimentation or investigation involving

fetal tissue from induced abortions.107 The statute at issue in that

litigation, A.R.S. § 36-2302(A), provides: A person shall not knowingly

J., concurring). Instead, he suggested that the prohibition was

unconstitutional because “under the guise of police regulation the state

has actually undertaken to discourage constitutionally privileged induced

abortions.” Id. at 1002, citing Thornburgh v. American College of

Obstetricians and Gynecologists, 106 S. Ct. 2169, 2178 (1986). The

concurring judge pointed out that the state had “failed to establish that

tissue derived from an induced abortion presents a greater threat to public

health or other public concerns than the tissue of human corpses [upon

which experimentation is allowed].” Id. Moreover, the state had not shown

a rational justification for prohibiting experimentation on fetal tissue from

an induced abortion, rather than a spontaneous one. Id.

101 Margaret S. v. Edwards, 794 F.2d 994, 999 (5th Cir. 1986).

102 Id.

103 Utah Code Ann. § 76-7.3-310.

104 Jane L. v. Bangerter, 61 F.3d 1493, 1501 (10th Cir.), rev’d on other grounds

sub nom., Leavitt v. Jane L., 518 U.S. 137 (1996).

105 2000 U.S. App. LEXIS 38596 (9th Cir. 2000).

106 Plaintiffs include numerous doctors and individuals suffering from

Parkinson’s disease who cannot receive transplants of fetal brain tissue

because of the statute.

107 2000 U.S. App. LEXIS 38596 (9th Cir. 2000).
APPENDIX E. 219

use any human fetus or embryo, living or dead, or any parts, organs,

or fluids of any such fetus or embryo resulting from an induced

abortion in any manner for any medical experimentation or scientific

or medical investigation purposes except as is strictly necessary to

diagnose a disease or condition in the mother of the fetus or embryo

and only if the abortion was performed because of such disease or

condition.108

An exception to this is found in A.R.S. § 36-2302(C), which reads:

This section shall not prohibit any routine pathological

examinations conducted by a medical examiner or hospital

laboratory provided such pathological examination is not

part of or in any way related to any medical or scientific

experimentation.109

The penalty for violation of A.R.S. § 36-2302, a class 5 felony, is one

and a half years in prison and fines up to $150,000.110

Plaintiffs argued that the statute prevented fetal tissue

transplantation treatment of Parkinson’s disease, certain fertility

treatments, and development of treatments for illness.111 The court

ultimately affirmed the decision of the lower court112 in holding that

the statute was unconstitutionally vague.113 Specifically, the

“distinction between experiment and treatment in the use of fetal

tissue is indeterminate, regardless of whether the tissue is obtained

after an induced abortion.”114 Furthermore, the court determined

that a criminal statute that serves to prohibit medical

experimentation but provides no guidance as to where to draw the

line between experiment and treatment does not provide doctors

108 A.R.S. § 36-2302(A)(2000).

109 A.R.S. § 36-2302(C)(2000).

110 See A.R.S. § 36-2303(2000).

111 2000 U.S. App. LEXIS 38596, *5 (2000).

112 Forbes v. Woods, 71 F. Supp.2d 1015, 1999 U.S. Dist. LEXIS 17025 (1999).

113 2000 U.S. App. LEXIS 38596, *12 (9th Cir. 2000).

114 2000 U.S. App. LEXIS 38596, *11 (9th Cir. 2000). The court determined

that the “knowingly” scienter requirement within the statute did not serve

as a clarification for the distinction.

115 2000 U.S. App. LEXIS 38596, *12 (2000).
220 MONITORING STEM CELL RESEARCH

with constructive notice, nor does it provide police, prosecutors,

juries, and judges with standards for application.115

Specific bans on embryo stem cell research are unlikely to

raise the same constitutional concerns. Part of the constitutional

deficiency in the Arizona, Illinois, Louisiana, and Utah cases was

that physicians offering health care services for their patients (such

as embryo donation from an infertile woman, preimplantation

genetic screening on an embryo, or treatment of a pregnant woman

for diabetes) would not know if the activity would be considered by

prosecutors to be experimental with respect to the embryo or fetus.

Moreover, the female patient’s reproductive freedom was implicated

statute. Stem cell researchers do not have those potential legal

arguments. There is a slight possibility, however, a reproductive

liberty challenge could be raised against a law banning reproductive

cloning.116

Conclusion

Bills on cloning and stem cell research are the subjects of

vast media attention and public debate. As technologies develop

that focus increasingly on the human embryo, lawmakers are having

difficulty coming to agreement on legislative policy in the absence

of societal accord about the moral and legal status of the embryo.

[Note from the President’s Council Staff: Please see the

accompanying Charts I and II detailing state laws and bills

relevant to this area. In the interest of space, Chart III, referenced

by the author, has not been included in this report. Additional

information is available on the Council’s website at

www.bioethics.gov in the transcript of the author’s presentation

to the Council at its July 24, 2003, meeting.]

116 Andrews, Lori B., “Is There A Right to Clone? Constitutional Challenges

to Bans on Human Cloning,” 11(3) Harvard Journal of Law and Technology

643 (1998).
APPENDIX E. 221

Chart I

State Cloning Legislation

State Reproductive

cloning ban

"Therapeutic"

cloning ban

Prohibition

of State

Funds

Permanent

ban Definition of "cloning"

Arkansas

Ark. Code §20-

16-1001 et. seq.

XX X

Asexual human

reproduction,

accomplished by

introducing the genetic

material from one or

more human somatic

cells into a fertilized or

unfertilized oocyte

whose nuclear material

has been removed or

inactivated so as to

produce a living

organism, at any stage

of development, that is

genetically virtually

identical to an existing

or previously existing

human organism

California

Cal. Bus. &

Prof. §16004,

16105; Cal.

Health &

Safety §§24185-

24187 (2003) X X

Nucleus transfer from a

human cell from

whatever source into a

human or nonhuman egg

cell from which the

nucleus has been

removed for the purpose

of, or to implant, the

resulting product to

initiate a pregnancy that

could result in the birth

of a human being.

Iowa

Iowa Code

§707B.1-.4

(2003)

XX X

Human asexual

reproduction,

accomplished by

introducing the genetic

material of a human

somatic cell into a

fertilized or unfertilized

oocyte whose nucleus

has been or will be

removed or inactivated,

to produce a living

organism with a human

or predominantly human

genetic constitution

Louisiana

La. Rev. Stat.

Ann.

§1299.36.1-.6

(2003)

Sunset

Provision: July

1, 2003

X

Does not

prohibit use of

state funds for

scientific

research or

cell-based

therapies that

do not involve

human cloning

X

Identical to California

Michigan

Mich. Comp.

Laws

§§333.26401-06,

333.16274,

16275, 20197,

750.430a (2003)

X X XX

Use of human somatic

cell nuclear transfer

technology (transferring

the nucleus of a human

somatic cell into an egg

from which the nucleus

has been removed or

rendered inert) to

produce a human

embryo
222 MONITORING STEM CELL RESEARCH

Chart I

State Cloning Legislation (Cont.'d)

Missouri

Mo. Rev. Stat.

§1.217 (2003)

X X

Replication of a

human person by

taking a cell with

genetic material and

cultivating such cell

through the egg,

embryo, fetal and

newborn stages of

development into a

new human person

North Dakota

N.D. Cent.

Code §§12.1-39-

01-02 (2003)

XX X

Human asexual

reproduction,

accomplished by

introducing the

genetic material of a

human somatic cell

into a fertilized or

unfertilized oocyte,

the nucleus of which

has been or will be

removed or

inactivated, to

produce a living

organism with a

human or

predominantly

human genetic

constitution

Rhode Island

R.I. Gen. Laws

§23-16.4-1 -.4-4

(2003) Sunset

provision: July

7, 2010

X

Cell transfer

and other

cloning

technologies

not included in

ban;

mitochondrial,

cytoplasmic

and gene

therapy not

prohibited for

research or

animal

creation

Use of somatic cell

nuclear transfer for

pregnancy

prohibited

(transferring the

nucleus of a human

somatic cell into an

oocyte from which

the nucleus has been

removed)

Virginia

Va. Code Ann.

§32.1-162.21-.22

(2003)

X X

Transferring the

nucleus from a

human cell from

whatever source into

an oocyte from

which the nucleus

has been removed or

rendered inert in

order to create a

human being
APPENDIX E. 223

CHART II

LAWS AND 2002-2003 BILLS

State Prohibit All

Human

Cloning

(including

therapeutic)

Prohibit

Reproductive

Cloning

Allow

Therapeutic

Cloning

Specifically

Ban

Destructive

Embryo

Research

Allow Embryo

Stem Cell

Research

AL X X

AZ X X

AR Law Law

CA X X Law X Law

CO X X

CT X X

DE X X

FL X X

GA

HI

ID

IL X X X

IN X X X

IA Law Law Law

KS X X X

KY X X

LA X X Law

ME

MD X

MA X X X

MI Law Law X

MN

MS

MO X X
224 MONITORING STEM CELL RESEARCH

CHART II

LAWS AND 2002-2003 BILLS (Cont.'d)

State Prohibit All

Human

Cloning

(including

therapeutic)

Prohibit

Reproductive

Cloning

Allow

Therapeutic

Cloning

Specifically

Ban

Destructive

Embryo

Research

Allow Embryo

Stem Cell

Research

MT

NE X X

NV

NH X X

NJ X X X X

NM

NY X X X X

NC

ND Law Law

OH

OK X X

OR X X

PA X

RI Law X

SC X X

SD

TN X X X

TX X X X

UT

VT X X

VA Law X

WA X X X

WV X X

WI X X X

WY

* The Laws that have already been enacted are indicated by the word "Law"

*The Bills are indicated by an "X"
APPENDIX F.

The Meaning of Federal Funding

PETER BERKOWITZ, J.D., PH.D.1

I. INTRODUCTION

How should the government approach the question of public

funding of activities that are deemed controversial by the American

people? Is it appropriate to make such decisions on moral grounds?

Can moral grounds for such decisions be avoided? If they can’t,

whose moral views, and of which sort, should govern, and with what

consequences for those in the minority?

Questions of this sort have been frequently discussed in the wake

of President Bush’s 2001 decision regarding federal funding of

embryonic stem cell research. In that decision, the President

permitted federal funds to be used, for the first time, to support

research on embryonic stem cells, but only those already in

existence. At the same time, he made it clear that there would be

no federal support for any research that involved or depended on

any future destruction of human embryos. In so doing, he was

upholding both the letter and the spirit of a Congressional

enactment, the 1996 Dickey Amendment, which prohibited the

creation of embryos for use in experiments, or the use of embryos in

research that led to their destruction.

President Bush’s decision has generated a great deal of

controversy. Most scientists and patient advocacy groups believe

that he made the wrong decision, and that the Dickey Amendment

is a terrible mistake. Among the objections one commonly hears to

the President’s policy are several that concern the meaning of federal

funding:

(1) By withholding federal funding for research that involved the

creation of new embryos or the future destruction of embryos in

existence, the President has effectively banned embryonic stem cell

research.

225

1

Peter Berkowitz teaches at George Mason University School of Law and is

a research fellow at Stanford’s Hoover Institution. He has served as a senior

consultant to the President’s Council on Bioethics.
226 MONITORING STEM CELL RESEARCH

(2) The decision was wrong because the President allowed his

personal moral views to govern federal policy. Or, along the same

lines, the congressional ban is wrong because it represents the

imposition of moral views—religiously based moral views at that—

to frustrate sound and beneficial public policy.

(3) The decision is morally incoherent, for if an act is so immoral

as to deserve the governmental disapproval implicit in withholding

funding, it should be accompanied by efforts to prohibit the activity

altogether.

Whatever the merits of the current law, or the President’s 2001

stem cell decision, these objections, once closely examined, cannot

pass muster. The first confuses a limitation on funding with the

imposition of a ban or prohibition. The second wrongly supposes

that legislating morals through federal budget decisions is always

or generally wrong. And the third incorrectly assumes that

government has an obligation to bring an end to all conduct it

believes immoral.

Explaining these errors requires an exploration of the meaning

of all government funding decisions. Such an exploration can not

decide the difficult question of the merits of the President’s stem

cell policy. It can, however, put to rest the objections built around

the claim that the policy somehow violates the letter or the spirit of

sound constitutional government.

II. FEDERAL FUNDING

A. Basic Considerations

The common objections to the President’s policy fail to come to

grips with what government funding in a liberal democracy really

means. Several fundamental features of our constitutional system

need to be emphasized.

First, no one and no activity has a constitutional right to federal

funding. There is no governmental obligation to fund most activities,

not even the most worthy, save for such matters as the Constitution

explicitly proclaims to be the responsibility of government, such as

national defense, the maintenance of federal courts, the holding of

elections, and so on. And even concerning these constitutional

essentials, it is an open question, to be resolved by our elected

representatives, of how government will choose to allocate taxpayer

dollars.
APPENDIX F. 227

Second, no individual or cause has a right to sit at the government

trough. Resources are scarce, and insufficient to support all worthy

activities. People with different causes and interests compete to

obtain them, and in order to succeed they are forced to bring their

case to members of Congress. Funds are distributed only through

the political process, within limits set by the Constitution, as the

result of deliberation, lobbying, deal-making, and the like.

Third, in a healthy democracy people will always have

disagreements about what activities should receive government

funding. Sometimes the disagreements will be intense, and

sometimes not. Sometimes the disagreements will include moral

disagreements, and sometimes not. Sometimes the political process

will generate a stable compromise on the issue, and sometimes one

side or the other decisively prevails.

Fourth, while the Constitution prohibits the government from

establishing religion, it does not interfere with the right of citizens

to form moral judgments based on their deeply held religious beliefs,

and to persuade a majority of their fellow citizens to enact legislation

informed by those moral judgments, provided of course that the

legislation does not interfere with other constitutionally guaranteed

rights.

Fifth, those who fail in the democratic process to obtain federal

funding will always feel that they did not get what they need or

want, but in the absence of a clear legal entitlement to such funding,

they cannot properly complain that the government has thereby

denied their rights or interfered with their liberty to exercise them.

Sixth, those who lose have several alternatives built into the

democratic process. They can try to persuade their representatives

in Congress to reconsider, they can vote in others more sympathetic

to their cause, they can seek to influence public opinion, or they can

seek non-government funding for their activities.

All of this is straight-forward and uncontroversial. It suggests

the legitimacy, indeed the routine character, of the President’s policy.

It might be regarded as the end of the story.

B. Are There Special Cases?

Although the framework laid out above may correctly describe

the situation for most or even all federal funding decisions, there

are moral and political reasons why people might regard, for

example, withholding of support for selected aspects of biomedical

research as a special case, an exception that demands a different

approach.

The nation strongly and overwhelmingly backs biomedical

research. And we generally leave the mapping of research strategies
228 MONITORING STEM CELL RESEARCH

to scientists and those who administer the institutions in which they

work. The entire biomedical enterprise in the US, including also the

training of the next generation of scientific researchers, has come

to depend heavily on government support. The public generally

favors this arrangement, and relies on government-funded research

for the treatment and for the cure of all still untreatable diseases,

such as cancer and Alzheimer’s disease.

Consequently, the decision to withhold public funds from any

particular piece of the biomedical research portfolio looks and feels,

both to scientists and to the public, like an intrusion of government

into a place where it does not belong, and it prompts harsh

accusations that government is engaging in censorship or even

outright prohibition of medically necessary scientific research. To

be sure, the FDA regularly imposes restrictions on research, but

mainly on grounds of safety. When, however, government’s objection

to research is moral in nature, it strikes scientists as a deprivation:

a restriction of freedom to inquire, a thwarting of worthy community

goals, an intrusion of morals into a sphere where they do not belong.

At the same time, it appears to those members of the public who

disagree with the decision as a failure by the government to abide

by its putative moral obligation to use its resources to explore all

fruitful areas of research in search of cures for dread diseases.

Moreover, there is reason to single out for special attention those

decisions about federal funding where powerful moral principles

are at loggerheads, and the nation is deeply and passionately

divided. This is the case of stem cell research. It poses a confrontation

between genuine and conflicting goods: on the one side, respect

for nascent human life and, on the other side, commitment to

unfettered scientific inquiry and to the fight against disabling and

deadly disease. The clash between those who hold that the moral

status of the embryo is no different from that of a fully developed

human being, and those who believe that the embryo is a clump of

cells, utterly devoid of moral worth is not resolved by appeal to

shared moral premises. This is because what the debate over stem

cell policy calls into question is how to apply our shared belief in

the rights and dignity of the individual.

Despite the powerful presumption in favor of federal funding of

biomedical research, and the moral stakes which both sides see as

exceedingly high, the controversy over federal funding of stem cell

research does not present a special case. In politics, though, how a

policy appears is important. For this reason, and despite the fact

that the common arguments condemning the president’s policy rest

on false assumptions or unreasonable expectations, and though they

may be mere rationalizations for the failure to win the policy battle,

it is worth examining their flaws fully.
APPENDIX F. 229

III. MORALS, FEDERAL FUNDING, AND LEGISLATION

A. Federal Funding

In the first place, federal funding is about resource distribution–

who and what will get how much of the nation’s scarce taxpayer

dollars. It is usually not about restricting basic rights. For example,

there is no constitutional right to the funding of biomedical research.

But often the question of whether government will or will not

fund an activity is about more than mere distribution. It is about

government shaping choices among various and competing goods

or undertakings. It is a statement of approval and encouragement

by government, a declaration by the nation that an activity or

undertaking is meritorious and has priority. Or, in the decision to

withhold funds, government policy can be a statement of disapproval

and discouragement, a declaration by the nation that a permitted

activity or undertaking lacks merit or has low priority.

Policy decisions about funding resemble policy decisions about

taxing. Both sorts of decisions create incentives and disincentives

by making activities more or less costly. The child tax credit, for

example, reduces the financial cost of child rearing. In so doing, it

strengthens families in two ways: it enables families to save money,

and it conveys an important message about the political importance

of the well-being of the family. Similarly, government funding of

research into disease and its prevention and treatment increases

the supply of these goods, and reflects our nation’s considered

judgment that the relief of physical suffering is a high national

priority.

While all law either requires, forbids, or permits, the provision or

withholding of funding and the use of tax incentives and

disincentives allows government to express a range of attitudes

toward that which it permits. In the United States, through such

decisions government strongly endorses charity and higher

education. It looks favorably on national service and the arts. It

shows a preference for marriage to cohabitation. It frowns upon

smoking. It is the distinction between permitting or tolerating an

activity (which is the case with embryo research and destruction)

and actively promoting it through governmental funding (which the

president’s policy on stem cells prohibits), that is crucial to

understanding the president’s stem cell research policy, but not only

to the stem cell controversy.

The question of federal funding routinely implicates questions

about the nation’s moral priorities among permissible activities. And
230 MONITORING STEM CELL RESEARCH

the question of moral priorities in politics is not so simple as a

question of good or bad; rather it is a question of better or worse.

One consequence of this is that in sorting out funding decisions it is

not a matter of one side introducing moral considerations. Most of

the time, both sides in disputes over policy are of necessity engaged

in making moral arguments.

This is true, and to an extraordinary degree, in the stem cell

controversy. Both sides–those who wish to defend the rights of

nascent human life, and those who wish to defend unfettered

scientific research directed at the relief of human suffering through

the cure of deadly disease–defend moral principles. To make matters

more difficult, both sides tend to defend those principles in their

absolute form. While the president’s position attempts to give weight

to both sides’ principles, both sides, because of the passion with

which each holds its principle, are dissatisfied. However, insofar as

the president’s approach reflects that adopted by the Dickey

Amendment, it follows a determination made by a majority of the

people’s representatives, and with that both sides should be

satisfied. But they aren’t.

But the dissatisfaction of both sides takes a recognizable form.

In general, because moral principles are so frequently at stake in

the fight for federal taxpayer dollars, funding decisions create

bitterness. This truer still, as in the stem cell debate, when the moral

principles are wielded in their absolute form, so that both sides can

claim defeat. If funding is withheld, those who believe the activity

is worthy can claim that their tax dollars, which they contribute in

the hope that they will serve the good of the country, are being held

back from what they deem a deserving or even overriding moral

purpose. This is the position taken by many scientists and

progressives with regard to the limitations imposed by the

President’s policy. If funding is provided, those who believe the

activity is immoral can claim that their tax dollars are being used to

advance a cause they believe is unworthy, or even abhorrent. This

is the position taken my some social conservatives who believe that

the limitations imposed by the President’s policy did not go nearly

far enough and pave the way, sooner rather than later to the routine

creation and destruction of human embryos for biomedical research.

Typically, both sides make moral claims, and one or the other–and

in the really tough cases such as stem cell policy, both sides–will

have to live with the fact that their moral principles are being rejected

(if not assaulted) by the government, in their own name and with

their own tax dollars.

Why should those who lose the political struggle put up with

this? For the simple reason that living in a liberal democracy means

sometimes being in the minority, even on questions of the utmost
APPENDIX F. 231

importance, but so long as the laws which one opposes are

consistent with the Constitution and enacted according to legally

appropriate procedures, one has an obligation to obey them.

B. Legislation

Is it really a legitimate aim of a liberal democracy to adopt laws

and take actions to shape the moral beliefs of its citizens? Perhaps

federal funding is the exception, and to the extent possible the moral

dimension should be eliminated from policy formation. Doesn’t

government in a liberal democracy have an obligation to remain

neutral toward competing conceptions of a good life, and so refrain

from enacting morals into law? Otherwise, doesn’t it impermissibly

infringe on people’s right to choose how to live their lives.

According to a common and sound criticism of this common view

of the liberal state, such neutrality is a chimera: it is impossible for

any government to remain neutral about morality and the nature of

a well-lived life, since the resolution of controversies over public

policy–for what purposes is the state permitted to classify citizens

by race? what is the meaning of marriage? to what extent may the

public schools engage in civic education?– always draw upon,

reinforce, or suppress a view about what is deserving, proper and

good. It is possible, as a matter of policy, to tolerate a wide variety

of choices and forms of life, but toleration itself is a moral principle

based on a certain interpretation of how to secure human freedom

and respect the dignity of the individual.

Some then object that because of its very foundational

commitments, our liberal democracy privileges the autonomous or

freely choosing life. And so in sense it does. But it need not and

should not do this unwittingly or surreptitiously. The mistake is to

think that liberal state stands or falls with the commitment to

neutrality. It doesn’t. It stands or falls with the commitment to

creating the conditions under which individuals can exercise political

freedom.

Law and public policy in a liberal democracy properly seek to

create conditions in which citizens can make informed and

responsible choices. It does this in a variety of ways. The first and

most taken for granted is through the establishment of public order.

It also does this through establishing a system of public schools,

promoting research in the arts and sciences, and enacting a wide

variety of social and economic legislation, all with a view to forming

a citizenry that is at home in, and capable of taking advantage of,

freedom. Legislation designed to encourage biomedical research

can be seen as creating circumstances in which we are better able

to enjoy the blessings of freedom. The same can be said of laws
232 MONITORING STEM CELL RESEARCH

designed to respect, and encourage respect, for nascent human life,

which can reasonably be understood as contributing to the

conditions under which individuals learn to respect humanity in

others and in themselves.

To be sure, even within the limits provided by law, government’s

encouragement of informed and responsible choice can easily

become a tool for the ill-conceived circumscribing of choice. Even

well meaning government efforts to help prepare citizens for liberty

and toleration can undermine both. Government funded education

can be dogmatic and ideological; government funded research may

be biased and unaccountable; government supported arts may

disseminate tawdry or jingoistic sentiments and images;

government funded programs directed at the family may fail to adapt

to changing times. Of course, these familiar abuses are not

arguments against government promoting the conditions that enable

citizens to take advantage of freedom. Rather, they are reasons for

proceeding with care, and with an appreciation of the complexities

of contemporary moral and political life.

IV. AMERICAN DILEMMAS

The president’s policy on stem cells is not the only funding

decision in contemporary American politics that has generated

controversy. Brief discussion of others sheds light on what is common

to all and what is distinctive to the stem cell debate.

Consider first the battle over abortion, which involves a long

standing struggle over the question of government funding for lawful

conduct. Shortly after entering office, President Bush ordered the

withholding of funding from international organizations that

performed abortions, a decision that was neither required of him

nor forbidden to him but within his discretion. The principle behind

this policy is common to his position on stem cell research:

government funds should not be used to destroy nascent human

life.

At home, a line of Supreme Court decisions stretching from 1977

to 1991dealing with abortion and government funding established

the principle that the constitution does not require government to

fund activities that the Constitution protects. In Maher v. Roe 432

U.S.464 (1977), the Court held 6-3 that Connecticut could provide

Medicaid benefits for childbirth while withholding benefits from

women who wished to have non-medically necessary abortions.

Justice Powell, writing for the majority, maintained that the right to

abortion announced in Roe v. Wade, “protects the woman from

unduly burdensome interference with her freedom to decide whether
APPENDIX F. 233

to terminate her pregnancy. It implies no limitation on the authority

of a State to make a value judgment favoring childbirth over abortion,

and to implement that judgment by the allocation of public funds.”

Powell’s analysis emphasized the “basic difference between state

interference with a protected activity and state encouragement of

an alternative.” In dissent, Justice Brennan disagreed vociferously.

He argued that the denial of funds unconstitutionally interfered with

the right of women to choose an abortion. Also in dissent, Justice

Marshall argued that by withholding funds for non-medically

necessary abortions, Connecticut was seeking “to impose a moral

viewpoint that no State may constitutionally enforce.”

In Harris v. McRae 448 U.S. 297 (1980), by a 5-4 margin, the Court

upheld the Hyde Amendment, which banned federal funding of

medically necessary abortions. The majority’s argument was much

the same as in Maher: “although government may not place

obstacles in the path of a woman’s exercise of her freedom of choice,

it need not remove those not of its own creation.” The government,

the majority reasoned, does not have an obligation to provide

taxpayer dollars so that individuals can exercise their individual

rights to the maximum. To this, Justice Brennan replied in dissent

that Hyde Amendment actually left poor women in a worse off

position. By refusing to provide poor women with funding for even

medically necessary abortions while subsidizing childbirth, the

government demonstrated profound disapproval for abortion and

thereby burdened the exercise of the right to privacy declared in

Roe.

In Rust v. Sullivan 500 U.S. 173 (1991), again by 5-4 margin, the

Court upheld federal regulations that barred health care

professionals who received federal funding from offering counseling

about abortion. Chief Justice Rehnquist, writing for the majority,

reiterated the Maher and Harris principle: “The Government has no

constitutional duty to subsidize an activity merely because the

activity is constitutionally protected.” In dissent, Justice Blackmun,

joined by Justices Stevens and Marshall, echoing the dissenters in

Maher and Harris, insisted that by conditioning federal funding on

the withholding of counseling about abortion, the government was

actually placing “formidable obstacles” in the path of women’s

exercise of their privacy rights.

While these cases appear closely analogous to the stem cell

controversy, they differ in a crucial respect. Although some of the

same forces are politically engaged, and although the moral issue

concerns the question of the inviolability of nascent human life, the

abortion cases would have been unlikely to come to the Court for

adjudication had the Court not declared in Roe v. Wade a

constitutional right to an abortion. For the argument made by those
234 MONITORING STEM CELL RESEARCH

who seek federally funded abortions is that by withholding funding,

the government is seeking to frustrate the exercise of a

constitutionally protected right. Absent such a right, there could be

no plausible legal claim. Indeed, absent an entitlement to

government sponsored health care benefits, there is no valid legal

claim that Medicare must pay for cosmetic surgery, sex-change

operations, contraceptive benefits, heart transplants, or any other

procedure one wants for oneself and can find a doctor to do. Only if

there were a constitutionally protected right not to be poor, not to

be without resources to fully take advantage of all the things that

we are legally entitled to pursue, could such a claim prevail as a

matter of law. While there is no such right, this is just the kind of

claim that dissenters in the Court’s cases concerning federal funding

and abortion defend. In short, because the Constitution provides

no special protection to biomedical research, the argument for legal

entitlement to funding of stem cell research proceeds on dramatically

weaker grounds than the rejected arguments in the abortion funding

cases.

Title VI of the Civil Rights Act of 1964 furnishes another example

of how government may withhold funds from practices it does not

outlaw. It provides that, “No person in the United States shall, on

the ground of race, color, or national origin, be excluded from

participation in, be denied the benefits of, or be subjected to

discrimination under any program or activity receiving Federal

financial assistance.” It is this provision that requires private

universities to avoid those racial classification in admissions and

hiring that would violate the prohibitions imposed on state action

by the equal protection clause of the 14th Amendment. Title VI is far

reaching, because most private universities rely heavily on

government funding for the support of basic research. And it

provides a way for the federal government to shape the moral

contours of what is largely private conduct, and bring that conduct

in line with fundamental constitutional principles. Of course private

institutions are free to continue to practice activities that disqualify

them for federal funding. All they have to do is refuse to take federal

funds.

Close in form to federal policy on stem cell research are social

security regulations regarding marriage and survivor benefits. For

example, although cohabitation without matrimony is not illegal,

indeed it is quite common, the federal government refuses to pay

social security survivor benefits to all but legal spouses. This is a

way for government to provide financial incentives for marriage.

And for government to take sides on the value of marriage,

proclaiming the union marked by it as good for individuals and good

for the polity. This is a policy decision that does not engender
APPENDIX F. 235

bitterness or controversy. It must be acknowledged that the

withholding of a reward could, under imaginable circumstances,

stigmatize those who choose to live together as a loving couple but

not to marry. But just as it can not plausibly be claimed today that

the child tax credit confers social disapprobation on married couples

without children, so too it cannot be plausibly claimed that unmarried

couples suffer social disapprobation because of government policy

that restricts the paying of social security survivor benefits to legal

spouses.

Or, from a different angle, consider the question of elementary

level and high school education. In 1923, in a landmark decision,

Meyer v. Nebraska 262 U.S. 390 (1923), the Supreme Court ruled that

parents have a right to educate their children in a foreign language.

In 1925, in a related case, Pierce v. Society of Sisters 268 U.S. 510

(1925), the Supreme Court ruled that parents have a right to educate

their children in private schools. But nobody concludes that the rights

that these cases protect prohibit states from policy decisions

encouraging public education. And nobody claims that the right of

parents to privately educate their children creates an entitlement to

have that private education funded by the government.

As these examples illustrate, the controversy over stem cells

should be seen as one among many political battles over the

allocation of limited federal funds. It is distinguished not by the

presence of moral principles, or the presence of moral principles on

both sides, but by the particular moral principles at stake, the

absoluteness with which they are wielded, and the intensity of the

passions their defense provokes.

V. CONCLUSION

When the question of federal funding is placed in perspective,it

can be seen that the common objections to the President’s policy on

stem cell research are misplaced.

First, by withholding federal funding for research that involved

the creation of new embryos or the future destruction of embryos,

the President did not effectively ban embryonic stem cell research.

His August 2001 decision for the first time provided federal funding

for stem cell research, and it permitted private individuals and

companies to pursue it.

Second, by basing his policy in part on moral considerations, the

President did not violate an obligation to keep morals out of politics,

because funding decisions, whichever way they go, typically and

unavoidably contain a moral component. Indeed, the moral
236 MONITORING STEM CELL RESEARCH

component often lies at the heart of the dispute and at the core of

the decision.

Third, by refusing to seek a blanket prohibition on an activity

from which he withheld funding on moral grounds, the President

did not make an incoherent decision. The complexities of a free

society frequently create situations in which it makes sense for

government to express doubt, anxiety or disapproval for an activity

it is unwilling or unable to outlaw.

None of this is to deny that the president’s policy on stem cell

research is open to criticism on the merits. It is only to claim that

the policy reflects a perfectly appropriate exercise of governmental

powers in a liberal democracy.
Appendix G.

Report on the Ethics of Stem Cell Research

PAUL LAURITZEN, PH.D.

Director, Program in Applied Ethics, Professor,

Department of Religious Studies, John Carroll University,

University Heights, Ohio

...the final stage is come when man by eugenics, by

prenatal conditioning, and by an education and

propaganda based on perfect applied psychology,

has obtained full control over himself. Human nature

will be the last part of nature to surrender to man.

(Lewis, 1947)

This sudden shift from a belief in Nurture, in the form

of social conditioning, to Nature, in the form of

genetics and brain physiology is the great

intellectual event, to borrow Nietzsche’s term, of the

late twentieth century. (Wolfe, 2001)

I begin with passages from an unlikely pair of authors because

although C. S. Lewis and Tom Wolfe are somewhat distant in time,

certainly different in temperament, and extravagantly different in

personal style, they share an imaginative capacity to envision the

possible consequences of modern technology. The technology that

occasioned Lewis’s reflections, “the aeroplane, the wireless, and

the contraceptive” may now seem quaint, but his warning about

turning humans into artifacts, that accompanied the passage quoted

above, is eerily prescient. Similarly, although he does not directly

take up stem cell research, Tom Wolfe’s reflections on brain imaging

technology, neuropharmacology, and genomics are worth noting in

relation to the future of stem cell research. In his inimitable way,

Wolfe summarizes his view of the implications of this technology in

the title of the essay from which the above passage comes. “Sorry,”

he says, “but your soul just died.”

The point of beginning with Lewis and Wolfe, then, is not that

I share their dire predictions about the fate to which they believe

technology propels us; instead, I begin with these writers because

237
238 MONITORING STEM CELL RESEARCH

they invite us to take an expansive view of technology. I believe

that such a perspective is needed and is in fact emerging in recent

work on stem cell research. This is not to say that the sort of

traditional analysis that has framed much of the debate on stem

cells, analysis that involves issues of embryo status, autonomy, and

informed consent, for example, is unhelpful; far from it. Nevertheless,

traditional moral analysis of stem cell research is nicely

complemented by a consideration of the “big picture” questions

that Lewis and Wolfe both wish to press. This report will therefore

seek to draw attention to the literature on stem cell research that

attends both to the narrow and to the expansive bioethical issues

raised by this research.

The Moral Status of the Embryo

There is little doubt that public reflection on stem cell research

in the United States has been affected by the extraordinarily volatile

cross-currents of the abortion debate. Although I will indicate below

several reasons why framing the stem cell debate as a subset of

that on abortion is problematic, nevertheless, in its current form,

stem cell research is debated in terms dictated by the abortion

controversy, and that has meant that questions about the status of

the embryo have been particularly prominent.1 For example, the

National Bioethics Advisory Commission (NBAC) described the

ethical issues raised by stem cell research as “principally related to

the current sources and/or methods of deriving these cells” (NBAC,

1999, 45). A policy brief from the American Association for the

Advancement of Science (AAAS) begins its discussion of the ethical

dispute over stem cell research by citing the disagreement over the

status of the embryo as the decisive variable leading to

fundamentally different views on this research (AAAS, 1999, 11).

The National Academy of Sciences’s report on stem cell research

claims that “the most basic [ethical] objection to embryonic stem

cell research is rooted in the fact that such research deprives a human

embryo of any further potential to develop into a complete human

being” (National Academy of Sciences, 2002, 44). The Ethics

Advisory Board of the Geron Corporation lists the moral status

question as the first moral consideration relevant to deciding the

acceptability of stem cell research (Geron Corporation Ethics

Advisory Board, 1999, 32). The list could go on.

Despite the fact that these statements all insist on the

importance of the status question, they also recognize that the

debate about the status of the early embryo is not new and that the

controversy over stem cell research does not, strictly speaking, raise

novel issues in this regard. Indeed, it is probably best to place the
APPENDIX G. 239

initial skirmishes over stem cell research in the context of moral

debates about human embryo research generally. In fact, it is worth

noting that the report of the NIH Human Embryo Research Panel

(HERP), published in 1994, explicitly identified the isolation of human

embryonic stem cells as one of thirteen areas of research with

preimplantation embryos that might yield significant scientific

benefit and that should be considered for federal funding (See NIH

HERP, 1994, ch. 2).

Although the recommendations of the HERP were never

implemented, the fact that a high-profile panel reviewed ex utero

preimplantation human embryo research and explicitly endorsed

stem cell research, meant that the panel report would affect the

policy debate about stem cell research, even though its

recommendation that the derivation and use of stem cells be

federally funded was not adopted. For one thing, the panel’s

anticipatory support for stem cell research assured that when human

stem cells were actually derived several years later, the debate that

ensued would be tied to the abortion controversy. As members of

the HERP panel have made clear, from the start, the work of the

panel was embroiled in controversy. For example, shortly after the

HERP was impaneled, thirty-two members of Congress wrote to

Harold Varmus, the director of NIH, to complain about the

composition of the panel. A lawsuit was filed in an attempt to

prevent the panel from meeting, and members of the panel received

threatening letters and phone calls (Green, 1994; Tauer, 1995; Hall,

2003).

Given the pro-life opposition to the HERP panel and its

recommendations, it is no real surprise that initial reactions to the

prospect of human stem cell research fell out along the fault lines of

abortion politics in the country. By and large, individuals and groups

opposed to abortion tended to be opposed to stem cell research,

and individuals and groups supportive of legalized abortion tended

to support stem cell research.2

 For example, the testimony that

Richard Doerflinger, the principal spokesperson for the U.S. Catholic

bishops on pro-life matters, offered before the Senate Appropriation

Subcommittee on Labor, Health, and Education in 1998 was

substantially the same as that he offered before the HERP in 1994

on stem cell research (Doerflinger, 1998, 1994). In both cases, the

fundamental issue was the status of the embryo. Given Catholic

teaching that the embryo must be treated as a person from

conception, no experimentation on the embryo can be allowed that

would not also be allowed on infants or children. Hence, the Catholic

church treats stem cell research as it has treated previous issues

involving the destruction of human embryos; it is condemned as

morally abhorrent.
240 MONITORING STEM CELL RESEARCH

In similar fashion, the arguments reviewed by the HERP panel

that supported embryo research generally in 1994, were mobilized

again four years later when stem cell research was the specific point

of contention; and again the focal point was embryo status.

Consequently, just as the HERP report opted for a “pluralistic” view

of the embryo that emphasized its developmental potential, so, too,

did the NBAC endorse the idea that the early embryo deserves

respect, but is not to be treated fully as a person.3

Moreover, the fact that the HERP defended its support of stem

cell research by stressing the developmental capacity of the embryo

also shaped the trajectory of much subsequent support for this work,

because insisting on respect for the embryo but denying its

personhood meant explaining how one could respect the embryo

while nevertheless destroying it. Daniel Callahan, for example,

posed this problem very strongly in response to the HERP report. If

“profound respect” for the embryo is compatible with destroying it,

he asked, “What in the world can that kind of respect mean?” It is,

he says, “an odd form of esteem, at once high-minded and altogether

lethal” (Callahan, 1995). Callahan was not alone in raising this issue

and attempts to answer his question continue to appear in the

literature (See Lebacqz, 2001; Meyer and Nelson, 2001; Ryan,

“Creating Embryos,” 2001; Steinbock, 2001, 2000).

In retrospect, then, it seems that the HERP report served almost

as choreography for the initial debates about stem cell research,

and, as a result, the steps in the debate closely followed those that

are familiar from the abortion controversy (On this point, see Hall

2003). The upshot, in my view, is that much of the debate has been

too narrowly focused and has a kind of repetitive and rigid quality

to it. As I noted above, for example, the Catholic church has

repeatedly claimed that the central issue raised by stem cell research

is that it involves the destruction of human embryos, embryos it

believes should be treated as persons.4

 For that reason, the rhetoric

with which the Catholic church condemns embryonic stem cell

research closely parallels that used to condemn abortion. Yet,

because the American bishops do not want to be perceived as antiscience, they have also repeatedly and uncritically praised adult

stem cell research, even though there are good reasons, given

Catholic concerns about social justice, to be concerned about the

pursuit of adult stem cell research. I will return to this point below,

but for now I wish simply to note that much of the opposition to

embryonic stem cell work has resembled Catholic opposition in

being circumscribed by questions of embryo status, narrowly

construed.

A similar constriction, however, is also apparent in the

preoccupations of supporters of stem cell research. Just as
APPENDIX G. 241

opponents of this research have ritualistically condemned the

destruction of early embryos but uncritically celebrated adult stem

cell work, supporters of embryonic stem cell research have typically

insisted on using embryos left over from IVF procedures, while

repudiating the use of embryos created solely for research. Indeed,

insisting on the distinction between so-called “spare” embryos and

“research” embryos and endorsing only the use of spare embryos

has been one way that supporters of embryo research have tried to

demonstrate their “respect” for the embryo. Yet, it is worth asking

whether the spare embryo/research embryo distinction does not,

to borrow Daniel Callahan’s image, provide a kind of “wafting

incense” to mask what supporters still find a disquieting smell

(Callahan, 1995).5

Although the debate about stem cell research might have been

framed in terms of the abortion controversy in any event, the HERP

report insured that the initial debate over stem cell work that

followed in aftermath of the public announcement of the work of

John Gearhart (Shamblott et al., 1998) and James Thomson in 1998

(Thomson et al.) would be navigated in the wake of the conflict over

abortion. As I indicated, the upshot is that the discussion about

stem cell research has been more cramped than it might otherwise

have been. The discussion has been too focused on the details of

embryological development; too focused on the differences between

those who view the early embryo as a person and those who do

not; and far too individualistically oriented. Before turning to ways

that the debate might be become less cramped, let me focus more

concretely on these difficulties.

The point about the debate being framed too individualistically

is nicely illustrated in an article on abortion by Lisa Sowle Cahill

entitled “Abortion, Autonomy, and Community” (Cahill, 1996). Cahill

begins this article by claiming that, in discussing the morality of

abortion, there is no way to avoid the question of the status of the

fetus. Nevertheless, she says, the debate about fetal status is almost

always conducted with the goal of determining the rights involved,

where rights are understood very individualistically. To the degree

that the fetus is acknowledged to have rights, those rights are pitted

against the rights of the pregnant women. Although Cahill doubts

that we can jettison the use of rights language altogether, if we are

going to use rights language, she says, we must “remove that

language from the context of moral and political liberalism” (361).

If we do so, we might be able to see that we have duties and

obligations to which we do not explicitly consent. As Cahill puts it,

“such obligations originate simply in the sorts of reciprocal

relatedness that constitutes being a human” (361).6

 For example,

moving away from an individualistic liberal view of the pregnant
242 MONITORING STEM CELL RESEARCH

woman as primarily or exclusively an autonomous moral agent might

lead us to recognize the obligations that individuals and communities

have to support her during and after a burdensome pregnancy (363).

We do not need to accept Cahill’s commitment to the Catholic

common good tradition to recognize the truth in her conclusion that

pitting the rights of the fetus against the rights of the pregnant

woman individualistically construed leads us to overlook important

social dimensions of the problem of abortion. It seems to me that

much the same dynamic is evident in the stem cell research debate.

Consider again the central argument that the Catholic church

has made against stem cell research. The Pontifical Academy for

Life suggests that the fundamental ethical issue is whether it is

morally licit to produce or use human embryos to derive embryonic

stem cells. The reasoning the Academy provides for concluding it

is not licit is worth reproducing in full. The Academy lists five points:

1. On the basis of a complete biological analysis,

the living human embryo is—from the moment of

the union of the gametes—a human subject with a

well defined identity, which from that point begins

its own coordinated, continuous and gradual

development, such that at no later stage can it be

considered as a simple mass of cells.

2. From this it follows that as a “human individual”

it has the right to its own life; and therefore every

intervention which is not in favor of the embryo is

an act which violates that right. ...

3. Therefore, the ablation of the inner cell mass (ICM)

of the blastocyst, which critically and irremediably

damages the human embryo, curtailing its

development, is a gravely immoral act and

consequently is gravely illicit.

4. No end believed to be good, such as the use of

stem cells for the preparation of other differentiated

cells to be used in what look to be promising

therapeutic procedures, can justify an intervention

of this kind. A good end does not make right an

action which in itself is wrong.

5. For Catholics, this position is explicitly confirmed

by the Magisterium of the Church which, in the

Encyclical Evangelium Vitae, with reference to the

Instruction Donum Vitae of the Congregation for the
APPENDIX G. 243

Doctrine of the Faith, affirms: “The Church has

always taught and continues to teach that the result

of human procreation, from the first moment of its

existence, must be guaranteed that unconditional

respect which is morally due to the human being in

his or her totality and unity in body and spirit: The

human being is to be respected and treated as a

person from the moment of conception; and therefore

from that same moment his rights as a person must

be recognized, among which in the first place is the

inviolable right of every innocent human being to

life’”(No. 60). (Pontifical Academy for Life, 2000;

emphasis in original) 

Notice that the core of the argument, namely points one and two, is

framed in terms of the rights of the individual embryo. We have

seen this emphasis already in noting Richard Doerflinger’s various

statements on stem cell research. Yet, notice also the claim that we

know the embryo to be an individual with rights on the basis of “a

complete biological analysis.” This is not, of course, the first time

that the Catholic church has made this claim. In the Declaration on

Procured Abortion, the Congregation for the Doctrine of the Faith

claimed that “modern genetic science” confirms the view that “from

the first instant, the programme is fixed as to what this living being

will be: a man, this individual-man with his characteristic aspects

already well determined” (Congregation, “Declaration on Procured

Abortion,”1974, 13). The Instruction on reproductive technology,

Donum Vitae, also makes this claim. “The conditions of science

regarding the human embryo provide a valuable indication for

discerning by the use of reason a personal presence at the moment

of the first appearance of a human life: how could a human individual

not be a human person?” (Donum Vitae, 13).

One reason the Catholic church has played such a major role in

framing the stem cell debate is that, in defending its position, it

combines the two claims we have just noted, neither of which is

explicitly religious. First, the early embryo is an individual person

with rights and, second, the fact that the embryo is an individual

person is confirmed by modern science. Indeed, a fair amount of

the literature that supports embryo research generally can be read

as an attempt to answer the question posed in Donum Vitae: How

can a human individual not be a human person?

Certainly Catholic writers who reject the church’s teaching on

the status of the embryo have responded directly to that question
244 MONITORING STEM CELL RESEARCH

(See Cahill, 1993; Farley, 2001; McCormick, 1994; Shannon, 2001;

Shannon and Walter, 1990), but so too have non-Catholics. For

example, in a statement issued by their ethics committee, what was

then called the American Fertility Society rejected the claim in

Donum Vitae that science supports the personhood of the embryo.

According to the ethics committee “... it remains fundamentally

inconsistent to assign the status of human individual to the human

zygote or early pre-embryo when compelling biological evidence

demonstrates that individuation, even in a primitive biologic sense,

is not yet established. Thus, homologues (identical) twins may result

from spontaneous cleavage of the pre-embryo at some point after

fertilization but prior to the completion of implantation. Furthermore,

during very early development, an embryo is not clearly established

and awaits the differentiation between the trophoblast and the

embryoblast” (American Fertility Society, 1988, 3S).

Arguably, writers like Mary Anne Warren and Bonnie Steinbock,

who distinguish between biological or genetic humanity and moral

humanity, are also at least indirectly answering the question posed

in Donum Vitae (Warren, 1997; Steinbock 2001, 1992). Yet, whether

writers are responding more or less directly to Catholic discourse,

or not at all, the important point is that the stem cell debate has

been remarkably preoccupied with the question of whether the early

embryo is an individual person and whether and how the minute

details of embryological development help us to answer this

question. This is one reason why a fair amount of the ethics literature

on the topic reads like a textbook on embryology.

I want to be clear here: I am not suggesting that the details of

embryological development are unimportant. The maxim from the

field of research ethics applies here as well: bad science is bad ethics.

My point is rather that the preoccupation with the details of early

embryogenesis may lock us even more rigidly into an individualistic

human rights framework than we are in debates about abortion. It

also leads us to frame the debate as fundamentally about one

question, and, indeed, it tempts us to treat the question as if there

is one and only one answer. In this frame of mind, once we have

that answer, there is not a lot more to talk about. Either the early

embryo is a person with the right to life, in which case embryonic

stem cell research is wrong, or the early embryo is not a person

with rights, and then there is no moral reason to object to stem cell

work. Gene Outka has made a similar point in his assessment of

stem cell literature. As Outka puts it, in its starkest form, the

crystallizing question is whether it is cogent to claim that embryonic

stem cell research is morally indistinguishable from murder (Outka,
APPENDIX G. 245

184). The problem with framing the question this way, he says, is

that it “encourages an unfortunate tendency to restrict evaluative

possibilities to a single either/or. Either one judges abortion and

the destruction of embryos to be transparent instances of treating

fetuses and embryos as mere means to other’s ends, or one judges

abortion and embryonic stem cell research to be, in themselves,

morally indifferent actions that should be evaluated solely in terms

of the benefit they bring to others.” (Outka, 2002, 184).

The frame of human rights reinforces this either/or because, as

I noted, a being is either a rights-bearing entity or it is not. I have

argued elsewhere, that this either/or tends to drive people to the

extremes. Either the embryo is a person or it is essentially a kind of

property (Lauritzen, 2001). Although I will not rehearse the argument

for rejecting the two extremes here, it is worth noting that the

rhetoric associated with each extreme does not appear to match

the practice of those who adopt the rhetoric or in fact to match the

considered moral judgments of most Americans on these issues.

I can illustrate my point in relation to the view that the early

embryo is a person with the right to life by describing a cartoon

that hangs on my office door (See Figure A). The cartoon depicts

protestors in front of a stem cell research lab condemning those

who work there as being anti-life. Down the street at the abortion

clinic, the workers are noting how quiet things have gotten at their

facility since the stem cell lab opened. The point of the cartoon, of

course, is that we may soon see protests and demonstrations of the

sort that are common at abortion clinics at facilities that conduct

stem cell research and that there is an irony in the fact that pro-life

advocates would be demonstrating against research being done to

find treatments for Alzheimer’s, Parkinson’s, and other devastating

illnesses. This is not entirely fair to the pro-life community, but it

makes a point.

In fact, I do not have trouble imaging protestors picketing stem

cell research facilities for, as we just noted, when stem cell research

and abortion are evaluated together and when the evaluative option

is a single either/or, then abortion and stem cell research may appear

indistinguishable from murder. Certainly the rhetoric of someone

like Richard Doerflinger has been consistent in condemning both

abortion and stem cell research as equivalent to murder. The cartoon

draws attention to this consistency, even while it questions the

commitment of pro-life advocates to scientific research designed to

promote the quality of life.
246 MONITORING STEM CELL RESEARCH

In one sense, then, the cartoon probes whether there is an

inconsistency between being pro-life and opposed to research an

Alzheimer’s disease, Parkinson’s disease, and other devastating

illnesses. I do not myself think that there is any inconsistency in

being pro-life and opposed to stem cell research, but the cartoon

does point in the direction of a fairly significant disconnect between

the rhetoric and the reality of those opposed to stem cell research

because they believe the early embryo is a person. To see this point,

imagine that, instead of a stem cell clinic, the cartoon depicted on

IVF clinic down the block from the abortion clinic and that the

workers at the abortion clinic are noting how quiet things have

gotten since the IVF clinic opened. The dramatic tension that made

the original cartoon funny would be missing from our revised cartoon

precisely because it is hard to imagine protestors disrupting the

work at IVF clinics. To be sure, the Catholic church and others have

argued that IVF is morally wrong, but the rhetoric condemning IVF

is exceptionally muted compared to that condemning abortion or

stem cell research. Nor has there been a concerted effort to put an

end to IVF practice in this country as there has been in the case of

abortion and stem cell research. Yet, if the embryo is a person from

conception, then participating in IVF as it is practiced in this country,

when early embryos are routinely frozen or discarded or both, is to

be complicit with murder. Why, then, are there no organized efforts

to shut down IVF clinics in this country?7

Indeed, opponents of stem cell research and cloning often write

as if these technologies raise the haunting specter of human embryo

research for the first time. The reality, of course, is that the existence

of in vitro fertilization depended entirely on embryo research and

that every variation or innovation in IVF protocols involves

experimentation on human embryos. Carol Tauer is one of the few

scholars who has pressed this point. As Tauer sees it:

...the entire history of the research leading to the

first successful IVF is the history of attempts to

fertilize oocytes in the laboratory. Eventually these

attempts succeeded, and the first IVF baby was

born, followed by thousands of others in the ensuing

decades.

The ethics literature contains scholarly

discussions as to whether it is ethically permissible

to make use of medical advances that result from

unethical research. This discussion sometimes

focuses on medical research conducted by the Nazis
APPENDIX G. 247

in concentration camps and institutions for retarded,

mentally ill, and handicapped persons. Yet I have

never seen reference to reproductive technologies

in this context. If the fertilization of embryos in

research is a practice that is abhorrent to many or

most people, then would it not be logical to question

the continuing use of the results of such research?

(Even the Catholic Church, which opposes the use

of IVF and most other forms of assisted reproduction,

does not invoke this argument to support its

opposition.) (Tauer, 2001, 153)

If the embryo research associated with IVF points to a problem

of consistency for those who oppose stem cell research because it

involves destroying persons, it is no less problematic for those who

support stem cell research but insist on respecting the embryo and

embrace the distinction between “research” and “spare” embryos.

For as Tauer points out, Robert Edwards, the scientist involved in

the first successful IVF procedure, began studying fertilization nearly

thirty years before Louise Brown was born in 1978, and the first

successful laboratory fertilization of human eggs took place a full

ten years before she was born. Tauer quotes Edwards’ report on

this work: “We fertilized many more eggs and were able to make

detailed examinations of the successive stages of fertilization. We

also took care to photograph everything because we would have to

persuade colleagues of the truth of our discoveries” (Tauer, 154).8

Nor was the creation of these “research” embryos done secretly:

Edwards and Steptoe published their work in the journal, Nature in

1970 (Edwards, Steptoe, and Purdy, 1970).

At the very least, then, there is something of an irony in the

fact that so much attention has been devoted to developing and

defending the distinctions between embryos created solely for

research and embryos left over from IVF procedures, because there

would be no embryos left over from IVF procedures had there not

been embryos created solely for research purposes to develop IVF

in the first place. Given this fact, and given that this fact is no great

secret even though it has not been discussed very much it appears

disingenuous to endorse the distinction between “research” and

“spare” embryos as a way of demonstrating respect for the early

embryo while nevertheless encouraging its destruction.

I have suggested that the fact that so much of the stem cell

debate has been framed in terms of whether the embryo is a person

with rights has been unfortunate because it has cast the debate in
248 MONITORING STEM CELL RESEARCH

sharply individualistic terms and has led to a preoccupation with

embryological development narrowly construed. In addition,

however, framing the debate in terms of embryo status and embryo

rights tends to exaggerate the differences among commentators in

contrast to their similarities. Consider, for example, the response of

conservative Judaism in the United States to this issue. Rabbi Elliot

Dorff has prepared a responsum on stem cell research for the

Rabbinical Assembly Committee on Jewish Law and Standards, and

his responsum is instructive.9

As responsa are, it is structured in terms of relevant questions:

in this case, two questions frame Dorff’s discussion. First, “may

embryonic stem cells from frozen embryos originally created for

purposes of procreation or embryonic germ cells from aborted

fetuses be used for research?” (Dorff, 2002, 1). Second, “may

embryonic stem cells from embryos created specifically for research,

either by combining donated sperm and eggs in a petri dish or by

cloning be used for research?” (1) I think it is noteworthy that the

very questions that frame Dorff’s analysis both reflect and perpetuate

a certain construction of the issue, but at this juncture, my point is

different: given the way the debate has been framed, what most

(non-Jewish) readers of Dorff’s analysis are likely to focus on is the

difference between his treatment of the early embryo and that of

others in the literature. Indeed, even where you might expect to

find and do in fact find on closer inspection similarities between

this Jewish analysis and Catholic reflection on this issue, the first

impression will be that of difference. The reason, of course, is that

our attention is drawn to Dorff’s analysis of the early embryo, and

Jewish views are sharply different about embryo status than those

of the Catholic church and other pro-life opponents of stem cell work.

For example, Dorff points out that, according to the Talmud,

during the first forty days of gestation, the embryo and the fetus are

considered as simply water. From the forty-first day until birth,

Jewish tradition considers the fetus as “the thigh of its mother.”

Moreover, says Dorff:

As it happens, modern science provides good

evidence to support the Rabbis’ understanding. As

Rabbi Immanuel Jakobovits noted long ago, the

Rabbis’ “forty days” is, by our obstetrical count,

approximately fifty-six days, for the Rabbis counted

from the woman’s first missed menstrual flow, while

doctors today count from the point of conception,

which is usually about two weeks earlier. By 56 days
APPENDIX G. 249

of gestation by obstetrical count the basic organs

have already appeared in the fetus. Moreover, we

now know that it is exactly at eight weeks of

gestation that the fetus begins to get bone structure

and therefore looks like something other than liquid.

Indeed, the Rabbis probably came to their conclusion

about the stages of development of the fetus

because early miscarriages indeed looked like

“merely water,” while those from 56 days on looks

like a thigh with flesh and bones. (16)

The contrast with Catholic teaching could hardly be more

striking. Not only are Jewish views of the status of the early embryo

notably different, but Jewish tradition claims scientific validation

for its view of the embryo, just as Catholic tradition does. Not

surprisingly, therefore, where Dorff answers both questions posed

by the responsum in the affirmative, Catholic tradition would answer

both negatively.

These differences are significant and must be attended to, but

it is worth asking whether focusing on these differences does not

obscure important similarities. Consider some of the similarities.

In sketching the Jewish view of stem cell research, Dorff notes that

certain theological commitments are central. He lists at least three

that would be strikingly similar to Catholic and other Christian

theological commitments.

• Our bodies are not ours; they belong to God and God

commands that we seek to preserve life and health.

• All human beings, regardless of ability or disability are

created in the image of God and are therefore to be valued

as such.

• Humans are not God. We are finite and fallible and this fact

ought to promote humility and urge caution.

Now if we focused merely on questions of embryo status, we

would miss entirely these similarities between Catholic and Jewish

views. More importantly, we would miss the fact that these

similarities may underwrite significant moral reflection on stem cell

research that is not rooted in concerns about the early embryo.

For example, Dorff notes that, given Jewish theological and legal

commitments, the provision of health care must be understood as a

communal responsibility. Thus, access to therapies developed
250 MONITORING STEM CELL RESEARCH

through stem cell research is a crucial issue of justice for the Jewish

community. This theme is echoed in Laurie Zoloth’s work on stem

cell research. As Zoloth puts it:

Research done always will mean research foregone.

Will this research help or avoid the problem of access

to health, given that poverty and poor health are so

desperately intertwined in this country?...How can

difficult issues of global justice and fair distribution

be handled in research involving private enterprise?

(Zoloth, 2001, 238)

Surely these are questions that any Catholic moral theologian

would gladly press.

Indeed, attending to the similarities between Zoloth’s work and

Catholic reflection on stem cell research brings us back to Lisa

Cahill’s observation about debates on abortion: they tend to be too

focused on questions of rights individually construed. When one

shifts the frame of analysis, new and different issues and new and

different ways to approach the same issues come into view. Notice,

for example, how close Zoloth and Cahill are on the issue of rights.

According to Zoloth, Jewish tradition foregrounds questions of

“obligations, duties, and just relationships to the other, rather than

the protection of rights, privacy, or ownership of the autonomous

self” (96). This leads Zoloth to ask: “Can the interests of the

vulnerable be heard in our debate?” (105). To be sure, the American

bishops have wanted to emphasize the vulnerability of the early

embryo when they have asked this question, but Catholic tradition,

like Jewish tradition, requires that we ask this question in a way

that is not captured when moral emphasis is merely about individual

rights and personal autonomy.

Or consider another shared sensibility that emerges if we move

away from questions of embryo status, namely a wariness about

the human tendency to hubris and overreaching. Zoloth put this

point eloquently in relation to the biblical story of the Tower of Babel.

She notes a rabbinic midrash on this text: “when a worker was killed,

no one wept, but when a brick fell, all wept.” Zoloth comments on

this midrash as follows:

It was this decentering of the human and reification

of the thing that was the catastrophe that felled the

enterprise... It is not just that they breached a limit

between what is appropriate to create and what is
APPENDIX G. 251

not, the process of the creation must be carefully

mediated, with deep respect for persons over the

temptations of the enterprise. Such a text elaborates

on the tension between repairing the world ... and

acts that claim that the world is ours to control

utterly (Zoloth, 2001, 106-107).

Beyond Questions of Embryo Status

This passage from Zoloth helps to illustrate the point I wish to

make in arguing that the stem cell debate has been too focused on

questions of embryo status and that we must move beyond status

questions if we are fully to do justice to the moral questions raised

by technological developments associated with stem cell work. For

concern about human efforts utterly to control the world is not a

moral worry narrowly tied to status questions.

Let me put this point in the form of a question that has not

typically been asked in the stem cell debate: Is adult stem cell

work as unproblematic as it is often assumed to be? That this is a

productive question is suggested by testimony of Francis Collins

before the President’s Council on Bioethics in December 2002.

Collins was asked to speak about the topic, “genetic enhancements:

current and future prospects” and he specifically addressed the issue

of pre-implantation genetic diagnosis (PGD). Although PGD is

usually understood to involve screening IVF embryos and discarding

unwanted ones, it is also possible to screen gametes. Because

gamete screening may not have broad utility, Collins did not discuss

the issue at length. He did, however, offer an interesting observation

about gamete selection. Focusing on gametes, he says, is useful

because it “isolates you away from some of the other compelling

arguments about moral status of the embryo and allows a sort of

cleaner discussion about what are the social goods or evils

associated with broad alterations in the sex ratio and inequities in

access to that technology” (Collins, 2002, 7). In other words, if in

the future we could screen gametes in the same way that we can

now screen embryos, most of the moral issues raised by PGD would

apply to gamete screening, even though gametes are not embryos.

Might we not make a similar claim about embryonic and adult stem

cell research? Do not many of the most pressing issues raised by

embryonic stem cell technology remain when our focus is adult stem

cell work rather than embryonic stem cell research?

The fact that we do not immediately answer yes to this question,

is testament to how decisively the debate about abortion has
252 MONITORING STEM CELL RESEARCH

structured the stem cell debate. Nevertheless, we need to see that

the answer to this question is yes and we need to see why.

Although I will not try to address all of the issues raised jointly

by embryonic and adult stem cell research, it is worth highlighting

several that I think require fuller discussion particularly with respect

to adult stem cell work than they have yet received.

Commodification Issues

Moral concerns about the commodification of gametes

and embryos have been discussed extensively in the

bioethics literature both in relation to reproductive

technology and in connection with embryonic stem cell work

(See Andrews and Nelkin, 2001; Annas, 1998; Corea, 1986;

Radin, 1996; Resnik, 2002; Ryan, Ethics and Economics,

2001). Suzanne Holland, for example, has discussed the

growing commodification of the human body in the biotech

age. She cites a series of articles published in the Orange

County Register that documents a vast for-profit market in

human body tissue (Holland, 2001, 266). The Register’s

investigative reporters documented that most nonprofit

tissue banks obtain tissue from cadavers donated by family

members of the deceased for altruistic reasons. Most

relatives are not told, and in fact have no idea, that donated

body parts will be sold for profit. As Holland puts it:

The “gift of life” is big business in America.

For a nonprofit tissue bank, one typical

donation can yield between $14,000 and

$34,000 in downstream sales, sometimes far

more than that. “Skins, tendons, heart

valves, veins, and corneas are listed at about

$110,000. Add bone from the same body,

and one cadaver can be worth about

$220,000.” Four of the largest nonprofit

tissue banks told the Orange County

Register that together they expected to

produce sales totaling $261 million in 2000.

(226)

Nor is the issue of downstream commodification restricted to

the sale of donated cadaveric tissue; it arises in relation to IVF

embryos donated for research. As Dorothy Nelkin and Lori Andrews

point out in their book, Body Bazaar, IVF patients are not generally
APPENDIX G. 253

told what the research involving their donated embryos will include.

Many will be unaware that their embryos will be used to develop

commercial stem cell lines (Nelkin and Andrews, 2001, 35).

It is significant that even the most vocal advocates of procreative

liberty and laissez-faire arrangements in reproductive matters recoil

from the prospect of selling human embryos. Yet, although the

commodification of tissue may be particularly troubling when it

involves embryos, if there is a problem with commodifying and

commercializing human tissue, it is a problem we confront with adult

stem cell research as well as with hES cell work.10 Lori Knowles

has made a similar point about being consistent in our moral

judgments about commodifying embryos. She notes that fears about

commodifying reproduction have led many to oppose the sale of

embryos and to reject the idea that couples who donate embryos

have any proprietary interest in the result of the research done with

their embryos. As Knowles puts it, “if it is wrong to commercialize

embryos because of their nature, then it is wrong for everyone. It is

simply inconsistent to argue that couples should act altruistically

because commercializing embryos is wrong, while permitting

corporations and scientists to profit financially from cells derived

by destroying those embryos” (Knowles, 1999, 40).

Knowles draws attention here to the fact that there is a tension

between our moral and legal traditions as they apply to

developments brought about by cloning, stem cell research, and

the existence of ex utero human embryos, among other technological

breakthroughs. For example, we patent embryonic stem cell lines,

thereby insuring massive profits for patent holders, while decrying

the commodification of embryonic life. Knowles is correct that there

is a tension between our profit-based medical research model and

our commitment to altruism and the access to health care that a

commitment to justice demands. Adult stem cell research, of course,

raises very similar issues, because the same tension exists between

the need for proprietary control of technology and the need for

affordable access. For example, patents have been sought and

granted for human adult stem cells work as well as for embryonic

stem cell technologies. According to a study on the patenting of

inventions related to stem cell research commissioned by the

European Group on Ethics in Science and New Technologies, as of

October 2001, two thousand twenty-nine patents were applied or

granted for stem cells and 512 patents were applied or granted for

embryonic stem cell work (Van Overwalle, 2002, 23; see also McGee

and Banger, 2002). As a result, access to therapies developed from
254 MONITORING STEM CELL RESEARCH

adult stem cell research is likely to be as serious an issue as access

to embryonic stem cell therapies.

Indeed, issues arising from commodification of both adult and

embryonic stem cells are likely to dominate the next phase of the

debate, if only because corporate interest in this work, both

nationally and internationally, is so strong (Hill, 2003). Noting that,

as of 2002, “a dozen biotech companies have entered the stem cell

industry and have invested millions of dollars,” David Resnik

suggests that the next stage of the stem cell debate will involve a

battle over property rights relating to stem cells (Resnik, 2002, 130-

31).

To be sure, the battle over property rights raises important legal

and policy question, but it also raises ethical questions as well. For

example, Resnik provides the following table of possible ethical

objections to patenting stem cells.

Although the ethical objections to commodifying stem cell work

can not be sorted out as neatly as this table suggests, Resnik

correctly identifies a variety of such objections. I will not try to

defend it here, but my own view is that the problem is not just that

stem cells and their products may be commodified, but that market

rhetoric may come to dominate the discussion (and practice) of

regenerative medicine in a way that is dehumanizing. That is,

Deontological Objections to

Property Rights in ES cells

Consequentialist Objections

to Property Rights in ES cells

ES cells (and their products)

should not be treated as

property; they should be

viewed as having inherent

dignity or respect.

Treating ES cells as

property will have dire

social consequences, such

as exploitation, destruction

of altruism, and loss of

respect for the value of

human life.

ES cells should not be

treated as private property;

they are res communis or

common property.

Treating ES cells as

property will undermine

scientific discovery and

technological innovation in

the field of regenerative

medicine. (Resnik, 2002, 139)
APPENDIX G. 255

market-rhetoric may lead us ultimately to think of humans as

artifacts. In short, I take very seriously Margaret Radin’s argument

that the rhetoric in which we conceive our world affects who and

what we are (Radin, 1996, 82).11 At the same time, however, stem

cell research is most likely to bear therapeutic fruit, if there is a

market in stem cells and their products. The pressing moral

question, then, is how do we promote the benefits that stem cell

research may yield without succumbing to a market rhetoric that

reduces humans to commodities?

Several writers have suggested that one answer may be to

promote greater governmental regulation. For example, Holland

argues for moving beyond a policy of restricting federal funding of

stem cell research but allowing an unregulated private market in

this field to active regulation to curb the private sector’s work on

stem cells. Lori Knowles suggests that the United States might

adopt a body like Canada’s Patented Medicine Prices Review Board

as a way to allow a market to function, but with oversight that would

provide access to potential cells for further research and price

controls of products to insure widespread access (Knowles, 1990,

40). George Annas has suggested that we need to establish a federal

Human Experimentation Agency to regulate in the area of human

experimentation. (Annas, 1998, 18). As Annas puts it:

Virtually all those who have studied the matter have

concluded that a broad-based public panel is needed

to oversee human experimentation in the areas of

genetic engineering, human reproduction,

xenografts, artificial organs, and other boundarycrossing experiments. (19)

Francis Fukuyama has argued that a new agency with a mandate to

regulate biotechnology on broad grounds and in both the public

and private sector may be needed (Fukuyama, 2002, 215). Vanessa

Kuhn argues that “it is time to put in place legislation that will deter

stakeholders from licensing their technology to one exclusive

distributor and thus creating a monopoly market, which would set

artificially-high prices and lead to less access for the sick especially

for the uninsured, the poor, and the elderly” (Kuhn, 2002). To that

end, Kuhn identifies four possibilities:

• Development of a new kind of patent.

• Set limits on exclusive licensing through compulsory

licensing.
256 MONITORING STEM CELL RESEARCH

• Lower the lifespan of hES cell patents

• Set stricter guidelines for hES patent utility (2)

I do not have the expertise to make policy recommendations,

but let me stress two points. First, the policy issues with regard to

commodifiying adult stem cell work will be as vexing as those

confronting regulation of embryonic stem cells. Second, although

these questions may at first appear to be strictly legal or largely

political matters, they involve serious value judgments about the

common good that are every bit as morally vital as questions about

the status of the embryo. I thus agree with Gene Outka, that not to

confront directly questions about how stem cell research will be

organized, financed, and overseen is a kind of ethical failure (Outka,

2002, 177). Obviously, for example, the institutional arrangements

for conducting stem cell research have implications for the questions

of justice we previously noted. The fact that so much stem cell

research is being done by private corporations insures future conflict.

On the one hand, corporations have fiduciary obligations to their

shareholders and will therefore seek to control access to stem cell

lines or therapies developed from those lines through patent

protection and licensing agreements. On the other hand, such a

system is likely to further widen the gap between the health care

haves and have-nots (See Lebacqz 2001; McLean 2001).

Moreover, as Karen Lebacqz notes, if justice is an important

consideration in deliberations about stem cell work, then it ought

to shape the research agenda. The example she gives to make this

point is worth noting. Just as with organ transplants, tissue rejection

may be a major problem for stem cell therapies. This is one reason

that the prospect of combining stem cell work with somatic cell

nuclear transfer (SCNT) has been so enticing. With this combination,

you could in theory develop tissue that would be completely

histocompatible. Nevertheless, according to Lebacqz, developing

stem cell therapies with SCNT is “highly questionable,” if justice is

a primary consideration. The reason is that unique cell lines would

need to be created for each patient, and that is likely to be very

expensive and thus unaffordable for many. Although it would

certainly be less expensive, the same would likely be true for adult

stem work. For that reason, rather than pursuing an individualized

approach to stem cell research, concerns about affordable access to

new therapies might urge the pursuit of universal donor cell lines.
APPENDIX G. 257

Embodiment, Boundary Issues, and Human Nature

In discussing the debate about embryo status, I focused

primarily on the contested question of whether the early embryo is

a person with the right to life. We saw that this question tends to

lead to the mobilization of minute details of embryological

development to support one’s view of the embryo. Yet, if attention

to embryo status tends to focus us on the microscopic, viewing stem

cell research through the lens of embryological development can

also have a kind of telescopic function through which larger issues

come into view.12 For example, Catherine Waldby and Susan Squier

argue in a forthcoming issue of the journal Configurations that

focusing on stem cells and embryonic life leads us fundamentally to

question what it means to be human. According to Waldby and

Squier:

Stem cell technologies have profound temporal

implications for the human life course, because they

can potentially utilize the earliest moments of

ontogenesis to produce therapeutic tissues to

augment deficiencies in aging bodies. Hence they

may effect a major redistribution of tissue vitality

from the first moments of life to the end of life. In

doing so however they demonstrate the perfect

contingency of any relationship between embryo

and person, the non-teleological nature of the

embryo’s developmental pathways. They show that

the embryo’s life is not proto-human, and that the

biology and biography of human life cannot be read

backwards into its moment of origin. (Waldby and

Squier, forthcoming)

The claim that there is a perfect contingency in the relationship

between embryo and person may at first appear to be just another

“microscopic” claim about embryo status, but it is clear that Waldby

and Squier mean to imply much more in asserting that the embryo’s

development is non-teleological. In effect, they reject the notion

that there is a meaningful trajectory to human life. What was killed,

they say, when stem cells were first derived from the inner cell mass

of a blastocyte was not a person, but a “biographical idea of human

life, where the narrative arc that describes identity across time has

been extended to include the earliest moments of ontogeny”

(Waldby and Squier, forthcoming).
258 MONITORING STEM CELL RESEARCH

That much more is at stake here than the question of whether

the embryo is a person is clear if we attend to the notion of a

trajectory of a human life. Gilbert Meilaender, for example, has

argued that our attitudes toward death and dying are importantly

shaped by our conception of what it means to have a life (Meilaender

1993). Indeed, according to Meilaender, two views of what it means

to have a life of what it means to be a person have been at war with

each other within the field of bioethics over the past thirty years

and these views underwrite sharply different views not just about

the issues of abortion or euthanasia that are implicated here but

with regard to practically every moral issue we might confront in

the field of bioethics.

On Meilaender’s view, having a life means precisely that there

is a trajectory that traces a “natural pattern” in embodied life that

“moves through youth and adulthood toward old age and, finally,

decline and death” (29). As he puts it elsewhere in this essay, “to

have a life is to be terra animata, a living body whose natural history

has a trajectory” (31). Although Meilaender develops the notion of

a natural trajectory of bodily life primarily to address the issue of

euthanasia and not stem cell research, his talk of “natural history,”

“natural pattern,” and “natural trajectory” draws attention to one

of the most significant issues raised by stem cell research and related

technologies. Does stem cell research undermine the very notion of

a human life constrained by natural bodily existence? The example

on which Meilaender focuses here is instructive for thinking about

the broad implications of stem cell research in this regard. If stem

cell therapies fundamentally alter our sense of a natural pattern to

aging would they not also fundamentally alter our sense of what it

means to be human? Meilaender’s answer is that such a change

would fundamentally affect what it means to be human precisely

because we are embodied creatures and for that reason our identity

is tied to the body and the body’s history.

Leon Kass has made a similar point recently in reflecting on

the prospect that regenerative medicine might significantly lengthen

the human life span (Kass, 2003). He, too, invokes the notion of a

natural trajectory, one that stem cell research may undermine.

Although it is possible to approach the prospect of extending the

human life-span in an abstract way, he says, to think of what such a

change would mean experientially is to recognize that “the ‘lived

time’ of our natural lives has a trajectory and a shape, its meaning

derived in part from the fact that we live as links in the chain of

generations” (13). Indeed, says Kass, without something like the

natural trajectory of bodily life that currently exists, the relationship
APPENDIX G. 259

between the generations would be decidedly different, and probably

not better. “A world of longevity,” writes Kass, “is increasingly a

world hostile to children” (13). Walter Glannon has argued that, at

the very least, increased longevity would increase competition for

scarce resources between older and younger generations. According

to Glannon, “it is at least intuitively plausible that an over populated

world with substantially extended human lives and scarce resources

could adversely affect the survival and reproductive prospects of

the young and harm them by thwarting their interest in being healthy

enough so that they could survive and procreate” (Glannon,

“Extending,” 347).13

Francis Fukuyama has also suggested some of the reasons why

increased longevity may imperil children, but he also notes that our

relationship to death may change as well (See 2002, ch. 4). “Death,”

he says, “may come to be seen not as a natural and inevitable aspect

of life, but a preventable evil like polio or measles. If so, then

accepting death will appear to be a foolish choice, not something to

be faced with dignity or nobility” (Fukuyama, 2002, 71).

Sometimes the question of the transformative possibilities that

come with stem cell research is raised even more starkly when the

question asked is not how may stem cell work affect what it means

to be human, but instead: Does stem cell research open the door to

a post human future? This is a point Waldby and Squier raise

explicitly when they discuss the combination of genetic engineering

and stem cell therapy. They suggest, for example, that

xenotransplantation forces us to confront the prospect of

transgressing species boundaries.14 The conclusion of their paper

is worth quoting in full:

Thus the ontological status of the embryo is not the

only thing in question. The ontological status of the

graft recipient must be negotiated, when the graft

involves genetically-engineered stem cells from

another species. And the ontological status of the

illnesses to which biomedical technology responds

is equally challenged, in an endless regression, as

the division between veterinary and human

medicine, or between zoonoses (diseases humans

can catch from animals) and what has recently been

dubbed humanooses, is called into question. This

increasingly permeable, increasingly constructed

barrier between human and animal presents us with

another form of life to negotiate, whose boundary
260 MONITORING STEM CELL RESEARCH

lies not between silicon and carbon, but rather

between steps in the evolutionary ladder or the

branching development tree of phylogenetic

lifeforms. Stem cell technologies thus challenge

both the temporal and spatial boundaries of human

life, both our biography and our biological niche,

giving a much broader meaning to the questioning

of embryonic personhood. (Waldby and Squier,

forthcoming)

Regrettably, with some notable exceptions, the ethical debate

about stem cell research has not taken up in a sustained way what

it would mean to pursue stem cell therapies that might significantly

undermine the notion of a natural human life or erode the boundary

between human and non-human species. When the issue is framed

in terms of the status of the embryo, the question tends to be

whether the research should be conducted at all. By contrast, when

the issue is framed in terms of adult stem cell work, the question is

not whether, but how and with what consequences. Yet, that is a

question we have not systematically answered. Given the potential

for good embedded in the prospects of adult stem cell research, it is

not surprising that there appears to be widespread and largely

uncritical acceptance of adult stem cell research. But, if the promise

of stem research is as revolutionary as is often claimed, we are going

to need a much more expansive discussion of stem cell research

both embryonic and adult than we have had heretofore. Obviously,

I cannot explore this more expansive horizon in any detail in this

report, but let me in closing suggest one direction we need to

explore.

To signal the decisive break that I think we may need from the

usual bioethics frame, I want to draw attention to Martha

Nussbaum’s recent article in the journal Daedalus entitled,

“Compassion & Terror” (Nussbaum, 2003). Discussing Euripides’

play, Trojan Women, Nussbaum reflects on the fact that the Greek

poets returned obsessively to the sacking of Troy and the acts of the

“rapacious and murderous Greeks.” She explores the poets’

compassionate imagining of the fate of Trojan women and children

to reflect on the conditions and limits of a compassionate vision.

Although Nussbaum is ultimately concerned about engendering a

compassionate vision for Americans in the face of terror and

particularly compassion for innocent women and children far from

our shores, her analysis of compassion is thought-provoking in

relation to stem cell research.
APPENDIX G. 261

Nussbaum notes that compassion is a complex emotion

requiring a series of judgments involving another person’s suffering

or lack of well-being. We must judge that someone has been harmed,

that the harm is serious, and that it was not deserved. Moreover,

says Nussbaum, Western tradition has stressed what could be called

the “judgment of similar possibilities.” In other words, “we have

compassion only insofar or we believe that the suffering person

shares vulnerabilities and possibilities with us” (Nussbaum, 2003,

15).

Now surely in just about everyone’s catalogue of human

vulnerabilities are illness, old age, and death. Yet, as we have just

seen, stem cell research might significantly transform the “human”

experience of illness and death, at least for some. If stem cell

therapies were to erode the notion of human nature or species

membership, might they not also erode some basic moral

sensibilities? Mary Midgley, for example, has argued that both the

notion of human nature and that of human rights are importantly

tied to membership in our species because rights are “supposed to

guarantee the kind of life that all specimens of Homo sapiens need”

(Midgley, 2000, 9).

Although Nussbaum avoids the language of human nature, it

is precisely this sort of point that she highlights when she argues

that compassion requires the belief that others share vulnerabilities

and possibilities with us. Indeed, like Midgley, Nussbaum ties the

notion of universal human rights to important human functions and

capabilities. The basic idea, she says, is to ask what constitutes

the characteristic activities of human beings. In other words:

‘What does the human being do, characteristically,

as suchand not, say, as a member of a particular

group, or a particular local community?’ To put it

another way, what are the forms of activity, of doing

and being, that constitute the human form of life and

distinguish it from other actual or imaginable forms

of life, such as the lives of animals and plants, or, on

the other hand, of immortal gods as imagined in

myths and legends (which frequently have precisely

the function of delimiting the human)? (Nussbaum,

1995, 72)

Nussbaum notes that this inquiry proceeds by examining a wide

variety of self-interpretations and that comparing characteristic

human activities with non-human activities and, through myths and

stories, comparing humans and the gods is particularly helpful. For
262 MONITORING STEM CELL RESEARCH

one thing, such an inquiry helps us to define limits that derive from

membership in the world of nature.

Indeed, although Nussbaum is particularly attentive to the wide

variety of cultural interpretations of what it means to be human,

she insists that to ground any essentialist or universal notion of

human rights, one must attend to human biology. Although her

account of the human is neither ahistorical nor a priori, it is linked

to an “empirical study of a species-specific form of life” (1995, 75).

When she develops her account of central human capabilities, she

begins with the body. She writes:

We live all our lives in bodies of a certain sort, whose

possibilities and vulnerabilities do not as such

belong to one human society rather than another.

These bodies, similar far more than dissimilar (given

the enormous range of possibilities) are our homes,

so to speak, opening certain options and denying

others, giving us certain needs and also certain

possibilities for excellence. The fact that any given

human being might have lived anywhere and

belonged to any culture is a great part of what

grounds our mutual recognitions; this fact, in turn,

has a great deal to do with the general humanness

of the body, its great distinctness from other bodies.

The experience of the body is culturally shaped, to

be sure; the importance we ascribe to its various

functions is also culturally shaped. But the body

itself, not culturally variant in its nutritional and other

related requirements, sets limits on what can be

experienced and valued, ensuring a great deal of

overlap. (Nussbaum, 1995, 76)

Nussbaum’s work both in identifying the judgments that

underwrite compassion and in tying an account of rights to human

function and capabilities that are presumably universal highlights

what is at stake, not merely with stem cell research but with a

growing list of biotechnological developments which appear to

destabilize the concept of human nature and which require that we

think carefully and hard about what it might mean for some humans

to have access to these technologies while other humans do not.15

At the very least, the combination of what Rabinow describes as

the biologicalization of identity around genetics rather than gender

and race with the possibility of manipulating that genetic identity

for those with the money or power to do so does not bode well for
APPENDIX G. 263

securing wide-spread compassion across economic or technological

divides. Even more important, however, is the recognition that the

very notion of human rights may ultimately rest on the idea (and

what, until recently has always been the reality) of a natural human

condition that is relatively stable.

I believe that Nussbaum is correct when she claims that

inquiring into characteristic human activities and comparing these

to non-human activities helps us to define limits and thereby to

promote human flourishing. Unfortunately, what Susan Squier calls

the “pluripotent rhetoric” of stem cell research is that of limitless

possibilities (Squier, Liminal Lives). The ultimate limit, of course, is

death and yet even this limit appears illusory in some visions of our

biotech future.

It is worth noting in closing that William Safire’s New Year’s

Day column at the dawn of the twenty-first century, in January 2000,

was entitled “Why Die?” The longing behind this question is neither

new nor unfamiliar. What is new is that this longing to escape the

vulnerabilities and limitations of the body is united with a technology

that holds out the prospect of fundamentally changing that body.

Yet, I agree with Gerald McKenny that we need to ask whether we

wish to accept and promote a view of bodily vulnerability as merely

an obstacle to human flourishing, which ought to be overcome at

any cost (McKenny, 1998, 223).

Although a longing for invulnerability is perhaps a

quintessentially human trait, and although the quest to reduce the

human suffering wrought by illness and disease is morally admirable,

there is no mistaking the hubris behind the question, Why Die?

Opponents of stem cell research have, from the start, argued that

there is a kind of idolatry in a science that would reduce the human

embryo to just so much biological material. What I have tried to

show in this report is that such concerns need not be limited to

those who think that the early embryo is fully a person. Nor should

this kind of concern be limited to those who oppose stem cell

research. I do not think the early embryo is a person and I believe

that both embryonic and adult stem cell research should go forward

under a system of strict regulation. Nevertheless, I confess to being

haunted by the passages with which I began and I believe that

future debates on stem cell research must take very seriously the

worries about commodification and the possibility of fundamentally

changing the trajectory of a human life.16
264 MONITORING STEM CELL RESEARCH

FIGURE A

Jimmy Margulies, Editorial Cartoonist

The Record, Hackensack, New Jersey

Cartoon used with permission of the artist.
APPENDIX G. 265

Endnotes

1 Given the current status of technology, deriving human embryonic stem cells

requires destroying embryos. If the cells could be derived without the destruction

of embryos or if parthenogenetically stimulated eggs produced stem cells, issue of

status would almost certainly fade. Nevertheless, serious ethical issues would still

remain. This is one reason I believe it is a mistake to focus narrowly on embryo

status.

2

 Gene Outka has argued that there is an “internal coherence” to views of the

embryo, issues of complicity, and views on adult stem cell research (Outka, 2002).

3

 Although the HERP report claimed that “it is not the role of those who help

form public policy to decide which of these views [of the embryo] is correct,” there

is little doubt that the panel adopted the pluralistic view. For that reason, most

commentators found the above claim disingenuous.

4

 Compare, for example, the various statements that Richard M. Doerflinger

has made on the U. S. bishops’ behalf. See Doerflinger, 1988, 1998, 1999, 2001.

Margaret Farley has argued that the Catholic preoccupation with abortion has eroded

its credibility on other important social issues, including stem cell research. (See

Farley, 2000).

5

 Another debate that is at least partly shaped by focusing on embryo status

revolves around the question of complicity. For example, supporters of stem cell

research may harbor a residual uneasiness about endorsing the destruction of human

embryos, at least if the number of articles in the literature explaining the concept of

complicity with wrongdoing is any indication. John Robertson, Ronald Green, and

Thomas Shannon, have all written on the issue of cooperation with evil in relation

to stem cell research. (Robertson, 1999; Green, 2002; Shannon, 2001; see also Kaveny,

2000; and Gilliam, 1997). To be sure, the issue of complicity or cooperation with

wrongdoing is a very traditional one in moral philosophy and theological ethics.

Still, if the early embryo does not deserve the respect accorded persons and if

destroying the embryo is compatible with respecting it, then deriving stem cells is

not an act of wrongdoing and issues of complicity do not arise.

6

 Cahill also emphasizes the way a liberal individualist view of the person

discounts the significance of embodiment. I will return to this point below.

7

 That both those who view the embryo as a person and those who do not but

who insist on respect for the embryo, have been remarkably cavalier with regard to

the use of embryos in IVF programs can be seen by the fact that there are currently

over 400,000 embryos frozen in the United States, a number we did not even know

until quite recently (Hoffman et al. in association with The Society for Assisted

Reproductive Technology and RAND, 2003).

8

 In a commentary published in Nature, in September 2001, Edwards writes:

“On the verge of clinical application, stem cells offer a startlingly fundamental

approach to alleviating severe incurable human maladies. Fondly believed to be a

recent development, they have in fact been part and parcel of human in-vitro

fertilization (IVF) from as long ago as 1962.”
266 MONITORING STEM CELL RESEARCH

9

 Dorff’s responsum was accepted by the Committee on Law and Standards by

a vote of twenty-two to one in March 2002. On the basis of Dorff’s responsum the

Rabbinical Assembly passed a resolution in April 2003 supporting stem cell research

for therapeutic purposes. (Resolution in Support of Stem Cell Research and

Education, April 2003: available at www.rabassembly.org.)

10 Alpers and Lo draw the distinction between commodification and

commercialization as follows: “The issue of commodification involves treating either

human beings or symbols of human life as merchandise or vendible goods. ...

Commercialization refers to the practice of realizing large profits from the

development and sale of techniques or products that involve distinctive human

material, such as embryos, eggs, or tissue” (Alpers and Lo, 1995).

11 Radin quotes Georg Lukács on the reification of commodities and the effects

on human consciousness. Lukács writes: “The transformation of the commodity

relation into a thing of ‘ghostly objectivity’ cannot therefore content itself with

reduction of all objects for the gratification of human needs to commodities. It

stamps its imprint upon the whole consciousness of man; his qualities and abilities

are no longer an organic part of his personality, they are things which he can ‘own’

or ‘dispose of’ like the various objects of the external world” (Radin, 1996, 82).

When we think about genes for enhancing memory or muscle mass, it is worth

keeping in mind Lukács’s claim that human qualities and abilities may come to be

thought of as objects for sale in the external world.

12 Erik Parens has noted the importance of attending to the big picture raised

by stem cell work and how the politics of abortion has obscured that picture. See

Parens, 2000.

13 In another essay, Glannon argues that substantially increasing the human

life span would profoundly affect issues of personal identity and thus a sense of

personal responsibility for one’s action. He ties his argument in interesting ways

to the biology of memory function (Glannon, “Identity”). For a classic philosophical

discussion of the problems associated with immortality, see Williams, 1973.

14 Although he is not discussing stem cell research explicitly, Paul Rabinow’s

discussion of technological change wrought during the last two decades is worth

noting. He writes: “In the United States, for example, in the last two decades,

while the most passionate value conflicts have raged around abortion, a general

reshaping of the sites of production of knowledge has been occurring. To cite the

biotechnology industry, the growing stock of genomic information, and the simple

but versatile and potent manipulative tools (exemplified by the polymerase chain

reaction) is to name a few key elements; a more complete list would include the

reshaping of American universities, the incessant acceleration in the computer

domains, and the rise of ‘biosociality’ as a prime locus of identity a biologicalization

of identity different from the older biological categories of the West (gender, age,

race) in that it is understood as inherently manipulable and re-formable” (Rabinow,

1999, 13). A couple of pages later, he writes: “My analysis points to the fact that

the basic understanding and practices of ‘bare life’ have been altered. The genome

projects (human, plant, animal, microorganismic) are demonstrating a powerful

approach to life’s constituent matter. It is now known that DNA is universal among

living beings. It is now known that DNA is extremely manipulable. One consequence

among many others is that the boundaries between species need to be rethought;

transgenic animals made neither by God nor by the long-term processes of evolution

now exist (16).
APPENDIX G. 267

15 For a science fiction exploration of this theme of selected genetic

enhancement, species boundary crossing see Octavia Butler, Dawn.

16 A number of people either helped with the preparation of this report or

provided feedback on an earlier draft. Thanks to Christa Adams, Diana Fritz Cates,

William FitzPatrick, James L. Lissemore, Charlie Ponyik, Mary Jane Ponyik, Kristie

Varga, Lisa Wells, and the ethics writers group at John Carroll University.

References

Alpers, Ann, and Bernard Lo. “Commodification and Commercialization in Human

Embryo Research.” Stanford Law and Policy Review, vol. 6, no. 2 (1995): 40.

American Association for the Advancement of Science and Institute for Civil Society.

Stem Cell Research and Applications Monitoring the Frontiers of Biomedical

Research. (November 1999).

American Fertility Society Ethics Committee. “Ethical Considerations of the New

Reproductive Technologies in Light of Instruction on the Respect for Human

Life in Its Origin and on the Dignity of Procreation.” Fertility and Sterility, vol.

49, (1988): Supplement.

Andrews, Lori, and Dorothy Nelkin. Body Bazaar: The Market for Human Tissue in

the Biotechnology Age. New York, NY: Crown Publishers, 2001.

Annas, George J. Some Choice: Law, Medicine, and the Market. New York: Oxford

University Press, 1998.

Butler, Octavia E. Dawn. Boston, MA: Warner Books, 1997.

Cahill, Lisa Sowle. “Abortion, Autonomy, and Community.” In Abortion: A Reader.

Ed. Lloyd Steffen (Cleveland: Pilgrim Press, 1996), 359-72.

______. “The Embryo and the Fetus: New Moral Contexts.” Theological Studies, vol.

54 (1993): 124-42.

Callahan, Daniel. Hastings Center Report, vol. 25, no. 1 (1995): 39.

Collins, Francis S. “Testimony before the Presidents Council on Bioethics.”

(December 13, 2002). Available at: http://www.bioethics.gov.

Congregation for the Doctrine of the Faith, “Declaration on Procured Abortion,”

1974.

______. “Donum vitae,” 1987.

Corea, Gena. The Mother Machine. New York: Harper and Row, 1985.

Doerflinger, Richard M. “Hearing on Stem Cell Research,” Testimony of Richard M.

Doerflinger on behalf of the Committee for Pro-Life Activities United States

Conference of Catholic Bishops before the Subcommittee on Labor, Health,

and Human Services, and Education Senate Appropriations Committee (July

18, 2001): available at http://www.usccb.org/prolife/issues/bioethic/

stemcelltest71801.htm.
268 MONITORING STEM CELL RESEARCH

______. “Public Comment before the National Bioethics Advisory Commission,”

National Conference of Catholic Bishops (April 16, 1999): available at http://

www.usccb.org/prolife/issues/bioethic/nbac.htm.

______. “Hearing on Legal Status of Embryonic Stem Cell Research,” Testimony of

Richard M. Doerflinger on behalf of the Committee for Pro-Life Activities,

National Conference of Catholic Bishops before the Senate Appropriations

Subcommittee on Labor, Health, and Education (January 26, 1999): available

at http://www.usccb.org/p;rolife/issues/bioethic/test99.htm.

______. “Testimony of Richard M. Doerflinger on behalf of the Committee for Pro-Life

Activities National Conference of Catholic Bishops before the Senate

Appropriations Subcommittee on Labor, Health and Education,” Hearing on

Embryonic Cell Research (December 2, 1998): available at http://www.usccb.

org/prolife/issues/bioethics/1202.htm.

______. “Public Comment: NIH Human Embryo Research Panel.” United States

Conference of Catholic Bishops (February 2, 1994).

______. “Ethical Problems in the Use of Tissues from Abortion Victims,” Public

Comment, Meeting of the Human Fetal Tissue Transplantation Research Panel,

(Bethesda, Maryland: National Institutes of Health, September 14, 1988):

available at http://www.usccb.org/prolife/issues/ fetalresearch/rd9 1488.htm.

Dorff, Elliot N. “Stem Cell Research.” Final Draft (August 2002).

Edwards, R. G. “IVF and the History of Stem Cells.” Nature, vol. 413 (September

27, 2001): 349-51.

Edwards, Steptoe, and Purdy. “Fertilization and Cloning In Vitro of Preovulation

Human Oocytes.” Nature 277 (1970): 1307-1309.

Farley, Margaret A. “Roman Catholic Views on Research Involving Human

Embryonic Stem Cells.” The Human Embryonic Stem Cell Debate: Science,

Ethics, and Public Policy. Cambridge, MA: The MIT Press, 2001. 113-18.

______. “The Church in the Public Forum: Scandal or Prophetic Witness?” The Catholic

Theological Society of America, Proceedings of the Fifty-fifth Annual

Convention, Vol. 55 (June 8-11, 2000): Presidential Address, 87-101.

FitzPatrick, William. “Surplus Embryos, Nonreproductive Cloning, and the Intend/

Foresee Distinction.” Hastings Center Report (May-June 2003): 29-36.

Fukuyama, Francis. Our Posthuman Future. New York: Farrar, Straus and Giroux,

2002.

Gearhart, John. “New Potential for Human Embryonic Stem Cells.” Science, vol.

282 (November 6, 1998): available at: http://www.sciencemag.org/

cgi/content/full/282/5391/1061.

Geron Ethics Advisory Board. “Research with Human Embryonic Stem Cells: Ethical

Considerations.” Hastings Center Report, vol. 29, no. 2 (March-April 1999).

Gilliam, Lynn. “Arguing by Analogy in the Fetal Tissue Debate.” Bioethics, vol. 11,

no. 5 (1997): 397-412.
APPENDIX G. 269

Glannon, Walter. “Extending the Human Life Span.” Journal of Medicine and

Philosophy, vol. 27, no. 3 (2002): 339-54.

______. “Identity, Prudential Concern, and Extended Lives.” Bioethics, vol. 16, no. 3

(2002): 266-83.

Green, Ronald M. “Benefiting from ‘Evil’: An Incipient Moral Problem in Human

Stem Cell Research.” Bioethics, vol. 16, no. 6 (2002): 544-56.

______. The Human Embryo Research Debates: Bioethics in the Vortex of Controversy.

New York: Oxford University Press, 2001.

______. “At the Vortex of Controversy.” Kennedy Institute of Ethics Journal vol. 4, no.

4 (1994): 345-56.

Hall, Stephen S. Merchants of Immortality: Chasing the Dream of Human Life

Extension. New York: Houghton Mifflin Company, 2003.

Hoffman, David I. et al. “Cryopreserved Embryos in the United States and Their

Availability for Research.” Fertility and Sterility, vol. 79, no. 5 (May 2003): 1063-

69.

Holland, Suzanne. “Contested Commodities at Both Ends of Life: Buying and Selling

Gametes, Embryos, and Body Tissues,” Kennedy Institute of Ethics Journal,

vol. 11, no. 3 (2001): 266.

Kass, Leon R. “Ageless Bodies, Happy Souls: Biotechnology and the Pursuit of

Perfection.” The New Atlantis, no. 1 (spring 2003). ______. Life, Liberty and the Defense of Dignity: The Challenge for Bioethics. San

Francisco, CA: Encounter Books, 2002.

Kaveny, M. Cathleen. “Appropriation of Evil: Cooperation’s Mirror Image,”

Theological Studies, vol. 61, no. 2 (June 2000): 280-313.

Knowles, Lori P. “Property, Progeny, and Patents.” Hastings Center Report, vol 29.

no. 2 (1999): 38.

Kuhn, Vanessa. “Stem Cells: Equity or Ownership?” The American Journal of

Bioethics, vol. 2, no. 1 (winter 2002): 2.

Lauritzen, Paul. “Neither Person nor Property: Embryo Research and the Status of

the Early Embryo,” America (March 26, 2001).

______. Cloning and the Future of Human Embryo Research. New York: Oxford

University Press, 2001.

______. “Expanding the Debate over Stem Cell Research,” Dialog: A Journal of Theology,

vol. 41, no. 3, (fall 2002): 238-39.

Lebacqz, Karen. “Stem Cells and Justice,” Dialog, vol. 41, no. 3 (fall 2002). ______. “On the Elusive Nature of Respect.” The Human Embryonic Stem Cell Debate:

Science, Ethics, and Public Policy. Cambridge, MA: The MIT Press, 2001. 149-

162.

Lewis, C. S. The Abolition of Man. New York: The Macmillan Company, 1947.
270 MONITORING STEM CELL RESEARCH

Macklin, Ph.D., Ruth. “Ethics, Politics, and Human Embryo Stem Cell Research.”

Women’s Health Issues, vol. 10, no. 3 (May/June 2000): 111-15.

Magnus, David, Arthur Caplan, and Glenn McGee, ed. In Who Owns Life? Amherst,

New York: Prometheus Books, 2002.

McCormick, R. A. “Who or What is the Preembryo?” In Corrective Vision:

Explorations in Moral Theology. Kansas City: Sheed & Ward, 1994.

McGee, Glenn, and Elizabeth Banger. “Ethical Issues in the Patenting and Control

of Stem Cell Research.” In Who Owns Life? Ed. David Magnus, Arthur Caplan,

and Glenn McGee. Amherst, New York: Prometheus Books, 2002. 243-264.

McKenny, Gerald P. “Enhancements and the Ethical Significance of Vulnerability.”

In Enhancing Human Traits: Ethical and Social Implications. Washington, D.C.:

Georgetown University Press, 1998. 222-37.

McKibben, Bill. Enough: Staying Human in an Engineered Age. New York: Henry

Holt and Company, 2003.

McLean, Margaret. “Stem Cells: Justice at the Gate.” Dialog, vol. 41, no. 3 (fall

2002).

Meilaender, Gilbert. “Terra es animata: On Having a Life.” Hastings Center Report,

vol. 23, no. 4 (July-August 1993): 25-32.

Meyer, Michael J., and Lawrence J. Nelson. “Respecting What We Destroy:

Reflections on Human Embryo Research.” Hastings Center Report, vol. 31, no.

1 (January/February 2001).

Midgley, Mary. “Biotechnology and Monstrosity: Why We Should Pay Attention to

the ‘Yuk Factor.’” Hastings Center Report, vol. 30, no. 5 (2000): 7-15.

National Academy of Sciences. Stem Cells and the Future of Regenerative Medicine.

Washington D.C.: National Academies Press, 2002.

National Bioethics Advisory Commission. Ethical Issues in Human Stem Cell

Research. vol. 1. Rockville, Maryland: NBAC, 1999.

Nelkin, Dorothy, and Lori B. Andrews. Body Bazaar: The Market for Human Tissue

in the Biotechnology Age. New York, NY: Crown Publishing, 2001.

National Institutes of Health, “Report of the Human Embryo Research Panel.”

(September 1994).

Nussbaum, Martha C. “Compassion and Terror.” Daedalus, vol. 128, no. 4 (winter

2003): 10-26.

______. “Human Capabilities, Female Human Beings.” Women, Culture, and

Development: A Study of Human Capabilities. Ed. Martha C. Nussbaum and

Jonathan Glover (Oxford: Clarendon Press, 1995), 61-104.

Outka, Gene. “The Ethics of Stem Cell Research.” Kennedy Institute of Ethics Journal

vol. 12, no. 2 (2002): 175-213.
APPENDIX G. 271

Parens, Ph.D., Erik. “Embryonic Stem Cells and the Bigger Reprogenetic Picture.”

Women’s Health Issues, vol. 10, no. 3 (May/June 2000): 116-20.

Pontifical Academy for Life. “On the Production and the Scientific and Therapeutic

Use of Human Embryonic Stem Cells.” Vatican (August 25, 2000): available at

Http://www.petersnet.net/browse/ 3021.htm.

Rabinow, Paul. French DNA: Trouble in Purgatory. Chicago, IL: The University of

Chicago Press, 1999.

Radin, Margaret Jane. Contested Commodities. Cambridge, MA: Harvard University

Press, 1996.

Resnik, David. “The Commercialization of Human Stem Cells: Ethical and Policy

Issues.” Health Care Analysis, vol. 10 (2002):127-54.

Robertson, John A. “Ethics and Policy in Embryonic Stem Cell Research.” Kennedy

Institute of Ethics Journal, vol. 9, no. 2 (1999): 109-36.

Roche, J.D., Patricia A. and Michael A. Grodin, M.D., F.A.A.P. “The Ethical Challenge

of Stem Cell Research.” Women’s Health Issues, vol. 10, no. 3 (May/June 2000):

136-39.

Ryan, Maura. “Creating Embryos for Research: On Weighing Symbolic Costs.”

Cloning and the Future of Human Embryo Research. Ed. Paul Lauritzen. New

York: Oxford University Press, 2001.

______. The Ethics and Economics of Assisted Reproduction. Washington, D.C.:

Georgetown University Press, 2001.

______. “The Politics and Ethics of Human Embryo and Stem Cell Research.” Women’s

Health Issues, vol. 10, no. 3 (May/June 2000): 105-10.

Safire, William. “Why Die?” The New York Times (January 1, 2000).

Shamblott, M. J. et al. “Derivation of Pluripotent Stem Cells from Cultured Human

Primordial Germ Cells.” Proceedings of the National Academy of Sciences,

vol. 95 (1998): 13726-31.

Shannon, T. A., and A. B. Walter. “Reflections on the Moral Status of the Preembryo.”

Theological Studies, vol. 51 (1990): 603-26.

Shannon, Thomas. “Human Embryonic Stem Cell Therapy.” Theological Studies,

vol. 62, (2001): 811-24.

Squier, Susan. Liminal Lives, unpublished manuscript.

Steinbock, Bonnie. “Respect for Human Embryos.” In Cloning and the Future of

Human Embryo Research. Ed. Paul Lauritzen. New York: Oxford University

Press, 2001.

______. “What Does ‘Respect for Embryos’ Mean in the Context of Stem Cell Research?”

Women’s Health Issues, vol. 10, no. 3 (May/June 2000): 127-30.

______. Life before Birth: The Moral and Legal Status of Embryos and Fetuses. New

York, NY: Oxford University Press, 1992.
272 MONITORING STEM CELL RESEARCH

Tauer, Carol. “Responsibility and Regulation: Reproductive Technologies, Cloning,

and Embryo Research.” In Cloning and the Future of Human Embryo Research.

Ed. Paul Lauritzen. Oxford University Press: New York, 2001. 153.

______. “Preimplantation Embryos, Research Ethics, and Public Policy.” Bioethics

Forum, vol. 11, no. 3 (1995): 30-37.

Thomson, James A. et al. “Embryonic Stem Cell Lines Derived from Human

Blastocysts.” Science, vol. 282 (November 6, 1998).

Van Overwalle, Geertrui. Study on the Patenting of Inventions Related to Human

Stem Cell Research. Luxembourg: Office for Official Publications of the European

Communities, 2002. 23.

Waldby, Ph.D., Catherine, and Susan Squier, Ph.D. “Ontogeny, Ontology, and

Phylogeny: Embryonic Life and Stem Cell Technologies.” Configurations

(Forthcoming).

Warren, Mary Anne. Moral Status: Obligations to Persons and Other Living Things.

New York, NY: Oxford University Press, 1997.

Williams, Bernard. Problems of the Self: Philosophical Papers 1956-1972. Cambridge,

England: Cambridge University Press, 1973.

Wolfe, Tom. Hooking Up. New York, NY: Farrar, Straus and Giroux, 2001.

Zoloth, Laurie. “Jordon’s Banks: A View from the First Years of Human Embryonic

Stem Cell Research.” In The Human Embryonic Stem Cell Debate. Ed. by

Suzanne Holland, Karen Lebacqz, and Laurie Zoloth. Cambridge, MA: The

MIT Press, 2001. 238.
Appendix H.

Human Embryonic Germ Cells:

June 2001 – July 2003

The Published Record

JOHN GEARHART, PH.D.

C. Michael Armstrong Professor,

 Institute for Cell Engineering,

Johns Hopkins University School of Medicine

There have been but two original research articles published on

human embryonic germ cell in the period covered by this report. It

is appropriate that only peer-reviewed articles be considered in this

report, as the field of stem cell research is rife with undocumented

or unsubstantiated claims. There have been several publications

on EG cells derived in mice and chick and comment will be made

on these reports as they may impact on the eventual use or study of

the human cells.

There is a concern, given the imprinting-based developmental

abnormalities observed in humans, and those that have been

produced experimentally in animals, that dysregulated gene

expression in stem cell derived tissues could pose a serious problem

in the use of such tissues for cell-based therapies. Genomic

imprinting is defined as an epigenetic modification of the DNA (other

than sequence) in the germ line that leads to the preferential

expression of a specific allele (monoallelic expression) of some genes

in the somatic cells of the offspring in a parental dependent manner.

Because a maternal gene allele may be a paternal allele in the next

generation, the imprinting must be reprogrammed in the germ line,

that is, the epigenetic ‘marks’ must be erased (during germ cell

development) and established in the newly formed embryo. The

timing of the erasure in humans is not known. Imprinting involves

methylation at specific sites in the DNA and most likely, other

changes as well.

There have been several reports from experiments with the mouse

that indicate that imprinting is abnormal in pluripotent stem cells

273
274 MONITORING STEM CELL RESEARCH

derived from mouse embryos. These studies include abnormal or

variable imprints in mouse EG cells, abnormal imprints in ES cells

derived from interspecific crosses, and abnormal gene expression

in mice derived from ES cell nuclear transfer.

The results of the study by Onyango et al. utilizing EG cell lines

derived at John Hopkins, clearly demonstrate that general

dysregulation of imprinted genes will not be a barrier to their use in

transplantation therapies. The report has determined that the EG

cells are not imprinted, that is, imprinting has been erased in the

primordial germ cells that gave rise to the EG cells, that the erasure

is maintained in the EG cells, but in all informative cases, they

observed the transcription of only a single allele in differentiated

cells derived from the human EG cells. These results, although on a

limited number of lines and only a few imprinted genes, would

indicate that these human EG cell lines will serve as reliable and

safe sources for the study of EG cell differentiation and, perhaps,

cells for cell-based interventions.

Another area of interest in genetic regulation within EG cells is that

of X inactivation, the mammalian method for equalization of the

dosage of X-linked genes in males and females. This equalization

is accomplished by the down regulation of the transcriptional output

of the X chromosomes in females, so that only one X is active in

diploid somatic cells of both sexes. Inactivation is initiated in female

blastocysts. Both X chromosomes in female primordial germ cells

are active. Migeon et al. (2001) have demonstrated that in the very

early stages of differentiation of cells from human EGs, only one X

chromosome is active, indicating normal genetic regulation has

occurred. In a report by Nesterova et al. (2002) on the use of mouse

female EG cells in the study of X chromosome inactivation/

reactivation during primordial germ cell migration and EG cell

formation. Both X chromosomes appear to be active in XX EG cells,

and presumably, one becomes inactive when cells differentiate from

the EG cells.

One of the goals of stem cell research is to provide sources of cells

for cell-based therapies. As a step in this direction, proof of concept

or proof of principle studies involve the use of human cells in animal

models of human disease or injury. Although few, if any, animal

models are true models for the human diseases, they are the closest

approximation that can be made. The first report on the use of cells

derived from stem cells of human embryonic sources was recently

published: Kerr et al. Human Embryonic Germ Cell Derivatives

Facilitate Motor Recovery of Rats with Diffuse Motor Neuron Injury,
APPENDIX H. 275

J. Neuroscience 23, June 15, 2003. This is the first demonstration

that a human pluripotent stem cell derived form embryonic or fetal

tissue can ameliorate a disease process in an animal model.

Neural progenitor cells, derived from human EG cells, were

introduced into the cerebrospinal spinal fluid of rats that had been

paralyzed as a result of infection with a neuroadapted Sindbis virus

that specifically targets motor neurons in the spinal cord. All animals

in which human cells were found had some degree of hindlimb

recovery. It was clear from the histology of the animals that the

human cells had differentiated into appropriate neural cell types

within the ventral horns, including motor neurons, the results

indicated that the major effect of the human cells was to protect

host neurons from death and to facilitate reafferentiation of motor

neuron cell bodies. Growth factors responsible for this recovery,

produced by the human cells, were identified as brain-derived

neurotrophic factor and transforming growth factor.

The significance of this experiment appears to be that for this disease

model, the human cells supply factors that facilitate motor neuron

recovery following viral damage. However, the cells did migrate to

the site of injury in the spinal cord, which differentiates them from

other cellular grafts, and then delivered the factors. Also, there was

considerable differentiation of the human cells into various neural

cells types in the cord.

In a review article on deriving glucose-responsive insulin-producing

cells from stem cells (Kaczorowski et al. 2002), mention is made on

the isolation of such cells from mouse and human ES cells and human

EG cells, but no new data was presented, only a reference to a paper

published in 2001.

EG cell studies in other species

Several studies on EG cells from other species have been published.

EG cells from the chick have been demonstrated to yield germ line

chimeras when transferred to early embryos (Park et al. 2003). EG

cell lines of the mouse were found to colonize not only the epiblast

but also the primary endoderm of the gastrulating embryo following

aggregation with 8-cell embryos (Durcova-Hills et al. 2003). This

observation was permitted as a result of using a transgenic construct

effect as a lineage marker for cells of the early embryo. Horii et al.

(2002) report the serum-free culture of mouse EG cells, a system

which inhibits the ‘spontaneous’ differentiation due to the presence
276 MONITORING STEM CELL RESEARCH

of various growth factors in the serum. The cells are, however, are

cultured on a feeder layer.

An area of great interest in EG cell derivation is the basis of the

underlying mechanism for the conversion, derivation or

transformation (terms reflecting our ignorance of the process) of

primordial germ cells to EG cells. Kimura et al. (2003) report that

the loss of a tumor suppressor gene in mice, PTEN (phosphatase

and tensin homology deleted form chromosome ten, aka MMAC1

and TEP1), leads to a high incidence of testicular teratomas and

enhances the production of EG cells from PGCs of the mutant mice.

While it is highly unlikely that the loss of this gene in the culture of

normal PGCs leads to the derivation of EG cells, it may implicate

downstream signaling pathways that are involved in cell cycle

progression, cell survival and cell migration as important players in

the process.
APPENDIX H. 277

References

Durcova-Hills, G., Wianny, F., Merriman, J., Zernicka-Goetz, M., and McLaren, A.

2003. Developmental fate of embryonic stem cells (EGCs), in vivo and in vitro.

Differentiation 71, 135-141.

Horii, T., Nagao, Y., Tokunaga, T., and Imai, H. 20003. Serum-free culture of murine

primordial germ cells and embryonic germ cells. Theriogen. 59, 1257-1264.

Kaczorowski, D.J., Patterson, E.S., Jastromb, W.E., Shamblott, M.J. 2002. Review

Article. Glucose-responsive insulin-producing cells from stem cells. Diabetes Metab.

Res. Rev. 18, 442-450.

Kerr, D.A., Lladó, J., Shamblott, M.J., Maragakis, N.J., Irani, D.N., Crawford, T.O,

Krishnan, C., Dike, S., Gearhart, J.D., and Rothstein, J.D. 2003. Human embryonic

germ cell derivatives facilitate motor recovery of rats with diffuse motor neuron

injury. J. Neurosci. 23, 5131-5140.

Kimura, T., Suzuki, A., Fujita, Y., Yomogida, K., Lomeli, H., Asada, N., Ikeuchi, M.,

Nagy, A., Mak, T.W., and Nakano, T. 2003. Development and Disease. Conditional

loss of PTEN leads to testicular teratoma and enhances embryonic germ cell

production. Development 130, 1691-1700.

Migeon, B.R., Chowdry, A.K., Dunston, J.A., and McIntosh, I. 2001. Identification of

TSIX, encoding an RNA antisense to human XIST, reveals differences from its murine

counterpart: Implications for X inactivation. Am. J. Human Genet. 69, 951-960.

Onyango, P., Jiang, S., Uejima, H., Shamblott, M.J., Gearhart, J.D., Cui, H., and

Feinberg, A.P. 2002. Monoallelic expression and methylation of imprinted genes in

human and mouse embryonic germ cell lineages. Proc. Natl. Acad. Sci. USA 99,

10599-10604.

Park, T.S., Hong, Y.H., Kwon, S.C., Lim, J.M., and Han, J.Y. 2003. Birth of germline

chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos.

Molec. Reprod, Develop. 65, 389-395.

Sapienza, C. 2002. Commentary. Imprinted gene expression, transplantation

medicine, and the “other” human embryonic stem cell. Proc. Natl. Acad. Sci. USA

99, 10243-10245.

Appendix I.

Current Progress in

Human Embryonic Stem Cell Research

TENNEILLE E. LUDWIG, PH.D., and

JAMES A. THOMSON, PH.D

Wisconsin National Primate Research Center, University of

Wisconsin-Madison

The immortality and potentially unlimited developmental

capacity of human embryonic stem (ES) [1] cells ignite the

imagination. After months or years of growth in culture dishes, these

cells retain the ability to form cell types ranging from heart muscle

to nerve to blood—possibly any cell in the body. Because of their

unique developmental potential, human ES cells have widespread

implications for human developmental biology, drug discovery, drug

testing, and transplantation medicine. Indeed, human ES cells

promise an essentially unlimited supply of specific cell types for in

vitro experimental studies and for transplantation therapies for

diseases such as heart disease, Parkinson’s disease, leukemia, and

diabetes.

The derivation of a human ES cell line destroys a human

embryo. Thus, the derivation of human ES cells resurrected a fierce

controversy over human embryo research in the United States, a

controversy originally created by the development of human in vitro

fertilization decades ago, but never completely resolved. In

particular, the derivation of human ES cells led to a re-examination

of the role of federal funding of human embryo research. In response

to the intense public interest, President George W. Bush reviewed

the potential of human ES cell research to improve the health of

Americans. In his national address on August 9, 2001, he stated

“Federal dollars help attract the best and brightest scientists. They

ensure new discoveries are widely shared at the largest number of

research facilities, and the research is directed toward the greatest

public good.” On that basis, he directed federal funding to “explore

the promise and potential of stem cell research,” including, for the

first time, human ES cell research. However, the President went on

to restrict federal funding to research that used only those human

279
280 MONITORING STEM CELL RESEARCH

ES cell lines derived prior to his address on August 9, 2001. This

paper reviews progress in human ES cell research in the wake of

that decision.

Human ES Cell Publication Summary

Since their initial derivation, only a limited number of

independent (i.e., derived from different embryos) human ES cell

lines that meet President Bush’s criteria have been used in published

research. Just nine human ES cell lines meeting President Bush’s

criteria are currently listed by the National Institutes of Health as

readily available for distribution to investigators. Despite limited

availability to date, research with human ES cells is proceeding.

Forty human ES cell primary research papers have been published

in peer-reviewed journals since the initial publication of human ES

cell isolation in 1998 (Table 1). Published human ES cell research

includes studies on the optimization of the culture environment,

characterization of human ES cells, modification of the ES cell

genome, and differentiation.

Table 1. Human ES Cell Research Publications

Area of interest Publications to

date Reference(s)

Derivation 5 [1-5]

Culture Optimization 6 [6-11]

 Feeder Layer

Alternatives/Replacements 4 [6-9]

 Media Analysis 1 [10]

 Freezing 1 [11]

Characterization 5 [12-16]

Modification 6 [17-22]

Differentiation into multiple

lineages 3 [23-25]

Differentiation into specific

lineages 15 [26-40]

 Neural 4 [26-29]

 Cardiac 5 [30-34]

 Endothelial (Vascular) 1 [35]

 Hematopoietic (Blood) 2 [36, 37]

 Pancreatic (Islet-like) 1 [38]

 Hepatic (liver) 1 [39]

 Trophoblast 1 [40]
APPENDIX I. 281

Culture Optimization for Human ES Cells.

Improvement of culture conditions to enable large-scale

production and reduce safety concerns has been a major research

focus. The first two research groups that described the derivation

of human ES cell lines examined long-term proliferation, karyotypic

stability, developmental potential, and cell surface marker

expression by ES cells [1, 2]. Because these first human ES cell

lines remain the most extensively characterized, most subsequent

research has utilized them. These human ES cell lines were derived

on mouse fibroblast feeder layers in the presence of fetal bovine

serum. The exposure to these and other sources of animal proteins

has raised concern that some yet unidentified pathogen(s) may have

been transferred to the ES cells by contact with cells or proteins

from other species, and that these pathogens could be transferred

to patients if these ES cells were to be used for transplantation

therapies. Thus, several research groups have been actively working

to reduce or eliminate non-human cells or proteins from human ES

cell culture.

Significant progress has been made in eliminating serum, and limited

progress has been made in eliminating fibroblasts from human ES

cell culture. Serum is a complex, poorly defined mixture of

components, and there is significant variation between lots [41].

Individual lots of serum, therefore, must be carefully screened for

their ability to sustain undifferentiated ES cell growth. If basic

fibroblast growth factor is added to a proprietary serum substitute

(Gibco BRL Knockout Serum Replacer), it supports human ES cells

and significantly reduces the batch variability associated with serum

[12]. However, this medium does not eliminate all serum products

from human ES cell culture medium, as it still contains a bovine

serum albumin component. With this same medium, human ES cells

can be cultured without direct contact with feeder layers if the

medium is first conditioned by exposure to mouse embryonic

fibroblasts [6]. However, the medium still contains bovine serum

products and is exposed to fibroblasts, therefore cross-species

contamination with pathogens remains a concern.

Recent reports demonstrate that human ES cells can be

maintained on feeder layers of human origin. Feeder layers obtained

from human bone marrow [9], fetal muscle or skin [7], adult human

fallopian tube epithelial cells [7], or human foreskin [8] support

human ES cell proliferation and maintenance of normal karyotype

and developmental potential. These results led to the growth of

human ES cells in the complete absence of non-human products [7].
282 MONITORING STEM CELL RESEARCH

New human ES cells derived under these conditions would eliminate

concerns about cross-species contamination by pathogens, but such

cell lines could not, at present, be supported by federal funding in

the United States. Growth on human feeder layers is a significant

advance because of reduced safety concerns; nonetheless, the

preparation of these feeders remains laborious and introduces a

significant source of biological variability. The complete elimination

of feeder layers and serum from human ES cell culture medium and

their replacement by defined, cloned products remains an important

goal for the future and is an active area of research for several groups.

Genetic Modification of Human ES Cells

Although the human genome project is essentially

completed, we are ignorant about the function of most human genes.

Human ES cells provide a powerful new model for identifying the

function of any human gene, and this requires efficient methods for

genetic modification of human ES cells. Genetic manipulation of

human ES cells is essential to elucidate gene function; direct the

differentiation of ES cells to specific lineages; purify desired

differentiated cell types from mixed populations of ES cell

derivatives; use the differentiated derivatives of ES cells as a vehicle

for gene therapy; and modulate the immune response to transplanted

ES cell derivatives.

Transfection methods routinely used for mouse ES cells

generally fail to transfect human ES cells efficiently, but there have

now been several approaches developed for human ES cells.

Transient [17] and stable [18] integration of plasmids into human

ES cells can be accomplished through specific transfection reagents,

the best reagents yielding stable (drug-selectable) transfection rates

of about 10–5. Recently more labor-intensive, HIV-based, lentivirus

vectors have been shown to transduce human ES cells at an

efficiency rate of over 90% [20, 21]. This should allow complex

mixtures of genes to be screened for specific phenotypic effects by

a process termed “expression cloning” [20].

Homologous recombination allows the defined modifications

of specific genes in living cells [42, 43] and has been used extensively

with mouse ES cells. However, the differences between mouse and

human ES cells delayed the development of homologous

recombination in human ES cells. Except for viral approaches, high

stable transfection efficiencies in human ES cells have been difficult

to achieve, and in particular, electroporation protocols established

for mouse ES cells do not work in human ES cells [22]. Also, in
APPENDIX I. 283

contrast to mouse ES cells, human ES cells proliferate inefficiently

from single cells, making screening procedures to identify rare

homologous recombination events difficult [12]. We have recently

developed modified electroporation protocols to overcome these

problems and have successfully targeted a ubiquitously expressed

gene (HPRT1), an ES cell-specific gene (POU5F1), and a tissuespecific gene (Tyrosine hydroxylase: TH) in human ES cells [22, 44].

The overall targeting frequencies for the three genes suggest that

homologous recombination is a broadly applicable technique in

human ES cells.

Homologous recombination in human ES cells will be

important for studying gene function in vitro and for lineage

selection. For therapeutic applications in transplantation medicine,

controlled modification of specific genes should be useful for

purifying specific ES cell-derived, differentiated cell types from a

mixed population [45]; altering the antigenicity of ES cell derivatives;

and giving cells new properties (such as viral resistance) to combat

specific diseases. Homologous recombination in human ES cells

might also be used for approaches combining therapeutic cloning

with gene therapy [46]. In vitro studies using homologous

recombination in human ES cells will be particularly useful for

learning more about the pathogenesis of diseases where mouse

models have proven inadequate. For example, HPRT-deficient mice

fail to demonstrate an abnormal phenotype, yet defects in this gene

cause Lesch-Nyhan disease in children [47]. In vitro neural

differentiation of HPRT-deficient human ES cells or transplantation

of ES cell-derived neural tissue to an animal model could help to

understand the pathogenesis of Lesch-Nyhan syndrome.

Human ES Cells as a Model of Early Human Development

The excitement surrounding the prospective role of human

embryonic stem (ES) cells in transplantation therapy has often

overshadowed a potentially more important role as a basic research

tool for understanding the development and function of human

tissues. The use of human ES cells is particularly valuable to derive

tissue for study that is difficult to obtain otherwise, and for which

animal models are inadequate.

Human ES cells offer a new and unique window into the

early events of human development, a period critical for

understanding infertility, birth defects, and miscarriage. Because

manipulation of the early post-implantation human embryo could

jeopardize the health of the resulting child, it has never been possible
284 MONITORING STEM CELL RESEARCH

to examine this important period of human development

experimentally. Nearly all of what is known about early human

development, especially in the early post-implantation period, is

based on very rare histological sections of human embryos, or on

an imperfect analogy to experimental studies in the mouse. The

mouse has been the mainstay of mammalian experimental

embryology because of its historical use, well-defined genetics, and

favorable reproductive characteristics. However, early mouse

development and early human development differ significantly. For

example, human and mouse embryos differ in the expression of

embryonic antigens; timing of embryonic genome expression;

formation, structure, and function of the fetal membranes and

placenta; and formation of an embryonic disc instead of an egg

cylinder. Thus, if one is interested in the development of a human

tissue known to differ significantly from the corresponding mouse

tissue, such as the yolk sac or the placenta, studying a human model

is desirable.

The first differentiation event in mammalian embryos is the

formation of the trophectoderm, the outer epithelial layer of the

blastocyst. The trophectoderm is crucial for implantation of the

embryo and gives rise to specialized populations of trophoblast cells

in the placenta [48, 49]. Mouse and human placentas differ in

structure and function, and these differences are clinically

significant. For example, the placental hormone chorionic

gonadotropin, which has an essential role in establishing and

maintaining human pregnancy, is not even produced by the mouse

placenta. When formed into chimeras with intact preimplantation

embryos, mouse ES cells rarely contribute to the trophoblast, and

the manipulation of external culture conditions has, to date, failed

to direct mouse ES cells to form trophoblast [50].

Spontaneously differentiated rhesus monkey [51] or human

ES cells [1] do secrete modest amounts of chorionic gonadotropin,

indicating the differentiation of trophoblast cells [52]. Recently it

was discovered that a single growth factor (BMP4) would induce

human ES cells to differentiate to a pure population of early

trophoblast [40]. These early human trophoblast cells have never

before been available for detailed study, and already this new

experimental model has provided information about the specific

genes that control the early development of the human placenta

[40]. The derivation of other early lineages from human ES cells in

vitro to provide a more complete understanding of early human

development is an active area of research.
APPENDIX I. 285

Cardiovascular Differentiation

Cardiovascular disease is the leading cause of death in the

United States, taking the lives of more people each year than the

next five leading causes of death combined [53]. Cardiovascular

disease and its related disorders affect more than 68 million

Americans, at a cost of more than 350 billion dollars annually. Heart

disease alone accounts for 229 billion dollars in health care costs

each year. Adult heart tissue cannot be expanded in culture, and

thus, there are no human heart cell lines available for research. The

limited amount of physiological research done directly on human

heart cells has generally relied on biopsy samples, which are small,

erratically available, and usually obtained from diseased hearts. In

contrast, human ES cells are already providing a reliable in vitro

supply of human heart cells for experimental study [30-34].

Animal models, such as the mouse, have historically been

used for the study of the heart. However, there are clinically

significant physiological differences between animal and human

cardiomyocytes that limit the usefulness of these models. For

example, the mechanisms regulating the QT polarization interval—

the time required for repolarization of the heart muscle between

beats—differ significantly between species. A prolonged QT

polarization interval in humans is related to ventricular arrhythmias

and cardiac arrest and has been a significant side effect of a wide

range of drugs in early human clinical trials. Drugs exhibiting this

serious side effect must be withdrawn from clinical trials, and such

drugs have been responsible for patients’ deaths. Because the

mechanisms that regulate repolarization of the heart muscle cells

differ appreciably between human and mouse models, screening

drugs on mouse hearts does not reliably detect this side effect. Yet,

because they do not divide in culture, human heart cells have not

been previously available for screening.

Human ES cells differentiate spontaneously to heart muscle

cells, and several research groups have reported the characterization

of these cells [23, 31, 54]. Human ES cells allowed to differentiate in

unattached clumps (termed “embryoid bodies”) form synchronized

contracting areas that express appropriate cardiac markers [23, 31,

32]. Co-culture of human ES cells with visceral, endoderm-like cells

also causes differentiation to cardiomyocytes [34], and 5-aza-2’-

deoxycytidine or density gradient separation allows some

enrichment of cardiomyocyte populations [32]. Human ES cellderived cardiomyocytes display many of the functional properties
286 MONITORING STEM CELL RESEARCH

of native cardiomyocytes, including the generation of synchronized

action potentials and response to cardioactive drugs [30-33]. Heart

cells exhibiting action potentials characteristic of nodal, atrial, and

ventricular cardiomyocytes are all present, and the human-specific

mechanisms regulating QT interval are functional [30]. Thus, human

ES cell-derived heart cells are already useful for drug screening,

and their use should make the drug development process quicker,

cheaper, and safer.

There is also a great interest in using human ES cell-derived

heart cells for transplantation, but this will likely be challenging.

Studies in animal models demonstrate that cell transplantation is

effective in increasing the myocyte population in damaged or

diseased cardiac tissue [55]. However, when heart cells die in a

heart attack, it is not because the heart cells themselves are

defective, but because the blood supply is cut off. Thus, to be

successful, transplanted heart cells would have to integrate

functionally with the surrounding heart cells, obtain a new blood

supply, and avoid immune rejection. Each of these problems has

potential solutions, but will require significant time and effort to

solve. Precursors of vascular tissue can also be derived from human

ES cells [35], and such cells may be useful in supporting cotransplanted heart cells.

Neural Differentiation

Because of the country’s aging population, neural

degenerative disorders such as Parkinson’s disease and Alzheimer’s

disease are becoming increasingly prevalent in the United States.

Historically, one of the difficulties in studying the pathogenesis of

neural disease has been the very limited access to the specific neural

cells involved in these diseases. Neural precursor (or stem) cells

cultured from fetal and adult brains have been extensively studied,

but appear to have limited developmental potential. For example,

the sustainable differentiation of neural stem cells to dopaminergic

neurons, the cell defective in Parkinson’s disease, has not yet been

achieved. Mouse ES cells, for example, differentiate efficiently to

dopaminergic neurons, and several groups are beginning to apply

approaches used with mouse ES cells to human ES cells. Human

ES cells should offer an improved supply of neural tissue, for both in

vitro experimental studies and transplantation therapies.

Human ES cell-derived embryoid bodies produce both neural

precursor cells and cells expressing markers of mature neurons and

glia [26-29]. The percentage of neural precursors can be enriched
APPENDIX I. 287

by alteration of culture conditions [26-28] or by purification using

cell surface markers [28]. Human ES cell-derived neural cells are

able to synthesize and respond to neurotransmitters, form synapses

and voltage-dependent ion channels capable of generating action

potentials, and generate electrical activity [28]. Some human ES

cell-derived neurons express tyrosine hydroxylase, the rate-limiting

enzyme involved in dopamine synthesis and a marker of

dopaminergic neurons [26, 27].

Human ES cell-derived neural precursors transplanted into

the mouse brain differentiate into all three types of central nervous

system cells (neurons, glia cells, and oliogodendrocytes) [26, 27].

These differentiated cells migrate, following host developmental

cues, into various areas of the brain (including cortex, hippocampus,

striatum olfactory bulb, septum, thalamus, hypothalamus, and

midbrain) [26, 27]. One of the concerns about using human ES cellderived neural cells in transplantation therapy is the fear that

undifferentiated ES cells may be transplanted with the differentiated

cells and form teratomas in the host. To date, transplantation of

isolated, human ES cell-derived neural precursor cells into mice has

not produced teratomas [26, 27], suggesting that appropriate

selection procedures can eliminate undifferentiated ES cell

contamination. However, longer-term testing is still needed to

address the teratoma formation issue more carefully.

Hematopoietic Differentiation

Human ES cells are already providing a sustainable source

of hematopoietic cells for in vitro studies [36, 37]. Hematopoietic

stem cells are by far the most studied adult stem cells, and bone

marrow transplants are the most common and effective form of stem

cell-based therapy. However, despite several decades of research

by hundreds of laboratories, hematopoietic stem cells have not yet

been successfully expanded in clinically useful amounts, and these

cells must instead be transferred directly from the donor. When

cultured in vitro, hematopoietic stem cells do not self-renew, but

instead differentiate to specific blood cells, and thus quickly

disappear. This makes the in vitro study of human hematopoiesis

difficult, as researchers must continually return to patients to obtain

hematopoietic stem cells from bone marrow, peripheral blood, or

placental cord blood. Human ES cells can differentiate into

hematopoietic precursor cells through co-culture with murine bone

marrow or yolk sac cells [36]. Enrichment of ES cell-derived

hematopoietic precursors is accomplished by treatment with

cytokines or BMP-4 [37]. Cell sorting using hematopoietic-specific
288 MONITORING STEM CELL RESEARCH

cell surface markers yields myeloid, erythroid, and megakaryocyte

precursors [36].

There are three major areas where human ES cell

hematopoiesis should impact human medicine. First, because

human ES cells can be expanded without limit, human

hematopoiesis can be studied without the need to continually return

to patients for tissue donations. The knowledge of these in vitro

studies is likely to improve therapies based on adult hematopoietic

stem cells. Second, human ES cell-derived blood cells could be used

either in bone marrow transplants, or as a source of blood products

such as red blood cells and platelets. And third, ES cell-derived

hematopoietic stem cells could aid in ES cell-based transplantation

therapies for other (non-hematopoietic) tissues. Transplantation of

ES cell-derived hematopoietic stem cells could be used to reduce or

eliminate immune rejection by creating hematopoietic chimerism

in patients receiving co-transplantation of other human ES cellderived tissues [45, 56-58].

Pancreatic Differentiation

Type 1 diabetes offers one of the most promising applications

of human ES cell-based transplantation therapy. The destruction of

pancreatic islet $-cells results in type 1 diabetes. $-cells produce

insulin, and as their numbers dwindle, the ability to appropriately

control blood glucose levels is lost. Even with current insulin

therapies, type 1 diabetes reduces a patient’s life expectancy by 10

to 15 years, and these patients often develop serious complications

such as blindness and kidney failure [59]. Recently, the

transplantation of $-cells from cadavers has proven to be an effective

treatment for some forms of uncontrollable diabetes, but the source

of tissue for transplantation is severely limiting and will never come

close to meeting the demands of over one million people with type

1 diabetes in the United States. Spontaneous in vitro differentiation

of human ES cells reveals a percentage of cells that produce insulin

and express other $-cells specific markers, offering hope of a scalable

source of $-cells for transplantation [38]

The challenges for using human ES cell-derived $-cells for

transplantation are significant and parallel those that face the entire

field of ES cell-based transplantation therapies. First, pancreatic

development is incompletely understood, and it is not yet possible

to direct ES cells to $-cells efficiently. However, given the pace of

advances in developmental biology over the last decade, it is likely

that in the next five to ten years, it will be possible to routinely
APPENDIX I. 289

generate clinically useful quantities of $-cells from human ES cells.

Second, integration into the body in a form that restores function of

the damaged tissue is essential. This is easier for $-cells than for

most cell types, as the function that must be restored is secretion of

insulin into the blood stream in response to high glucose, and this

function does not require a complex physical connection between

the transplanted and host tissues. Indeed, the clinical trials using

cadaver-derived $-cells have transplanted the cells into the liver,

and the cells function in that site. Third, transplanted $-cells must

not be rejected by the immune system. Although the transplantation

of $-cells has been clinically successful, the severe

immunosuppressive therapy required may make the procedure

inappropriate for the average diabetic patient. Importantly, $-cells

derived from adult stem cells from the patient, or even from ES cells

derived through “therapeutic cloning” using a nucleus from the

patient, would not solve the immune rejection problem for diabetes.

Type 1 diabetes is an autoimmune process, and unless that immune

response is altered the very process that made the patient diabetic

in the first place would destroy transplanted $-cells genetically

identical to the patient’s. Finally, neoplastic transformation of the

transplanted cells is a serious concern for any cell-based therapy in

which the cells are first cultured extensively. All actively dividing

cells accumulate mutations over time, and the potential exists that

enough mutations could accumulate to make some cells tumor cells.

None of the challenges facing ES cell-based transplantation

therapies are insurmountable, and indeed, type 1 diabetes is an

excellent candidate for treatment using this approach. However,

the challenges do underscore both the importance of careful

preclinical testing, particularly in non-human primates, and the

amount of work still to be done before people’s lives will be improved

by these therapies.

Conclusions

Since their initial derivation, there has been significant

progress in culture optimization, characterization, genetic

modification, and differentiation of human ES cells. However, ethical

and political controversy continues to impede progress in human

ES cell research. The decision by President George W. Bush,

restricting federal funding to human ES cell lines derived before

August 9, 2001, created a distribution bottleneck that is just now

beginning to be resolved. Although these initial cell lines may

support much of the basic research now being conducted, the very

first cell lines were originally derived for research purposes, with
290 MONITORING STEM CELL RESEARCH

the expectation that future cell lines would more appropriately

address legitimate safety concerns for therapeutic applications. In

spite of the slow start, the diversity of investigators already

contributing to human ES cell research is, nonetheless, promising

and suggests that the initial lag phase for the human ES cell field is

already coming to an end and that an exponential growth phase is

beginning. During the next year or two, it is likely that the

purification of specific, therapeutically useful human ES cell

derivatives, such as dopaminergic neurons, will be published, and

that defined culture conditions eliminating the need for both feeder

layers and non-human proteins will be developed. When these

events occur, President Bush’s compromise will be particularly

damaging to the field, and there will be an even greater need to

derive new cell lines.

References

1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ,

Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts.

Science 1998; 282: 1145-1147.

2. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem

cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol

2000; 18: 399-404.

3. Lanzendorf SE, Boyd CA, Wright DL, Muasher S, Oehninger S. Use of

human gametes obtained from anonymous donors for the production of human

embryonic stem cell lines. Fertility and Sterility 2001; 76: 132-137.

4. He Z, Huang S, Li Y, Zhang Q. Human embryonic stem cell lines

preliminarily established in China. Zhonghua Yi Xue Za Zhi 2002; 82: 1314-1318.

5. Amit M, Itskovitz-Eldor J. Derivation and spontaneous differentiation of

human embryonic stem cells. J Anat 2002; 200: 225-232.

6. Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK.

Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol

2001; 19: 971-974.

7. Richards M, Fong CY, Chan WK, Wong PC, Bongso A. Human feeders

support prolonged undifferentiated growth of human inner cell masses and

embryonic stem cells.[comment]. Nature Biotechnology 2002; 20: 933-936.

8. Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R, ItskovitzEldor J. Human Feeder Layers for Human Embryonic Stem Cells. Biol Reprod 2003;

68: 2150-2156.

9. Cheng L, Hammond H, Ye Z, Zhan X, Dravid G. Human adult marrow cells

support prolonged expansion of human embryonic stem cells in culture. Stem Cells

2003; 21: 131-142.
APPENDIX I. 291

10. Lim JW, Bodnar A. Proteome analysis of conditioned medium from mouse

embryonic fibroblast feeder layers which support the growth of human embryonic

stem cells. Proteomics 2002; 2: 1187-1203.

11. Reubinoff BE, Pera MF, Vajta G, Trounson AO. Effective cryopreservation

of human embryonic stem cells by the open pulled straw vitrification method. Hum

Reprod 2001; 16: 2187-2194.

12. Amit M, Carpenter MK, Inokuma MS, Chiu C, Harris CP, Waknitz MA,

Itskovitz-Eldor J, Thomson JA. Clonally derived human embryonic stem cell lines

maintain pluripotency and proliferative potential for prolonged periods of in vitro

culture. Developmental Biology 2000; 227: 271-278.

13. Henderson JK, Draper JS, Baillie HS, Fishel S, Thomson JA, Moore H,

Andrews PW. Preimplantation human embryos and embryonic stem cells show

comparable expression of stage-specific embryonic antigens. Stem Cells 2002; 20:

329-337.

14. Drukker M, Katz G, Urbach A, Schuldiner M, Markel G, Itskovitz-Eldor J,

Reubinoff B, Mandelboim O, Benvenisty N. Characterization of the expression of

MHC proteins in human embryonic stem cells. Proc Natl Acad Sci U S A 2002; 99:

9864-9869.

15. Sathananthan H, Pera M, Trounson A. The fine structure of human

embryonic stem cells. Reprod Biomed Online 2002; 4: 56-61.

16. Moore FL, Jaruzelska J, Fox MS, Urano J, Firpo MT, Turek PJ, Dorfman

DM, Pera RA. Human Pumilio-2 is expressed in embryonic stem cells and germ cells

and interacts with DAZ (Deleted in AZoospermia) and DAZ-like proteins. Proceedings

of the National Academy of Sciences of the United States of America 2003; 100: 538-

543.

17. Lebkowski JS, Gold J, Xu C, Funk W, Chiu CP, Carpenter MK. Human

embryonic stem cells: culture, differentiation, and genetic modification for

regenerative medicine applications. Cancer J 2001; 7: S83-93.

18. Eiges R, Schuldiner M, Drukker M, Yanuka O, Itskovitz-Eldor J, Benvenisty

N. Establishment of human embryonic stem cell-transfected clones carrying a marker

for undifferentiated cells. Current Biology 2001; 11: 514-518.

19. Pfeifer A, Ikawa M, Dayn Y, Verma I. Transgenesis by lentiviral vectors:

Lack of gene silencing in mammalian embryonic stem cells and preimplantation

embryos. PNAS 2002; 99: 2140-2145.

20. Ma Y, Ramezani A, Lewis R, Hawley R, Thomson JA. High-level sustained

transgene expression in human embryonic stem cells using lentiviral vectors. Stem

Cells 2003; 21: 111-117.

21. Gropp M, Itsykson P, Singer O, Ben-Hur T, Reinhartz E, Galun E, Reubinoff

BE. Stable genetic modification of human embryonic stem cells by lentiviral vectors.

Mol Ther 2003; 7: 281-287.

22. Zwaka TP, Thomson JA. Homologous recombination in human embryonic

stem cells. Nat Biotechnol 2003; 21: 319-321.
292 MONITORING STEM CELL RESEARCH

23. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M,

Soreq H, Benvenisty N. Differentiation of human embryonic stem cells into embryoid

bodies compromising the three embryonic germ layers. Mol Med 2000; 6: 88-95.

24. Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N. Effects

of eight growth factors on the differentiation of cells derived from human embryonic

stem cells. Proc Natl Acad Sci U S A 2000; 97: 11307-11312.

25. Goldstein RS, Drukker M, Reubinoff BE, Benvenisty N. Integration and

differentiation of human embryonic stem cells transplanted to the chick embryo.

Dev Dyn 2002; 225: 80-86.

26. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA. In vitro

differentiation of transplantable neural precursors from human embryonic stem cells.

Nat Biotechnol 2001; 19: 1129-1133.

27. Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, BenHur T. Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001;

19: 1134-1140.

28. Carpenter MK, Inokuma MS, Denham J, Mujtaba T, Chiu CP, Rao MS.

Enrichment of neurons and neural precursors from human embryonic stem cells.

Exp Neurol 2001; 172: 383-397.

29. Schuldiner M, Eiges R, Eden A, Yanuka O, Itskovitz-Eldor J, Goldstein RS,

Benvenisty N. Induced neuronal differentiation of human embryonic stem cells. Brain

Research 2001; 913: 201-205.

30. He JQ, Ma Y, Lee Y, Thomson JA, Kamp TJ. Human Embryonic Stem Cells

Develop Into Multiple Types of Cardiac Myocytes. Action Potential Characterization.

Circ Res 2003; 5: 5.

31. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Livne

E, Binah O, Itskovitz-Eldor J, Gepstein L. Human embryonic stem cells can

differentiate into myocytes with structural and functional properties of

cardiomyocytes. J Clin Invest 2001; 108: 407-414.

32. Xu C, Police S, Rao N, Carpenter MK. Characterization and enrichment of

cardiomyocytes derived from human embryonic stem cells. Circ Res 2002; 91: 501-

508.

33. Kehat I, Gepstein A, Spira A, Itskovitz-Eldor J, Gepstein L. High-resolution

electrophysiological assessment of human embryonic stem cell-derived

cardiomyocytes: a novel in vitro model for the study of conduction. Circ Res 2002;

91: 659-661.

34. Mummery C, Ward-Van Oostwaard D, Doevendans P, Spijker R, Van Den

Brink S, Hassink R, Van Der Heyden M, Opthof T, Pera M, De La Riviere AB, Passier

R, Tertoolen L. Differentiation of human embryonic stem cells to cardiomyocytes:

role of coculture with visceral endoderm-like cells. Circulation 2003; 107: 2733-2740.

35. Levenberg S, Golub JS, Amit M, Itskovitz-Eldor J, Langer R. Endothelial

cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2002; 99:

4391-4396.
APPENDIX I. 293

36. Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA.

Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc

Natl Acad Sci U S A 2001; 98: 10716-10721.

37. Chadwick K, Wang L, Li L, Menendez P, Murdoch B, Rouleau A, Bhatia M.

Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic

stem cells. Blood 2003; 17: 17.

38. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M.

Insulin production by human embryonic stem cells. Diabetes 2001; 50: 1691-1697.

39. Rambhatla L, Chiu CP, Kundu P, Peng Y, Carpenter MK. Generation of

hepatocyte-like cells from human embryonic stem cells. Cell Transplant 2003; 12: 1-

11.

40. Xu RH, Chen X, Li DS, Li R, Addicks GC, Glennon C, Zwaka TP, Thomson

JA. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat

Biotechnol 2002; 20: 1261-1264.

41. McKiernan SH, Bavister BD. Different lots of bovine serum albumin inhibit

or stimulate in vitro development of hamster embryos. In Vitro Cell Dev Biol 1992;

28A: 154-156.

42. Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS.

Insertion of DNA sequences into the human chromosomal beta-globin locus by

homologous recombination. Nature 1985; 317: 230-234.

43. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting

in mouse embryo-derived stem cells. Cell 1987; 51: 503-512.

44. Zwaka TP, Thomson JA. Homologous recombination in human embryonic

stem cells. In: Lanza R (ed.) The handbook of embryonic stem cells. Vol 2: Embryonic

stem cells. San Deigo: Elsevier science; 2003: (in press).

45. Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from

human embryonic stem cell lines. Stem Cells 2001; 19: 193-204.

46. Rideout WM, 3rd, Hochedlinger K, Kyba M, Daley GQ, Jaenisch R.

Correction of a genetic defect by nuclear transplantation and combined cell and

gene therapy. Cell 2002; 109: 17-27.

47. Finger S, Heavens RP, Sirinathsinghji DJ, Kuehn MR, Dunnett SB. Behavioral

and neurochemical evaluation of a transgenic mouse model of Lesch-Nyhan

syndrome. Journal of Neurological Science 1988; 86: 203-213.

48. Fisher SJ. The placenta dilemma. Semin Reprod Med 2000; 18: 321-326.

49. Cross JC. Genetic insights into trophoblast differentiation and placental

morphogenesis. Semin Cell Dev Biol 2000; 11: 105-113.

50. Beddington RS, Robertson EJ. An assessment of the developmental

potential of embryonic stem cells in the midgestation mouse embryo. Development

1989; 105: 733-737.
294 MONITORING STEM CELL RESEARCH

51. Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA,

Hearn JP. Isolation of a primate embryonic stem cell line. Proceedings of the National

Academy of Sciences, U.S.A. 1995; 92: 7844-7848.

52. Muyan M, Boime I. Secretion of chorionic gonadotropin from human

trophoblasts. Placenta 1997; 18: 237-241.

53. American Heart Association. Heart disease and stroke statistics — 2003

update. Dallas, Tex: American Heart Association; 2002.

54. Mummery C, Ward D, van den Brink CE, Bird SD, Doevendans PA, Opthof

T, Brutel de la Riviere A, Tertoolen L, van der Heyden M, Pera M. Cardiomyocyte

differentiation of mouse and human embryonic stem cells. J Anat 2002; 200: 233-

242.

55. Soonpaa MH, Daud AI, Koh GY, Klug MG, Kim KK, Wang H, Field LJ.

Potential approaches for myocardial regeneration. Ann N Y Acad Sci 1995; 752: 446-

454.

56. Gandy KL, Weissman IL. Tolerance of allogeneic heart grafts in mice

simultaneously reconstituted with purified allogeneic hematopoietic stem cells.

Transplantation 1998; 65: 295-304.

57. Spitzer TR, Delmonico F, Tolkoff-Rubin N, McAfee S, Sackstein R, Saidman

S, Colby C, Sykes M, Sachs DH, Cosimi AB. Combined histocompatability leukocyte

antigen-matched donor bone marrow and renal transplantation for multiple myeloma

with end-stage renal disease: The induction of allograft tolerance through mixed

lymphohematopeoietic chimerism. Transplantation 1999; 68: 480-484.

58. Wekerle T, Sykes M. Mixed chimerism as an approach for the induction of

transplantation tolerance. Transplantation 1999; 68: 459-467.

59. The effect of intensive treatment of diabetes on the development and

progression of long-term complications in insulin-dependent diabetes mellitus. The

Diabetes Control and Complications Trial Research Group. N Engl J Med 1993; 329:

977-986.
Appendix J.

Multipotent Adult Progenitor Cells: An Update

CATHERINE M. VERFAILLIE, M.D.

Division of Hematology, Department of Medicine,

and Stem Cell Institute,

University of Minnesota

INTRODUCTION:

In this paper, we want to provide updated information

regarding a rare cell population, we have named, multipotent adult

progenitor cells or MAPC. In 2001-2002, we published a series of

papers demonstrating that while attempting to select and culture

mesenchymal stem cells (MSC) from human and subsequently

mouse and rat bone marrow (BM), we accidentally identified a rare

population of cells that has characteristics unlike most adult somatic

stem cells in that they appear to proliferate without senescence,

and have pluripotent differentiation ability in vitro and in vivo 1,2.

Phenotype of Bone Marrow MAPC: MAPC can be cultured

from human, mouse and rat bone marrow (BM). Unlike MSC, MAPC

do not express major histocompatibiliy (MHC)- class I antigens, do

not express, or express only low levels of, the CD44 antigen, and

are CD105 (also endoglin, or SH2) negative 1,2. Unlike hematopoietic

stem cells (HSC), MAPC do not express CD45, CD34, and cKit 1,2,

but like HSC, MAPC express Thy1, AC133 (human MAPC) and Sca1

(mouse) albeit at low levels 1,2 .In the mouse, MAPC express low

levels of stage specific embryonic antigen (SSEA)-1, and express

low levels of the transcription factors Oct4 and Rex1, known to be

important for maintaining embryonic stem (ES) cells undifferentiated

3

 and to be down-regulated when ES cells undergo somatic cell

commitment and differentiation 2.

MAPC can also be isolated from other tissues, and other species:

We also showed that MAPC can be cultured from mouse

brain and mouse muscle 4. Of note, the differentiation potential and

expressed gene profile of MAPC derived from the different tissues

295
296 MONITORING STEM CELL RESEARCH

appears to be highly similar. These studies used whole brain and

muscle tissue as the initiating cell population, therefore containing

more than neural cells and muscle cells, respectively. The

implications of this will be discussed below. Studies are ongoing to

determine if cultivation of MAPC from other organs is possible, and

whether culture of MAPC, like ES cells, is mouse-strain dependent.

Initial studies suggest that a population of MAPC-like cells can also

be cultured from bone marrow from cynomologous monkeys

(unpublished observations)(studies done by our collaborator Felipe

Prosper, University of Navarra, Pamplona, Spain) and from bone

marrow of dogs (unpublished observations)(studies done at the

University of Minnesota).

Non-senescent nature of MAPC:

Unlike most adult somatic stem cells, MAPC proliferate

without obvious signs of senescence, and have active telomerase.

In humans, the length of MAPC telomeres is 3-5kB longer than in

neutrophils and lymphocytes, and telomere length is not different

when MAPC are derived from young or old donors 1. This suggests

that MAPC are derived from a population of cells that either has

active telomerase in vivo, or that is highly quiescent in vivo, and

therefore has not yet incurred telomere shortening in vivo. In human

MAPC cultures we have not yet seen cytogenetic abnormalities. As

human MAPC are however undergoing symmetrical cell divisions,

it remains possible that despite lack of gross cytogentic changes,

minor mutations accumulate over time. We are therefore planning

to use comparative genomic hybridization to address the question

at what time genetic abnormalities occur, if they do. Initial results

from gene array analysis suggest that MAPC, like ES cells, have a

large number of DNA repair genes expressed (unpublished

observations), which may protect them from more frequent genetic

abnormalities in view of the fact that they undergo multiple

sequential symmetrical cell divisions.

However, several subpopulations of mouse MAPC, and to a

lesser extent rat MAPC, have become aneuploid, even though

additional subpopulations thawed subsequently were

cytogenetically normal. Aneuploidy is seen more frequently once

mouse (and rat) MAPC have been expanded for >60-70 population

doublings and following repeated cryopreservtions and thawing

episodes. This characteristic of mouse MAPC is not dissimilar from

other mouse cell populations, including mouse ES cells.
APPENDIX J. 297

Stringent culture conditions required for maintenance of the

undifferentiated state of MAPC:

Culture of MAPC is, however, technically demanding. Major

factors that play a role in successful maintenance of MAPC include

cell density, CO2 concentration and pH of the medium, lot of fetal

calf serum that is used, and even the type of culture plastic that is

used. Control of cell density appears to be species specific: mouse,

rat and perhaps cynomologous monkey MAPC need to be maintained

at densities between 500 and 1,000 cells / cm2, whereas human

and perhaps dog MAPC need to be maintained between 1,500 and

3,000 cells/ cm2

. The reason why MAPC tend to differentiate to the

default MSC lineage when maintained at higher densities is not

known. However, for MAPC to have clinical relevance, this will need

to be overcome. Gene array and proteomics studies are ongoing to

identify the contact and / or soluble factors that may be responsible

for causing differentiation when MAPC are maintained at higher

densities. These very demanding technical skills can however be

“exported” from the University of Minnesota as, after training at

the University of Minnesota, investigators at the University of Tokai,

Japan (manuscript submitted) and investigators at the University

of Gent, Belgium have successfully isolated MAPC from human bone

marrow, and investigators at the University of Navarra, Spain, have

successfully isolated MAPC from rat bone marrow.

IN VITRO DIFFERENTIATION POTENTIAL OF MAPC:

We published last year that human, mouse and rat MAPC

can be successfully differentiated into typical mesenchymal lineage

cells, including osteoblasts, chondroblasts, adipocytes and skeletal

myoblasts 1. In addition, human, mouse and rat MAPC can be

induced to differentiate into cells with morphological, phenotypic

and functional characteristics of endothelial cells 5, and

morphological, phenotypic and functional characteristics of

hepatocytes 6.

Neuroectodermal differentiation:

Since then, we have also been able to induce differentiation

of MAPC from mouse bone marrow into cells with morphological,

phenotypic and functional characteristics of neuroectodermal cells7.

Differentiation of MAPC to cells with neuroectodermal

characteristics occurred by initial culture in the presence of basic

fibroblast growth factor (bFGF) as the sole cytokine, followed by
298 MONITORING STEM CELL RESEARCH

culture with FGF-8b and sonic hedgehog (SHH), and then brain

derived neurotrophic factor (BDNF) 8,9. Differentiation using these

sequential cytokine stimuli was associated with activation of

transcription factors known to be important in neural commitment

in vivo and differentiation from NSC and mES cells in vitro. Cells

staining positive for astrocyte, oligodendrocyte and neuronal

markers were detected. Neuron-like cells became polarized, and as

has been described in most studies in which ES cells or NSC were

differentiated in vitro to a mid-brain neuroectodermal fate using

FGF8 and SHH, approximately 25% of cells stained positive for

dopaminergic markers, 25% for serotonergic markers, and 50% for

GABA-ergic markers. Subsequent addition of astrocytes induced

further maturation and prolonged survival of the MAPC-derived

neuron-like cells, which now also acquired electrophysiological

characteristics consistent with neurons, namely voltage gated

sodium channels and synaptic potentials 10,11.

Muscle differentiation:

In addition, we now have convincing evidence that MAPC

can differentiate into cells with phenotypic as well as functional

characteristics of smooth muscle cells (manuscript in preparation).

Interestingly, the lineage that continues to be elusive is cardiac

myoblasts, despite the fact that mouse MAPC injected in the

blastocyst contribute to the cardiac muscle 2. Although a number of

in vitro differentiation conditions induce expression of Nkx2.5,

GATA4, and myosin heavy chain mRNA and proteins 12-14, we have

been unable to induce differentiation of MAPC to cells with the

typical functional characteristic of cardiac myoblasts, i.e.

spontaneous rhythmic contractions or beating, a differentiation path

that is almost a default differentiation pathway for mouse ES cells.

The reason for the lack of functional cardiac myoblast properties is

currently unknown.

Another important cell lineage that has not yet been generated is

insulin-producing cells, even though initial studies suggest that

differentiation to cells expressing at least early pancreatic and

endocrine pancreas transcription factors can be obtained.

In vitro differentiation of MAPC as model system for gene

discovery:

A last comment regarding in vitro differentiation of MAPC

is that, in contrast to differentiation of ES cells in vitro, the final

differentiated cell product derived from MAPC is commonly >70-

80% pure. This should allow using these in vitro differentiation
APPENDIX J. 299

models for gene and drug discovery. For instance, in a recently

published study 15 we compared the expressed gene profile in human

MAPC induced to differentiate to osteoblasts and chondroblasts,

two closely related cell lineages. We could demonstrate that

although a large number of genes are co-regulated when MAPC

differentiate to these two lineages, specificity in differentiation can

readily be detected. For instance a number of known and yet to be

fully characterized transcription factor mRNAs were differentially

expressed during the initial phases of differentiation. Studies are

ongoing to further define the role of these genes in lineage specific

differentiation. These studies exemplify however the power of this

model system to study lineage specific differentiation in vitro.

DEGREE OF PLURIPOTENCY OF MAPC:

We have shown that transfer of 10-12 mouse MAPC into

mouse blastocysts results in the generation of chimeric mice. When

10-12 MAPC, expanded for 50-55 population doublings, were injected

approximately 80% of offspring were chimeric, with the degree of

chimerisms varying between 1–40% 4. Cells found in different organs

acquire phenotypic characteristics of the tissue. For instance MAPC

derived cells detected in the brain of chimeric animals differentiate

appropriately into region specific neurons, as well as astrocytes and

oligodendrocytes 16. More recent studies using MAPC from later

population doublings have shown that the frequency of chimerism

decreases when MAPC are maintained for longer time in culture,

even though animals with chimerism of more than 70% could be

obtained (unpublished observations). These studies indicate that

like ES cells, MAPC can give rise to most if not all somatic cell types

of the mouse. Whether MAPC can do this without help of other cells

in the inner cell mass, i.e. can generate a mouse by tetraploid

complementation 17, is not yet known. Also not yet known is whether

MAPC contribute to the germ line when injected in the blastocyst.

POST-NATAL CONTRIBUTION TO TISSUES:

Neither human nor mouse MAPC injected into the muscles

of severe combined immunodeficient (SCID) mice have led to the

development of teratomas (unpublished observations). Likewise,

we have not yet detected donor-derived tumor formation following

IV injection of human or mouse MAPC in NOD-SCID animals.

However, when mouse undifferentiated MAPC are administered IV

to NOD-SCID mice, engraftment in the hematopoietic system as well

as epithelia of gut, liver and lung is seen 2

. Preliminary studies using

human MAPC suggest that a similar pattern of engraftment may
300 MONITORING STEM CELL RESEARCH

occur, even though the level of contribution to blood, liver, gut and

lung is lower (unpublished observations). Noteworthy is the fact

that neither mouse nor human MAPC appear to contribute to other

tissues when injected IV, except to endothelium (see below).

Although PCR analysis for human DNA in human – mouse

transplants or for b-galactosidase in mouse-mouse transplants

yielded positive signals in many tissues, we believe that this is

mainly due to contaminating blood cells. When tissues were carefully

examined for tissue specific differentiated MAPC progeny, we could

not detect MAPC-progeny in brain, skeletal muscle, cardiac muscle,

skin or kidneys. Lack of engraftment in brain, skeletal and cardiac

muscle may be due to the fact that transplants were done in noninjured animals, where the blood brain barrier is intact, and where

little or no cell turnover is expected in muscle. More difficult to

explain is the absence of MAPC-derived progeny in skin, possibly

the organ with the greatest cell turnover. Studies are ongoing to

trace the homing behavior of MAPC following infusion in non-injured

animals and injured animals, which may shed light on these

observations.

In vivo differentiation into skeletal muscle:

Muguruma et al have also shown that undifferentiated

human MAPC injected in the muscle of non-obese diabetic (NOD)-

SCID mice differentiate into cells that stain positive for muscle

transcription factors and muscle cytoskeletal proteins (manuscript

submitted). Similar results were seen in Minnesota. We also found

that pre-treatment of human MAPC with 5-azacytidine, required to

induce muscle differentiation in vitro, enhanced the degree of

engraftment of human cells in mouse muscle, suggesting that predifferentiation of MAPC may under certain circumstances enhance

the level of engraftment (unpublished observations).

Contribution to endothelium in vivo:

When endothelial cells generated from human MAPC by

incubation in vitro with vascular endothelial growth factor (VEGF) 5

were infused in animals in which a tumor had been implanted

underneath the skin, we detected enhanced tumor growth and found

that up to 30% of the tumor vasculature was derived from the human

endothelial cells. Likewise, wounds in the ears of these animals as

a result of ear tagging contained human endothelial cells. One of

the animals developed a host-tumor, an occurrence seen frequently

in aging NOD-SCID mice. We detected contribution of MAPC-derived

endothelium to tumor vessels 2

. Likewise, one of the NOD-SCID
APPENDIX J. 301

mice that received human MAPC developed a host thymic

lymphoma. Human MAPC, like mouse MAPC, appeared to

differentiate into endothelial cells that contribute to tumor

angiogenesis.

Engraftment of MAPC in stroke model:

In yet another in vivo study 18 we evaluated the effect of

human MAPC in a rat stroke model. Cortical brain ischemia was

produced in male rats by permanently ligating the right middle

cerebral artery distal to the striatal branch. Animals were placed

on cyclosporine-A and 2 weeks later, 2x105 human MAPC were

injected around the infarct zone. As controls, animals received

normal saline or MAPC conditioned medium. Limb placement test

and tactile stimulation test were blindly assessed 1 week before

brain ischemia, 1 day before transplantation, and at 2 and 6 weeks

after grafting. The limb placement test included eight subtests

described by Johansson and coworkers 19. In a tactile stimulation

test 20, a small piece of adhesive tape was rapidly applied to the

radial aspect of each forepaw. The rats were then returned to their

home cages, and the order of the tape removal (i.e., left versus right)

was recorded. Three to five trials were conducted on each test day.

Each trial was terminated when the tapes were removed from both

forepaws or after 3 min. Animals were subsequently sacrificed to

determine the fate of the human cells injected in the brain. After 2

and 6 weeks, animals that received human MAPC scored statistically

significantly better in the limb placement test as well as tactile

stimulation test compared with animals that received only

cyclosporine-A (CSA), or were injected with normal saline or MAPC

conditioned medium. The level of recuperation of motor and sensory

function was 80% of animals without stroke. When the brain was

examined for the presence and differentiation of human MAPC to

neuroectodermal cells, we found that human MAPC were present,

but remained rather immature. Therefore, we cannot attribute the

motor and sensory improvement to region specific differentiation to

neuronal cells and integration of neurons derived from MAPC in the

host brain. Rather the improvement must be caused by trophic

effects emanated by the human MAPC to either improve

vascularization of the ischemic area, to support survival of the

remaining endogenous neurons, or to recruit neuronal progenitors

from the host brain. These possibilities are currently being evaluated.
302 MONITORING STEM CELL RESEARCH

POSSIBLE MECHANISMS UNDERLYING THE PHENOMENON OF

MULTIPOTENT ADULT PROGENITOR CELLS:

Currently we do not fully understand the mechanism(s)

underlying the culture selection of MAPC. We have definitive data

to demonstrate that the pluripotency of MAPC is not due to coculture of several stem cells.

Pluripotency cannot be attributed to multiple stem cells:

First, using retroviral marking studies we have definitive

proof that a single cell can differentiate in vitro to cells of mesoderm,

both mesenchymal and non-mesenchymal, neuroectoderm and

hepatocyte-like cells, and this for human 1,6, mouse and rat MAPC

2,6. Second, we have shown that a single mouse MAPC is sufficient

for generation of chimeric animals 2. Indeed, we published that 1/3

animals born from blastocysts in which a single MAPC was injected

were chimeric with chimerism degrees varying between 1 and 45%.

This rules therefore out that the pluripotent nature of these cells is

due to co-existence in culture of multiple somatic stem cells.

Cell fusion is not likely explanation:

A second possibility for the greater degree of differentiation

potential would be that cells undergo fusion and acquire via this

mechanism greater pluripotency. Fusion has been shown to be

responsible for apparent ES characteristics of marrow and neural

stem cells 21,22 that had been cocultured with ES cells in vitro, and

more recently for the apparent lineage switch of bone marrow cells

to hepatocytes when hematopoietic cells were infused in animals

with hereditary tyrosinemia due to lack of the fumarylacetoacetate

hydroxylase (FAH) gene 23[Wang et al, Nature 2003]. In the former

two studies, the majority of genes expressed in the marrow or neural

cell that fused with ES cells were silenced, and the majority of the

genes expressed in ES cell were persistently expressed. Likewise

for the bone marrow-hepatocyte fusion, the majority of genes

expressed normally in hematopoioetic cells (except the FAH gene)

were silenced whereas genes expressed in hepatocytes

predominated. Finally, the cells generated were in general tetraploid

or aneuploid.

We do not believe that this phenomenon underlies the

observation that MAPC are pluripotent. Cultivation and

differentiation in vitro (in general, except the final differentiation
APPENDIX J. 303

step for neuroectoderm) does not require that MAPC are co-cultured

with other cells, making the likelihood that MAPC are the result of

fusion very low. Smith et al suggested in a recent commentary that

MAPC could be caused by fusion of multiple cell types early on

during culture leading to reprogramming of the genetic information

and pluripotency [REF]. However, we have no evidence that MAPC

are tetraploid or aneuploid early during culture, making this

possibility less likely. Nevertheless, studies are ongoing to rule this

out. The in vivo studies were not set up to fully be capable of ruling

out this possibility.

However, a number of findings suggest that fusion may not

likely be the cause for the engraftment seen postnatally, nor the

chimerism in the blastocyst injection experiment. The frequency of

the fusion event described for the ES-BM, ES-NSC, and HSChepatocyte fusion was in general very low, i.e. 1/100,000 cells.

Expansion of such fused cells could only be detected when drug

selection was applied in the in vitro systems, and withdrawal of

NTBC (2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione) in

the FAH mouse model was used to select for cells expressing the

FAH gene. The percent engraftment seen in our post-natal transplant

models was in the range of 1% - 9%. The frequency of chimerism

seen in blastocyst injection studies ranged between 33% and 80%

when 1 and 1 and 10-12 MAPC were injected, respectively. These

frequencies are significantly higher than what has been described

for fusion events with ES cells in vitro, and in the HSC-hepatocyte

fusion studies in vivo.

Furthermore, in contrast to what was described in the papers

indicating that fusion may be responsible for apparent plasticity, all

in vivo studies done with MAPC were done without selectable

pressure, mainly in non-injured animals. Therefore, it is less likely

that the pluripotent behavior of MAPC in vivo is due to fusion

between the MAPC and the tissues where they engraft / contribute

to. However, specific studies are currently being designed to formally

rule this out.

Primitive ES-like cells that persist vs. de-differentiation:

Currently, we do not have proof that MAPC exist as such in

vivo. Until we have positive selectable markers for MAPC, this

question will be difficult to answer. If the cell exists in vivo, one

might hypothesize that it is derived for instance from primordial

germ cells that migrated aberrantly to tissues outside the gonads

during development. It is, however, also possible that removal of
304 MONITORING STEM CELL RESEARCH

certain (stem) cells from their in vivo environment results in

“reprogramming” of the cell to acquire greater pluripotency. The

studies on human MAPC suggest that such a cell that might undergo

a degree of reprogramming is likely a protected (stem) cell in vivo,

as telomere length of MAPC from younger and older donors is similar,

and significantly longer than what is found in hematopoietic cells

from the same donor. The fact that MAPC can be isolated from

multiple tissues might argue that stem cells from each tissue might

be able to be reprogrammed. However, as was indicated above, the

studies in which different organs were used as the initiating cell

population for generation of MAPC did not purify tissue specific

cells or stem cells. Therefore, an alternative explanation is that the

same cells isolated from bone marrow that can give rise to MAPC in

culture might circulate, and be collected from other organs. However,

we have until now been unsuccessful in isolating MAPC from blood

or from umbilical cord blood, arguing against this phenomenon.

Finally, cells selected from the different organs could be the same

cells resident in multiple organs, such as MSC that are present in

different locations, or cells associated with tissues present in all

organs such as for instance blood vessels. Studies are ongoing to

determine which of these many possibilities is correct.

CONCLUSION:

We believe that MAPC would have clinical relevance

whether they exist in vivo, or are created in vitro. However,

understanding the nature of the cell will have impact on how one

would approach their clinical use. If they exist in vivo, it will be

important to learn where they are located, and to determine whether

their migration, expansion and differentiation in a tissue specific

manner can be induced and controlled in vivo. If they are a culture

creation, understanding the mechanism underlying the

reprogramming event will be important as that might allow this

phenomenon to happen on a more routine and controlled basis.

Either way, a long road lies ahead before MAPC might be

applicable in clinical trials. Hurdles to be overcome include

development of robust culture systems that will allow

automatization. Like of other stem cells, including ES cells, we will

need to determine in preclinical models whether undifferentiated

vs. lineage committed vs. terminally differentiated cells should be

used to treat a variety of disorders. If lineage committed or terminally

differentiated cells will be needed, robust clinical scale

differentiation cultures will need to be developed. Furthermore,

studies will need to be performed to demonstrate whether
APPENDIX J. 305

potentially contaminating undifferentiated MAPC will interfere with

engraftment, and / or differentiate inappropriately in vivo. Likewise,

studies aimed at determining what level of HLA-mismatch will be

tolerated in transplantations, whether tolerization via hematopoietic

engraftment from MAPC will be required. As is also the case for

other extensively cultured cells, we will need to further determine

if prolonged expansion leads to genetic abnormalities in cells that

might lead to malignancies when transplanted in vivo.

As a final remark, MAPC appear to have pluripotent potential

both in vitro and in vivo. Furthermore, they appear to proliferate

without obvious senescence when maintained under very

stringently controlled culture conditions. Because of these reasons,

some have argued that they might be a viable alternative to ES cells.

However, at this stage of the research, I feel that such a conclusion

is premature. Whether MAPC have equal longevity as ES cells, and

have the ability to create all >200 cell types in the body is still not

known. Moreover, there appear to be certain cell types that are more

readily generated from ES cells compared with MAPC, such as for

instance cardiac myoblasts, whereas it appears for instance more

easy to generate hepatocyte like cells from MAPC than ES cells.

Therefore, I continue to strongly believe that strict comparative

studies between the two cell populations are needed to determine

the true potential of the cells, and that the scientific insights gained

from these studies should be used to determine which of the cells

will be suitable for use in the clinical setting.

REFERENCES

1. Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM.

Purification and ex vivo expansion of postnatal human marrow mesodermal

progenitor cells. Blood. 2001;98:2615-2625

2. Jiang Y, Jahagirdar B, Reyes M, Reinhardt RL, Schwartz RE, Chang

H-C, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Kaushal S,

Largaespada DL, Verfaillie CM. Pluripotent nature of adult marrow derived

mesenchymal stem cells. Nature. 2002;418:41-49

3. Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4

defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet.

2000;24:372-376

4. Jiang Y, Vaessen B, Lenvik T, Blackstad M, Reyes M, Verfaillie CM.

Multipotent progenitor cells can be isolated from post-natal murine bone marrow,

muscle and brain. Exp Hematol. 2002;30(8):896-904
306 MONITORING STEM CELL RESEARCH

5. Reyes M, Dudek A, Jahagirdar B, Koodie K, Marker PH, Verfaillie CM.

Origin of endothelial progenitors in human post-natal bone marrow. J Clin Invest.

2002;109:337-346

6. Schwartz RE, Reyes M, Koodie L, Jiang Y, Blackstad M, Lund T,

Lenvik T, Johnson S, Hu W-S, Verfaillie CM. Multipotent adult progenitor cells

from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest.

2002;109(10):1291-1302

7. Jiang Y, Henderson D, Blackstadt M, Chen A, Miller FF, Verfaillie CM.

Neuroectodermal differentiation from mouse multipotent adult progenitor cells.

Proc. Natl. Acad. Sci U S A. 2003;In Press

8. Ling Z, Potter E, Lipton J, Carvey P. Differentiation of mesencephalic

progenitor cells into dopaminergic neurons by cytokines. Exp Neurol.

1998;149:411-423

9. Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD. Efficient

generation of midbrain and hindbrain neurons from mouse embryonic stem cells.

Nat Biotechnol. 2000;18:675-679

10. Wagner J, Akerud P, Castro DS, Holm PC, Canals JM, Snyder EY,

Perlmann T, Arenas E. Induction of a midbrain dopaminergic phenotype in Nurr1-

overexpressing neural stem cells by type 1 astrocytes. Nat Biotech. 1999;17:653-

659

11. Song H, Stevens CF, Gage FH. Astroglia induce neurogenesis from

adult neural stem cells. Nature. 2002;417:39-44

12. Tanaka M, Chen Z, Bartunkova S, Yamasaki N, Izumo S. The cardiac

homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential

for heart development. Development. 1999;126:1269-1280

13. Laverriere AC, MacNeill C, Mueller C, Poelmann RE, Burch JB,

Evans T. GATA-4/5/6, a subfamily of three transcription factors transcribed in

developing heart and gut. J Biol Chem. 1994;269:23177-23184

14. Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selected

cardiomyocytes from differentiating embronic stem cells form stable intracardiac

grafts. J Clin Invest. 1996;98:216-224

15. Qi H, Aguiar DJ, Williams SM, La Pean A, Pan W, Verfaillie CM.

Identification of genes responsible for osteoblast differentiation from human

mesodermal progenitor cells. Proc. Natl Acad. Sci U S A. 2003 Mar18;100(6):3305-

3310

16. Keene CD, Ortiz-Gonzalez XR, Jiang Y, Largaespada DA, Verfaillie

CM, Low WC. Neural differentiation and incorporation of bone marrow-derived

multipotent adult progenitor cells after single cell transplantation into blastocyst

stage mouse embryos. Cell Transplant. 2003;12(3):201-213
APPENDIX J. 307

17. Wang ZQ, Kiefer F, Urbanek P, Wagner EF. Generation of completely

embryonic stem cell-derived mutant mice using tetraploid blastocyst injection.

Mech Dev. 1997;62:137-145

18. Zhao L-R, Duan W-M, Reyes M, Keene CD, Verfaillie CM, Low WC.

Human bone marrow stem cells exhibit neural phenotypes and ameliorate

neurological deficits after grafting into the ischemic brain of rats. Exp Neurol.

2002;174:11-20

19. Ohlsson AL, Johansson BB. Environment influences functional

outcome of cerebral infarction in rats. Stroke. 1995;4:644-649

20. Netto CA, Hodges JD, Sinden JD, LePeillet E, Kershaw T, Schallert

T, Whishaw IQ. Bilateral cutaneous stimulation of the somatosensory system in

hemidecorticate rats. Behav. Neurosci. 1984;98:518-540

21. Ying QY, Nichols J, Evans EP, Smith AG. Changing potency by

spontaneous fusion. Nature. 2002;416:545-548

22. Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y,

Meyer EM, More Ll, Petersen BE, Scott EW. Bone marrow cells adopt the

phenotype of other cells by spontaneous cell fusion. Nature. 2002;416:542-545

23. Lagasse E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M,

Osborne L, Wang X, Finegold M, Weissman IL, Grompe M. Purified hematopoietic

stem cells can differentiate into hepatocytes in vivo. Nat Med. 2000;6:1229-1234

Appendix K.

Adult Stem Cells

DAVID A. PRENTICE, PH.D.

Professor of Life Sciences at Indiana State University,

 Terre Haute, Indiana

Within just a few years, the possibility that the human body

contains cells that can repair and regenerate damaged and diseased

tissue has gone from an unlikely proposition to a virtual certainty.

Adult stem cells have been isolated from numerous adult tissues,

umbilical cord, and other non-embryonic sources, and have

demonstrated a surprising ability for transformation into other tissue

and cell types and for repair of damaged tissues. This paper will

examine the published literature regarding the identity of adult stem

cells and possible mechanisms for their observed differentiation into

tissue types other than their tissue of origin. Reported data from

both human and animal studies will be presented on the various

tissue sources of adult stem cells and the differentiation and repair

abilities for each source, especially with regards to current and

potential therapeutic treatments.

Adult stem cells have received intense scrutiny over the past

few years due to surprising discoveries regarding heretofore

unknown abilities to form multiple cell and tissue types, as well as

the discovery of such cells in an increasing number of tissues. The

term “adult stem cell” is somewhat of a misnomer, because the cells

are present even in infants and similar cells exist in umbilical cord

and placenta. More accurate terms have been proposed, such as

tissue stem cells, somatic stem cells, or post-natal stem cells.

However, because of common usage this review will continue to

use the term adult stem cell.

This paper will review the literature related to adult stem

cells, including current and potential clinical applications (with

apologies to the many who are not cited, due to the exponential

increase in papers regarding adult stem cells and the limitations of

this review.) The focus will be on human adult stem cells, but will

309
310 MONITORING STEM CELL RESEARCH

also include results from animal studies which bear on the potential

of adult stem cells to be used therapeutically for patients.

This paper will not attempt to review the literature related

to hematopoietic stem cells, i.e., the bone marrow stem cell that is

the immediate precursor for blood cells, and the formation of typical

blood cells. Nor will this paper review the substantial literature

regarding clinical use of bone marrow or bone marrow stem cell

transplants for hematopoietic conditions such as various cancers

and anemias, nor the striking clinical results seen for conditions

such as scleromyxedema, multiple sclerosis, systemic lupus,

arthritis, Crohn’s disease, etc.1

 In these instances, the stem cells

are used primarily to replace the hematopoietic system of the patient,

after ablation of the patient’s own bone marrow hematopoietic

system. Finally, multipotent adult progenitor cells (MAPC’s), a bone

marrow stem cell that has shown significant abilities at proliferation

in culture and differentiation into other body tissues,2 have been

reviewed by Dr. Catherine Verfaillie in a separate paper for the

President’s Council on Bioethics, and the reader is directed to that

review for more information.

Key questions regarding adult stem cells are: (1) their

identity, (2) their tissue source of origin, (3) their ability to form other

cell or tissue types, and (4) the mechanisms behind such changes

in differentiation and effects on tissues and organs. Historically only

a few stem cells were recognized in humans, such as the

hematopoietic stem cell which produces all of the blood cell types,

the gastrointestinal stem cell associated with regeneration of the

gastrointestinal lining, the stem cell responsible for the epidermal

layer of skin, and germ cell precursors (in the adult human, the

spermatogonial stem cell.) These stem cells were considered to

have very limited repertoires, related to replenishment of cells within

their tissue of origin. These limitations were considered to be a

normal part of the developmental paradigm in which cells become

more and more restricted in their lineage capabilities, leading to

defined and specific differentiated cells in body tissues. Thus,

discovery of stem cells in other tissues, or with the ability to cross

typical lineage boundaries, is both exciting and confusing because

such evidence challenges the canonical developmental paradigm.

STEM CELL MARKERS

Identification of cells typically relies on use of cell surface

markers—cellular differentiation (CD) antigens—that denote the

expression of particular proteins associated with genomic activity
APPENDIX K. 311

related to a particular differentiation state of the cell. Identification

also has relied on morphological and molecular indications of

function, such as expression of specific enzymes. Since stem cells

by definition have not yet taken on a specific differentiated function,

their identification has relied primarily on use of cell surface markers,

and only secondarily on production of differentiated products in

various tissues. One stated goal has been to isolate a single putative

adult stem cell, characterized fully by specific markers and molecular

characteristics, and then to follow the differentiation of this single

cell (and/or its progeny) to show that it indeed has multipotent or

pluripotent capabilities (clonogenic ability). For bone marrow stem

cells, selection of putative adult stem cells has usually excluded

typical markers for hematopoietic lineages (lin-

), CD45, CD38, with

inclusion or exclusion of the hematopoietic marker CD34 and

inclusion of the marker c-kit (CD117). Other proposed markers for

adult stem cells are AC133-2 (CD133), which is found on many stem

cell populations,3 and C1qRp, the receptor for complement molecule

C1q,4 found on a subset of CD34+/- human stem cells from bone

marrow and umbilical cord blood. When transplanted into

immunodeficient mice, C1qRp-positive human stem cells formed not

only hematopoietic cells but also human hepatocytes. Other

methods of isolation and identification include the ability of putative

stem cells to exclude fluorescent dyes (rhodamine 123, Hoechst

33342), allowing isolation by fluorescence-activated cell sorter

(FACS) of a “side population” of cells within a tissue that have stem

cell characteristics. Expression of the Bcrp1 gene (ABCG2 gene in

humans) is apparently responsible for this dye exclusion, and could

provide a common molecular expression marker for stem cells5. A

study of expressed genes from a single cell-derived colony of human

mesenchymal stem cells identified transcripts from numerous cell

lineages,6 and a similar attempt at profiling the gene expression of

human neural stem cell in culture with leukemia inhibitory factor

(LIF) has been done,7 perhaps providing an expressed molecular

milieu which could identify candidate stem cells. Attempts to

determine the complete molecular signature of gene expression

common to human and mouse stem cells have shown over 200

common genes between hematopoietic and neural stem cells, with

some considerable overlap with mouse embryonic stem cells as

well.8 The function of many of these genes is as yet unknown, but

may provide distinctive markers for identification of adult stem cells

in different tissues.

However, dependence on particular markers for prospective

identification and isolation of adult stem cells seems unreliable. In

particular, the use of specific hematopoietic markers such as the
312 MONITORING STEM CELL RESEARCH

presence or absence of CD34, has yielded mixed results in terms of

the identification of putative stem cells. There is evidence that the

expression of CD34 and CD133 can actually change over time, and

its expression may be part of a cycling phenomenon among human

hematopoietic and mesenchymal stem cells in the bone marrow and

peripheral blood, and perhaps in other tissues,9 i.e., an isolated CD34+

cell may become CD34-, and then reacquire CD34 expression.

Likewise, a systematic analysis of the cell surface markers and

differentiation potential of supposedly distinct isolated populations

of human bone marrow stem cells revealed no differences in practice

between the cell populations.10 Moreover, an analysis of genetic

and ultrastructural characteristics of human mesenchymal stem cells

undergoing differentiation and dedifferentiation has revealed

reversibility in the characteristics studied.11 Thus, any attempt to

isolate a single type of adult stem cell for study may not actually

capture the intended cell, or may, by using a particular set of isolation

or growth conditions, alter its gene expression. This idea has been

elaborated by Thiese and Krause,12 who note that this “uncertainty

principle” means any attempt to isolate and characterize a cell

necessarily alters its environment, and thereby potentially its gene

expression, identity, and potential ability to differentiate along

various lineages. Likewise, the stochastic nature of cell

differentiation in such dynamic and interacting systems means that

attempts to delineate differentiation pathways must include

descriptions of each parameter associated with the conditions used,

and still may lead only to a probabilistic outcome for differentiation

of a stem cell into a particular tissue. Blau et al.13 have raised the

question of whether there may be a “universal” adult stem cell,

residing in multiple tissues and activated dependent on cellular

signals, e.g., tissue injury. When recruited to a tissue, the stem cell

would take its cues from the local tissue milieu in which it finds

itself (including the soluble growth factors, extracellular matrix, and

cell-cell contacts.) Examples of such environmental influences on

fate choice have been noted previously.14 Thus, it may not be

surprising to see examples of cells isolated using the same marker

set showing disparate differentiative potentials,15,16,17,18 based on the

context of the isolation or experimental conditions, or to see cells

with different marker sets showing similar differentiation. In the

final analysis, description of a “stem cell”, its actual tissue of origin,

and even its differentiation ability, may be a moving target

describable only within the context of the particular experimental

paradigm used, and may require asking the correct questions in

context of the cell’s identity and abilities not clonally but rather

within a population of cells, and within a certain environment.12,19
APPENDIX K. 313

Given the uncertainties involved in isolating and identifying

particular adult stem cells, Moore and Quesenberry20 suggest that

we consider an adult stem cell’s functional ability to be, at a

minimum, taking on the morphology and cell markers of a

differentiated tissue, supplemented by any further functional activity

and interaction within a tissue. Certainly a physiological response

by improvement of function in a damaged organ system is an

indication of a functional response.19,20 As will be discussed later,

the function and therapeutic benefit may not necessarily require

direct differentiation and integration of an adult stem cell into a

desired tissue, but could be accomplished by stimulation of

endogenous cells within the tissue.

DIFFERENTIATION MECHANISMS

Several possible mechanisms have been proposed for

differentiation of adult stem cells into other tissues. One mechanism

that has received attention lately is the possibility of cell fusion,

whereby the stem cell fuses with a tissue cell and takes on that

tissue’s characteristics. In vitro experiments using fusion of somatic

cells with embryonic stem cells and embryonic germ cells21 have

demonstrated that the cell hybrid can take on characteristics of the

more primitively developed cell. However, given that such

characteristics of spontaneous cell fusion hybrids in vitro have been

known for quite some time,22 and that a cell fusion hybrid does not

explain in vitro differentiation of adult stem cells unexposed to

tissues, the experiments could not verify this as a possible

mechanism for adult stem cell differentiation. More recently, in vivo

experiments have shown that for liver,23 formation of a cell fusion

hybrid is a viable explanation for some of the differentiation as well

as repair of liver damage seen in these experiments. In an in vitro

experiment where human mesenchymal stem cells were co-cultured

with heat-shocked small airway epithelial cells, a mixed answer

was obtained—some of the stem cells differentiated directly into

epithelial cells, while others formed cell fusion hybrids to repair the

damage.24 The ability to form cell hybrids in some tissues may be a

useful mechanism for repair of certain types of tissue damage or for

delivery of therapeutic genes to a tissue.25 The reprogramming of

cellular gene expression via hybrids is not unlike a novel method

reported recently for transdifferentiation of somatic cells. In this

method, fibroblasts were soaked in the cytoplasm and nucleoplasm

of a lysed, differentiated T lymphocyte cell, taking up factors from

the exposed “soup” of the cellular contents of the differentiated cell,

and began expressing functional characteristics of a T cell.26
314 MONITORING STEM CELL RESEARCH

In contrast to the results discussed above, other experiments

have shown no evidence that cell fusion plays a role in differentiation

of adult stem cells into other tissue types. For example, using human

subjects it was shown that human bone marrow cells differentiated

into buccal epithelial cells in vivo without cell fusion,27 and human

cord blood stem cells formed hepatocytes in mouse liver without

evidence of cell fusion.28 In these cases it appears that the adult

stem cells underwent changes in gene expression and directly

differentiated into the host tissue cell type, integrating into the

tissue. It is likely that the mechanism of adult stem cell

differentiation may vary depending on the target tissue, or possibly

on the state of the adult stem cell used, especially given that normal

functioning liver typically shows cell fusion hybrids, with cell fusion

functioning as a mechanism for most of the differentiation and repair

in tissues such as liver, and direct differentiation

(transdifferentiation) into other cell types functioning in other

tissues. Much remains to be determined regarding the mechanisms

associated with adult stem cell differentiation.

Keeping in mind the uncertainties noted above for

identification of a particular adult stem cell and its initial tissue of

origin, the majority of this review will focus on some of the evidence

for adult stem cell differentiation into other tissues. The cells will

be categorized based on general tissue of isolation, with the primary

emphasis on human adult stem cells, supplemented with information

from animal studies.

BONE MARROW STEM CELLS

Bone marrow contains at least two, and likely more,2,29

discernable stem cell populations. Besides the hematopoietic stem

cell which produces blood cell progeny, a cell type termed

mesenchymal or stromal also exists in marrow. This cell provides

support for hematopoietic and other cells within the marrow, and

has also been a focus for possible tissue repair.30 Isolation is typically

based on some cell surface markers, but also primarily on the ability

of these cells to form adherent cell layers in culture. Human

mesenchymal stem cells have been shown to differentiate in vitro

into various cell lineages including neuronal cells,31,32 as well as

cartilage, bone, and fat lineages.33 In vivo, human adult

mesenchymal stem cells transferred in utero into fetal sheep can

integrate into multiple tissues, persisting for over a year. The cells

differentiated into cardiac and skeletal muscle, bone marrow stromal

cells, fat cells, thymic epithelial cells, and cartilage cells. Analysis

of a highly purified preparation of human mesenchymal stem cells34
APPENDIX K. 315

indicated that they could proliferate extensively in culture,

constitutively expressing the telomerase enzyme, and even after

extensive culture retained the ability to differentiate in vitro into

bone, fat, and cartilage cells. Isolated colonies of the cells formed

bone when injected into immunodeficient mice. Expanding on their

previous in vitro work with rat and human mesenchymal/stromal

stem cells, Woodbury et al.

35 performed molecular analyses of rat

stromal stem cells and found that the cells express genes associated

with all three primary germ layers—mesodermal, ectodermal, and

endodermal—as well as a gene associated for germinal cells. The

gene expression pattern was also seen in a clonal population of

cells, indicating that it was not due to an initial mixed population of

cells, but was the typical gene expression pattern of the stromal

cells. The results suggested that the stromal stem cells were already

multidifferentiated and that switching to a neuronal differentiation

pattern involved quantitative regulation of existing gene expression

patterns. Koc et al.36 have used infusion of allogeneic donor

mesenchymal stem cells in an attempt to correct some of the skeletal

and neurological defects associated with Hurler syndrome

(mucopolysaccharidosis type-IH) and metachromatic leukodystrophy

(MLD). A total of 11 patients received donor mesenchymal stem

cells, expanded from bone marrow aspirate. Four patients showed

significant improvements in nerve conduction velocities, and all

patients showed maintenance or slight improvement in bone mineral

density.

Bone marrow-derived cells in general have shown ability to

form many tissues in the body. For example, bone marrow-derived

stem cells in vivo appear able to form neuronal tissues,18,37 and a

single adult bone marrow stem cell can contribute to tissues as

diverse as marrow, liver, skin, and digestive tract.16 One group has

now developed a method for large-scale generation of neuronal

precursors from whole adult rat bone marrow.38 In this procedure,

treatment of unfractionated bone marrow in culture with epidermal

growth factor and basic fibroblast growth factor gave rise to

neurospheres with cells expressing neuronal markers.

In vivo studies using fluorescence and genetic tracking of

adult stem cells in animals, and tracking of the Y chromosome in

humans, has shown that bone marrow stem cells can contribute to

numerous adult tissues. Follow-up of patients receiving adult bone

marrow stem cell transplants has allowed tracking of adult stem

cells within humans, primarily by identification of Y chromosomebearing cells in female patients who had received bone marrow stem

cells from male donors. Biopsy or postmortem samples show that
316 MONITORING STEM CELL RESEARCH

some of the transplanted bone marrow stem cells could form liver,

skin, and digestive tract cells,39 as well as participate in the

generation of new neurons within the human brain.40 Bone marrow

stem cells have also been shown to contribute to Purkinje cells in

the brains of adult mice41 and humans42. Generation of this particular

type of neural cell is significant in that new Purkinje cells do not

normally appear to be generated after birth.

Regeneration or replacement of dead or damaged cells is

the primary goal of regenerative medicine and one of the prime

motivations for study of stem cells. It is thus of significant interest

that bone marrow stem cells have shown the ability to produce

therapeutic benefit in animal models of stroke. In mice, fluorescencetracked bone marrow derived stem cells expressed neuronal

antigens and also incorporated as endothelial cells, possibly

producing therapeutic benefit by allowing increased blood flow to

damaged areas of the brain.43 In rats, intravenous (IV) administration

of rat44 or human45 bone marrow stromal cells resulted in significant

behavioral recovery after stroke. Interestingly, only a small

percentage of the stromal stem cells appeared to incorporate into

the damaged brain as neuronal cells (1-5% in the case of the human

marrow stromal cells), but the levels of neurotrophin growth factors

within the brains increased and were possibly the signal for repair

of damaged brain tissue, perhaps by stimulation of endogenous

neuronal precursors. It is also of interest that the marrow stromal

cells were injected IV and not intracerebrally, indicating that the

stem cells somehow “homed” to the site of tissue damage. Most

studies showing adult stem cell differentiation into other tissues

show an increased incorporation of cells, or even an absolute

requirement for differentiation, relying on tissue damage to initiate

the differentiation. This may indicate that without a “need” for

replacement and repair, there is little or no activation of adult stem

cells. The recruitment and homing of adult stem cells to damaged

tissues are fascinating but relatively unexplained phenomena. One

report46 indicates that recruitment of quiescent stem cells from bone

marrow to the circulation requires release of soluble c-kit ligand

(stem cell factor), but the range of factors necessary for recruitment

and homing to organs other than bone marrow is unknown at this

time and warrants increased investigation.

Bone marrow stem cells have also shown the ability to

participate in repair of damaged retinal tissues. When bone marrow

stem cells were injected into the eyes of mice, they associated with

retinal astrocytes and extensively incorporated into the vascular

(blood vessel) network of the eye.47 The cells could also rescue and
APPENDIX K. 317

maintain normal vasculature in the eyes of mice with a degenerative

vascular disease. In another animal study, bone marrow derived

stem cells were observed to integrate into injured retina and

differentiated into retinal neuronal cells.48 Stromal stem cells have

also shown capability in mice to repair spinal cord which was

demyelinated.49 One of the problems related to spinal cord injury is

loss of the protective myelin sheath from spinal cord after injury. A

mixed bone marrow stem cell fraction was injected into the area of

damage in the spinal cord, and remyelination of the area was seen.

In another mouse study, marrow stromal cells injected into injured

spinal cord formed guiding strands within the cord;50 interestingly,

the effect was more pronounced when the stromal cells were injected

1 week after injury rather than immediately after injury.

Because bone marrow stem cells are of mesodermal lineage,

it is not surprising that they show capabilities at forming other

tissues of mesodermal origin. Human marrow stromal cells, which

have been shown to form cartilage cells, have been used in an in

vitro system to define many of the molecular events associated with

formation of cartilage tissue.51 Bone marrow derived stem cells have

also been shown capable of regenerating damaged muscle tissue.52

In an elegant study following genetically marked bone marrow stem

cells in mice, LaBarge and Blau were able to document multiple

steps in the progression of the stem cells to form muscle fibers and

repair muscle damage.53 The ability of human bone marrow derived

stem cells to form muscle cells and persist in the muscle was recently

documented. In this case, a patient had received a bone marrow

transplant at age 1, and developed Duchenne muscular dystrophy

at age 12. Biopsies at age 14 showed donor nuclei integrated within

0.5-0.9% of the muscle fibers of the patient, indicating the ability of

donated marrow cells to persist in tissue over long periods of time.54

Bone marrow stem cells have also shown capability at

forming kidney cells. Studies following genetically marked bone

marrow stem cells in rats55 and mice56 showed that the stem cells

could form mesangial cells to repopulate the glomerulus of the

kidney. In the mouse study, formation of cell fusion products was

ruled out as a mechanism for differentiation of the bone marrow

stem cells. Other animal studies have shown contribution of bone

marrow stem cells to repair of damaged renal tubules in the

kidney;57,58 taken together, animal studies indicate that bone marrow

stem cells can participate in restoring damaged kidney tissue.59

Liver was one of the earliest tissues recognized as showing

potential contribution to differentiated cells by bone marrow stem
318 MONITORING STEM CELL RESEARCH

cells. Bone marrow stem cells have been induced to form

hepatocytes in culture60 and liver-specific gene expression has been

induced in vitro in human bone marrow stem cells.61 In vivo, bone

marrow stem cells were able to incorporate into liver as hepatocytes

and rescue mice from a liver enzyme deficiency, restoring normal

liver function.62 Bone marrow stem cells also repopulated liver after

irradiation of mice to destroy their bone marrow.63 Examination of

livers of female patients who had received male bone marrow

transplants, and male patients who had received female liver

transplants, showed that similar repopulation of liver from bone

marrow stem cells could take place in humans.64 Examination of

the kinetics of liver repopulation by bone marrow stem cells in a

mouse model indicated that the replacement was slow, with only

small numbers of cells replaced by the bone marrow stem cells.65

As noted previously, two recent studies have found that

replenishment of liver by bone marrow stem cells occurs primarily

via cell fusion hybrid formation, even in repair of liver damage.23 A

side-population of stem cells has been identified in mouse liver,

similar to that seen in bone marrow. This hepatic side-population,

which contributes to liver regeneration, can be replenished by sidepopulation bone marrow stem cells.66

Pancreas and liver arise from adjacent endoderm during

embryological development, and show relatedness in some gene

expression and interconversion in some instances. Bone marrow

derived cells have shown the ability to form pancreatic cells in animal

studies. Mouse bone marrow stem cells containing a genetic

fluorescent marker that is only expressed if insulin is expressed were

transplanted into irradiated female mice.67 Within 6 weeks of

transplant, fluorescent donor cells were observed in pancreatic

islets; donor cells identified in bone marrow and peripheral blood

did not show fluorescence. In vitro, the bone marrow derived cells

showed glucose-dependent insulin secretion as well. Bone marrow

derived stem cells have also demonstrated the ability to induce

regeneration of damaged pancreas in the mouse.68 Mice with

experimentally induced hyperglycemia from pancreatic damage

were treated with bone marrow derived stem cells expressing the

c-kit marker. Interestingly, only a low percentage of donor cells

were identified as integrating into the regenerating pancreas, with

most of the regeneration due to induced proliferation and

differentiation of endogenous pancreatic cell precursors, suggesting

that the bone marrow stem cells provided growth signals for the

tissue regeneration.
APPENDIX K. 319

Heart, as a mesodermally-derived organ, is a likely candidate

for regeneration with bone marrow derived stem cells. Numerous

references now document the ability of these adult stem cells to

contribute to regeneration of cardiac tissue and improve performance

of damaged hearts. In animal studies, for example, rat69, mouse70,71,72

and human73,74 stem cells have been identified as integrating into

cardiac tissue, forming cardiomyocytes and/or cardiac blood vessels,

regenerating infarcted heart tissue, and improving cardiac function.

In mice, bone marrow derived stem cells injected into old animals

seems capable of restoring cardiac function,75 apparently through

increased activity for cardiac blood vessel formation. One fascinating

study using xenogeneic (cross-species) transplants suggests that

stromal cells may show immune tolerance by the host.76 Mouse

marrow stromal cells were transplanted into fully immunocompetent

rats, and contributed formation of cardiomyocytes and cardiac

vessels. Even after 13 weeks, the mouse cells were not rejected by

the rat hosts. Evidence has accumulated from postmortem studies

that bone marrow stem cells can contribute to cardiomyocytes after

damage to the human heart as well.77,78 The evidence has led

numerous groups to use bone marrow derived stem cells in treatment

of patients with damaged cardiac tissue.79,80,81,82 Results from these

clinical trials indicate that bone marrow derived stem cells, including

cells from the patients themselves, can regenerate damaged cardiac

tissue and improve cardiac performance in humans. In terms of

restoring angiogenesis and improving blood circulation, results in

patients are not limited to the heart. Tateishi-Yuyama et al.83 have

shown that bone marrow derived stem cells from the patients

themselves can improve blood circulation in gangrenous limbs, in

many cases obviating the need for amputation.

Bone marrow derived adult stem cells have also been found

to contribute to various other adult tissues. Animal studies indicate

evidence that bone marrow stem cells can contribute as progenitors

of lung epithelial tissue84, and mesenchymal stem cells can home to

damaged lung tissue, engraft, and take on an epithelial morphology,

participating in repair and reduction of inflammation.85 Bone marrow

derived stem cells also have been shown to contribute to

regeneration of gastrointestinal epithelia in human patients.86 A

recent study in mice has indicated that bone marrow stem cells can

also participate in cutaneous healing, contributing to repair of skin

after wounding.87
320 MONITORING STEM CELL RESEARCH

PERIPHERAL BLOOD STEM CELLS

There is abundant evidence that bone marrow stem cells

can leave the marrow and enter the circulation, and specific

mobilization of bone marrow stem cells is used to harvest stem cells

more easily for various bone marrow stem cell treatments.88

Therefore, it is not surprising that adult stem cells have been isolated

from peripheral blood. Mobilized stem cells in peripheral blood have

been administered intravenously in a rat model of stroke,

ameliorating some of the behavioral deficits associated with the

damaged neural tissue89, leading to a proposal that stem cell

mobilization in patients might be used as a treatment for stroke in

humans.90 Mobilized stem cells have also been used in cardiac

regeneration in mice72. Two recent studies have found that human

peripheral blood stem cells exhibiting pluripotent properties can

be isolated from unmobilized human blood. One study showed that

the isolated cells were adherent, similar to marrow mesenchymal

cells, and could be induced to differentiate into cells from all three

primary germ layers, including macrophages, T lymphocytes,

epithelial cells, neuronal cells, and liver cells.91 The other study

showed induction of the peripheral blood stem cells could produce

hematopoietic, neuronal, or cardiac cells in culture.92 In the latter

study, undifferentiated stem cells were negative for both major

histocompatability antigens (MHC) I and II, expressed high levels

of the Oct-4 gene (usually associated with pluripotent capacity in

other stem cells), and could form embryoid body structures in

culture.

NEURONAL STEM CELLS

One extremely interesting finding of the past few years has

been the discovery of neuronal stem cells, indicating that cell

replenishment was possible within the brain (something previously

considered impossible.) Neuronal stem cells have been isolated

from various regions of the brain including the more-accessible

olfactory bulb93 as well as the spinal cord94, and can even be

recovered from cadavers soon after death.95 Evidence now exists

that neuronal stem cells can produce not only neuronal cells but

also other tissues, including blood and muscle.96,97,98,99,100,101 Animal

studies have shown that adult neural stem cells can participate in

repair of damage after stroke, either via endogenous neuronal

precursors102 or transplanted neural stem cells.103 Evidence indicates

that endogenous neurons and astrocytes may also secrete growth

factors to induce differentiation of endogenous precursors.104 In
APPENDIX K. 321

addition, two studies now provide suggestive evidence that neural

stem cells/neural progenitor cells may show low immunogenicity,

being immunoprivileged on transplant,105 and raising the possibility

for use of donor neural stem cells to treat degenerative brain

conditions.

Pluchino et al.106 recently used adult neural stem cells to

test potential treatment of multiple sclerosis lesions in the brain.

Using a mouse model of chronic multiple sclerosis—experimental

immune encephalitis—they injected neural stem cells either

intravenously or intracerebrally into affected mice. Donor cells

entered damaged, demyelinated regions of the brain and

differentiated into neuronal cells. Remyelination of brain lesions

and recovery from functional impairment were seen in the mice.

Neural stem cells have also been used to investigate potential

treatments for Parkinson’s disease. Using experimentally-lesioned

animals as models for Parkinson’s disease, human neural stem cells

have been observed to integrate and survive for extended periods

of time.107 Dopaminergic cells (the cells degenerated in Parkinson’s

disease) can be induced in these systems,108 and neural stem cells

are capable of rescuing and preventing the degeneration of

endogenous dopaminergic neurons,109,110 also producing improved

behavioral performance in the animals. In these studies, the data

suggest that the transplanted neural stem cells did not participate

to a large extent in direct formation of dopaminergic neurons, but

rather secreted neuroprotective factors and growth factors that

stimulated the endogenous neural cells. In this respect, infusion of

transforming growth factor into the brains of Parkinson’s mice

induced proliferation and differentiation of endogenous neuronal

precursors in mouse brain.111 Following this potential for stimulation

of endogenous neuronal cells, Gill et al. recently reported on a Phase

I trial in which glial derived neurotrophic factor (GDNF) was infused

into the brains of five Parkinson’s patients.112 After one year there

was a 61% increase in the activities of daily living score, and an

increase in dopamine storage observed in the brain. In a tantalizing

clinical application with direct injection of neural stem cells, a

Parkinson’s patient was implanted with his own neural stem cells,

resulting in an 80% reduction in symptoms at one year after

treatment.113 Further clinical trials are underway.

The olfactory ensheathing glial (OEG) cell from olfactory bulb

has been used extensively in studies regarding spinal cord injury

and axon regrowth. Human OEG cells can be expanded in number

in culture and induced to produce all three main neural cell types.93

Transplant of the cells into animal models of spinal cord injury has
322 MONITORING STEM CELL RESEARCH

shown that the cells can effect remyelination of demyelinated spinal

cord axons,114 and provide functional recovery in paraplegic rats,115

including in transected spinal cords.116 Another study has found

that infusion of growth factors such as GDNF can stimulate functional

regeneration of sensory axons in adult rat spinal cord.117

Interestingly, one group has made use of the similarities between

enteric glial cells and OEG cells, and shown that transplanted enteric

glial cells can also promote regeneration of axons in the spinal cord

of adult rats.118 Clinical trials are underway to test the abilities of

OEG cells in spinal cord injury patients. Finally, a significant

impediment to recovery from spinal cord injury is the formation of a

glial/astrocyte scar at the site of injury, which can prevent growth

of axons no matter what the source of the cells. Menet et al. have

shown, using a mutant mouse model, that much of the scar can be

prevented by inhibition of glial fibrillary acidic protein and

vimentin.119 In mutant mice that lacked these genes, there was

increased sprouting of axons and functional recovery after spinal

cord injury. Thus, endogenous neural cell growth and reconnection

might suffice for repair of damage if inhibitory mechanisms can be

removed from neural systems.

hNT CELLS

Embryonal carcinoma (EC) cells can be derived from

teratocarcinomas of adult patients, and show multipotent

differentiation abilities in culture. From one such isolation, a “tamed”

(non-tumorigenic) line of cells with neuronal generating capacity

has been developed, termed hNT (NT-2) cells. Because of their

capacity to generate neuronal cells, these cells have been studied

for possible application in regeneration of neuronal tissues. The

hNT neurons show the ability to generate dopaminergic neurons,120

and have shown some benefit of transplantation in animal models

of amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease).121 Early

clinical trials using hNT neurons transplanted into stroke patients

have shown initial positive results.122

MUSCLE STEM CELLS

Muscle contains satellite cells that normally participate in

replacement of myoblasts and myofibers. There are also indications

that muscle additionally may harbor other stem cells, either as

hematopoietic migrants from bone marrow and peripheral blood, or

as intrinsic stem cells of muscle tissue. Muscle appears to contain

a side population of stem cells, as seen in bone marrow and liver,

with the ability to regenerate muscle tissue.123 Muscle derived stem
APPENDIX K. 323

cells have been clonally isolated and used to enhance muscle and

bone regeneration in animals.124 An isolated population of musclederived stem cells has also been shown to participate in muscle

regeneration in a mouse model of muscular dystrophy.125 Stimulation

of muscle regeneration from muscle-derived stem cells, as observed

in other tissues, is greatly increased after injury of the tissue.126,127

An interesting use of muscle-derived stem cells has been the

regeneration and strengthening of bladder in a rat model of

incontinence.128 Because of the similar nature of muscle cells

between skeletal muscle and heart muscle, muscle-derived stem

cells have also been proposed for use in repairing cardiac damage,129

with evidence that mechanical beating is necessary for full

differentiation of skeletal muscle stem cells into cardiomyocytes.130

At least one group has used skeletal muscle cells for clinical

application to repair cardiac damage in a patient, with positive

results.131

LIVER STEM CELLS

As noted before, there are similarities between liver and

pancreas which could facilitate interconversion of cells between the

two tissues. This concept has been demonstrated using genetic

engineering to add a pancreatic development gene to liver cells,

converting liver to pancreas.132 Rat liver stem cells have been

converted in vitro into insulin-secreting pancreatic cells.133 When

transplanted into immundeficient mice which are a model for

diabetes, the converted liver stem cells were able to reverse

hyperglycemia in the mice. One other interesting observation

regarding liver stem cells has been the possible formation of

myocytes in the heart by liver stem cells. A clonal cell line derived

from adult male rat liver and genetically tagged was injected into

female rats, and marked, Y-chromosome bearing myocytes were

identified in the host hearts after six weeks.134

PANCREATIC STEM CELLS

Interconversion between pancreas and liver has also been

demonstrated starting with pancreatic stem cells, in which mouse

pancreatic cells repopulated the liver and corrected metabolic liver

disease.135 For pancreas, however, the possibility of solutions to the

scourge of diabetes has been a driving force in efforts to define a

stem cell that could regulate insulin in a normative, glucosedependent fashion. The success of the Edmonton protocol,136 where

cadaveric pancreatic islets are transplanted into patients, has

provided a glimmer of hope, but more readily-available sources of
324 MONITORING STEM CELL RESEARCH

insulin-secreting cells are needed. Fortunately, there seems to be

no shortage of potential candidates that can form insulin-secreting

cells. The pancreas itself appears to contain stem/progenitor cells

that can regenerate islets in vitro and in vivo. Studies indicate that

these pancreatic stem cells can functionally reverse insulindependent diabetes in mice.137 Similar pancreatic stem cells have

been isolated from humans and shown to form insulin-secreting cells

in vitro,

138 the hormone glucagon-like peptide-1 appears to be an

important inducing factor of pancreatic stem cell differentiation.

Interestingly, the same hormone could induce mouse intestinal

epithelial cells to convert into insulin-producing cells in vitro, and

the cells could reverse insulin-dependent diabetes when implanted

into diabetic mice.139 Besides pancreatic and intestinal stem cells,

other adult stem cell types showing the ability to secrete insulin

and regenerate damaged pancreas include bone marrow57,58 and

liver.133 Genetic engineering of rat liver cells to contain the pancreatic

gene PDX-1 has also been used to generate insulin-secreting cells

in vitro; the cells could also restore normal blood glucose levels when

injected into mice with experimentally-induced diabetes.140

CORNEAL LIMBAL STEM CELLS

Corneal limbal stem cells have become commonly used for

replacement of corneas, especially in cases where cadaveric donor

corneas are insufficient. Limbal cells can be maintained and cell

number expanded in culture,141 grown on amniotic membranes to

form new corneas, and transplanted to patients with good success.142

A recent report indicates that human corneal stem cells can also

display properties of functional neuronal cells in culture.143 Another

report found that limbal epithelial cells or retinal cells transplanted

into retina of rats could incorporate and integrate into damaged

retina, but did not incorporate into normal retina.144

MAMMARY STEM CELLS

Reports have indicated that mammary stem cells also exist.

Isolated cells from mouse could be propagated in vitro and

differentiated into all three mammary epithelial lineages.145 Clonallypropagated cells were induced in culture to generate complex threedimensional structures similar to that seen in vivo. Transcriptional

profiling indicated that the mammary stem cells showed similar gene

expression profiles to those of bone marrow stem cells. In that

respect, there is a report that human and mouse mammary stem

cells exist as a side population, as seen for bone marrow, liver, and

muscle stem cells.146 When propagated in culture, the isolated
APPENDIX K. 325

mammary side population stem cells could form epithelial ductal

structures.

SALIVARY GLAND

A recent report indicates that stem cells can be isolated by

limiting dilution from regenerating rat salivary gland and propagated

in vitro.

147 Under differing culture conditions, the cells express genes

typical of liver or pancreas, and when injected into rats can integrate

into liver tissue.

SKIN

Multipotent adult stem cells have been isolated from the

dermis and hair follicle of rodents.148 The cells play a role in

maintenance of epidermal and hair follicle structures, can be

propagated in vitro, and clonally isolated stem cells can be induced

to form neurons, glia, smooth muscle, and adipocytes in culture.

Dermal hair follicle stem cells have also shown the ability to reform

the hematopoietic system of myeloablated mice.149

TENDON

A recent report notes the isolation of established stem celllike lines from mouse tendon. The cells exhibited a mesenchymal

morphology, and expressed genes related to osteogenic,

chondrogenic, and adipogenic potential, similar to that seen in bone

marrow mesenchymal stem cells.150

SYNOVIAL MEMBRANE

Stem cells from human synovial membrane (knee joint) have

been isolated which show multipotent abilities for differentiation,

including evidence of myogenic potential.151 These stem cells were

used in a mouse model of Duchenne muscular dystrophy to test their

ability to repair damaged muscle. Stem cells injected into the

bloodstream could engraft and incorporate into muscle, taking on a

muscle phenotype, and with evidence of muscle repair.152

HEART

Beltrami et al. analyzed the hearts of post-mortem patients

who succumbed 4-12 days after heart attack, and found evidence of

dividing myocytes in the human heart. While it is unclear from the

study whether the cells were originally cardiomyocytes or were other
326 MONITORING STEM CELL RESEARCH

stem cells which had homed to damaged heart tissue, such as bone

marrow stem cells, the evidence indicated dividing cells within the

heart.153

CARTILAGE

Human cartilage biopsies placed into culture show apparent

dedifferentiation into primitive chondrocytes with mesenchymal

stem cell appearance.154 These chondrocytes have been used for

transplants to repair articular cartilage damage, and in treatment

of children with osteogenesis imperfecta.155,156,157

THYMIC PROGENITORS

Bennett et al. have reported the isolation of thymic epithelial

progenitor cells.158 Ectopic grafting (under the kidney capsule) of

the cells into mice allowed production of all thymic epithelial cell

types, as well as attraction of homing T lymphocytes. In separate

experiments, Gill et al. also isolated a putative thymic progenitor

cell from mice and were able to use these cells to reform miniature

thymuses when the cells were transplanted under mouse kidney

capsule.159

DENTAL PULP STEM CELLS

Stem cells have been isolated from human adult dental pulp

that could be clonally propagated and proliferated rapidly.160 Though

there were some similarities with bone marrow mesenchymal stem

cells, when injected into immunodeficient mice the adult dental pulp

stem cells formed primarily dentin-like structures surrounded by

pulpy interstitial tissue. Human baby teeth have also been identified

as a source of stem cells, designated SHED cells (Stem cells from

Human Exfoliated Deciduous teeth).161 In vitro, SHED cells could

generate neuronal cells, adipocytes, and odontoblasts, and after

injection into immunodeficient mice, the cells were indicated in

formation of bone, dentin, and neural cells.

ADIPOSE (FAT) DERIVED STEM CELLS

One of the more interesting sources identified for human

stem cells has been adipose (fat) tissue, in particular liposuctioned

fat. While there is some debate as to whether the cells originate in

the fat tissue or are perhaps mesenchymal or peripheral blood stem

cells passing through the fat tissue, they represent a readily-available

source for isolation of potentially useful stem cells. The cells can be
APPENDIX K. 327

maintained for extended periods of time in culture, have a

mesenchymal-like morphology, and can be induced in vitro to form

adipose, cartilage, muscle, and bone tissue.162 The cells have also

shown the capability of differentiation into neuronal cells.163

UMBILICAL CORD BLOOD

Use of umbilical cord stem cells has seen increasing interest,

as the cells have been recognized as a useful source for

hematopoietic transplants similar to bone marrow stem cell

transplants, including for treatment of sickle cell anemia.164 Cord

blood shows decreased graft-versus-host reaction compared to bone

marrow,165 perhaps due to high interleukin-10 levels produced by

the cells.166 Another possibility for the decreased rejection seen

with cord blood stem cell transplants is decreased expression of

the beta-2-microglobulin on human cord blood stem cells.167 Cord

blood can be cryopreserved for over 15 years and retain significant

functional potency.168 Cord blood stem cells also show similarities

with bone marrow stem cells in terms of their potential to

differentiate into other tissue types. Human cord blood stem cells

have shown expression of neural markers in vitro,

169 and intravenous

administration of cord blood to animal models of stroke has produced

functional recovery in the animals.89,170 Infusion of human cord blood

stem cells has also produced therapeutic benefit in rats with spinal

cord injury,171 and in a mouse model of ALS.172 A recent report noted

establishment of a neural stem/progenitor cell line derived from

human cord blood that has been maintained in culture over two

years without loss of differentiation ability.173 Several reports also

note the production of functional liver cells from human cord blood

stem cells.174 Additional differentiative properties of human umbilical

cord blood stem cells are likely to be discovered as more investigation

proceeds on this source of stem cells.

UMBILICAL CORD MESENCHYME (WHARTON’S JELLY)

While most of the focus regarding umbilical cord stem cells

has focused on the cord blood, there are also reports that the matrix

cells from umbilical cord contain potentially useful stem cells. Using

pigs, this matrix from umbilical cord, termed Wharton’s jelly, has

been a source for isolation of mesenchymal stem cells. The cells

express typical stem cell markers such as c-kit and high telomerase

activity, have been propagated in culture for over 80 population

doublings, and can be induced to form neurons in vitro.

175 When

transplanted into rats, the cells expressed neuronal markers and
328 MONITORING STEM CELL RESEARCH

integrated into the rat brain, additionally without any evidence of

rejection.176

AMNIOTIC STEM CELLS

Amniotic fluid has also been found to contain stem cells

that can take on neuronal properties when injected into brain.177

These stem cells were recently isolated from human amniotic fluid,178

and were found to express Oct-4, a gene typically associated with

expression in pluripotent stem cells.

MESANGIOBLASTS

Mesangioblasts are a multipotent stem cell that has been

isolated from large blood vessels such as dorsal aorta.179 The cells

show long term proliferative capacity in culture as well as the

capability of differentiation into most mesodermally derived types

of tissue. In a recent report, the cells were injected into the

bloodstream of mice that are a model for muscular dystrophy,180 and

participated in repair of the muscle tissue.

Adult stem cells in other tissues very likely exist, but this

survey of many of the known adult stem cells and their capacities

for differentiation and tissue repair can serve as a beginning point

for discussion regarding the progress as well as potential of adult

stem cells. Some final thoughts on current and potential utilization

of adult stem cells follow.

ADULT STEM CELL MOBILIZATION FOR TISSUE REPAIR

An important point to consider as we look ahead regarding

utilization of adult stem cells for tissue repair is that it may be

unnecessary first to isolate and culture stem cells before injecting

them back into a patient to initiate tissue repair. Rather, it may be

easier and preferable to mobilize endogenous stem cells for repair

of damaged tissue. Initial results regarding this possibility have

already been seen in some animal experiments, in which bone

marrow and peripheral blood stem cells were mobilized with

injections of growth factors and participated in repair of heart and

stroke damage.72,89,90 The ability to mobilize endogenous stem cells,

coupled with natural or perhaps induced targeted homing of the

cells to damaged tissue, could greatly facilitate use of adult stem

cells in simplified tissue regeneration schemes.181
APPENDIX K. 329

GENE THERAPY APPLICATIONS WITH ADULT STEM CELLS

Adult stem cells can provide an efficient vehicle for gene

therapy applications, and engineered adult stem cells may allow

increased functionality, proliferative capacity, or stimulatory

capability to these cells. The feasibility of genetically engineering

adult stem cells has been shown, for example, in the use of bone

marrow stem cells containing stably inserted genes. The engineered

stem cells when injected into mice could still participate in formation

and repair of differentiated tissue, such as in lung.182 As another

example, engineered stem cells containing an autoantigen, to induce

immune tolerance of T cells to insulin-secreting cells, were shown

to prevent onset of diabetes in a mouse model of diabetes,183 a

strategy that may be useful for various human autoimmune diseases.

Introduction of the PDX-1 gene into liver stem cells stimulated

differentiation into insulin-producing cells which could normalize

glucose levels when transplanted into mice with induced diabetes.140

Simply engineering cells to increase their proliferative capacity can

have a significant effect on their utility for tissue engineering and

repair. For example, McKee et al.184 engineered human smooth

muscle cells by introducing human telomerase, which greatly

increased their proliferative capacity beyond the normal lifespan of

smooth muscle cells in culture, while allowing retention of their

normal smooth muscle characteristics. These engineered smooth

muscle cells were seeded onto biopolymer scaffolds and allowed to

grow into smooth muscle layers, then seeded with human umbilical

vein endothelial cells. The resulting engineered arterial vessels

could be useful for transplants and bypass surgery. Similarly, human

marrow stromal cells that were engineered with telomerase

increased their proliferative capacity significantly, but also showed

enhanced ability at stimulating bone formation in experimental

animals.185 Genetically-engineered human adult stem cells have

already been used in successful treatment of patients with genetic

disease. Bone marrow stem cells, from infants with forms of severe

combined immunodeficiency syndrome (SCID), were removed from

the patients, a functional gene inserted, and the engineered cells

reintroduced to the same patients. The stem cells homed to the

bone marrow, engrafted, and corrected the defect.186,187,188

Adult stem cells could also be used to deliver stimulatory

or protective factors to tissues and endogenous stem cells. This

would utilize the innate homing ability of adult stem cells, but would

not necessarily rely on differentiation of the stem cells to participate

in tissue replenishment. For example, Benedetti et al. utilized the
330 MONITORING STEM CELL RESEARCH

homing capacity of neural stem cells in brain by engineering mouse

neural stem cells with the gene for interleukin-4. Transfer into brain

glioblastomas in mice led to the survival of most of the mice, and

imaging analysis documented the progressive disappearance of

large tumors.189 Likewise, engineered mesenchymal stem cells were

transplanted into the brains of mice that are a model of NiemannPick disease; the enzyme acid sphingomyelinase is lost in the

disease, resulting in neurological damage and early death. The

mesenchymal stem cells were engineered to overexpress the

missing enzyme. When injected into brains of the mouse model,

the mice showed a delay in onset of neurological abnormalities and

an extension of lifespan, suggesting that the stem cells delivered

and secreted the necessary enzyme to the brain tissue.190 Musclederived stem cells that were engineered to express the growth factor

bone morphogenetic protein-2 were used to stimulate bone healing

in mice with skull bone defects. While the muscle-derived stem

cells did show differentiation as bone cells, the results indicated

that the critical factor was delivery of the secreted growth factor by

the stem cells to the areas of bone damage, allowing much more

rapid healing than in control animals.191 As noted previously, neural

stem cells show an ability to rescue degenerating neurons, including

the dopaminergic neurons whose loss is associated with Parkinson’s

disease. The delivery of neuroprotective substances is postulated

as the most likely explanation for this phenomenon, rather than

substantial differentiation by the injected neural stem cells.109 In

support of this hypothesis, when neural stem cells were specifically

engineered to overexpress a neurotrophic factor similar to glial

derived neurotrophic factor, degeneration of dopaminergic neurons

was prevented.110

STIMULATING ENDOGENOUS CELLS

The indications from the previous examples suggest that

direct stimulation of endogenous stem cells within a tissue may be

the easiest, safest, and most efficient way to stimulate tissue

regeneration. Such stimulation need not rely on any added stem

cells. This approach would circumvent the need to isolate or grow

stem cells in culture, or inject any stem cells into the body, whether

the cells were derived from the patient or another source. Moreover,

direct stimulation of endogenous tissue stem cells with specific

growth factors might even preclude any need to mobilize stem cells

to a site of tissue damage. A few experimental results suggest that

this approach might be possible. One group has reported that use

of glial derived neurotrophic factor and neurotrophin-3 can stimulate

regeneration of sensory axons in adult rat spinal cord.117
APPENDIX K. 331

Administration of transforming growth factor to the brains of

Parkinson’s mice stimulated proliferation and differentiation of

endogenous neuronal stem cells and produced therapeutic results

in the mice,111 and infusion of glial derived neurotrophic factor into

the brains of Parkinson’s patients resulted in increased dopamine

production within the brain and therapeutic benefit to the patients.112

And, Zeisberg et al. have found that bone morphogenetic protein-7

(BMP-7) can counteract deleterious cell changes associated with

tissue damage. In this latter study, a mouse model of chronic kidney

damage was used. Damage to the tissue causes a transition from

epithelial to mesenchymal cell types in the kidney, leading to fibrosis.

The transition appears to be initiated by the action of transforming

growth factor beta-1 on the tissues, and BMP-7 was shown to

counteract this signaling in vitro. Systemic administration of BMP7 in the mouse model reversed the transition in vivo and led to repair

of severely damaged renal tubule epithelial cells.192 These

experiments indicate that direct stimulation of tissues by the correct

growth factors could be sufficient to prevent or repair tissue damage.

The key to such treatments would be identification of the correct

stimuli specific to a tissue or cell type.

In summary, our current knowledge regarding adult stem

cells has expanded greatly over what was known just a few short

years ago. Results from both animal studies and early human clinical

trials indicate that they have significant capabilities for growth,

repair, and regeneration of damaged cells and tissues in the body,

akin to a built-in repair kit or maintenance crew that only needs

activation and stimulation to accomplish repair of damage. The

potential of adult stem cells to impact medicine in this respect is

enormous.
332 MONITORING STEM CELL RESEARCH

Adult Stem Cells—Addendum (October 2003)

For the President’s Council on Bioethics

David A. Prentice

Since initial submission of the commissioned paper, numerous

additional published references have documented the abilities of

adult stem cells to stimulate regeneration of damaged tissues. Just

a few of the most significant are mentioned here. Mesenchymal

stem cells engineered to express the Akt1 gene, when transplanted

into mice, demonstrated the ability to repair and restore performance

of infarcted heart, essentially to a normal state.a

 Another clinical

trial in addition to those mentioned in the paper has shown

significant improvement in patients with heart damage, with

reduction in the area of damage and improved heart function after

adult stem cell treatment.b Three more published articles support

the existence of a stem cell in the heart and its participation in

cardiac regeneration.c Stroke damage in rats was repaired using

human neural stem cellsd and prostate was regenerated in vivo in

mice using adult stem cells.e Another report indicates that human

mixed bone marrow stem cells can contribute significant amounts

of lung tissue in patientsf and pluripotent stem cells were discovered

in the mouse inner earg, which can form all 3 primary germ layers

and might lead to potential therapies for hearing loss. Finally, bone

marrow stem cells were discovered to have a protective as well as

regenerative role in diabetes.h

ADDENDUM REFERENCES

a

Mangi AA et al., “Mesenchymal stem cells modified with Akt prevent remodeling

and restore performance of infarcted hearts”, Nature Medicine 9, 1195-1201, Sept

2003

bBritten MB et al., “Infarct remodeling after intracoronary progenitor cell treatment

in patients with acute myocardial infarction”, Circulation 108, 2212-2218, Nov 2003

c

Urbanek K et al., “Intense myocyte formation from cardiac stem cells in human

cardiac hypertrophy”, Proceedings of the National Academy of Sciences USA 100,

10440-10445, 2 Sept 2003; Beltrami AP et al., “Adult cardiac stem cells are multipotent

and support myocardial regeneration”, Cell 114, 763-776, 19 Sept 2003; Oh H et al.,

“Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion

after infarction”, Proceedings of the National Academy of Sciences USA 100, 12313-

12318, 14 Oct 2003

dJeong S-W et al., “Human neural stem cell transplantation promotes functional

recovery in rats with experimental intracerebral hemorrhage”, Stroke 34, 2258-2263,

Sept 2003
APPENDIX K. 333

e

Xin L et al., “In vivo regeneration of murine prostate from dissociated cell populations

of postnatal epithelia and urogenital sinus mesenchyme”, Proceedings of the National

Academy of Sciences USA 100, 11896-11903, 30 Sept 2003

f

Suratt BT et al., “Human pulmonary chimerism after hematopoietic stem cell

transplantation”, American Journal of Respiratory and Critical Care Medicine 168,

318-322, 2003

gLi H et al., “Pluripotent stem cells from the adult mouse inner ear”, Nature Medicine

9, 1293-1299, Oct 2003

hLi FX et al., “The development of diabetes in E2f1/E2f2 mutant mice reveals

important roles for bone marrow-derived cells in preventing islet cell loss”,

Proceedings of the National Academy of Sciences USA 100, 12935-12940, 28 Oct 2003

REFERENCES

1 See, for example: Feasel AM et al., “Complete remission of scleromyxedema

following autologous stem cell transplantation”, Archives of Dermatology 137, 1071-

1072; Aug 2001; Mancardi GL et al.; “Autologous hematopoietic stem cell

transplantation suppresses Gd-enhanced MRI activity in MS”; Neurology 57, 62-68;

10 July 2001; Traynor AE et al.; “Hematopoietic stem cell transplantation for severe

and refractory lupus”; Arthritis Rheumatology 46, 2917-2923; November 2002;

Wulffraat NM et al.; “Prolonged remission without treatment after autologous stem

cell transplantation for refractory childhood systemic lupus erythematosus”; Arthritis

Rheumatology 44, 728-731; March 2001; Burt RK et al., “High-dose immune

suppression and autologous hematopoietic stem cell transplantation in refractory

Crohn disease”, Blood 101, 2064-2066; 2003.

2

 Jiang Y et al; “Pluripotency of mesenchymal stem cells derived from adult marrow”;

Nature 418, 41-49; 4 July 2002.

3

 Yu Y et al., “AC133-2, a novel isoform of human AC133 stem cell antigen”, Journal

of Biological Chemistry 277, 20711-20716; 7 June 2002.

4

 Danet GH et al., “C1qRp defines a new human stem cell population with

hematopoietic and hepatic potential”, Proceedings of the National Academy of

Sciences USA 99, 10441-10445; 6 Aug 2002.

5

 Zhou S et al., “The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety

of stem cells and is a molecular determinant of the side-population phenotype”,

Nature Medicine 7, 1028-1034; Sept 2001.

6

 Tremain N et al; “MicroSAGE Analysis of 2,353 Expressed Genes in a Single Cell

derived Colony of Undifferentiated Human Mesenchymal Stem Cells Reveals mRNAs

of Multiple Cell Lineages.” Stem Cells 19, 408-418; 2001.

7 Wright LS et al., “Gene expression in human neural stem cells: effects of leukemia

inhibitory factor”, Journal of Neurochemistry. 86, 179-195; July 2003.

8

 Ivanova NB et al., “A stem cell molecular signature”, Science 298, 601-604; 18 Oct

2002; Ramalho-Santos M et al., “ “Stemness”: Transcriptional profiling of embryonic

and adult stem cells”, Science 298, 597-600; 18 Oct 2002.
334 MONITORING STEM CELL RESEARCH

9

 Dao MA et al., “Reversibility of CD34 expression on human hematopoietic stem

cells that retain the capacity for secondary reconstitution”, Blood 101, 112-118; 1

Jan 2003; Handgretinger R et al., “Biology and plasticity of CD133+ hematopoietic

stem cells”, Annals of the New York Academy of Sciences 996, 141-151; 2003; Huss

Ret al., “Evidence of peripheral blood-derived, plastic-adherent CD34–/low

hematopoietic stem cell clones with mesenchymal stem cell characteristics”, Stem

Cells 18, 252-260; 2000; Huss R. “Isolation of primary and immortalized CD34–

hematopoietic and mesenchymal stem cells from various sources”, Stem Cells 18, 1-

9; 2000.

10 Lodie TA et al; “Systematic analysis of reportedly distinct populations of

multipotent bone marrow-derived stem cells reveals a lack of distinction”; Tissue

Engineering 8, 739-751; 2002.

11 Tagami M et al., “Genetic and ultrastructural demonstration of strong reversibility

in human mesenchymal stem cell”, Cell and Tissue Research 312, 31-40; Apr 2003.

12 Theise ND and Krause DS; “Suggestions for a New Paradigm of Cell Differentiative

Potential”, Blood Cells, Molecules, and Diseases 27, 625–631; 2001; Thiese ND and

Krause DS, “Toward a new paradigm of cell plasticity”, Leukemia 16, 542-548; 2002.

13 Blau HM et al., “The evolving concept of a stem cell: entity or function?”, Cell 105,

829-841; 29 June 2001.

14 Tsai RYL and McKay RDG, “Cell Contact Regulates Fate Choice by Cortical Stem

Cells.” Journal of Neuroscience 20, 3725-3735; May 2000.

15 Wagers AJ et al.; “Little evidence for developmental plasticity of adult

hematopoietic stem cells”; Science 297, 2256-2259; 27 Sept 2002.

16 Krause DS et al.; “Multi-Organ, Multi-Lineage Engraftment by a Single Bone

Marrow-Derived Stem Cell”; Cell 105, 369-377; 4 May 2001.

17 Castro RF et al., “Failure of bone marrow cells to transdifferentiate into neural

cells in vivo”, Science 297, 1299; 23 Aug 2002.

18 Brazelton, TR et al.; “From marrow to brain: expression of neuronal phenotypes in

adult mice”; Science 290, 1775-1779; 1 Dec 2000.

19 Moore BE and Quesenberry PJ, “The adult hemopoietic stem cell plasticity debate:

idols vs new paradigms”, Leukemia 17, 1205-1210; 2003.

20 Alison MR et al.; “Plastic adult stem cells: will they graduate from the school of

hard knocks?”, Journal of Cell Science 116, 599-603; 2003.

21 Tada M et al., “Nuclear reprogramming of somatic cells by in vitro hybridization

with ES cells”, Current Biology 11, 1553-1558; 2 Oct 2001; Terada N et al., “Bone

marrow cells adopt the phenotype of other cells by spontaneous cell fusion”, Nature

416, 542-545; 4 Apr 2002; Ying Q-L et al., “Changing potency by spontaneous fusion”,

Nature 545-548; 4 Apr 2002; Tada M et al., “Embryonic germ cells induce epigenetic

reprogramming of somatic nucleus in hybrid cells”, EMBO Journal 16, 6510-6520;

1997.

22 See, for example: Sorieul S and Ephrussi B, “Karyological demonstration of

hybridization of mammalian cells in vitro” Nature 190, 653–654; 1961; Littlefield JW,

“Selection of hybrids from matings of fibroblasts in vitro and their presumed
APPENDIX K. 335

recombinants”, Science 145, 709-710; 14 Aug 1964; Weiss MC and Green H, “Humanmouse hybrid cell lines containing partial complements of human chromosomes

and functioning human genes”, Proceedings of the National Academy of Sciences

USA 58, 1104-1111; Sept 1967; Ladda RL and Estensen RD, “Introduction of a

heterologous nucleus into enucleated cytoplasms of cultured mouse L-cells”,

Proceedings of the National Academy of Sciences USA 67, 1528-1533; Nov 1970; Köhler

G Milstein C, “Continuous culture of fused cells secreting antibody of predefined

specificity” Nature 256, 495-497, 1975.

23 Wang X et al.; “Cell fusion is the principal source of bone-marrow-derived

hepatocytes”; Nature 422, 897-901; 24 April 2003; Vassilopoulos G et al.;

“Transplanted bone marrow regenerates liver by cell fusion”; Nature 422, 901-904;

24 April 2003.

24 Spees JL et al., “Differentiation, cell fusion, and nuclear fusion during ex vivo

repair of epithelium by human adult stem cells from bone marrow stroma”,

Proceedings of the National Academy of Sciences USA 100, 2397-2402; 4 Mar 2003.

25 Blau HM, “Stem-cell fusion: A twist of fate”, Nature 419, 437; 3 Oct 2002.

26 Collas P and Håkelien A-M, “Reprogramming Somatic Cells for Therapeutic

Applications”; Journal of Regenerative Medicine 4, 7-13; February 2003; Håkelien

AM et al; “Reprogramming fibroblasts to express T-cell functions using cell extracts”;

Nature Biotechnology 20, 460-466; May 2002; Håkelien AM and Collas P, “Novel

approaches to transdifferentiation”, Cloning and Stem Cells 4, 379-387; 2002.

27 Tran SD et al.; “Differentiation of human bone marrow-derived cells into buccal

epithelial cells in vivo: a molecular analytical study”; Lancet 361, 1084-1088; 29

March 2003.

28 Newsome PN et al., “Human cord blood-derived cells can differentiate into

hepatocytes in the mouse liver with no evidence of cellular fusion”, Gastroenterology

124, 1891-1900; June 2003.

29 Mazurier F et al; “Rapid myeloerythroid repopulation after intrafemoral

transplantation of NOD-SCID mice reveals a new class of human stem cells”; Nature

Medicine 9, 959-963; July 2003.

30 Bianco P et al., “Bone marrow stromal stem cells: nature, biology, and potential

applications”, Stem Cells 19, 180-192; 2001.

31 Sanchez-Ramos J et al., “Adult bone marrow stromal cells differentiate into neural

cells in vitro”, Experimental Neurology 164, 247-256; 2000.

32 Woodbury D et al; “Adult rat and human bone marrow stromal cells differentiate

intoneurons” Journal of Neuroscience Research 61, 364-370; 2000.

33 Pittenger MF et al., “Multilineage potential of adult human mesenchymal stem

cells”, Science 284, 143-147; 2 Apr 1999.

34 Gronthos S et al., “Molecular and cellular characterisation of highly purified stromal

stem cells derived from human bone marrow”, Journal of Cell Science 116, 1827-

1835; 2003.
336 MONITORING STEM CELL RESEARCH

35 Woodbury D et al., “Adult bone marrow stromal stem cells express germline,

ectodermal, endodermal, and mesodermal genes prior to neurogenesis”, Journal of

Neuroscience Research 96, 908-917; 2002.

36 Koc ON et al., Allogeneic mesenchymal stem cell infusion for treatment of

metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH), Bone Marrow

Transplant 215-222; Aug 2002.

37 Mezey, E et al.; “Turning blood into brain: Cells bearing neuronal antigens

generated in vivo from bone marrow”; Science 290, 1779-1782; 1 Dec 2000.

38 Kabos P et al.; “Generation of neural progenitor cells from whole adult bone

marrow”; Experimental Neurology 178, 288-293; December 2002.

39 Körbling MK et al.; “Hepatocytes and epithelial cells of donor origin in recipients

of peripheral-blood stem cells”; New England Journal of Medicine 346, 738-746; 7

March 2002.

40 Mezey E et al.; “Transplanted bone marrow generates new neurons in human

brains”; Proceedings of the National Academy of Sciences USA 100, 1364-1369; 4 Feb

2003.

41 Priller J et al; “Neogenesis of cerebellar Purkinje neurons from gene-marked bone

marrow cells in vivo”; Journal of Cell Biology 155, 733-738; 26 Nov 2001.

42 Weimann JM et al., “Contribution of transplanted bone marrow cells to Purkinje

neurons in human adult brains”, Proceedings of the National Academy of Sciences

USA 100, 2088-2093; 18 Feb 2003.

43 Hess DC et al., “Bone marrow as a source of endothelial cells and NeuN-expressing

cells after stroke”, Stroke 33, 1362-1368; May 2002.

44 Chen J et al.; “Therapeutic benefit of intravenous administration of bone marrow

stromal cells after cerebral ischemia in rats”; Stroke 32, 1005-1011; April 2001.

45 Li Y et al.; “Human marrow stromal cell therapy for stroke in rat”; Neurology 59,

514-523; August 2002.

46 Heissig B et al, “Recruitment of stem and progenitor cells from the bone marrow

niche requires MMP-9 mediated release of kit-ligand” Cell 109, 625-637; 31 May

2002.

47 Otani A et al; “Bone marrow derived stem cells target retinal astrocytes and can

promotes or inhibit retinal angiogenesis”; Nature Medicine 8, 1004-1010; Sept 2002.

48 Tomita M et al; “Bone marrow derived stem cells can differentiate into retinal

cells in injured rat retina”; Stem Cells 20, 279-283; 2002.

49 Sasaki M et al., “Transplantation of an acutely isolated bone marrow fraction

repairs demyelinated adult rat spinal cord axons,” Glia 35, 26-34; July 2001.

50 Hofstetter CP et al., “Marrow stromal cells form guiding strands in the injured

spinal cord and promote recovery”, Proceedings of the National Academy of Sciences

USA 99, 2199-2204; 19 February 2002.
APPENDIX K. 337

51 Sekiya I et al., “In vitro cartilage formation by human adult stem cells from bone

marrow stroma defines the sequence of cellular and molecular events during

chondrogenesis”, Proceedings of the National Academy of Sciences USA 99, 4397-

4402; 2 Apr 2002.

52 Ferrari G et al; “Muscle regeneration by bone marrow-derived myogenic

progenitors”; Science 279, 1528-1530; Mar 6, 1998.

53 LaBarge MA and Blau HM, “Biological progression from adult bone marrow to

multinucleate muscle fiber in response to injury”; Cell 111, 589-601; 15 November

2002

54 Gussoni E. et al, “Long term persistence of donor nuclei in a Duchenne Muscular

dystrophy patient receiving bone marrow transplantation” Journal of Clinical

Investigation 110, 807-814; Sept 2002.

55 Ito T et al., “Bone marrow is a reservoir of repopulating mesangial cells during

glomerular remodeling”, Journal of the American Society of Nephrology 12, 2625-

2635; 2001.

56 Masuya M et al.; “Hematopoietic origin of glomerular mesangial cells”; Blood 101,

2215-2218; 15 March 2003.

57 Kale S et al., “Bone marrow stem cells contribute to repair of the ischemically

injured renal tubule”, Journal of Clinical Investigation 112, 42-49; July 2003.

58 Lin F et al; “Hematopoietic stem cells contribute to the regeneration of renal

tubules after renal ischemia-reperfusion injury in mice”; Journal of the American

Society of Nephrology 14, 1188-1199; 2003.

59 Poulsom R et al; “Bone marrow stem cells contribute to healing of the kidney”;

Journal of the American Society of Nephrology 14, S48-S54; 2003.

60 Miyazaki M et al.; “Improved conditions to induce hepatocytes from rat bone

marrow cells in culture”; Biochemical and Biophysical Research Reports 298, 24-30;

October 2002.

61 Fiegel HC et al., “Liver-specific gene expression in cultured human hematopoietic

stem cells”, Stem Cells 21, 98-104; 2003.

62 Lagasse et al., “Purified hematopoietic stem cells can differentiate into hepatocytes

in vivo”, Nature Medicine 6, 1229-1234; November 2000.

63 Theise, N et al.; “Derivation of hepatocytes from bone marrow cells in mice after

radiation-induced myeloablation”; Hepatology 31, 235-240; Jan. 2000.

64 Theise, N et al.; “Liver from bone marrow in humans”; Hepatology 32, 11-16; July

2000; Alison, M et al.; “Cell differentiation: hepatocytes from non-hepatic adult stem

cells”; Nature 406, 257; 20 July 2000.

65 Wang X et al., “Kinetics of liver repopulation after bone marrow transplantation”,

American Journal of Pathology 161, 565-574; Aug 2002.

66 Wulf GG et al., “Cells of the hepatic side population contribute to liver regeneration

and can be replenished by bone marrow stem cells”, Haematologica 88, 368-378;

2003.
338 MONITORING STEM CELL RESEARCH

67 Ianus A et al.; In vivo derivation of glucose competent pancreatic endocrine cells

from bone marrow without evidence of cell fusion; Journal of Clinical Investigation

111, 843-850; March 2003.

68 Hess D et al., “Bone marrow-derived stem cells initiate pancreatic regeneration”,

Nature Biotechnology 21, 763-770; July 2003.

69 Bittira B et al., “In vitro preprogramming of marrow stromal cells for myocardial

regeneration”, Annals of Thoracic Surgery 74, 1154-1160; 2002.

70 Orlic D et al.; “Bone marrow cells regenerate infarcted myocardium”; Nature 410,

701-705; April 5, 2001.

71 Jackson KA et al.; “Regeneration of ischemic cardiac muscle and vascular

endothelium by adult stem cells”; Journal of Clinical Investigation 107, 1395-1402;

June 2001.

72 Orlic D et al., “Mobilized bone marrow cells repair the infarcted heart, improving

function and survival”; Proceedings of the National Academy of Sciences USA 98,

10344-10349, 28 August 2001.

73 Toma C et al.; “Human mesenchymal stem cells differentiate to a cardiomyocyte

phenotype in the adult murine heart”; Circulation, 105, 93-98; 1/8 January 2002.

74 Kocher AA et al.; “Neovascularization of ischemic myocardium by human bonemarrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling

and improves cardiac function”; Nature Medicine 7, 430-436; April 2001.

75 Edelberg, JM et al; “Young adult bone marrow derived endothelial precursor cells

restore aging impaired cardiac angiogenic function”; Circulation Research 90, e89-

e93; 2002.

76 Saito T et al; “Xenotransplant cardiac chimera: immune tolerance of adult stem

cells”; Annals of Thoracic Surgery 74, 19-24; 2002.

77 Laflamme MA et al; “Evidence for cardiomyocyte repopulation by extracardiac

progenitors in transplanted human hearts”, Circulation Research 90, 634-640; 5 Apr

2002.

78 Deb A et al., “Bone marrow-derived cardiomyocytes are present in adult human

heart”, Circulation 107, 1207-1209; 11 March 2003.

79 Perin EC et al., “Transendocardial, Autologous Bone Marrow Cell Transplantation

for Severe, Chronic Ischemic Heart Failure”, Circulation 107, r75-r83; 2003.

80 Stamm C et al.; “Autologous bone-marrow stem-cell transplantation for myocardial

regeneration”; The Lancet 361, 45-46; 4 Jan 2003.

81 Tse H-F et al.; “Angiogenesis in ischaemic myocardium by intramyocardial

autologous bone marrow mononuclear cell implantation”; The Lancet 361, 47-49; 4

January 2003.

82 Strauer BE et al.; “Repair of infarcted myocardium by autologous intracoronary

mononuclear bone marrow cell transplantation in humans”; Circulation 106, 1913-

1918; 8 October 2002.
APPENDIX K. 339

83 Tateishi-Yuyama E et al.; “Therapeutic angiogenesis for patients with limb

ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a

randomised controlled trial”; Lancet 360, 427-435; 10 August 2002.

84 Kotton DN et al.; “Bone marrow-derived cells as progenitors of lung alveolar

epithelium”; Development 128, 5181-5188; December 2001.

85 Ortiz LA et al., “Mesenchymal stem cell engraftment in lung is enhanced in

response to bleomycin exposure and ameliorates its fibrotic effects”, Proceedings

of the National Academy of Sciences USA 100: 8407-8411; 8 July 2003.

86 Okamoto R et al; “Damaged epithelia regenerated by bone marrow-derived cells

in the human gastrointestinal tract”; Nature Medicine 8, 1011-1017; Sept 2002.

87 Badiavas EV, “Participation of Bone Marrow Derived Cells in Cutaneous Wound

Healing”, Journal Of Cellular Physiology 196, 245–250; 2003.

88 Kessinger A, Sharp JG, “The whys and hows of hematopoietic progenitor and

stem cell mobilization”, Bone Marrow Transplant 31, 319-329; Mar 2003.

89 Willing AE et al., “Mobilized peripheral blood cells administered intravenously

produce functional recovery in stroke”, Cell Transplantation 12, 449-454; 2003.

90 Borlongan CV and Hess DC, “G-CSF-mobilized human peripheral blood for

transplantation therapy in stroke”, Cell Transplantation 12, 447-448; 2003.

91 Zhao Y et al.; “A human peripheral blood monocyte-derived subset acts as

pluripotent stem cells”; Proceedings of the National Academy of Sciences USA 100,

2426-2431; 4 March 2003.

92 Abuljadayel IS, “Induction of stem cell-like plasticity in mononuclear cells derived

from unmobilised adult human peripheral blood”, Current Medical Research and

Opinion 19, FastTrack PREPRINT: 355–375; 18 June 2003 (doi: 10.1185/

030079903125001901).

93 Pagano SF et al; “Isolation and Characterization of Neural Stem Cells from the

Adult Human Olfactory Bulb”; Stem Cells 18, 295-300; 2000.

94 Shihabuddin S et al.; “Adult spinal cord stem cells generate neurons after

transplantation in the adult dentate gyrus”; Journal of Neuroscience 20, 8727-8735;

December 2000.

95 Palmer TD et al., “Progenitor cells from human brain after death”, Nature 411, 42-

43; 3 May 2001.

96 Bjornson CRR et al., “Turning brain into blood: a hematopoietic fate adopted by

adult neural stem cells in vivo”, Science 283, 534-537; 22 Jan 1999.

97 Galli R et al; “Neural stem cells: An overview”; Circulation Research 92, 598-602;

Feb 2003.

98 Clarke DL et al.; “Generalized potential of adult neural stem cells”; Science 288,

1660-1663, 2 June 2000.
340 MONITORING STEM CELL RESEARCH

99 Galli R et al; “Skeletal myogenic potential of human and mouse neural stem cells”;

Nature Neuroscience 3, 986-991; Oct 2000.

100 Rietze RL et al. “Purification of a pluripotent neural stem cell from the adult

mouse brain”, Nature 412, 736-739; 16 Aug 2001.

101 Englund U et al; “Grafted neural stem cells develop into functional pyramidal

neutrons and integrate into host cortical circuitry”; Proceedings of the National

Academy of Sciences USA 99, 17089-17094; 24 Dec 2002. 102 Arvidsson A et al.; “Neuronal replacement from endogenous precursors in the

adult brain after stroke”; Nature Medicine 8, 963-970; Sept 2002.

103 Riess P et al.; “Transplanted neural stem cells survive, differentiate, and improve

neurological motor function after experimental traumatic brain injury”; Neurosurgery

51, 1043-1052; Oct 2002.

104 Chang MY et al., “Neurons and astrocytes secrete factors that cause stem cells

to differentiate into neurons and astrocytes, respectively”, Molecular Cellular

Neuroscience 23, 414-426; July 2003.

105 Hori J et al., “Neural progenitor cells lack immunogenicity and resist destruction

as allografts”, Stem Cells 21, 405-416; 2003; Klassen H et al., “The immunological

properties of adult hippocampal progenitor cells”, Vision Research 43, 947-956; Apr

2003.

106 Pluchino S et al.; “Injection of adult neurospheres induces recovery in a chronic

model of multiple sclerosis”; Nature 422, 688-694; 17 April 2003.

107 Liker MA et al.; “Human neural stem cell transplantation in the MPTP-lesioned

mouse”; Brain Research 971, 168-177; May 2003.

108 Kim TE et al; “Sonic hedgehog and FGF8 collaborate to induce dopaminergic

phenotypes in the Nurr1-overexpressing neural stem cells”; Biochemical and

Biophysical Research Communications 305, 1040-1048; 2003.

109 Ourednik J et al.; “Neural stem cells display an inherent mechanism for rescuing

dysfunctional neurons”; Nature Biotechnology 20, 1103-1110; Nov 2002.

110 Åkerud P et al.; “Persephin-overexpressing neural stem cells regulate the function

of nigral dopaminergic neurons and prevent their degeneration in a model of

Parkinson’s disease”; Molecular and Cellular Neuroscience 21, 205-222; Nov 2002.

111 Fallon J et al.; “In vivo induction of massive proliferation,directed migration, and

differentiation of neural cells in the adult mammalian brain,” Proceedings of the

National Academy of Sciences USA 97, 14686-14691; 19 December 2000.

112 Gill SS et al., “Direct brain infusion of glial cell line-derived neurotrophic factor in

Parkinson disease”, Nature Medicine 9, 589-595; May 2003.

113 Lévesque M and Neuman T, “Autologous transplantation of adult human neural

stem cells and differentiated dopaminergic neurons for Parkinson disease: 1-year

postoperative clinical and functional metabolic result”, American Association of

Neurological Surgeons annual meeting, Abstract #702; 8 April 2002.
APPENDIX K. 341

114 Barnett et al.; “Identification of a human olfactory ensheathing cell that can effect

transplant-mediated remyelination of demyelinated CNS axons,” Brain 123, 1581-

1588, Aug 2000.

115 Ramón-Cueto A et al., “Functional recovery of paraplegic rats and motor axon

regeneration in their spinal cords by olfactory ensheathing glia,” Neuron 25, 425-

435; February 2000.

116 Ramón-Cueto A et al., “Long-distance axonal regeneration in the transected adult

rat spinal cord is promoted by olfactory ensheathing glia transplants”, The Journal

of Neuroscience 18, 3803–3815; 15 May 1998.

117 Ramer MS et al.; “Functional regeneration of sensory axons into the adult spinal

cord,” Nature 403, 312-316; Jan 20, 2000.

118 Jiang S et al; “Enteric glia promote regeneration of transected dorsal root axons

inot spinal cord of adult rats”; Experimental Neurology 181, 79-83; 2003.

119 Menet V et al., “Axonal plasticity and functional recovery after spinal cord injury

in mice deficient in both glial fibrillary acidic protein and vimentin genes”,

Proceedings of the National Academy of Sciences USA 100, 8999-9004; 22 July 2003.

120 Iacovitti L et al., “Differentiation of human dopamine neurons from an embryonic

carcinomal stem cell line”, Brain Research 912, 99-104; 2001.

121 Garbuzova-Davis S et al; “Positive effect of transplantation of hNT neurons (Ntera

2/D1 cell-line) in a model of familial amyotrophic sclerosis”; Experimental Neurology

174, 169-180; Apr 2002; Garbuzova-Davis S et al; “Intraspinal implantation of hNT

neurons into SOD1 mice with apparent motor deficit”; Amyotrophic Lateral Sclerosis

and Other Motor Neuron Disorders 2, 175-180; Dec 2001.

122 Kondziolka MD et al; “Transplantation of cultured human neuronal cells for patients

with stroke”; Neurology 55, 565-569; 2000; Meltzer CC et al., “Serial

[18F]Fluorodeoxyglucose Positron Emission Tomography after Human Neuronal

Implantation for Stroke”, Neurosurgery 49, 586-592; 2001; Nelson PT et al., “Clonal

human (hNT) neuron grafts for stroke therapy”, American Journal of Pathology 160,

1201-1206; Apr 2002.

123 Asakura A et al., “Myogenic specification of side population cells in skeletal

muscle”, Journal of Cell Biology 159, 123–134; 14 Oct 2002.

124 Lee JY et al., Clonal isolation of muscle-derived cells capable of enhancing muscle

regeneration and bone healing”, Journal of Cell Biology 150, 1085-1099; 4 Sept 2000.

125 Torrente Y et al.; “Intraarterial injection of muscle-derived CD34+Sca-1+ stem

cells restores dystrophin in mdx mice”; Journal of Cell Biology 152, 335-348; January

22, 2001.

126 Qu-Petersen Z et al.; “Identification of a novel population of muscle stem cells in

mice: potential for muscle regeneration”; Journal of Cell Biology 157, 851-864; 27

May 2002.

127 Polesskaya A et al., “Wnt signaling induces the myogenic specification of resident

CD45+ adult stem cells during muscle regeneration”, Cell 113, 841-852; 27 June

2003.
342 MONITORING STEM CELL RESEARCH

128 Lee JY et al., “The effects of periurethral muscle-derived stem cell injection on

leak point pressure in a rat model of stress urinary incontinence”, International

Urogynecology Journal of Pelvic Floor Dysfunction 14, 31-37; Feb 2003.

129 Atkins, B et al.; “Intracardiac Transplantation of Skeletal Myoblasts Yields Two

Populations of Striated Cells In Situ”; Annals of Thoracic Surgery 67, 124-129; 1999 130 Iijima Y et al., “Beating is necessary for transdifferentiation of skeletal musclederived cells into cardiomyocytes”, FASEB Journal 17, 1361-1363; July 2003 (full

text Epub 8 May 2003, doi:10.1096/fj.02-1048fje).

131 Menasché P et al. “Myoblast transplantation for heart failure.” Lancet 357, 279-

280; 27 January 2001.

132 Horb ME et al., “Experimental conversion of liver to pancreas”, Current Biology,

13, 105–115; 21 Jan 2003.

133 Yang L et al.; “In vitro trans-differentiation of adult hepatic stem cells into

pancreatic endocrine hormone-producing cells”; Proceedings of the National

Academy of Sciences USA, 99, 8078-8083; 11 June 2002.

134 Malouf, NN et al.; “Adult-derived stem cells from the liver become myocytes in

the heart in vivo”; American Journal of Pathology 158, 1929-1935; June 2001.

135 Wang, X et al; “Liver repopulation and correction of metabolic liver disease by

transplanted adult mouse pancreatic cells” American Journal of Pathology 158, 571-

579; Feb 2001.

136 Shapiro AMJ et al., “Islet transplantation in seven patients with type 1 diabetes

mellitus using a glucocorticoid-free immunosuppressive regimen”, New England

Journal of Medicine 343, 230-238; 27 July 2000; Ryan EA et al., “Clinical outcomes

and insulin secretion after islet transplantation with the Edmonton protocol”,

Diabetes 50, 710-719; Apr 2001.

137 Ramiya VK et al.; “Reversal of insulin-dependent diabetes using islets generated

in vitro from pancreatic stem cells,” Nature Medicine 6, 278-282, March 2000.

138 Bonner-Weir S et al.; “In vitro cultivation of human islets from expanded ductal

tissue”; Proceedings of the National Academy of Sciences USA 97, 7999-8004; 5 July

2000; Gmyr V et al., “Adult human cytokeratin 19-positive cells reexpress insulin

promoter factor 1 in vitro: Further evidence for pluripotent pancreatic stem cells in

humans”; Diabetes 49, 1671-1680; Oct 2000; de la Tour D et al.; “Beta-cell

differentiation from a human pancreatic cell line in vitro and in vivo”; Molecular

Endocrinology 15, 476-483, Mar 2001; Abraham et al.; “Insulinotropic hormone

glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor

cells into insulin-producing cells”; Endocrinology 143, 3152-3161; August 2002.

139 Suzuki A et al.; “Glucagon-like peptide 1 (1-37) converts intestinal epithelial cells

into insulin-producing cells”; Proceedings of the National Academy of Sciences USA

100, 5034-5039; 29 April 2003.

140 Ferber S et al; “Pancreatic and duodenal homeobox gene 1 induces expression of

insulin genes in liver and ameliorates streptozocin induced hyperglycemia”; Nature

Medicine 6, 568-572; May 2000.
APPENDIX K. 343

141 Meller D et al., “Ex vivo preservation and expansion of human limbal epithelial

stem cells on amniotic membrane cultures”, British Journal of Ophthalmology 86,

463-471; Apr 2002.

142 Tsai et al.; “Reconstruction of damaged corneas by transplantation of autologous

limbal epithelial cells.”; New England Journal of Medicine 343, 86-93, 2000; Schwab

IR et al.; “Successful transplantation of bioengineered tissue replacements in

patients with ocular surface disease”; Cornea 19, 421-426; July 2000; Tsubota K et

al.; “Treatment of severe ocular-surface disorders with corneal epithelial stem-cell

transplantation”; New England Journal of Medicine 340, 1697-1703; 3 June 1999;

Henderson TR et al., “The long term outcome of limbal allografts: the search for

surviving cells”, British Journal of Ophthalmology 85, 604-609; May 2001.

143 Seigel GM et al., “Human corneal stem cells display functional neuronal

properties”, Molecular Vision 9, 159-163; 30 Apr 2003.

144 Chacko DM et al., “Transplantation of ocular stem cells: the role of injury in

incorporation and differentiation of grafted cells in the retina”, Vision Research 43,

937-946; Apr 2003.

145 Dontu G et al., “In vitro propagation and transcriptional profiling of human

mammary stem/progenitor cells”, Genes and Development 17, 1253-1270; 15 May

2003.

146 Alvi AJ et al., “Functional and molecular characterisation of mammary side

population cells”, Breast Cancer Research 5, R1-R8; 2003.

147 Okumura K et al., “Salivary gland progenitor cells induced by duct ligation

differentiate into hepatic and pancreatic lineages”, Hepatology 38, 104-113; July

2003.

148 Toma JG et al; “Isolation of multipotent adult stem cells from the dermis of

mammalian skin”; Nature Cell Biology 3, 778-784; 3 Sept 2002; Oshima H et al.,

“Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell

104, 233-245; 2001; Taylor G et al., “Involvement of follicular stem cells in forming

not only the follicle but also the epidermis”, Cell 102, 451-461; 2000.

149 Lako M et al.; “Hair follicle dermal cells repopulate the mouse haematopoietic

system”; Journal of Cell Science 115, 3967-3974; Sept 2002.

150 Salingcarnboriboon R et al., “Establishment of tendon-derived cell lines exhibiting

pluripotent mesenchymal stem cell-like property”, Experimental Cell Research 287,

289-300; 15 July 2003.

151 De Bari C et al., “Multipotent mesenchymal stem cells from adult human synovial

membrane”, Arthritis Rheumatism 44, 1928-1942; 2001.

152 De Bari C et al.; “Skeletal muscle repair by adult human mesenchymal stem cells

from synovial membrane”; Journal of Cell Biology 160, 909-918; 17 March 2003.

153 Beltrami, AP et al.; “Evidence That Human Cardiac Myocytes Divide after

Myocardial Infarction”; New England Journal of Medicine 344, 1750-1757; 7 June

2001.

154 Robinson D et al., “Characteristics of cartilage biopsies used for autologous

chondrocytes transplantation”, Cell Transplant 10, 203-208; Mar-Apr 2001.
344 MONITORING STEM CELL RESEARCH

155 Horwitz EM et al., “Transplantability and therapeutic effects of bone marrowderived mesenchymal cells in children with osteogenesis imperfecta”, Nature

Medicine 5, 309–313; 1999.

156 Horwitz EM et al., “Isolated allogeneic bone marrow-derived mesenchymal cells

engraft and stimulate growth in children with osteogenesis imperfecta: Implications

for cell therapy of bone”, Proceedings of the National Academy of Sciences USA 99,

8932-8937; 25 June 2002.

157 Horwitz EM et al., “Clinical responses to bone marrow transplantation in children

with severe osteogenesis imperfecta”, Blood 97, 1227-1231; 1 March 2001.

158 Bennett AR et al., “Identification and characterization of thymic epithelial

progenitor cells”, Immunity 803-814; June 2002.

159 Gill J et al; “Generation of a complete thymic microenvironment by MTS24+

thymic epithelial cells”; Nature Immunology 3, 635–642; 1 Jul 2002.

160 Gronthos S et al., “Postnatal human dental pulp stem cells (DPSCs) in vitro and in

vivo”, Proceedings of the National Academy of Sciences USA 97, 13625-13630; 5 Dec

2000.

161 Miura M et al; “SHED: Stem cells from human exfoliated deciduous teeth”;

Proceedings of the National Academy of Sciences USA 100, 5807-5812; 13 May 2003.

162 Zuk PA et al; “Multilineage cells from human adipose tissue: implications for

cell-based therapies” Tissue Engineering 7, 211-228; 2001; Halvorsen Y-DC et al.,

“Extracellular matrix mineralization and osteoblast gene expression by human

adipose tissue–derived stromal cells”, Tissue Engineering 7, 729-741; 2001; Erickson

GR et al., “Chondrogenic potential of adipose tissue-derived stromal cells in vitro

and in vivo”, Biochemical and Biophysical Research Communications 290, 763-769;

2002.

163 Zuk PA et al., “Human adipose tissue is a source of multipotent stem cells”,

Molecular Biology of the Cell 13, 4279-4295; Dec 2002; Safford KM et al., “Neurogenic

differentiation of murine and human adipose-derived stromal cells”, Biochemical

and Biophysical Research Communications 294, 371–379; 2002.

164 See, for example: Kurtzberg J et al., “Placental blood as a source of hematopoietic

stem cells for transplantation into unrelated recipients”, New England Journal of

Medicine 335, 157-166; 18 July 1996; Laughlin MJ et al., “Hematopoietic engraftment

and survival in adult recipients of umbilical-cord blood from unrelated donors”,

New England Journal of Medicine 344, 1815-1822; 14 June 2001; Gore L et al.;

“Successful cord blood transplantation for sickle cell anemia from a sibling who is

human leukocyte antigen-identical: implications for comprehensive care”, Journal

of Pediatriatic Hematology and Oncology 22, 437-440; Sep-Oct 2000.

165 Rocha V et al., “Graft-versus-host disease in children who have received a

cordblood or bone marrow transplant from an HLA-identical sibling”, New England

Journal of Medicine, 342, 1846-1854; 22 June 2000.

166 Rainsford E, Reen DJ, “Interleukin 10, produced in abundance by human newborn

T cells, may be the regulator of increased tolerance associated with cord blood

stem cell transplantation”, British Journal of Haematology 116, 702-709; Mar 2002.
APPENDIX K. 345

167 Beerheide W et al., “Downregulation of ß2-microglobulin in human cord blood

somatic stem cells after transplantation into livers of SCID-mice: an escape

mechanism of stem cells?”, Biochemical and Biophysical Research Communications

294, 1052–1063; 2002.

168 Broxmeyer HE et al., “High-efficiency recovery of functional hematopoietic

progenitor and stem cells from human cord blood cryopreserved for 15 years”,

Proceedings of the National Academy of Sciences USA 100, 645-650; 12 Jan 2003.

169 Sanchez-Ramos J et al., “Expression of neural markers in human umbilical cord

blood”, Experimental Neurology 171, 109-115; 2001.

170 Chen J et al., “Intravenous administration of human umbilical cord blood reduces

behavioral deficits after stroke in rats”, Stroke 32, 2682-2688; Nov 2001.

171 Saporta S et al., “Human umbilical cord blood stem cells infusion in spinal cord

injury: engraftment and beneficial influence on behavior”, Journal of Hematotherapy

& Stem Cell Research 12, 271-278; 2003.

172 Garbuzova-Davis S et al., “Intravenous administration of human umbilical cord

blood cells in a mouse model of amyotrophic lateral sclerosis: distribution, migration,

and differentiation”, Journal of Hematotherapy & Stem Cell Research 12, 255–270;

2003.

173 Buzanska L et al., “Neural stem cell line derived from human umbilical cord blood

- morphological and functional properties”, Journal of Neurochemistry 85, Suppl 2,

33; June 2003.

174 Kakinuma S et al; “Human Umbilical Cord Blood as a Source of Transplantable

Hepatic Progenitor Cells”; Stem Cells 21, 217-227; 2003; Ishikawa F et al;

“Transplantied human cord blood cells give rise to hepatocytes in engrafted mice”;

Annals of the New York Academy of Sciences 996, 174-185; 2003; Wang X et al.,

“Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient

mice that received transplants of highly purified human hematopoietic stem cells”,

Blood 101, 4201-4208; 15 May 2003.

175 Mitchell K et al; “Matrix Cells from Wharton’s Jelly Form Neurons and Glia”;

Stem Cells 21, 51-60; 2003.

176 Weiss ML et al., “Transplantation of porcine umbilical cord matrix cells into the

rat brain”, Experimental Neurology Article in Press, Corrected Proof; available online

11 July 2003.

177 Okawa H et al., “Amniotic epithelial cells transform into neuron-like cells in the

ischemic brain”, NeuroReport 12, 4003-4007; 2001.

178 Prusa A-R et al., “Oct-4-expressing cells in human amniotic fluid: a new source

for stem cell research?”, Human Reproduction 18, 1489-1493; 2003.

179 Minasi MG et al., “The meso-angioblast: a multipotent, self-renewing cell that

originates from the dorsal aorta and differentiates into most mesodermal tissues”,

Development 129, 2773-2783; 2002.

180 Sampaolesi M et al., “Cell therapy of alpha-sarcoglycan null dystrophic mice

through intra-arterial delivery of mesoangioblasts”, Science Published online 10

July 2003 (doi: 10.1126/science.1082254).
346 MONITORING STEM CELL RESEARCH

181 Jensen GS and Drapeau C, “The use of in situ bone marrow stem cells for the

treatment of various degenerative diseases”; Medical Hypotheses 59, 422-428; 2002.

182 Grove JE et al; “Marrow-Derived Cells as Vehicles for Delivery of Gene Therapy

to Pulmonary Epithelium”; Stem Cells, 645-651; July 2002.

183 Steptoe RJ et al.; “Transfer of hematopoietic stem cells encoding autoantigen

prevents autoimmune diabetes”; Journal of Clinical Investigation 111, 1357-1363;

May 2003.

184 McKee JA et al; “Human arteries engineered in vitro”; EMBO Reports 4, 633-638;

May 2003.

185 Shi S et al., “Bone formation by human postnatal bone marrow stromal cells is

enhanced by telomerase expression”, Nature Biotechnology 20, 587-591; June 2002;

Simonsen JL et al., “Telomerase expression extends the proliferative life-span and

maintains the osteogenic potential of human bone marrow stromal cells”, Nature

Biotechnology 20, 592-596; June 2002.

186 Cavazzana-Calvo M et al., “Gene therapy of human severe combined

immunodeficiency (SCID)-X1 disease”, Science 288, 669–672; 2000.

187 Hacein-Bey-Abina A et al., “Sustained correction of X-linked severe combined

immunodeficiency by ex vivo gene therapy”, New England Journal of Medicine 346,

1185-1193; 18 Apr 2002.

188 Aiuti A et al., “Correction of ADA-SCID by stem cell gene therapy combined with

nonmyeloablative conditioning”, Science 296, 2410-2413; 28 June 2002.

189 Benedetti S et al., “Gene therapy of experimental brain tumors using neural

progenitor cells”, Nature Medicine 6, 447-450; Apr 2000.

190 Jin, HK et al; “Intracerebral transplantation of mesenchymal stem cells into acid

sphingomyelinase-deficient mice delays the onset of neurological abnormalities and

extends their life span”; Journal of Clinical Investigation 109, 1183-1191; May 2002.

191 Lee JY et al., “Effect of bone morphogenetic protein-2-expressing muscle-derived

cells on healing of critical-sized bone defects in mice”, The Journal of Bone and

Joint Surgery 83-A, 1032-1039; July 2001.

192 Zeisberg M et al., “BMP-7 counteracts TGF-ß1-induced epithelial-to-mesenchymal

transition and reverses chronic renal injury”, Nature Medicine 7, 964-968; July 2003.
Appendix L.

Stem Cells and Tissue Regeneration:

Lessons from Recipients of Solid Organ

Transplantation

SILVIU ITESCU, M.D.

Director of Transplantation Immunology, Departments of

Medicine and Surgery, Columbia University, New York, NY

CONTENTS

1. OVERVIEW

2. IMMUNOBIOLOGY OF ORGANTRANSPLANTATION

The Human Leukocyte Antigens (HLA)

T Cell Recognition Of Antigen Presented By HLA

Molecules

Thymic Education Of T Cells

T Cell Recognition Of Alloantigens

Tolerance Induction

3. IMMUNOSUPPRESSIVE AGENTS COMMONLY USED IN

ORGAN TRANSPLANT RECIPIENTS: BENEFITS AND

ADVERSE OUTCOMES

4. STEM CELL TRANSPLANTATION AND

IMMUNOSUPPRESSION

Materno-Fetal Tolerance

Immunogenic Characteristics Of Embryonic And

Adult Stem Cells

Tolerogenic Effects Of Stem Cell Transplantation

1. OVERVIEW

The Major Histocompatibility Complex (MHC) is located on the short

arm of chromosome 6 in humans and encodes the alloantigens

known as Human Leukocyte Antigens (HLA), polymorphic cell

surface molecules which enable the immune system to recognize

both self and foreign antigens. The class II HLA molecules (HLADR, HLA-DP, and HLA-DQ) are usually found only on antigen347
348 MONITORING STEM CELL RESEARCH

presenting cells such as B lymphocytes, macrophages, and dendritic

cells of lymphoid organs, and initiate the immune response to foreign

proteins, including viruses, bacteria, and foreign HLA antigens on

transplanted organs.

Following binding of foreign proteins, class II HLA on antigenpresenting cells activate CD4+ T cells, which in turn activate

cytotoxic CD8+ T cells to recognize the same foreign antigen bound

to HLA class I (HLA-A, HLA-B, and HLA-C, molecules found on the

surface of all cells) and destroy the target. The actual recognition of

foreign HLA transplantation antigens by T cells is referred to as

allorecognition. Two distinct pathways of allorecognition have been

described, direct and indirect. The direct pathway involves receptors

on the host T cells that directly recognize intact HLA antigens on

the cells of the transplanted organ. The indirect pathway requires

an antigen-presenting cell that internalizes the foreign antigen and

presents it via its own HLA class II molecule on the surface of an

antigen-presenting cell to the CD4+ helper T cells.

Once recognition has taken place, an important cascade of events

is initiated at the cellular level, culminating in intracellular release

of ionized calcium from intracellular stores. The calcium binds with

a regulatory protein called calmodulin, forming a complex that

activates various phosphatases, particularly calcineurin. Calcineurin

dephosphorylates an important cytoplasmic protein called nuclear

factor of activated T cells (NFAT), resulting in its migration to the

nucleus and induction of the production of various cytokines such

as IL-2. These cytokines recruit other T cells to destroy the

transplanted organ, ultimately resulting in rejection and loss of the

graft.

Immunosuppressive regimens used to prevent allograft rejection

are aimed at inhibiting the various arms of the immune response,

typically require multiagent combinations, and need to be

maintained for the duration of life. The currently used

armamentarium confers significant side-effect risks, including

infectious and neoplastic complications. Moreover, despite success

at preventing early allograft rejection, long-term survival of

transplanted organs remains difficult to achieve and novel methods

to achieve long-term tolerance are being actively sought.

Stem cells obtained from embryonic or adult sources differ from

other somatic cells in that they express very low levels of HLA

molecules on their cell surfaces. This endows these cell types with

the theoretical potential to escape the standard mechanisms of

immune rejection discussed above. However, under conditions that

enable cellular differentiation in vitro and in vivo each of these stem

cell populations acquires high level expression of HLA molecules,

suggesting that their long-term survival following transplantation
APPENDIX L. 349

in vivo may be limited by typical immune rejection phenomena.

Recent experimental data, however, provide striking counterintuitive

examples that stem cells from both embryonic and adult sources

may evade the recipient’s immune system and result in long-term

engraftment in the absence of immunosuppression despite

acquisition of surface HLA molecule expression. These observations

may have significant impact on the emerging field of regenerative

medicine.

2. IMMUNOBIOLOGY OF ORGAN TRANSPLANTATION

The Human Leukocyte Antigens (HLA)

Differences between individuals which enable immune recognition

of non-self from self are principally due to the extreme polymorphism

of genes in the Major Histocompatibility Complex (MHC) on

chromosome 6 in man which encode the cell surface HLA molecules.

These molecules are cell surface glycoproteins whose biologic

function is to bind antigenic peptides (epitopes) derived from

viruses, bacteria, or cancer cells, and present them to T cells for

subsequent immune recognition. Each HLA gene includes a large

number of alleles and the peptide binding specificity varies for each

different HLA allele. The 1996 WHO HLA Nomenclature Committee

report lists more than 500 different HLA class I and class II alleles.

Crystallographic x-ray studies have demonstrated that the

hypervariable regions encoded by polymorphic regions in the alleles

correspond to HLA binding pockets which engage specific “anchor”

residues of peptide ligands. One HLA molecule will recognize a

range of possible peptides, whereas another HLA molecule will

recognize a different range of peptides. Consequently, no two

individuals will have the same capability of stimulating an immune

response, since they do not bind the same range of immunogenic

peptides. It is estimated that >99% of all possible peptides derived

from foreign antigens are ignored by any given HLA molecule. Since

in the absence of HLA polymorphism a large number of immunogenic

peptides would not be recognized, the extensive HLA polymorphism

in the population reduces the chance that a given virus or bacterium

would not be recognized by a sizable proportion of the population,

reducing the likelihood of major epidemics or pandemics.

T Cell Recognition Of Antigen Presented By HLA Molecules

Since HLA molecules regulate peptide display to and activation of

the immune system, considerable effort has been devoted to

understanding the molecular basis of peptide-HLA interactions.
350 MONITORING STEM CELL RESEARCH

These issues are important for defining the biology of T cell antigen

recognition and the properties of a protein that make it immunogenic

or non-immunogenic. Specific antigen recognition by T cells is

dependent on recognition by the T cell receptor of a threedimensional complex on the surface of antigen-presenting cells

(APC) comprised of the HLA molecule and its bound peptide. The

peptides are produced by complex antigen processing machineries

within the APC (i.e. proteolytic enzymes, peptide transporters and

molecular chaperones) which generate a pre-selected peptide pool

for association with the HLA molecules. The different types of T

cells require different HLA molecules for antigen presentation, socalled “HLA restriction” phenomena. T cell receptors on CD8+

cytotoxic T cells (CTLs) bind peptides presented by HLA class I

molecules, whereas CD4+ T helper cells (Th) recognize peptides

bound to HLA class II molecules. Of the 8-13 amino acid residues of

a bound peptide within a class I or II HLA molecule, only three to

four amino acid side chains are accessible to the T cell receptor,

and a similar number of amino acids are involved in binding to the

HLA molecule.

Thymic Education Of T Cells

T cells mature in the thymus to appropriately respond to foreign

pathogens without inadvertently attacking the host. Under the

influence of various thymic resident cells and factors they elaborate,

maturing T cells fall into two categories: those that are able to

discriminate between self and non-self and can appropriately

respond to foreign pathogens without inadvertently attacking the

host, and those which are unable to appropriately discriminate

between self and non-self. Dendritic cells have been implicated in

the deletion, or inhibition, of T cells reactive to self-antigens,

particularly in the thymus during T cell development or in peripheral

lymphoid organs. The process of self/non-self discrimination by the

maturing T cells is dependent on thymic dendritic cell (DC)

presentation of self-antigens in the context of self-HLA molecules.

When maturing thymic T cells are highly reactive with self-antigen/

HLA complexes, they are deleted so that potentially autoreactive T

cells will not be released into the periphery. If a particular foreign

antigen can be presented in such a way in the thymus as to fool the

maturing T cells into believing that the antigen is part of self tissue,

then T cells capable of reacting with this antigen will also be

eliminated. Indeed, it has been demonstrated that when mouse

thymic DC present transgenically introduced foreign antigens to

developing T cells, the mature peripheral T cell repertoire of the

mouse lacks T cells capable of reacting with the specific foreign
APPENDIX L. 351

antigen, i.e. it is tolerant to the foreign antigen. This has raised the

possibility that injection of dendritic cells into an allogeneic recipient

might induce tolerance to a subsequent allograft by causing deletion

or inhibition of alloreactive T cells.

T Cell Recognition Of Alloantigens

Recognition of foreign, or allogeneic, HLA antigens by the recipient

immune system is the major limitation to the survival of solid organ

grafts. The central role of HLA molecules in allograft rejection is

due to their role as restriction elements for T cell recognition of donor

antigens and the extensive polymorphism displayed by the HLA

molecules, which elicit host immune responses. Although progress

has been made in the short-term survival of transplants, chronic

immunologic rejection remains an impediment to long-term survival.

The primary cause of acute rejection of transplanted organs is

so-called “direct” recognition of whole allogeneic HLA antigens by

receptors on the surface of recipient T cells. The direct recognition

pathway involves recognition by recipient T cells of donor HLA class

I and class II molecules, resulting in the generation of cytotoxic and

helper T lymphocytes which play a pivotal role in the rejection

process. In contrast, chronic rejection of transplanted organs results

from so-called “indirect recognition” of donor HLA peptides derived

from the allogeneic HLA molecules shed by the donor tissue. These

foreign HLA molecules are taken up and processed by recipient

antigen presenting cells (APC), and peptide fragments of the

allogeneic HLA molecules containing polymorphic amino acid

residues are bound and presented by recipient’s (self) HLA

molecules to recipient (self) T cells. Although direct and indirect

recognition of alloantigen generally leads to adverse graft outcome,

tolerance induction may occur following exposure of the recipient

to donor alloantigens prior to transplantation. Since this strategy is

based on the nature and dose of the antigen as well as the route of

administration, understanding how to control the balance between

activation and unresponsiveness mediated by the direct and/or

indirect recognition of alloantigen is a an area of active research

which could lead to development of new therapies to prolong graft

survival.

Indirect allorecognition has been implicated in recurrent rejection

episodes in various transplantation models of cardiac, kidney and

skin grafts. Determinants on donor HLA molecules can be divided

into two main categories: (a) the dominant allodeterminants that

are efficiently processed and presented to alloreactive T cells during

allograft rejection; and (b) the cryptic allodeterminants that are

potentially immunogenic but do not normally induce alloreactive
352 MONITORING STEM CELL RESEARCH

responses, presumably due to incomplete processing and/or

presentation. Indirect recognition of allo-HLA peptides is important

for the initiation and spreading of the immune response to other

epitopes within the allograft. So-called “spreading” of indirect T

cell responses to other allo-HLA epitopes expressed by graft tissue

is strongly predictive of recurring episodes of rejection. Tolerance

induction to the dominant donor determinants represents potential

effective strategy for blocking indirect alloresponses and ensuring

long-term graft survival in animal models.

Tolerance Induction

Advances in surgical methods and current immunosuppressive

therapies have led to significant improvement in short-term graft

survival, however long-term survival rates remain poor. For example,

whereas both kidney and heart allografts have one-year graft

survival rates of 85 to 95 percent, only about 50% of transplanted

hearts survive five years and only about 50% of kidney grafts survive

ten years. Thus, despite being able to achieve short-term success,

these relatively poor long-term graft survival rates demonstrate the

limitations of the current clinical immunosuppressive regimens to

enable long-term immune evasion by the graft. Consequently, a

major goal of transplantation immunobiologists is to induce donorspecific tolerance, allowing the long-term survival of human

allografts without the need of HLA-compatibility and without the

continuous recipient immunosupression leading to the concomitant

risks of infection, malignancy, and/or other specific drug side effects.

This would theoretically improve long-term graft survival, reduce

or eliminate the continuing need for expensive, toxic and non-specific

immunosuppressive therapy and enhance the quality of life.

Insight into some of the mechanisms involved in tolerance

induction has been gained from pre-clinical and clinical studies in

numerous animal models and in patients, particularly those with

liver allografts which typically do not induce a prominent immune

response leading to rejection. One possible mechanism by which

liver transplantation results in allograft tolerance tolerance may be

that the donor or “passenger” lymphoid cells in the transplanted

liver emigrate and take up residence in the recipient’s immune

organs, such as the thymus or lymph nodes. Donor lymphocytes at

these sites might “re-educate” the recipient immune system so that

the donor organ is not recognized as foreign. In an attempt to initiate

a similar process in other organ recipients, transfusions of donor

blood or bone marrow have been used to enhance solid organ graft

survival in animal models and in clinical trials. These studies are

currently ongoing in various organ systems.
APPENDIX L. 353

Molecular understanding of the cellular immune response has

led to new strategies to induce a state of permanent tolerance after

transplantation. Several approaches have shown promise, including

the use of tolerizing doses of class I HLA-molecules in various forms

for the induction of specific unresponsiveness to alloantigens, and

the use of synthetic peptides corresponding to HLA class II

sequences. Other approaches include alteration in the balance of

cytokines that direct the immune response away from the TH1 type

of inflammatory response and graft rejection to the TH2 type of

response that might lead to improved graft survival, and the use of

agents to induce “co-stimulatory blockade” of T cell activation. This

latter approach is based on the concept that blockade of a “second

signal” to the T cell enables the signal provided to the T cell receptor

by the HLA-peptide complex to induce antigen specific tolerance.

The experimental use of human dendritic cells as tolerogenic

agents has been limited due to the low frequency of circulating

dendritic cells in peripheral human blood, the limited accessibility

to human lymphoid organs, and the terminal state of differentiation

of circulating human dendritic cells making their further expansion

ex vivo difficult. Dendritic cells are migratory cells of sparse, but

widespread, distribution in both lymphoid and non-lymphoid tissues.

Although the earliest precursors are ultimately of bone marrow

origin, the precise lineage of dendritic cells is controversial and

includes both myeloid-derived and lymphoid-derived populations.

Recent work has revealed that an expanded population of mature

human dendritic cells can be derived from non-proliferating

precursors in vitro is by culturing bone-marrow derived cells with a

combination of cytokines. This method of enrichment for human

dendritic cells from a precursor population can result in the

production of dendritic cells that are tolerogenic to foreign antigens.

Whether such cells could be useful when co-administered with an

allograft transplant remains to be determined. Nevertheless, it is

clear that considerable progress has been made in the past few

years using approaches to manipulate the immune response to

enable routine donor-specific tolerance, and there is reason to be

optimistic that with better understanding of molecular and cellular

mechanisms this goal could be attained.
354 MONITORING STEM CELL RESEARCH

3. IMMUNOSUPPRESSIVE AGENTS COMMONLY USED IN

ORGAN TRANSPLANT RECIPIENTS: BENEFITS AND ADVERSE

OUTCOMES

Cyclosporine

Cyclosporine has been the single most important factor associated

with improved outcomes after organ transplantation over the past

two decades. CyA binds to a cytosolic cell protein, cyclophilin (CyP).

The CyA-CyP complex then binds to calcineurin and subsequently

blocks interleukin-2 (IL-2) transcription. The binding of IL-2 to the

IL-2 receptors on the surface of T lymphocytes is a key stimulant in

promoting lymphocyte proliferation, activation, and ultimately

allograft rejection. A review of the first decade of experience with

heart transplantation revealed a total of 379 cardiac allograft

recipients worldwide; actuarial survival rates in this cohort of

patients at 1 year and 5 years were 56% and 31% respectively; the

main causes of death being acute rejection and the side effects of

immunosuppression. With the introduction and widespread use of

CyA over the next decade, survival rates dramatically improved to

85% and 75% at 1 and 5 years respectively. Similar results were

obtained with other organ transplants, including kidney and lung.

The major adverse effects of CyA are nephrotoxicity,

hypertension, neurotoxocity and hyperlipidemia; less common side

effects include hirsuitism, gingival hyperplasia and liver dysfunction.

CyA nephrotoxicity can manifest as either acute or chronic renal

dysfunction. It is important to note that a number of drugs commonly

used in transplant patients, such as aminoglycosides, amphotericin

B and ketoconazole can potentiate the nephrotoxicity induced by

CyA. More than half the patients receiving CyA will require treatment

for hypertension within the first year following transplantation.

Corticosteroids also potentiate the side effects of CyA such as

hypertension, hyperlipidemia and hirsuitism. Frequent monitoring

of the serum level is essential to minimize the adverse effects. One

of the major limitations of the original oil-based CyA formulation

(Sandimmune) is its variable and unpredictable bioavailability. In

the mid-90s Neoral was introduced, a new microemulsion formula

of CyA, which has greater bioavailability and more predictable

pharmacokinetics than Sandimmune.

Tacrolimus

Tacrolimus (FK506) is a macrolide antibiotic that inhibits T-cell

activation and proliferation and inhibits production of other
APPENDIX L. 355

cytokines. The product of Streptomyces tsurubaensis fermentation,

FK 506 was first discovered in 1984 and first used in clinical studies

in 1988 at the University of Pittsburgh. While the mechanism of action

of tacrolimus is similar to that of CyA, and comparative clinical trials

have suggested similar efficacy, it has been suggested that some

groups of patients may benefit from tacrolimus rather than CyA as

primary immunosuppressive therapy. Unlike CyA, hirsuitism and

gingival hyperplasia occur infrequently with tacrolimus; thus,

tacrolimus-based therapy may improve compliance and quality of

life in female and pediatric transplant recipients. It should be noted

that alopecia has been documented with tacrolimus, but is known

to improve with dose reductions. The decreased incidence of

hypertension and hyperlipidemia with tacrolimus makes it preferable

to CyA in patients with difficult to treat hypertension or

hyperlipidemia. A final indication for tacrolimus has been as a rescue

immunosuppressant in cardiac transplant recipients on CyA with

refractory rejection or intolerance to immunosuppression (severe side

effects). Since tacrolimus is metabolized using the same cytochrome

P450 enzyme system as CyA, drug interactions are essentially the

same. Thus, drugs that induce this system may increase the

metabolism of tacrolimus, thereby decreasing its blood levels.

Conversely, drugs that inhibit the P450 system decrease the

metabolism of tacrolimus, thereby increasing its blood levels. It is

important to note that some studies have indicated a higher

incidence of nephrotoxicity with tacrolimus as compared to CyA.

Azathioprine and Mycophenolate Mofetil (MMF)

Despite being available for more than 35 years, azathioprine is still

a useful agent as an immunosuppressive agent. Following

administration, azathioprine is converted into 6-mercaptopurine,

with subsequent transformation to a series of intracellularly active

metabolites. These inhibit both an early step in de novo purine

synthesis and several steps in the purine salvage pathway. The net

effect is depletion of cellular purine stores, thus inhibiting DNA and

RNA synthesis, the impact of which is most marked on actively

dividing lymphocytes responding to antigenic stimulation. In

currently used immunosuppressive protocols, azathioprine is used

as part of a triple therapy regimen along with CyA or tacrolimus

and prednisone. Mycophenolate mofetil (MMF), which is rapidly

hydrolyzed after ingestion to mycophenolic acid, is a selective,

noncompetitive, reversible inhibitor of onosine monophosphate

dehydrogenase, a key enzyme in the de novo synthesis of guanine

nucleotides. Unlike other marrow-derived cells and parenchymal

cells that use the hypoxanthine-guanine phosphoribosyl transferase
356 MONITORING STEM CELL RESEARCH

(salvage) pathway, activated lymphocytes rely predominantly on the

de novo pathway for purine synthesis. This functional selectivity

allows lymphocyte proliferation to be specifically targeted with less

anticipated effect on erythropoiesis and neutrophil production than

is seen with azathioprine.

Early studies in human kidney and heart transplant recipients

showed that MMF, when substituted for azathioprine in standard

triple-therapy regimens, is well tolerated and more efficacious than

azathioprine. In a large, double-blind, randomized multicenter study

comparing MMF versus azathioprine (with CyA and prednisone)

involving 650 patients, the MMF group was associated with

significant reduction in mortality as well as a reduction in the

requirement for rejection treatment. However, there was noted to

be an increase in the incidence of opportunistic viral infections in

the MMF group. The overall greater efficacy of MMF compared to

azathioprine has resulted in MMF generally replacing azathioprine

in triple immunosuppressive protocols together with steroids and

cyclosporine in most solid organ recipients.

Corticosteroids

Steroids are routinely used in almost all immunosuppressive

protocols after organ transplantation. The metabolic side effects of

steroids are well known and lead to significant morbidity and

mortality in the post-transplant period. Almost 90% of organ

recipients continue to receive prednisone at 1-year post-transplant

and 70% at three-years post-transplant. A recent review of over 1800

patients from a combined registry outlined the morbid complications

that patients suffer within the first year after transplantation. Many

of these complications are known side effects of prednisone,

including hypertension (16%), diabetes mellitus (16%),

hyperlipidemia (26%), bone disease (5%) and cataracts (2%). It is

thereby obvious that avoidance of steroids may decrease morbidity

and mortality after organ transplantation. Two general approaches

are used to institute prednisone-free immunosuppression: early and

late withdrawal.

Withdrawal of prednisone during the first month post-transplant

has resulted in long-term success of steroid withdrawal in 50–80%

of patients. In these studies, the use of antilymphocyte antibody

induction therapy appears to increase the likelihood of steroid

withdrawal. Several centers have reported their results with

immunosuppressive regimens that did not include steroids in the

early post-transplant period. Studies reporting high success rates

of 80% have used specific enrolment criteria, such as excluding

patients with recurrent acute rejections or those with female gender.
APPENDIX L. 357

Review of numerous studies demonstrate that steroid free

maintenance immunosuppression is possible in atleast 50% of

patients, is as safe as triple drug therapy and may reduce some of

the long-term complications of steroids. Owing to the fact that the

majority of acute rejection episodes occur in the first three months

post-transplant, steroid withdrawal is made after this time period,

resulting in long-term success in about 80% of patients. Generally,

there is no need for conventional induction agents when late

withdrawal of steroids is done.

Anti-Lymphocyte Antibody Therapy

Despite the extensive use of induction therapy using anti-lymphocyte

antibody in solid organ transplantation, their exact role is unclear.

There is no doubt that routine use of these agents is unwarranted

as the generalized immunosuppression induced by then increased

the risk of infections and malignancy. Despite the lack of consistent

data supporting the routine use of induction therapy with antilymphocyte antibody agents, there is a role in certain select

situations. Specifically, patients with early post-operative renal or

hepatic dysfunction may benefit especially by the avoidance of

cyclosporine therapy while using these induction agents. Antilymphocyte antibody therapy can provide effective

immunosuppression for atleast 10 to 14 days without CyA or

tacrolimus therapy. It has also been suggested that patients with

overwhelming postoperative bacterial infections or diabetics with

severe postoperative hyperglycemia may benefit from the

comparatively low doses of corticosteroids required during antilymphocyte induction therapy.

The two main types of induction agents have been either the

polyclonal antilymphocyte or antithymocyte globulins and more

recently the murine monoclonal antibody OKT3. While these agents

have been shown to be effective in terminating acute allograft

rejection and in treating refractory rejection, the results of

comparative studies of outcomes with and without monoclonal

induction therapy have varied, with most studies demonstrating an

effect on rejection that is maintained only while antibody therapy is

ongoing. Without repeated administration, these agents only delay

the time to a first rejection episode without decreasing the overall

frequency or severity of rejection. More importantly, their use has

been associated with an increased risk of short-term (infections)

and long-term (lympho-proliferative disorders) complications. A

complication specific to OKT3 is the development of a “flu-like

syndrome” characterized by fever, chills and mild hypotension,

typically seen with the first dose.
358 MONITORING STEM CELL RESEARCH

Since antilymphocyte antibodies are produced in nonhuman

species, their use is associated with the phenomenon of

sensitization, leading to decreased effectiveness with repeated use

as well as the possibilty of serum sickness. The development of

sensitization has been linked with an increased risk of acute vascular

rejection. While this association has not been reported by other

centers using OKT3 prophylaxis, it is believed that the development

of immune-complex disease, inadequate immunosuppression due

to decreased OKT3 levels or that OKT3 sensitization may be a marker

for patients at higher risk for humoral rejection may be responsible

for this phenomenon.

Interleukin-2 Receptor Inhibition

A new class of drugs has been developed which targets the high

affinity IL-2 receptor. This receptor is present on nearly all activated

T cells but not on resting T cells. In vivo activation of the high-affinity

IL-2 receptor by IL-2 promoted the clonal expansion of the activated

T cell population. A variety of rodent monoclonal antibodies directed

against the α chain of the receptor have been used in animals and

humans to achieve selective immunosuppression by targeting only

T-cell clones responding to the allograft. Chimerisation or

humanisation of these monoclonal antibodies resulted in antibodies

with a predominantly human framework that retained the antigen

specificity of the original rodent monoclonal antibodies. A fully

humanized anti-IL2R monoclonal antibody, daclizumab, and a

chimeric anti-IL-2R monoclonal antibody, basiliximab, have

undergone successful phase III trials demonstrating their efficacy

in the immunoprophylaxis of patients undergoing renal and cardiac

transplantation.

Both agents have immunomodulatory effects that are similar to

those of other monoclonal antibody-based therapies (i.e., induction

of clonal anergy rather than clonal deletion). The advantages of these

agents include their lack of immunogenicity, long half-lives, ability

to repeat dosing, and short-term safety profile. Daclizumab appears

to be an effective adjuvant immunomodulating agent in cardiac

allograft recipients. It has advantages over conventional induction

therapy as it is more selective and can be used for prolonged and

potentially repeated periods. Studies with larger cohorts are needed

to further study the short-term and long-term survival benefits for

patients following organ transplantation and should determine the

optimal dosing schedules of these new agents.
APPENDIX L. 359

4. STEM CELL TRANSPLANTATION AND IMMUNOSUPPRESSION

Materno-Fetal Tolerance

As outlined above, when tissues from an HLA-disparate donor are

transplanted into a recipient they are always recognized as foreign,

and immunosuppression is required to prevent rejection. An

important exception to this is observed in pregnant women who

tolerate their unborn fetus despite the fact that it expresses a full

set of non-maternal HLA antigens inherited from the father. The

mechanisms by which embryonic tissue demonstrates immune

privilege during prenatal development have not yet been fully

elucidated, however it is evident that interactions between fetus

and mother differ substantially from the events triggered by a

classical allograft. Consequently, much work is being dedicated to

the emerging field of materno-fetal immunobiology in order to enable

the development of innovative strategies to induce tolerance and

prevent allogeneic graft rejection.

When maternal T cells encounter the fetus they demonstrate

adaptive tolerance. In part this may be due to the absence of

expression of MHC class II antigens and low levels of expression of

MHC class I antigens on fetal cells. However, this can only partly

explain the state of prolonged maternal tolerance since induction of

HLA class I and II molecules inevitably occurs as the fetus matures

and differentiates, yet rejection still does not occur. Consequently,

non-fetal aspects of the placental barrier must be of critical

importance in maintaining prolonged tolerance to the fetus. An

important mechanism may relate to upregulation of the human nonclassical HLA class Ib antigen, designated HLA-G, by the

syncytiotrophoblast. HLA-G molecules bind to inhibitory receptors

on natural killer cells and subsequently protect against maternal

rejection responses. The placenta produces high levels of the antiinflammatory cytokine interleukin 10 which stimulates HLA-G

synthesis while concomitantly downregulating MHC class I antigen

production, thus contributing to the tolerance-inducing local

environment. The trophoblast also produces high levels of the

enzyme indoleamine 2,3-dioxygenase, which catabolizes tryptophan,

an essential amino acid necessary for rapid T cell proliferation.

Annexin II, found in isolated placental membranes in vitro is present

in placental serum, exerts immunosuppressive properties, and

additionally contributes to fetal allograft survival. Together, these

features indicate that materno-fetal tolerance results from a

combination of transiently reduced antigenicity of the fetus in
360 MONITORING STEM CELL RESEARCH

combination with a complex tolerance-inducing milieu at the

placental barrier.

Immunogenic Characteristics Of Embryonic And Adult Stem Cells

Murine and human embryonic stem (ES) cells do not express HLA

class I and II antigens, and demonstrate reduced surface expression

of co-stimulatory molecules important for T cell activation.

Transplantation of murine ES cells demonstrates long-term graft

survival despite the fact that these cells do acquire HLA class II

antigen expression after in vivo differentiation. Since they are able

to accomplish long-term engraftment without the need for

immunosuppression, their inability to induce an immune response

is not likely to be the result of escaping immune surveillance, but

rather due to their ability to colonize the recipient thymus and induce

intrathymic deletion of alloreactive recipient T cells.

Recently, a population of cells has been described in human adult

bone marrow that has similar functional characteristics to embryonic

stem cells in that they have high self-regenerating capability and

capacity for differentiation into multiple cell types, including muscle,

cartilage, fat, bone, and heart tissue. While such cells, termed adult

mesenchymal stem cells (MSC), appear to have a more restricted

self-renewal capacity and differentiation potential than ES cells, their

functional characteristics may be sufficient for clinically meaningful

tissue regeneration. A striking recent observation is that MSC can

broadly inhibit T-cell proliferation and activation by various types of

antigenic stimulation, including allogeneic stimuli. MSCs have been

shown to inhibit both naive and memory T cell responses in a dosedependent fashion and affect cell proliferation, cytotoxicity, and the

number of interferon gamma (IFN-gamma)-producing T cells. MSCs

appear to inhibit T cell activation through direct contact, and do not

require other regulatory cellular populations. Similarly to ES cells,

adult bone marrow-derived mesenchymal stem cells (MSCs) do not

express HLA class II molecules, and only low levels of HLA class I

molecules. Despite the fact that MSC can be induced to express

surface HLA class II molecules by in vitro culture with cytokines

such as interferon-gamma, their ability to inhibit T cell activation

results in induction of T cell non-responsiveness to the MSC

themselves, endowing them with potential survival advantages in

the setting of transplantation.

Tolerogenic Effects Of Stem Cell Transplantation

Extending the approaches discussed above using donor-derived

blood transfusions to induce a tolerogenic state to the subsequent
APPENDIX L. 361

organ, the most promising clinical strategy for tolerance induction

at present is the use of donor-derived hematopoietic stem cells in

conjunction with reduced myeloablative conditioning. The objective

of this therapy is to achieve a state of so-called mixed chimerism, or

the permanent co-existence of donor- and recipient-derived blood

cells comprising all the different hematopoietic lineages in the same

host. This approach has been tested in a variety of small and large

animal settings and currently available data suggest that stable

engraftment of donor bone marrow reliably renders the host tolerant

to donor antigens and subsequently to any cellular or solid organ

graft of the same donor.

The two underlying mechanisms by which creation of a mixedchimeric host results in tolerance induction are (1) thymic deletion

of potentially donor-specific alloreactive T cells, and (2) nonthymic

peripheral mechanisms, such as blocking costimulatory T cell

activation, which facilitate the process of donor bone-marrow or stem

cell engraftment. However, despite the efficacy of an approach using

fully HLA-mismatched stem cells in an allogeneic host to induce

tolerance to a subsequent organ allograft, the host is placed at a

high risk of substantial morbidity and mortality due to toxicity of

the myeloablative conditioning regimen and potential for graftversus-host disease, or immune-mediated attack of the host by the

implanted allogeneic stem cells.

In an attempt to overcome these potential limiting toxicities,

investigators have suggested the use of either adult bone marrowderived mesenchymal stem cells or preimplantation-derived

embryonic stem (ES) cells for induction of mixed chimerism. The

theoretical advantages of these cell types is their low level of surface

expression of HLA class I and II antigens, and reduced surface

expression of co-stimulatory molecules important for T cell

activation. Rat preimplantation stage derived embryonic-like stem

cells have been shown to successfully engraft in the recipient bone

marrow without the need for pre-conditioning therapies such as

irradiation, cytotoxic drug regimens or T cell depletion. Long-term

partial mixed chimerism by use of rat preimplantation stage derived

embryonic-like stem cells did not trigger graft-versus-host reactions,

in contrast to the high frequency of this complication in the clinical

setting of allogeneic hematopoietic stem cell transplantation. Of

most interest, the induced partial chimerism enabled the recipient

animals to be tolerant to a subsequent heart allograft. Allograft

acceptance required the presence of an intact thymus, and rat ES

cells were present in the recipient thymus.

Similar results have been reported following transplantation of

human adult bone marrow-derived mesenchymal stem cells (MSC)

into fetal sheep early in gestation, before and after the expected
362 MONITORING STEM CELL RESEARCH

development of immunologic competence. In this xenogeneic

system, human MSC engrafted, differentiated in a site-specific

manner, and persisted in multiple tissues for as long as 13 months

after transplantation, including the thymus. Since MSCs do not

present alloantigen and do not require MHC expression to exert

their inhibitory effect on alloimmune reactivity, the possibility exists

that they could theoretically be derived from a donor irrespective of

their HLA type and used to inhibit T-cell responses to transplantation

antigens of an unrelated third party. In initial human clinical studies,

the use human adult bone marrow-derived mesenchymal stem cells

has been shown to successfully enable engraftment of subsequently

infused allogeneic bone marrow in transplant recipients, reduce the

risk of graft-versus-host disease, and reduce the need for

concomitantly administered immunosuppression. Whether similar

results will be obtained when combining adult bone marrow-derived

mesenchymal stem cells with solid organ allografts remains to be

determined, and this is an area of active research for clinical

transplant immunobiologists. Of broader relevance, if the results

relating to long-term engraftment and survival of adult bone marrowderived MSC are confirmed and extended in human clinical studies,

they will have broad implications for the field of tissue and organ

regeneration.

SELECTED REFERENCES

1. Billingham RE, Brent L, Medawar PB (1953) Actively acquired tolerance of

foreign cells. Nature 172:603-606.

2. Auchincloss H Jr (2001) In search of the elusive holy grail: the mechanisms

and prospects for achieving clinical transplantation tolerance. Am J Transplant

1:6-12.

3. Wekerle T, Kurtz J, Ito H, Ronquillo JV, Dong V, Zhao G, Shaffer J, Sayegh MH,

Sykes M (2000) Allogeneic bone marrow transplantation with co-stimulatory

blockade induces macrochimerism and tolerance without cytoreductive host

treatment. Nat Med 6:464-469.

4. Watkins DI (1995) The evolution of major histocompatibility class I genes in

primates. Crit Rev Immunol 15:1-29.

5. Salter-Cid L, Flajnik MF (1995) Evolution and developmental regulation of the

major histocompatibility complex. Crit Rev Immunol 15:31-75.

6. Fowlkes BJ, Ramsdell F (1993) T-cell tolerance. Curr Opin Immunol 5:873-879.

7. Marrack P, Kappler J (1990) T cell tolerance. Semin Immunol 2:45-49.
APPENDIX L. 363

8. Van Parijs L, Abbas AK (1998) Homeostasis and self-tolerance in the immune

system: turning lymphocytes off. Science 280:243-249.

9. Davies JD, Martin G, Phillips J, Marshall SE, Cobbold SP, Waldmann H (1996) T

cell regulation in adult transplantation tolerance. J Immunol 157:529-533.

10. Thomson AW, Lu L (1999) Dendritic cells are regulators of immune reactivity:

implications for transplantation. Transplantation 68:1-8.

11. Brent L (1997) Fetally and neonatally induced immunologic tolerance. In: Brent

L (ed) A history of transplantation immunology. Academic, San Diego.

12. Matzinger P (1994) Tolerance, danger, and the extended family. Annu Rev

Immunol 12:991-1045.

13. Janeway CAJ (1992) The immune system evolved to discriminate infectious

nonself from noninfectious self. Immunol Today 13:11-16.

14. Mellor AL, Munn DH (2001) Extinguishing maternal immune responses during

pregnancy: implications for immunosuppression. Semin Immunol 13:213-218.

15. Fändrich F, Lin X, Chai GX, Schulze M, Ganten D, Bader M, Holle J, Huang DS,

Parwaresch R, Zavazava N, Binas B (2002) Preimplantation-stage stem cells

induce allogeneic graft tolerance without supplementary host conditioning.

Nat Med 8:171-178.

16. Liechty KW, et al (2000). Human mesenchymal stem cells engraft and

demonstrate site-specific differentiation after in utero transplantation in sheep.

Nat Med 6(11):1282-6.

17. Krampera M, et al (2002). Bone marrow mesenchymal stem cells inhibit the

response of naive and memory antigen-specific T cells to their cognate peptide.

Blood, 101(9):3722-3729.

18. Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O (2003).

Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and

mitogenic responses independently of the major histocompatibility

complex.Scand J Immunol. 57(1):11-20.

Appendix M.

Potential Use of Cellular Therapy

for Patients With Heart Disease

SILVIU ITESCU, M.D.

Departments of Medicine and Surgery, Columbia University,

 New York, NY

Summary

Congestive heart failure remains a major public health

problem, and is frequently the end result of cardiomyocyte apoptosis

and fibrous replacement after myocardial infarction, a process

referred to as left ventricular remodelling. Cardiomyocytes undergo

terminal differentiation soon after birth, and are generally considered

to irreversibly withdraw from the cell cycle. In response to ischemic

insult, adult cardiomyocytes undergo cellular hypertrophy, nuclear

ploidy, and a high degree of apoptosis. A small number of human

cardiomyocytes retain the capacity to proliferate and regenerate in

response to ischemic injury, however whether these cells are derived

from a resident pool of cardiomyocyte stem cells or from a renewable

source of circulating bone marrow-derived stem cells that home to

the damaged myocardium is at present not known. Replacement

and regeneration of functional cardiac muscle after an ischemic insult

to the heart could be achieved by either stimulating proliferation of

endogenous mature cardiomyocytes or resident cardiac stem cells,

or by implanting exogenous donor-derived or allogeneic cells such

as fetal or embryonic cardiomyocyte precursors, bone marrowderived mesenchymal stem cells, or skeletal myoblasts. The newly

formed cardiomyocytes must integrate precisely into the existing

myocardial wall in order to augment synchronized contractility and

avoid potentially life-threatening alterations in the electrical

conduction of the heart. A major impediment to survival of the

implanted cells is altered immunogenicity by prolonged ex vivo

culture conditions. In addition, concurrent myocardial

revascularization is required to ensure viability of the repaired region

and prevent further scar tissue formation. Human adult bone marrow

contains endothelial precursors which resemble embryonic

365
366 MONITORING STEM CELL RESEARCH

angioblasts and can be used to induce infarct bed neovascularization

after experimental myocardial infarction. This results in protection

of cardiomyocytes against apoptosis, induction of cardiomyocyte

proliferation and regeneration, long-term salvage and survival of

viable myocardium, prevention of left ventricular remodelling and

sustained improvement in cardiac function. It is reasonable to

anticipate that cell therapy strategies for ischemic heart disease

will need to incorporate (1) a renewable source of proliferating,

functional cardiomyocytes, and (2) angioblasts to generate a network

of capillaries and larger size blood vessels for supply of oxygen and

nutrients to both the chronically ischemic endogenous myocardium

and to the newly-implanted cardiomyocytes.

Introduction

Congestive heart failure remains a major public health

problem, with recent estimates indicating that end-stage heart

failure with two-year mortality rates of 70-80% affects over 60,000

patients in the US each year 1

. In Western societies heart failure is

primarily the consequence of previous myocardial infarction 2. As

new modalities have emerged which have enabled significant

reduction in early mortality from acute myocardial infarction,

affecting over 1 million new patients in the US annually, there has

been a paradoxical increase in the incidence of post-infarction heart

failure among the survivors. Current therapy of heart failure is

limited to the treatment of already established disease and is

predominantly pharmacological in nature, aiming primarily to inhibit

the neurohormonal axis that results in excessive cardiac activation

through angiotensin- or norepinephrine-dependent pathways. For

patients with end-stage heart failure treatment options are extremely

limited, with less than 3000 being offered cardiac transplants

annually due to the severely limited supply of donor organs 3,4, and

implantable left ventricular assist devices (LVADs) being expensive,

not proven for long-term use, and associated with significant

complications 5-7. Clearly, development of approaches that prevent

heart failure after myocardial infarction would be preferable to those

that simply ameliorate or treat already established disease.

Heart Failure After Myocardial Infarction Results From

Progressive Ventricular Remodelling. Heart failure after myocardial

infarction occurs as a result of a process termed myocardial

remodelling. This process is characterized by myocyte apoptosis,

cardiomyocyte replacement by fibrous tissue deposition in the

ventricular wall 8-10, progressive expansion of the initial infarct area

and dilation of the left ventricular lumen 11,12. Another integral
APPENDIX M. 367

component of the remodelling process appears to be the

development of neoangiogenesis within the myocardial infarct scar

13,14, a process requiring activation of latent collagenase and other

proteinases 15. Under normal circumstances, the contribution of

neoangiogenesis to the infarct bed capillary network is insufficient

to keep pace with the tissue growth required for contractile

compensation and is unable to support the greater demands of the

hypertrophied, but viable, myocardium. The relative lack of oxygen

and nutrients to the hypertrophied myocytes may be an important

etiological factor in the death of otherwise viable myocardium,

resulting in progressive infarct extension and fibrous replacement.

Since late reperfusion of the infarct vascular bed in both humans

and experimental animal models significantly benefits ventricular

remodelling and survival 16-18, we have postulated that methods to

successfully augment vascular bed neovascularization might

improve cardiac function by preventing loss of hypertrophied, but

otherwise viable, cardiac myocytes.

Inability Of Damaged Myocardium To Undergo Repair Due To Cell

Cycle Arrest Of Adult Cardiomyocytes. Cardiomyocytes undergo

terminal differentiation soon after birth, and are thought by most

investigators to irreversibly withdraw from the cell cycle. Analysis

of cardiac myocyte growth during early mammalian development

indicates that cardiac myocyte DNA synthesis occurs primarily in

utero, with proliferating cells decreasing from 33% at mid-gestation

to 2% at birth 19. While ventricular karyokinesis and cytokinesis are

coupled during fetal growth, resulting in increases in mononucleated

cardiac myocytes, karyokinesis occurs in the absence of cytokinesis

for a transient period during the post-natal period, resulting in

binucleation of ventricular myocytes without an overall increase in

cell number. A similar dissociation between karyokinesis and

cytokinesis characterizes the primary adult mammalian cardiac

response to ischemia, resulting in myocyte hypertrophy and increase

in nuclear ploidy rather than myocyte hyperplasia 20,21. Moreover, in

parallel with an inability to progress through cell cycle, ischemic

adult cardiomyocytes undergo a high degree of apoptosis.

When cells proliferate, the mitotic cycle progression is tightly

regulated by an intricate network of positive and negative signals.

Progress from one phase of the cell cycle to the next is controlled by

the transduction of mitogenic signals to cyclically expressed proteins

known as cyclins and subsequent activation or inactivation of several

members of a conserved family of serine/threonine protein kinases

known as the cyclin-dependent kinases (cdks) 22. Growth arrest

observed with such diverse processes as DNA damage, terminal
368 MONITORING STEM CELL RESEARCH

differentiation, and replicative senescence is due to negative

regulation of cell cycle progression by two functionally distinct

families of Cdk inhibitors, the Ink4 and Cip/Kip families 19. The cell

cycle inhibitory activity of p21Cip1/WAF1 is intimately correlated

with its nuclear localization and participation in quaternary

complexes of cell cycle regulators by binding to G1 cyclin-CDK

through its N-terminal domain and to proliferating cell nuclear

antigen (PCNA) through its C-terminal domain 23-26. The latter

interaction blocks the ability of PCNA to activate DNA polymerase,

the principal replicative DNA polymerase 27. For a growth-arrested

cell to subsequently enter an apoptotic pathway requires signals

provided by specific apoptotic stimuli in concert with cell-cycle

regulators. For example, caspase-mediated cleavage of p21, together

with upregulation of cyclin A–associated cdk2 activity, have been

shown to be critical steps for induction of cellular apoptosis by either

deprivation of growth factors 28 or hypoxia of cardiomyocytes 29.

Throughout life, a mixture of young and old cells is present

in the normal myocardium. Although most myocytes seem to be

terminally differentiated, there is a fraction of younger myocytes

(15–20%) that retains the capacity to replicate 30. Moreover, recent

observations have suggested that some human ventricular

cardiomyocytes also have the capacity to proliferate and regenerate

in response to ischemic injury 31,32. The dividing myocytes can be

identified on the basis of immunohistochemical staining of

proliferating nuclear structures such as Ki67 and cell surface

expression of specific surface markers, including c-kit (CD117).

Whether these cells are derived from a resident pool of

cardiomyocyte stem cells or are derived from a renewable source of

circulating bone marrow-derived stem cells that home to the

damaged myocardium remains to be determined. More importantly,

the signals required for homing, in situ expansion and differentiation

of these cells are, at present, unknown. Gaining an understanding

of these issues would open the possibility of manipulating the

biology of endogenous cardiomyocytes in order to augment the

healing process after myocardial ischemia.

Strategies For The Use Of Cellular Therapy To Improve Myocardial

Function. Replacement and regeneration of functional cardiac

muscle after an ischemic insult to the heart could be achieved by

either stimulating proliferation of endogenous mature

cardiomyocytes or resident cardiac stem cells, or by implanting

exogenous donor-derived or allogeneic cardiomyocytes. The newly

formed cardiomyocytes must integrate precisely into the existing

myocardial wall in order to augment contractile function of the
APPENDIX M. 369

residual myocardium in a synchronized manner and avoid alterations

in the electrical conduction and syncytial contraction of the heart,

potentially resulting in life-threatening consequences. In addition,

whatever the source of the cells used, it is likely that concurrent

myocardial revascularization must also occur in order to ensure

viability of the repaired region and prevent further scar tissue

formation. The following section discusses various methods of using

cellular therapies to replace damaged myocardium or re-initiate

mitosis in mature endogenous cardiomyocytes, including

transplanted bone marrow-derived cardiomyocyte or endothelial

precursors, fetal cardiomyocytes, and skeletal myoblasts.

Potential Role For Bone Marrow-Derived Or Embryonic

Cardiomyocyte Lineage Stem Cells In Myocardial Repair/

Regeneration. Over the past several years, a number of studies

have suggested that stem cells can be used to generate

cardiomyocytes ex vivo for potential use in a range of cardiovascular

diseases 33-37. Multipotent bone marrow-derived mesenchymal stem

cells have been identified in adult murine and human bone marrow

functionally by their ability to differentiate to lineages of diverse

mesenchymal tissues, including bone, cartilage, fat, tendon, and

both skeletal and cardiac muscle 36, and phenotypically by their

expression of specific surface markers and lack of hematopoietic

lineage markers such as CD34 or CD45 35. It is well established that

murine embryonic stem (ES) cells can give rise to cardiomyocytes

in vitro and in vivo 38,39. Recently, Kehat et al. were able to

demonstrate that human embryonic stem cells can also differentiate

in vitro into cells with characteristics of cardiomyocytes 37. However,

there are striking differences in the human and murine stem cell

models, and this needs to be taken into account when extrapolating

results of mouse experiments to the human condition. For example,

human ES cells have a very low efficiency of differentiation to

cardiomyocytes compared with murine ES cells, and a considerably

slower time course (a median of 11 days vs 2 days). Whether these

differences reflect true variations between species, or differences

in the experimental protocols, remains to be determined.

Irrespective whether the cardiomyocyte lineage stem cell

precursors are obtained from adult bone marrow or embryonic

sources, the newly generated cardiomyocytes appear to resemble

normal cardiomyocytes in terms of phenotypic properties, such as

expression of actinin, desmin and troponin I, and function, including

positive and negative chronotropic regulation of contractility by

pharmacological agents and production of vasoactive factors such

as atrial and brain natriuretic peptides. However, in vivo evidence
370 MONITORING STEM CELL RESEARCH

for functional cardiac improvement following transplantation of adult

bone marrow-derived or ES-derived cardiomyocytes has been

exceedingly difficult to show to date. In part this may be because

the signals required for cardiomyocyte differentiation and functional

regulation are complex and poorly understood. For example,

phenotypic and functional differentiation of mesenchymal stem cells

to cardiomyocyte lineage cells in vitro requires culture with

exogenously added 5-azacytidine 33,34. Alternatively, the poor

functional data obtained to date may reflect immune-mediated

rejection of cells which have been modified during the ex vivo culture

process or poor viability due to the lack of a sufficient vascular supply

to the engrafted cells (see below).

Potential Role For Autologous Skeletal Myoblasts In Myocardial

Repair. An alternative approach to replacing damaged myocardium

involves the use of autologous skeletal myoblasts 40. The procedure

involves harvesting a patient’s skeletal muscle cells, expanding the

cells in a laboratory, and re-injecting the cells into the patient’s heart.

Perceived advantages of the approach include ease of access to the

cellular source, the fact that immunosuppression is not needed, and

the lack of ethical dilemmas associated with the use of allogeneic

or embryonic cells. It has also been argued that using relatively

ischemia-resistant skeletal myoblasts rather than cardiomyocytes

might enable higher levels of cell engraftment and survival in

infarcted regions of the heart, where cardiomyocytes would probably

perish 41.

Successful engraftment of autologous skeletal myoblasts

into injured myocardium has been reported in multiple animal

models of cardiac injury. These studies have demonstrated survival

and engraftment of myoblasts into infarcted or necrotic hearts 40,

differentiation of the myoblasts into striated cells within the

damaged myocardium 40, and improved myocardial functional

performance 40,42,43. Other studies have shown that the survival of

transplanted myoblasts can be improved by heat shock pretreatment

44 , and have confirmed that the benefits of skeletal myoblast transfer

are additive with those of conventional therapies, such as

angiotensin-converting enzyme inhibition 45. More recently, the

procedure has been reported anecdotally to result in improved

myocardial function in humans 46. On the basis of these preliminary

results, clinical trials have begun both in Europe and in the United

States. In addition to demonstrating functional improvement in

large, prospective series, questions that remain to be addressed

include whether the skeletal myoblasts can make meaningful

electromechanical connections to the surrounding endogenous
APPENDIX M. 371

cardiomyocytes through gap junctions, whether the cellular mass

will contract in concert, and whether electrical impulses will be

transmitted to the myoblast tissue without inducing significant

tachyarrhythmias.

Poor Survival Of Cells Transplanted Into Damaged Myocardium

After Ex Vivo Culture. A major limitation to successful cellular

therapy in animal models of myocardial damage has been the

inability of the introduced donor cells to survive in their host

environment, whether such transplants have been congenic

(analogous to the autologous scenario in humans) or allogeneic. It

has become clear that a major impediment to survival of the

implanted cells is the alteration of their immunogenic character by

prolonged ex vivo culture conditions. For example, whereas

myocardial implantation of skeletal muscle in the absence of tissue

culture does not induce any adverse immune response and results

in grafts showing excellent survival for up to a year, injection of

cultured isolated (congenic) myoblasts results in a massive and rapid

necrosis of donor myoblasts, with over 90% dead within the first

hour after injection 47-50. This rapid myoblast death appears to be

mediated by host natural killer (NK) cells 50 which respond to

immunogenic antigens on the transplanted myoblasts altered by

exposure to tissue culture conditions 48. It seems likely that a similar

mechanism of host NK cell-mediated rejection will apply also to

transplanted ES-derived, cultured cardiomyocytes 51 since massive

death of injected donor cells is recognized as a major problem with

transplanted cardiomyocytes, especially in the inflammatory

conditions that follow infarction 52. In this regard, the report that

cultured mesenchymal stem cells obtained from adult human bone

marrow are not rejected on transfer to other species is intriguing 36,

needs confirmation in humans, and requires detailed investigation

into possible tolerogenic mechanisms.

Concomitant Induction Of Vascular Structures Augments Survival

and Function Of Cardiomyocyte Precursors. An additional

explanation for the poor survival of transplanted cardiomyocytes or

skeletal myoblasts may be that viability and prolonged function of

transplanted cells requires an augmented vascular supply. Whereas

many transplanted cardiomyocytes die by apoptosis, cultured

cardiomyocytes that incorporate more vascular structures in vivo

demonstrate significantly greater survival52. Moreover, in situations

where transplanted cardiomyocyte precursors contained an

admixture of cells also giving rise to vascular structures, survival

and function of the newly formed cardiomyocytes has been

significantly augmented.
372 MONITORING STEM CELL RESEARCH

In one study, direct injection of whole rat bone marrow into

a cryo-damaged heart resulted in neovascularization, cardiomyocyte

regeneration and functional improvement 34. More recently, systemic

delivery of highly purified bone marrow-derived hematopoietic stem

cells in lethally irradiated mice contributed to the formation of both

endothelial cells and long-lived cardiomyocytes in ischemic hearts 53.

Most strikingly, significant improvement in cardiac function of mice

who had previously undergone LAD ligation was demonstrated after

direct myocardial injection of syngeneic bone marrow-derived stem

cells, defined on the basis of c-kit (CD117) expression 54. This

population of cells contains a mixture of cellular elements in addition

to cardiomyocyte precursors, including CD117-positive endothelial

progenitors (see below). These cells were found to proliferate and

differentiate into myocytes, smooth muscle cells and endothelial

cells, resulting in the partial regeneration of the destroyed

myocardium and prevention of ventricular scarring. Together, these

findings raise the intriguing possibility that for long-term in vivo

viability and functional integrity of stem cell-derived cardiomyocytes

it may be necessary to induce neovascularization by coadministration of endothelial cell progenitors (see below).

Endothelial Precursors And Formation Of Vascular Structures

During Embryogenesis. In order to develop successful methods

for inducing neovascularization of the adult heart, one needs to

understand the process of definitive vascular network formation

during embryogenesis. In the pre-natal period, hemangioblasts

derived from the human ventral aorta give rise to cellular elements

involved in both vasculogenesis, or formation of the primitive

capillary network, and hematopoiesis 55,56. In addition to

hematopoietic lineage markers, embryonic hemangioblasts are

characterized by expression of the vascular endothelial cell growth

factor receptor-2, VEGFR-2, and have high proliferative potential with

blast colony formation in response to VEGF 57-60. Under the regulatory

influence of various transcriptional and differentiation factors,

embryonic hemangioblasts mature, migrate and differentiate to

become endothelial lining cells and create the primitive vasculogenic

network. The differentiation of embryonic hemangioblasts to

pluripotent stem cells and to endothelial precursors appears to be

related to co-expression of the GATA-2 transcription factor, since

GATA-2 knockout embryonic stem cells have a complete block in

definitive hematopoiesis and seeding of the fetal liver and bone

marrow61. Moreover, the earliest precursor of both hematopoietic

and endothelial cell lineage to have diverged from embryonic ventral

endothelium has been shown to express VEGF receptors as well as
APPENDIX M. 373

GATA-2 and alpha4-integrins 62. Subsequent to capillary tube

formation, the newly-created vasculogenic vessels undergo

sprouting, tapering, remodelling, and regression under the direction

of VEGF, angiopoietins, and other factors, a process termed

angiogenesis. The final component required for definitive vascular

network formation to sustain embryonic organogenesis is influx of

mesenchymal lineage cells to form the vascular supporting

structures such as smooth muscle cells and pericytes.

Characterization Of Endothelial Progenitors, Or Angioblasts, In

Human Adult Bone Marrow. In studies using various animal models

of peripheral ischemia a number of groups have shown the potential

of adult bone marrow-derived elements to induce neovascularization

of ischemic tissues 63-69. In the most successful of these 63, bone

marrow-derived cells injected directly into the thighs of rats who

had undergone ligation of the left femoral artery and vein induced

neovascularization and augmented blood flow in the ischemic limb

as documented by laser doppler and immunohistochemical analyses.

Although the nature of the bone marrow-derived endothelial

progenitors was not precisely identified in these studies, the

cumulative reports indicated that this site may be an important

source of endothelial progenitors which could be useful for

augmenting collateral vessel growth in ischemic tissues, a process

termed therapeutic angiogenesis.

In more recent studies, our group has identified such

endothelial progenitors in human adult bone marrow 70. By

employing both in vitro and in vivo experimental models we have

sought to precisely identify the surface characteristics and biological

properties of these bone marrow-derived endothelial progenitor

cells. Following G-CSF treatment, mobilized mononuclear cells were

harvested and CD34+ cells were separated using anti-CD34 mAb

coupled to magnetic beads. 90-95% of CD34+ cells co-expressed

the hematopoietic lineage marker CD45, 60-80% co-expressed the

stem cell factor receptor CD117, and <1% co-expressed the

monocyte/macrophage lineage marker CD14. By quadruple

parameter analysis, the VEGFR-2 positive cells within the

CD34+CD117bright subset displayed phenotypic characteristics of

endothelial progenitors, including co-expression of Tie-2, as well as

AC133, but not markers of mature endothelium such as ecNOS, vWF,

E-selectin, and ICAM. Sorting CD34+ cells on the basis of CD117

bright or dim expression demonstrated that GATA-2 mRNA and

protein levels were significaantly higher in the CD117bright
374 MONITORING STEM CELL RESEARCH

population, indicating that human adult bone marrow contains cells

with an angioblast-like phenotype.

Since the frequency of circulating endothelial cell precursors

in animal models has been shown to be increased by either VEGF 71

or regional ischemia 63-66, phenotypically-defined angioblasts were

examined for proliferative responses to VEGF and to factors in

ischemic serum. CD117brightGATA-2hi angioblasts demonstrated

significantly higher proliferative responses relative to CD117dim

GATA-2lo bone marrow-derived cells from the same donor following

culture for 96 hours with either VEGF or ischemic serum. The

expanded angioblast population consisted of large blast cells,

defined by forward scatter, which continued to express immature

markers, including GATA-2 and CD117bright but not markers of mature

endothelial cells, including eNOS or E-selectin, indicating blast

proliferation without differentiation under these culture conditions.

However, culture on fibronectin with endothelial growth medium

resulted in outgrowth of monolayers with endothelial morphology

and functional and phenotypic features characteristic of endothelial

cells, including uniform uptake of acetylated LDL, and co-expression

of CD34, factor VIII, and eNOS. Thus, G-CSF treatment of adult

humans mobilizes into the peripheral circulation a bone-marrow

derived population with phenotypic and functional characteristics

of embryonic angioblasts, as defined by specific surface phenotype,

high proliferative responses to VEGF and cytokines in ischemic

serum, and ability differentiate into endothelial cells by culture in

medium enriched with endothelial growth factors.

Human Angioblasts Induce Neovascularization Of The Myocardial

Infarct Zone. Intravenous injection of freshly-obtained human

angioblasts into athymic nude rats who had undergone ligation of

the left anterior descending (LAD) coronary artery resulted in infarct

zone infiltration within 48 hours. Few human cells were detected in

unaffected areas of hearts with regional infarcts or in myocardium

of sham-operated rats. Histologic examination at two weeks postinfarction revealed that injection of human angioblasts was

accompanied by a significant increase in infarct zone

microvascularity, cellularity, and numbers of capillaries, and by

reduction in matrix deposition and fibrosis in comparison to controls.

Neovascularization was significantly increased within both the

infarct zone and in the peri-infarct rim in rats receiving angioblasts

compared with controls receiving saline or other cells which

infiltrated the heart (e.g. CD34- cells or saphenous vein endothelial

cells, SVEC). The neovascularization induced by human angioblasts
APPENDIX M. 375

was due to both an increase in capillaries of human origin as well

as of rat origin, as defined by monoclonal antibodies with specificity

for human or rat CD31 endothelial markers. Capillaries of human

origin, defined by co-expression of DiI fluorescence and human CD31,

but not rat CD31, accounted for 20-25% of the total myocardial

capillary vasculature, and was located exclusively within the central

infarct zone of collagen deposition. In contrast, capillaries of rat

origin, as determined by expression of rat, but not human, CD31,

demonstrated a distinctively different pattern of localization, being

absent within the central zone of collagen deposition and abundant

both at the peri-infarct rim between the region of collagen deposition

and myocytes, and between myocytes.

Human Angioblasts Protect Hypertrophied Endogenous

Cardiomyocytes Against Apoptosis. By concomitantly staining rat

tissues for the myocyte-specific marker desmin and performing DNA

end-labeling using the TUNEL technique, temporal examinations

demonstrated that the infarct zone neovascularization induced by

injection of human angioblasts prevented an eccentrically-extending

pro-apoptotic process evident in saline controls. Thus, at two weeks

post-infarction, myocardial tissue of LAD-ligated rats who received

saline demonstrated 6-fold higher numbers of apoptotic myocytes

compared with that from rats receiving intravenous injections of

human angioblasts. Moreover, these myocytes had distorted

appearance and irregular shape. In contrast, myocytes from LADligated rats who received human angioblasts had regular, oval shape,

and were significantly larger than myocytes from control rats.

Human Angioblasts Induce Sustained Regeneration/ Proliferation

Of Endogenous Cardiomyocytes. In addition to protection of

hypertrophied myocytes against apoptosis, human angioblastdependent neovascularization resulted in a striking induction of

regeneration/proliferation of endogenous rat cardiomyocytes at the

peri-infarct rim 72. At two weeks after LAD ligation, rats receiving

human angioblasts demonstrated numerous “fingers” of

cardiomyocytes of rat origin, as determined by expression of rat MHC

class I molecules, extending from the peri-infarct region into the

infarct zone. The islands of cardiomyocytes at the peri-infarct rim

in animals receiving human angioblasts contained a high frequency

of rat myocytes with DNA activity, as determined by dual staining

with mAbs reactive against cardiomyocyte-specific troponin I and

rat Ki67. In contrast, in animals receiving saline there was a high

frequency of cells with fibroblast morphology and reactivity with

rat Ki67, but not troponin I, within the infarct zone. The number of

cardiomyocytes progressing through cell cycle at the peri-infarct
376 MONITORING STEM CELL RESEARCH

region of rats receiving human angioblasts was 40-fold higher than

that at sites distal to the infarct, 20-fold higher than that found in

non-infarcted hearts, and 5-fold higher than that at the peri-infarct

rim of animals receiving saline 72.

Neovascularization Of Acutely Ischemic Myocardium By Human

Angioblasts Prevents Ventricular Remodelling And Causes

Sustained Improvement In Cardiac Function. By 15 weeks postinfarction, rats receiving human angioblasts demonstrated markedly

smaller scar sizes together with increased mass of viable

myocardium within the anterior free wall. Whereas collagen

deposition and scar formation extended almost through the entire

left ventricular wall thickness in controls, with aneurysmal dilatation

and typical EKG abnormalities, the infarct scar extended only to 20-

50% of the left ventricular wall thickness in rats receiving CD34+

cells. Moreover, pathological collagen deposition in the non-infarct

zone was markedly reduced in rats receiving CD34+ cells. At 15

weeks, the mean proportion of scar/normal left ventricular

myocardium was 13% in rats receiving CD34+ cells compared with

36-45% for each of the other groups studied (saline, CD34-, SVEC).

Remarkably, by two weeks after injection of human

angioblasts, and in a parallel time-frame with the observed neovascularization, left ventricular ejection fraction (LVEF) recovered

by a mean of 22%. This effect was long-lived, with LVEF recovering

by a mean of 34% at the end of follow-up, 15 weeks post injection.

Neither CD34- cells nor SVEC demonstrated similar effects. At 15

weeks post-infarction, mean cardiac index in rats injected with

human angioblasts was only reduced by 26% relative to normal rats,

whereas for each of the other groups it was reduced by 48-59%.

Together, these results indicate that the neovascularization,

reduction in peri-infarct myocyte apoptosis and increase in

cardiomyocyte regeneration/proliferation observed at two weeks

prevented myocardial replacement with fibrous tissue and caused

sustained improvement in myocardial function.

Potential Use Of Angioblasts In Combination With Cardiomyocyte

Progenitors For Repair and Regeneration of Ischemic Myocardium.

While increasing capillary density through angioblast-dependent

neovascularization is a promising approach for preventing apoptotic

death and inducing regeneration of endogenous cardiomyocytes

following acute myocardial infarction, the role of angioblast therapy

for the treatment of congestive heart failure following chronic

ischemia is at present unknown. Nevertheless, it is reasonable to

anticipate that cellular therapies for congestive heart failure due to
APPENDIX M. 377

ischemic cardiomyopathy will need to address two interdependent

processes: (1) a renewable source of proliferating, functional

cardiomyocytes, and (2) development of a network of capillaries and

larger size blood vessels for supply of oxygen and nutrients to both

the chronically ischemic, endogenous myocardium and to the newlyimplanted cardiomyocytes. To achieve these endpoints it is likely

that co-administration of angioblasts and mesenchymal stem cells

will be needed in order to develop regenerating cardiomyocytes,

vascular structures, and supporting cells such as pericytes and

smooth muscle cells. Future studies will need to address the timing,

relative concentrations, source and route of delivery of each of these

cellular populations in animal models of acute and chronic

myocardial ischemia.

In addition to synergistic cellular therapies, it is likely that

optimal regimens for the treatment of acute and chronically ischemic

hearts will require a combined approach employing additional

pharmacological strategies. For example, augmentation in

myocardial function might be achieved by combining infusion of

human angioblasts and cardiomyocyte progenitors together with

beta blockade, ACE inhibition or AT1-receptor blockade to reduce

angiotensin II-dependent cardiac fibroblast proliferation and

collagen secretion 73-76. Understanding the potential of defined

lineages of stem cells or undifferentiated progenitors, and their

interactions with pharmacological interventions, will lead to better

and more focussed clinical trial designs using each cell type

independently or in combination, depending on which particular

clinical indication is being targeted.
378 MONITORING STEM CELL RESEARCH

REFERENCES

1. Effects of enalapril on mortality in severe congestive heart failure. Results of the

Cooperative North Scandinavian Enalapril Survival Study (CONSENSUS)

Trial Study Group. N Engl J Med 1987; 316(23):1429-1435.

2. Mahon NG, O’Roke C, Codd MB, et al. Hospital mortality of acute myocardial

infarction in the thrombolytic era. Heart 1999;81:478-82.

3. Hognes JR. In The Artificial Heart: Prototypes, Policies and Patients. Washingtion,

DC: National Academy Press; 1991:1-312.

4. Annual Report of the US Scientific Registry for Organ Transplantation and the

Organ Procurement and Transplantation Network - 1990. Washingtion, DC:

US Department of Health and Human Services: 1990.

5. Frazier OH, Rose EA, Macmanus Q, et al. Multicenter clinical evaluation of the

Heartmate 1000 IP left ventricular assist device. Ann Thorac Surg 1992;

102:578-587.

6. McCarthy PM, Rose EA, Macmanus Q, et al. Clinical experience with the Novacor

ventricular assist system. J Thorac Cardiovasc Surg. 1991; 102:578-587.

7. Oz MC, Argenziano M, Catanese KA, et al. Bridge experience with long-term

implantable left ventricular assist devices. Are they an alternative to

transplantation? Circulation. 1997; 95:1844-1852.

8. Colucci WS. Molecular and cellular mechanisms of myocardial failure. Am J

Cardiol 1997;80:15L-25L.

9. Ravichandran LV and Puvanakrishnan R. In vivo labeling studies on the

biosynthesis and degradation of collagen in experimental myocardial

myocardial infarction. Biochem Intl 1991;24:405-414.

10. Agocha A, Lee H-W, Eghali-Webb M. Hypoxia regulates basal and induced DNA

synthesis and collagen type I production in human cardiac fibroblasts:

effects of TGF-beta, thyroid hormone, angiotensis II and basic fibroblast

growth factor. J Mol Cell Cardiol 1997;29:2233-2244H.

11. Pfeffer JM, Pfeffer MA, Fletcher PJ, Braunwald E. Progressive ventricular

remodeling in rat with myocardial infarction. Am J Physiol 1991;

260:H1406-14.

12. White HD, Norris RM, Brown MA, Brandt PWT, Whitlock RML, Wild CJ. Left

ventricular end systolic volume as the major determinant of survival after

recovery from myocardial infarction. Circulation 1987; 76:44-51.

13. Nelissen-Vrancken H, Debets J, Snoeckx L, Daemen M, Smits J. Time-related

normalization of maximal coronary flow in isolated perfused hearts of rats

with myocardial infarction. Circulation 1996;93:349-355.

14. Kalkman EAJ, Bilgin YM, van Haren P, van Suylen R-J, Saxena PR, Schoemaker

RG.Determinants of coronary reserve in rats subjected to coronary artery

ligation or aortic banding. Cardiovasc Res 1996.
APPENDIX M. 379

15. Heymans S, Luutun A, Nuyens D, et al. Inhibition of plasminogen activators or

matrix metalloproteinases prevents cardiac rupture but impairs

therapeutic angiogenesis and causes cardiac failure. Nat Med 1999;5:1135-

1142.

16. Hochman JS and Choo H. Limitation of myocardial infarct expansion by

reperfusion independent of myocardial salvage. Circulation 1987;75:299-

306.

17. White HD, Cross DB, Elliot JM, et al. Long-term prognostic importance of patency

of the infarct-related coronary artery after thrombolytic therapy for

myocardial infarction. Circulation 1994;89:61-67.

18. Nidorf SM, Siu SC, Galambos G, Weyman AE, Picard MH. Benefit of late coronary

reperfusion on ventricular morphology and function after myocardial

infarction. J Am Coll Cardiol 1992;20:307-313.

19. MacLellan WR and Schneider MD. Genetic dissection of cardiac growth control

pathways Annu. Rev. Physiol. 2000. 62:289-320.

20. Soonpaa MH, Field LJ. Assessment of cardiomyocyte DNA synthesis in normal

and injured adult mouse hearts. Am J Physiol 272, H220-6 (1997).

21. Kellerman S, Moore JA, Zierhut W, Zimmer HG, Campbell J, Gerdes AM. Nuclear

DNA content and nucleation patterns in rat cardiac myocytes from different

models of cardiac hypertrophy. J Mol Cell Cardiol 24, 497-505 (1992).

22. Hill MF, Singal PK. Right and left myocardial antioxidant responses during heart

failure subsequent to myocardial infarction. Circulation 1997 96:2414-20.

23. Li, Y., Jenkins, C.W. , Nichols, M.A. and Xiong, Y. (1994) Cell cycle expression

and p53 regulation of the cyclin-dependent kinase inhibitor p21. Oncogene,

9, 2261-2268

24. Steinman, R.A. , Hoffman, B. , Iro, A. , Guillouf, C. , Liebermann, D.A. and ElHouseini, M.E. (1994) Induction of p21 (WAF1/CIP1) during differentiation.

Oncogene, 9, 3389-3396.

25. Halevy, O. , Novitch, B.G. , Spicer, D.B. , Skapek, S.X. , Rhee, J. , Hannon, G.J. ,

Beach, D. and Lassar, A.B. (1995) Correlation of terminal cell cycle arrest

of skeletal muscle with induction of p21 by MyoD. Science, 267, 1018-

1021.

26. Andres, V. and Walsh, K. (1996) Myogenin expression, cell cycle withdrawal and

phenotypic differentiation are temporally separable events that precedes

cell fusion upon myogenesis. J. Cell Biol., 132, 657-666.

27. Tsurimoto, T. PCNA Binding Proteins. Frontiers in Bioscience, 4:849-858, 1999.

28. Levkau B, Koyama H, Raines EW, Clurman BE, Herren B, Orth K, Roberts JM,

Ross R. Cleavage of p21cip1/waf1 and p27 kip1 mediates apoptosis in

endothelial cells through activation of cdk2: role of a caspase cascade.

Mol Cell. 1998;1:553–563.

29. Adachi S, et al. Cyclin A/cdk2 activation is involved in hypoxia-induced apoptosis

in cardiomyocytes Circ Res. 88:408, 2001.
380 MONITORING STEM CELL RESEARCH

30. Anversa P, and Nadal-Ginard B. Myocyte renewal and ventricular remodelling

Nature 415, 240 - 243, 2002.

31. Kajstura J, Leri A, Finato N, di Loreto N, Beltramo CA, Anversa P. Myocyte

proliferation in end-stage cardiac failure in humans. Proc Natl Acad Sci

USA 95, 8801-8805 (1998).

32. Beltrami AP, et al. Evidence that human cardiac myocytes divide after myocardial

infarction. N Engl J Med 344, 1750-7 (2001).

33. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from

marrow stromal cells in vitro. J Clin Invest 1999, 103:697-705.

34. Tomita S, Li R-K, Weisel RD, et al. Autologous transplantation of bone marrow

cells improves damaged heart function. Circulation 1999; 100:II-247.

35. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human

mesenchymal stem cells. Science 1999, 284:143-147.

36. Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells

engraft and demonstrate site-specific differentiation after in utero

transplantation in sheep. Nat Med 2000, 6:1282-6.

37. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Livne E,

Binah O, Itskovitz-Eldor J, Gepstein L. Human embryonic stem cells can

differentiate into myocytes with structural and functional properties of

cardiomyocytes. J Clin Invest. 2001; 108: 407-14.

38. Klug MG, Soonpaa MH, Koh GY, Field LJ (1996) Genetically selected

cardiomyocytes from differentiating embryonic stem cells form stable

intracardiac grafts. J Clin Invest 98:216-224.

39. Hescheler J, Fleischmann BK, Wartenberg M, Bloch W, Kolossov E, Ji G, Addicks

K, Sauer H (1999) Establishment of ionic channels and signalling cascades

in the embryonic stem cell-derived primitive endoderm and cardiovascular

system. Cells Tissues Organs 165:153-164.

40. DA Taylor, BZ Atkins, P Hungspreugs, TR Jones, MC Reedy, KA Hutcheson, DD

Glower, WE. Regenerating functional myocardium: improved performance

after skeletal myoblast transplantation. Nature Medicine 1998, 4: 929-933.

41. L Field: Future therapy for cardiovascular disease. In Proceedings of the NHLBI

Workshop Cell Transplantation: Future Therapy for Cardiovascular

Disease? Columbia, MD; 1998.

42. CE Murry, RW Wiseman, SM Schwartz, SD Hauschka: Skeletal myoblast

transplantation for repair of myocardial necrosis. J Clin Invest 1996, 98:

2512-2523.

43. BZ Atkins, MT Hueman, JM Meuchel, MJ Cottman, KA Hutcheson, DA Taylor:

Myogenic cell transplantation improves in vivo regional performance in

infarcted rabbit myocardium. J Heart Lung Transpl 1999, 18: 1173-1180.

44. Suzuki K, Smolenski RT, Jayakumar J, Murtuza B, Brand NJ, Yacoub MH (2000)

Heat shock treatment enhances graft cell survival in skeletal myoblast

transplantation to the heart. Circulation 102:III216-221.
APPENDIX M. 381

45. Pouzet B, Ghostine S, Vilquin J-T, Garcin I, Scorsin M, Hagege AA, Duboc D,

Schwartz K, Menasche P (2001) Is skeletal myoblast transplantation

clinically relevant in the era of angiotensin-converting enzyme inhibitors?

Circulation 104:I223-228.

46. Menasche P, Hagege AA, Scorsin M, Pouzet B, Desnos M, Duboc D, Schwartz K,

Vilquin J-T, Marolleau J-P (2001) Myoblast transplantation for heart failure.

Lancet 347:279.

47. Beauchamp JR, Morgan JE, Pagel CN, Partridge TA (1999) Dynamics of myoblast

transplantation reveal a discrete minority of precursors with stem celllike properties as the myogenic source. J Cell Biol 144:1113-1122.

48. Smythe GM, Grounds MD (2000) Exposure to tissue culture conditions can

adversely affect myoblast behaviour in vivo in whole muscle grafts:

implications for myoblast transfer therapy. Cell Transplant 9:379-393.

49. Smythe GM, Hodgetts SI, Grounds MD (2001) Problems and solutions in myoblast

transfer therapy. J Cell Mol Med 5:33-47.

50. Hodgetts SI, Beilharz MW, Scalzo T, Grounds MD (2000) Why do cultured

transplanted myoblasts die in vivo? DNA quantification shows enhanced

survival of donor male myoblasts in host mice depleted of CD4+ and CD8+

or NK1.1+ cells. Cell Transplant 9:489-502.

51. Maier S, Tertilt C, Chambron N, Gerauer K, Huser N, Heidecke C-D, Pfeffer K

(2001) Inhibition of natural killer cells results in acceptance of cardiac

allografts in CD28-/- mice. Nature Med 7:557-562.

52. Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE (2001) Cardiomyocyte

grafting for cardiac repair: graft cell death and anti-death strategies. J

Mol Cell Cardiol 33:907-921.

53. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW, Entman ML,

Michael LH, Hirschi KK, Goodell MA (2001) Regeneration of ischemic

cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest

107:1395-1402.

54. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted

myocardium. Nature 2001, 410:701-705.

55. Tavian M, Coulombel L, Luton D, San Clemente H, Dieterlen-Lievre F, Peault B.

Aorta-associated CD34 hematopoietic cells in the early human embryo.

Blood 1996;87:67-72.

56. Jaffredo T, Gautier R, Eichmann A, Dieterlen-Lievre F. Intraaortic hemopoietic

cells are derived from endothelial cells during ontogeny. Development

1998;125:4575-4583.

57. Kennedy M, Firpo M, Choi K, Wall C, Robertson S, Kabrun N, Keller G. A common

precursor for primitive erythropoiesis and definitive haematopoiesis.

Nature 1997;386:488-493.

58. Choi K, Kennedy M, Kazarov A, Papadimitriou, Keller G. A common precursor

for hematopoietic and endothelial cells. Development 1998;125:725-732.
382 MONITORING STEM CELL RESEARCH

59. Elefanty AG, Robb L, Birner R, Begley CG. Hematopoietic-specific genes are not

induced during in vitro differentiation of scl-null embryonic stem cells.

Blood 1997;90:1435-1447.

60. Labastie M-C, Cortes F, Romeo P-H, Dulac C, Peault B. Molecular identity of

hematopoietic precursor cells emerging in the human embryo. Blood

1998;92:3624-3635.

61. Tsai FY, Keller G, Kuo FC, Weiss M, Chen J, Rosenblatt M, Alt FA, Orkin SH. An

early hematopoietic defect in mice lacking the transcription factor GATA2. Nature 1994;371:221-225.

62. Ogawa M, Kizumoto M, Nishikawa S, Fujimoto T, Kodama H, Nishikawa SI. Blood

1999;93:1168-1177.

63. Asahara, T. et al. Isolation of putative progenitor cells for endothelial

angiogenesis. Science 1997; 275:964-967.

64. Folkman, J. Therapeutic angiogenesis in ischemic limbs. Circulation 1998; 97:108-

110.

65. Takahashi, T. et al. Ischemia- and cytokine-induced mobilization of bone marrowderived endothelial progenitor cells for neovascularization. Nat. Med. 1999;

5:434-438.

66. Kalka, C. et al. Transplantation of ex vivo expanded endothelial progenitor cells

for therapeutic neovascularization. Proc. Natl. Acad. Sci. USA 2000;

97:3422-3427.

67. Rafii S, Shapiro F, Rimarachin J, Nachman R, Ferris B, Weksler B, Moore AS,

Asch AS. Isolation and characterization of human bone marrow

microvascular endothelial cells: hematopoietic progenitor cell adhesion.

Blood 1994;84:10-19.

68. Shi Q, Rafii S, Wu MH-D, et al. Evidence for circulating bone marrow-derived

endothelial cells. Blood 1998;92:362-367.

69. Lin Y, Weisdorf DJ, Solovey A, Hebbel RP. Origins of circulating endothelial cells

and endothelial outgrowth from blood. J Clin Invest 2000;105:71-77.

70. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma

S, Edwards NM, Itescu S. Neovascularization of ischemic myocardium by

human bone-marrow-derived angioblasts prevents cardiomyocyte

apoptosis, reduces remodeling and improves cardiac function. Nature Med.

2001; 7: 430-6.

71. Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, Inai Y, Silver M,

Isner JM. VEGF contributes to postnatal neovascularization by mobilizing

bone marrow-derived endothelial progenitor cells. EMBO J 1999; 18:3964-

3972.

72. Kocher A, Schuster M, Szabolcs M, Itescu S. Cardiomyocyte regeneration after

neovascularization of ischemic myocardium by human bone marrowderived angioblasts. Nature Medicine 2002, in press.
APPENDIX M. 383

73. McEwan PE, Gray GA, Sherry L, Webb DJ, Kenyon CJ. Differential effects of

angiotensin II on cardiac cell proliferation and intramyocardial perivascular

fibrosis in vivo. Circulation 1998;98:2765-2773.

74. Kawano H, Do YS, Kawano Y, Starnes V, Barr M, Law RE, Hsueh WA. Angiotensin

II has multiple profibrotic effects in human cardiac fibroblasts. Circulation

2000;101:1130-1137.

75. Pfeffer MA, Braunwald E, Moye LA, et al. Effect of captopril on mortality and

morbidity in patients with left ventricular dysfunction after myocardial

infarction. Results of the survival and ventricular enlargement trial. The

SAVE investigators. N Engl J Med 1992;327:669-677.

76. Pitt B, Segal R, Martinez FA, et al. Randomised trial of losartan versus captopril

in patients over 65 with heart failure (Evaluation of Losartan in the Elderly

Study, ELITE). Lancet 1997;349:747-752.

Appendix N.

The Biology of Nuclear Cloning and the Potential of

Embryonic Stem Cells for Transplantation Therapy

RUDOLF JAENISCH, M.D.

Whitehead Institute, 9 Cambridge Center, Cambridge, MA

SUMMARY

An emerging consensus is that somatic cell nuclear transfer

(SCNT) for the purpose of creating a child (also called “reproductive

cloning”) is not acceptable for both moral and scientific reasons. In

contrast, SCNT with the goal of generating an embryonic stem cell

line (“therapeutic cloning”) remains a controversial issue. Although

therapeutic cloning holds the promise of yielding new ways of

treating a number of degenerative diseases, it is not acceptable to

many because the derivation of an embryonic stem cell line from

the cloned embryo (an essential step in this process) necessarily

involves the loss of an embryo and hence the destruction of potential

human life.

In this article, I will develop two main arguments that are based

on the available scientific evidence. 1) In contrast to an embryo

derived by in vitro fertilization (IVF), a cloned embryo has little if

any potential to ever develop into a normal human being. This is

because, by circumventing the normal processes of gametogenesis

and fertilization, nuclear cloning prevents the proper reprogramming

of the clone’s genome, which is a prerequisite for development of

an embryo to a normal individual. It is unlikely that these biological

barriers to normal development can be solved in the foreseeable

future. Therefore, from a biologist’s point of view, the cloned human

embryo, used for the derivation of an embryonic stem cell and the

subsequent therapy of a needy patient, has little if any potential to

create a normal human life. 2) Embryonic stem cells developed from

a cloned embryo are functionally indistinguishable from those that

have been generated from embryos derived by in vitro fertilization

(IVF). Both types of embryonic stem cells have an identical potential

to serve as a source for therapeutically useful cells.

385
386 MONITORING STEM CELL RESEARCH

It is crucial that the ongoing debate on the possible therapeutic

application of SNCT is based on biological facts. The goal of this

article is to provide such a basis and to contribute to a more rational

discussion that is founded on scientific evidence rather than on

misconceptions or misrepresentations of the available scientific data.

I. Introduction

It is important to distinguish between “reproductive cloning” and

“nuclear transplantation therapy” (also referred to as “SCNT” or

“therapeutic cloning”). In reproductive cloning a cloned embryo is

generated by transfer of a somatic nucleus into an enucleated egg

with the goal to create a cloned individual. In contrast, the purpose

of nuclear transplantation therapy is to generate an embryonic stem

cell line (referred to as “ntES cells”) that is “tailored” to the needs

of a patient who served as the nuclear donor. The ntES cells could

be used as a source of functional cells that would be suitable for

treating an underlying disease by transplantation.

There is now experience from cloning of seven different

mammalian species that is relevant for three main questions of public

interest: 1) Would a cloned human embryo be “normal”? 2) Could

the problems currently seen with cloning be solved in the foreseeable

future? 3) Would ES cells derived from a cloned human embryo be

“normal” and useful for cell therapy? The arguments advanced in

this article are strictly based on molecular and biological evidence

that has been obtained largely in the mouse. I will not attempt to

review the cloning literature but only refer to selected papers on

cloned mice. The relevant literature on cloning of mammals can be

found in recent reviews (Byrne and Gurdon, 2002; Gurdon, 1999;

Hochedlinger and Jaenisch, 2002b; Oback and Wells, 2002; Rideout

et al., 2001; Wilmut, 2001; Young et al., 1998).

II. Most cloned animals die or are born with abnormalities

The majority of cloned mammals derived by nuclear transfer (NT)

die during gestation, and those that survive to birth frequently

display “Large Offspring Syndrome”, a neonatal phenotype

characterized by respiratory and metabolic abnormalities and

enlarged and dysfunctional placentas (Rideout et al., 2001; Young

et al., 1998). In order for a donor nucleus to support development

into a clone, it must be reprogrammed to a state compatible with
APPENDIX N. 387

embryonic development. The transferred nucleus must properly

activate genes important for early embryonic development and also

suppress differentiation-associated genes that had been transcribed

in the original donor cell. Inadequate “reprogramming”1

 of the donor

nucleus is most likely the principal reason for developmental failure

of clones. Since few clones survive to birth, the question remains

whether survivors are fully normal or merely the least affected

animals carrying through to adulthood despite harboring subtle

abnormalities that originate in faulty reprogramming but that are

not severe enough to interfere with survival to birth or beyond.

III. Reprogramming of the genome during normal development

and after nuclear transfer

The fundamental difference between nuclear cloning and normal

fertilization is that the nucleus used in nuclear cloning comes from

a somatic (body) cell that has not undergone the developmental

events required to produce the egg and sperm. Nuclear cloning

involves the transplantation of a somatic nucleus into the oocyte

from which the nucleus has been removed. However, the genes in

the somatic nucleus are not in the same state as those in the fertilized

egg because nuclear transplantation short-cuts the complex process

of egg and sperm maturation which involves extensive

“reprogramming” of the genome, a process that shuts some genes

off and leaves others on. Reprogramming during gametogenesis

prepares the genome of the two mature gametes with the ability to

activate faithfully the genetic program that ensures normal

embryonic development when they combine at fertilization (Fig 1a).

This reprogramming of the genome begins at gastrulation, when

primordial germ cells (PGCs) are formed, and continues during

differentiation into mature gametes resulting, in a radically different

chromatin configuration of sperm and oocyte (Rideout et al., 2001).

Experiments have shown that uniparental embryos (embryos

whose genomes are derived solely from either the maternal or

paternal parent) do not develop normally. Uniparental embryos first

seem normal; they direct cleavage (early development to the

blastocyst stage) despite profound differences in their epigenetic

organization (Reik et al., 2001). However, uniparental embryos fail

soon after the implantation of the embryo into the wall of the uterus,

indicating that both parental genomes are needed and functionally

complement each other beginning at this later step of

embryogenesis. Presumably, the different epigenetic organization

of the two genomes is crucial for achieving normal development.
388 MONITORING STEM CELL RESEARCH

Moreover, it has been well established that the imbalance of

imprinted 2

 gene expression represents an important cause of

embryonic failure.

In order for cloned embryos to complete development, genes

normally expressed during embryogenesis but silent in the somatic

donor cell, must be reactivated. This complex process of epigenetic3

remodeling (i.e., the reconfiguration of the genome by turning on

and turning off specific genes) that occurs during gametogenesis in

normal development ensures that the genome of the zygote can

faithfully activate early embryonic gene expression (Fig 1a). In a

cloned embryo, reprogramming, which in normal gametogenesis

requires months to years to complete, must occur in a cellular context

radically different from gametogenesis and within the short interval

(probably within hours) between transfer of the donor nucleus into

the egg and the time when zygotic transcription becomes necessary

for further development. Given these radically different conditions,

one can envisage a spectrum of different outcomes to the

reprogramming process ranging from (i) no reprogramming of the

genome, resulting in immediate death of the NT embryo; through

(ii) partial reprogramming, allowing initial survival of the clones,

but resulting in an abnormal phenotype and/or lethality at various

stages of development; to (iii) faithful reprogramming producing fully

normal animals (Fig 1b). The phenotypes observed over the past

five years in cloned embryos and newborns suggest that complete

reprogramming is the exception, if it occurs at all.

IV. Development of clones depends on the differentiation-state

of the donor nucleus

The majority of cloned embryos fail at an early step of embryonic

development, soon after implantation in the wall of the uterus, an

early step of embryonic development (Hochedlinger and Jaenisch,

2002b; Rideout et al., 2001). Those that live to birth often display

common abnormalities irrespective of the donor cell type (Table 1).

In addition to symptoms referred to as “Large Offspring Syndrome”,

neonate clones often suffer from respiratory distress and kidney,

liver, heart or brain defects (Cibelli et al., 2002). However, the

abnormalities characteristic of cloned animals are not inherited by

their offspring (Tamashiro et al., 2002), indicating that epigenetic

aberrations (i.e., failure of genome reprogramming) rather than

genetic aberrations (changes in the sequences within the DNA) are

the cause.
APPENDIX N. 389

The efficiency of creating cloned animals is strongly influenced

by the differentiation-state of the donor nucleus (Table 1). In the

mouse, for example, only 1-3% of cloned blastocysts derived from

somatic donor nuclei, e.g., those prepared from fibroblasts or

cumulus cells, will develop to adult cloned animals (Hochedlinger

and Jaenisch, 2002b). In certain cases, such as those using

terminally differentiated B or T cell donor nuclei, the efficiency of

cloning is so low as to preclude the direct derivation of cloned

animals. In stark contrast to these examples, cloning using donor

nuclei prepared from embryonic stem (ES) cells is significantly more

efficient (between 15 and 30 %, Table 1). This correlation with

differentiation-state suggests that embryonic nuclei require less

reprogramming of their genome, ostensibly because the genes

essential for embryonic development are already active and need

not be reprogrammed. In fact, the nucleus of an embryonic cell such

as an ES cell may well have the same high efficiency to generate

postnatal mice after nuclear transfer as the nucleus prepared from

a recently fertilized egg (Table 1, compare Fig. 4). Nonetheless,

most if not all mice that have been cloned from ES cell donor nuclei,

in contrast to mice derived through natural fertilization from the

zygote, are abnormal, indicating that the processes of

gametogenesis (development of sperm and of egg) and fertilization

endows the zygote nucleus with the ability to direct normal

development. In summary, these data indicate that the potential of

a nucleus to generate a normal embryo is lost progressively with

development.

V. Adult cloned animals: how normal are they?

The observation that apparently healthy adult cloned animals have

been produced in seven mammalian species (albeit at low efficiency)

is being used by some as a justification for attempting to clone

humans. In fact, even those that survive to adulthood, such as Dolly,

may succumb relatively early in adulthood because of numerous

health problems. Insights into the mechanisms responsible for clone

failure before and after birth have come from molecular and biological

analyses of mouse clones that have reached (i) the blastocyst stage,

(ii) the perinatal period and (iii) adulthood.

(i) Most clones fall short of activating key embryonic genes and fail

early.

As stated above in order for clones to develop, the genes that are

normally expressed during embryogenesis, but are silent in the

somatic donor cell, must be reactivated (Hochedlinger and Jaenisch,
390 MONITORING STEM CELL RESEARCH

2002b; Rideout et al., 2001). It is the failure to activate key

“embryonic” genes that are required for early development that

leads to the demise of most clones just after implantation. Recently,

a set of about 70 key embryonic genes termed “Oct-4 like” genes

have been identified that are active in early embryos but not in

somatic donor cells. Importantly, the failure to faithfully activate this

set of genes can be correlated with the frequent death of cloned

animals during the immediate post-implantation period (Bortvin et

al., 2003). These results define “faulty reprogramming” as the cause

of early demise of cloned embryos as the failure to reactivate key

embryonic genes that are silent in the donor cell.

(ii) Newborn clones misexpress hundreds of genes.

Clones that survive to birth suffer from serious problems, many of

which appear to be due to an abnormal placenta. The most common

phenotypes observed in animals cloned from either somatic or ES

cell nuclei are fetal growth abnormalities such as increased placental

and birth weight. This has suggested that surviving clones had

accurately reprogrammed the “Oct-4 like” genes that are essential

for the earliest stages of development, i.e. those immediately

following implantation of the embryo into the uterus. The abnormal

phenotype of those clones that do survive through these early stages

and develop to birth indicates that other genes that are important

for later stages of development but are not essential for early survival

are not correctly reprogrammed. To assess the extent of abnormal

expression of various genes in the cells of clones, global gene

expression has been assessed by microarray analysis of RNA

prepared from the placentas and livers of neonatal cloned mice, i.e.,

clones that survived development and were viable at birth; these

clones had been derived by nuclear transfer (NT) of nuclei prepared

either from cultured ES cells or from freshly isolated cumulus cells

(somatic cells that surround the egg) (Humpherys et al., 2002). Direct

comparison of gene expression profiles of over 10,000 genes (of the

30,000 or so in the mammalian genome) showed that for both classes

of cloned neonatal mice, approximately 4% of the expressed genes

in their placentas differed dramatically in expression levels from

those in controls, and that the majority of abnormally expressed

genes were common to both types of clones. When imprinted genes,

a class of genes that express only one allele (either from maternal

or paternal origin), were analyzed, between 30 and 50% were not

correctly activated. These data represent strong molecular evidence

that cloned animals, even those that survive to birth, suffer from

serious gene expression abnormalities.
APPENDIX N. 391

(iii) Cloned animals develop serious problems with age

The generation of adult and seemingly healthy adult cloned animals

has been taken as evidence that normal cloned animals can be

generated by nuclear transfer, albeit with low efficiency. Indeed, a

routine physical and clinical laboratory examination of 24 cloned

cows of 1 to 4 years of age failed to reveal major abnormalities (Lanza

et al., 2001). Cloned mice of a corresponding age as that of the cloned

cows (2 - 6 months in mice vs. 1 - 4 years in cows) also appear

“normal” by superficial inspection. However, when cloned mice were

aged, serious problems, not apparent at younger ages, became

manifest. One study found that the great majority of cloned mice

died significantly earlier than normal mice, succumbing with immune

deficiency and serious pathological alterations in multiple organs

(Ogonuki et al., 2002). Another study found that aged cloned mice

became overweight with major metabolic disturbances (Tamashiro

et al., 2002). Thus, serious abnormalities in cloned animals may often

become manifest only when the animals age.

Firm evidence about aging and “normalcy” of cloned farm

animals is incomplete or anecdotal because cloned animals of these

species are still comparatively young (relative to their respective

normal life span). For example, the premature death of Dolly (Giles

and Knight, 2003) is entirely consistent with serious abnormalities

in cloned sheep that become manifest only at later ages. Also, two

of the analyzed cloned cows developed disease soon after the study

on “healthy and normal cattle” (Lanza et al., 2001) had appeared:

one animal developed an ovarian tumor and another one suffered

brain seizures (J. Cibelli, pers. comm.). While it cannot be ruled out

that these are “spontaneous” maladies unconnected with the

cloning procedure, a more likely alternative is that these problems

were direct consequences of the nuclear transfer procedure.

(iv) Are there any “normal” clones?

It is a key question in the public debate whether it is ever possible

to produce a normal individual by nuclear cloning, even if only with

low efficiency. The available evidence suggests that it may be

difficult if not impossible to produce normal clones for the following

reasons: 1) As summarized above, all analyzed clones at birth

showed dysregulation of hundreds of genes. The development of

clones to birth and beyond despite widespread epigenetic

abnormalities suggests that mammalian development can tolerate

dysregulation of many genes. 2) Some clones survive to adulthood

by compensating for gene dysregulation. Though this

“compensation” assures survival, it may not prevent maladies to
392 MONITORING STEM CELL RESEARCH

become manifest at later ages. Therefore, most if not all clones are

expected to have at least subtle abnormalities that may not be so

severe as to result in an obvious phenotype at birth but will cause

serious problems later as seen in aged mice. Clones may just differ

in the extent of abnormal gene expression: if the key “Oct-4 like”

genes are not activated, clones die immediately after implantation.

If those genes are activated, the clone may survive to birth and

beyond.

As schematically shown in Fig. 2, the two stages when the

majority of clones fail are immediately after implantation and at birth.

These are two critical stages of development that may be particularly

vulnerable to faulty gene expression. Once cloned newborns have

progressed through the critical perinatal period, various

compensatory mechanisms may counterbalance abnormal

expression of other genes that are not essential for the subsequent

postnatal survival. However, the stochastic occurrence of disease

and other defects at later age in many or most adult clones implies

that such compensatory mechanisms do not guarantee “normalcy”

of cloned animals. Rather, the phenotypes of surviving cloned

animals may be distributed over a wide spectrum from abnormalities

causing sudden demise at later postnatal age or more subtle

abnormalities allowing survival to advanced age (Fig. 2). These

considerations illustrate the complexity of defining subtle gene

expression defects and emphasize the need for more sophisticated

test criteria such as environmental stress or behavior tests. However,

the available evidence suggests that truly normal clones may be

the exception.

It should be emphasized that “abnormality” or “normalcy” is

defined here by molecular and biological criteria that distinguish

cloned embryos or animals from control animals produced by sexual

reproduction. The most informative data for the arguments presented

above come from the mouse. There is, however, every reason to

believe that these difficulties associated with producing mice and a

variety of other mammalian embryos by nuclear transplantation will

also afflict the process of human reproductive cloning (Jaenisch and

Wilmut, 2001).

(v) Is it possible to overcome the problems inherent in reproductive

cloning?

It is often argued that the “technical” problems in producing normal

cloned mammals will be solved by scientific progress that will be

made in the foreseeable future. The following considerations argue

that this may not be so.
APPENDIX N. 393

A principal biological barrier that prevents clones from being

normal is the “epigenetic” difference (such as distinct patterns of

DNA methylation4

) between the chromosomes inherited from mother

and from father, i.e. the difference between the “maternal” and the

“paternal” genome of an individual. Such methylation of specific

DNA sequences is known to be responsible for shutting down the

expression of nearby genes. Parent-specific methylation marks are

responsible for the expression of imprinted genes and cause only

one copy of an imprinted gene, derived either from sperm or egg, to

be active while the other allele is inactive (Ferguson-Smith and

Surani, 2001). When sperm and oocyte genomes are combined at

fertilization, the parent-specific marks established during oogenesis

and spermatogenesis persist in the genome of the zygote (Fig 3A).

Of interest for this discussion is that within hours after fertilization,

most of the global methylation marks (with the exception of those

on imprinted genes) are stripped from the sperm genome whereas

the genome of the oocyte is resistant to this active demethylation

process (Mayer et al., 2000). This is because the oocyte genome is

in a different “oocyte-appropriate” epigenetic state than the sperm

genome. The oocyte genome becomes only partially demethylated

within the next few days by a passive demethylation process. The

result of these post-fertilization changes is that the two parental

genomes are epigenetically different (as defined by the patterns of

DNA methylation) in the later stage embryo and remain so in the

adult in imprinted as well as non-imprinted sequences.

In cloning, the epigenetic differences that are established during

gametogenesis may be erased because both parental genomes of

the somatic donor cell are introduced into the egg from the outside

and are thus exposed equally to the demethylation activity present

in the egg cytoplasm (Fig 3B). This predicts that imprinted genes

should be particularly vulnerable to inappropriate methylation and

associated dysregulation in cloned animals. The results summarized

earlier are consistent with this prediction. For cloning to be made

safe, the two parental genomes of a somatic donor cell would need

to be physically separated and separately treated in an “oocyteappropriate” and a “sperm-appropriate” way, respectively. At

present, it seems that this is the only rational approach to guarantee

the creation of the epigenetic differences that are normally

established during gametogenesis. Such an approach is beyond our

present abilities. These considerations imply that serious biological

barriers exist that interfere with faithful reprogramming after nuclear

transfer. It is a safe conclusion that these biological barriers represent

a major stumbling block to efforts aimed at making nuclear cloning

a safe reproductive procedure for the foreseeable future.
394 MONITORING STEM CELL RESEARCH

It has been argued that the problems in mammalian cloning are

similar to those encountered with IVF 30 years ago: Thus, following

this argument, the methods of culture and embryo manipulations

just would need to be improved to develop reproductive cloning

into a safe reproductive technology that is as acceptable as IVF.

This argument appears to be fundamentally flawed. It is certainly

correct that merely “technical” problems needed to be solved to

make IVF efficient and safe. It is important to distinguish between

the perfection of technical skills to imitate a biological event and

the development of wholly new science to overcome the blocks to

events that have severe biological restrictions. Nuclear cloning faces

serious biological barriers that cannot be addressed by mere

adjustments in experimental technique. Indeed, since the birth of

Dolly no progress has been made in solving any of the underlying

biological issues of faulty gene reprogramming and resulting

defective development.

VI. Therapeutic applications of SCNT

(i) Reproductive cloning vs. therapeutic cloning

In spite of the biological and ethical barriers associated with

reproductive cloning, nuclear transfer technology has significant

therapeutic potential that is within our grasp. There is an enormous

distinction between the goals and the end product of these two

technologies. The purpose of reproductive cloning is to generate a

cloned embryo that is then implanted in the uterus of a female to

give rise to a cloned individual. In contrast, the purpose of nuclear

transplantation therapy is to generate an embryonic stem cell line

that is derived from a patient (referred to as “ntES cells”) and can

be used subsequently for tissue replacement.

Many scientists recognize the potential of NtES cells for organ

transplantation (for recent review see (Hochedlinger and Jaenisch,

2003). This procedure is currently complicated by immune rejection

due to immunological incompatibility. Thus, virtually all organ

transplants undertaken at present involve the use of donor organs

that are recognized as foreign by the immune systems of the recipient

and thus are targeted for destruction by these immune systems. To

treat this “host versus graft” disease, immunosuppressive drugs

are routinely given to transplant recipients in order to suppress this

organ rejection. Such immunosuppressive treatment has serious side

effects including increased risks of infections and malignancies. In

principle, ES cells can be created from a patient’s nuclei using

nuclear transfer. Because ntES cells will be genetically identical to
APPENDIX N. 395

the patient’s cells, the risks of immune rejection and the requirement

for immunosuppression are eliminated. Moreover, ES cells provide

a renewable source of replacement tissue allowing for repeated

therapy whenever needed. Finally, if ES cells are derived from a

patient carrying a known genetic defect, the mutation in question

can be corrected in the ntES cells using standard gene targeting

methods before introducing these ES cells (or derived tissue-specific

stem cells) back into the patient’s body.

(ii) Combining nuclear cloning with gene and cell therapy

In a “proof of principle” experiment, nuclear cloning in combination

with gene and cell therapy has been used to treat a mouse genetic

disorder that has a human counterpart (Figure 4). To do so, the wellcharacterized Rag2 mutant mouse was used as “patient” (Rideout

et al., 2002). This mutation causes severe combined immune

deficiency (SCID), because the enzyme that catalyzes immune

receptor rearrangements in lymphocytes is non-functional.

Consequently, these mice are devoid of mature B and T cells, a

disease resembling human Omenn syndrome (Rideout et al., 2002).

In a first step, somatic (fibroblast) donor cells were isolated from

the tails of Rag2-deficient mice and their nuclei were injected into

enucleated eggs. The resultant embryos were cultured to the

blastocyst stage and isogenic ES cells were isolated. Subsequently,

one of the mutant Rag2 alleles was targeted by homologous

recombination in ES cells to restore normal Rag2 gene structure and

function. In order to obtain somatic cells for treatment, these

genetically repaired ES cells were differentiated into embryoid

bodies and further into hematopoietic precursors by expressing

HoxB4, a transcription factor that is responsible for programming

the behavior of the hematopoietic stem cells, i.e., those cells that

are able to generate the full range of red and white cells in the blood.

Resulting hematopoietic precursors were transplanted into

irradiated Rag2-deficient animals in order to treat the disease caused

by their Rag2 mutation. Initial attempts to engraft these cells were,

however, unsuccessful because of an increased level of natural killer

(NK) cells in the Rag mutant host. ES cell derived hematopoietic

cells express low levels of the MHC antigens and thus are a preferred

target for NK mediated destruction. Elimination of NK cells by

antibody depletion or genetic ablation allowed the ntES cells to

efficiently populate the myeloid and to a lesser degree the lymphoid

lineages of these mice. Functional B and T cells that had undergone

proper rearrangements of their immunoglobulin and T cell receptor

alleles as well as serum immunoglobulins were detected in the

transplanted mutants. Hence, important cellular components of the
396 MONITORING STEM CELL RESEARCH

immune system were restored in mice that previously were unable

to produce these cells.

This experiment demonstrated that embryonic stem (ES) cells

derived by NT from somatic cells of a genetically afflicted individual

can be combined with gene therapy to treat the underlying genetic

disorder. Because Rag2 deficiency causes an increase in NK activity

and necessitated the elimination of NK cells prior to transplantation

in the above-described experiments, some have concluded that “The

experiment failed to show success with therapeutic cloning”

(Coalition and Ethics, 2003) and that “This indicates that the only

successful therapy using cloned embryos would be through

‘reproductive’ cloning, to produce born clones who can serve as

tissue donors for patients” (Prentice, 2002). This is a troubling

misinterpretation of the data. (i) It has been shown that ES cellderived hematopoietic cells can successfully engraft and rescue

lethally irradiated mice indicating that increased NK activity is a

peculiarity of Rag2-deficiency (Kyba et al., 2002). Therefore, it would

seem that for most diseases, no anti-NK treatment would be required

to assure engraftment of ES cell-derived somatic cells. (ii) It is correct

that treatment of a human patient with Omenn syndrome, which is

equivalent to Rag2 deficiency, by SCNT may also require anti-NK

treatment to transiently reduce NK activity. This would allow the

transplanted cells to engraft as in the mouse experiment. Once

these cells are successfully engrafted, there is every reason to

believe that such anti-NK treatment would no longer be necessary.

In conclusion, the mouse experiment indicates that, unlike the

situation with reproductive cloning, no biological barriers exist that

in principle prevent the use of SCNT to treat human diseases. The

technical issues in using SCNT and human stem cells for therapeutic

purposes need, however, to be solved, but there are no indications

at present that these represent formidable problems that will resist

relatively rapid solution.

VII. Faulty reprogramming after nuclear transfer: does it interfere

with the therapeutic potential of ES cells derived by SCNT?

As summarized above, most if not all cloned animals are abnormal

because of faulty reprogramming after nuclear transfer. Does this

epigenetic dysregulation affect the potential of ntES cells to generate

functional somatic cells that can be used for cell therapy? To address

this question, I will first compare the in vivo development of embryos

with the in vitro process of ES cell derivation from explanted
APPENDIX N. 397

embryos. This will be followed by discussing the epigenetic state

of the ES cell genome. Finally, I will contrast the phenotype of cloned

mice derived from ES cell donor nuclei with that of chimeric mice

generated by injection of ES cells into blastocysts.

(i) The phenotype of an embryo is determined by its donor nucleus

As mentioned repeatedly above, embryos can be derived from the

fertilized egg or from a somatic nucleus by SCNT. The potential of

the resulting blastocyst, when implanted into the womb, to develop

into a fetus and a postnatal animal depends strictly on the nature of

the donor nucleus (Fig 5): (i) When derived from the zygote, most

embryos develop to birth and generate a normal animal; (ii) Similarly,

most blastocysts cloned from an embryonic stem cell donor nucleus

develop to birth but, in contrast to the normally fertilized embryo,

the great majority of the cloned animals will be abnormal (“Large

offspring syndrome”) (Eggan et al., 2001; Humpherys et al., 2001);

(iii) The great majority of cloned blastocysts derived from somatic

donor nuclei such as fibroblasts or cumulus cells will die soon after

implantation and only a few clones will survive to birth and these

too will be abnormal, suffering once again from the Large offspring

syndrome (Wakayama and Yanagimachi, 2001); (iv) Finally, the

likelihood of cloned blastocysts derived from another type of somatic

donor nuclei - those present in terminally differentiated lymphoid

cells - to generate a cloned animal is extremely low and has not

been achieved except by using a two step procedure involving the

intermediate generation of embryonic stem cells (Hochedlinger and

Jaenisch, 2002a). These observations suggest that a blastocyst

retains an “epigenetic memory” of its donor nucleus. This memory

determines its potential for fetal development: while a fertilized

embryo develops normally, any embryo derived by SCNT will be

abnormal though the efficiency of a given clone to develop to birth

is strongly influenced by the differentiation state of the donor cell

(see Table 1). In other words, the cloned embryo after implantation

into the womb will be abnormal because the cloned blastocyst

retained an epigenetic memory of its donor nucleus and this causes

faulty fetal development. This epigenetic memory is erased when a

blastocyst, either derived by nuclear cloning or from the fertilized

egg, is explanted into tissue culture and grown into an embryonic

stem cell. Erasure of the epigenetic memory has major consequences

for the “normalcy” of embryonic stem cells.

(ii) The derivation of embryonic stem cells is a highly selective process

that erases the “epigenetic memory” of the donor nucleus

Embryonic stem cells, regardless of whether they have been

generated from a fertilized egg or by SCNT, are derived from the
398 MONITORING STEM CELL RESEARCH

cells of a blastocyst that have been explanted and propagated in

tissue culture. Of the blastocyst cells that are explanted in this way,

those that derive from the portion of the blastocyst termed the inner

cell mass (ICM) initially express “key” embryonic genes such as

Oct-4. However, soon after explantation, most ICM cells extinguish

Oct-4 expression and cease proliferating (Buehr et al., 2003). Only

one or a few of the ICM-derived cells will eventually re-express Oct4 and these few Oct-4-positive cells are those that resume rapid

proliferation, yielding the cell populations that we designate as

“embryonic stem” cells. These cells represent a cell population that

has no equivalent in the normal embryo and may be considered a

tissue culture artifact, though a useful one (Fig. 6).

The important point for this discussion is that the propagation of

blastocyst cells in vitro results in a rare population of surviving cells

that have erased the “epigenetic memory” of the donor nucleus.

This process results ultimately in ES cells that have, regardless of

donor nuclear origin, an identical developmental potential. In other

words, ES cells derived from embryos produced by normal

fertilization and those produced from cloned embryos are

functionally indistinguishable (Hochedlinger and Jaenisch, 2002b;

Rideout et al., 2002; Wakayama et al., 2001). Because the ES cells

that derive from normally fertilized embryos are able to participate

in the generation of all normal embryonic tissues, we can conclude

that the ES cells derived from cloned embryos have a similar

potential to generate the full range of normal tissues.

(iii) ES cells, epigenetic instability and therapeutic potential

Epigenetic instability appears to be a consistent characteristic of

ES cells. This was shown when individual ES cells were analyzed

for expression of imprinted genes: even cells in a recently subcloned

ES cell line differed strongly in the expression of genes such as H19

or Igf2. The variable expression was correlated with the DNA

methylation status of the genes, which switched from an

unmethylated to a methylated state between sister cells (Humpherys

et al., 2001). This was a surprising result in view of the known

potential of ES cells to generate terminally differentiated cells that

function normally after transplantation into an animal. Possible

explanations include (i) that epigenetic instability in ES cells is a

consequence of propagation of cells in tissue culture or (ii) that

epigenetic instability is a prerequisite for cells to be pluripotent,

i.e., this instability may be a manifestation of a plasticity in the gene

expression program that is required to enable the ES cells to generate

a wide variety of differentiated cell lineages.
APPENDIX N. 399

Whatever the explanation for the observed epigenetic instability

of ES cells may be, it supports the view that the process of generating

ES cells erases all epigenetic memory of the donor nucleus and, as

a consequence of the selection process, generates epigenetic

instability in the selected cells. In other words, epigenetic instability

appears to be an intrinsic characteristic of ES cells regardless of

whether derived by SCNT or from a fertilized egg. This is consistent

with the conclusion that both types of ES cells have an equivalent

potency to generate functional cells in culture and, in the longer

term, fully normal differentiated tissues upon implantation of these

cells in vivo.

(iv) ES cells form normal chimeras but abnormal nuclear clones

As outlined above, faulty reprogramming leads to abnormal

phenotypes of cloned mice derived from ES cell donor nuclei. Why

is faulty reprogramming and epigenetic instability a problem for

reproductive cloning but not for therapeutic applications? The main

reason for this seeming paradox is that, in contrast to reproductive

cloning, the therapeutic application of NT does not require the

formation of a fetus. Therapeutic applications involve the ability of

cloned ES cells to form a single tissue or organ, not to recapitulate

all of fetal development. For example, normal fetal development

requires faithful expression of the imprinted genes. As outlined

above, nuclear cloning causes between 30% and 50% of imprinted

genes to be dysregulated consistent with the notion that disturbed

imprinting is a major contributing factor to clone failure. As most

imprinted genes have no known function in the postnatal animal,

the dysregulation of imprinting would not be expected to impede

functionality of in vitro differentiated ES cells because this process

does not require the formation of a fetus. Therefore, the functionality

of mature cells derived in culture from ES cells would not depend

on the faithful reprogramming of the imprinted genes. Dysregulation

of some imprinted genes such as Igf2 are known, however, to cause

disease in the adult. Thus, it will be important to test whether

dysregulation of such genes has adverse effects on the function of

somatic cells derived from ES cells.

When injected into a blastocyst, ES cells form normal chimeras.

It appears that the presence of surrounding “normal” cells, i.e. cells

that are derived from a fertilized embryo, prevents an abnormal

phenotype of the chimera such as the “Large Offspring Syndrome”

that is typical for cloned animals. Any therapeutic application

creates, of course, a chimeric tissue where cells derived from ntES

cells are introduced into a diseased adult individual and interact

with surrounding “normal” host cells. Therefore, no phenotypic
400 MONITORING STEM CELL RESEARCH

abnormalities, such as those seen in cloned animals, would be

expected in patients transplanted with cells derived from ntES cells.

VIII. SCNT for cell therapy: destruction of potential human life?

A key concern raised against the application of the nuclear

transplantation technology for tissue therapy in humans is the

argument that the procedure involves the destruction of potential

human life. From a biological point of view, life begins with

fertilization when the two gametes are combined to generate a new

embryo that has a unique combination of genes and has a high

potential to develop into a normal baby when implanted into the

womb. A critical question for the public debate on SCNT is this one:

is the cloned embryo equivalent to the fertilized embryo?

In cloning, the genetic contribution is derived from one individual

and not from two. Obviously, the cloned embryo is the product of

laboratory-assisted technology, not the product of a natural event.

From a biological point of view, nuclear cloning does not constitute

the creation of new life, rather the propagation of existing life

because no meiosis, genetic exchange and conception are involved.

Perhaps more important is, however, the overwhelming evidence

obtained from the cloning of seven different mammalian species.

As summarized above, the small fraction of cloned animals that

survive beyond birth, even if they appear “normal” upon superficial

inspection, are likely not so. The important conclusion is that a cloned

human embryo would have little if any potential to develop into a

normal human being. With other words, the cloned human embryo

lacks essential attributes that characterize the beginning of normal

human life.

Taking into account the potency of fertilized and cloned embryos,

the following scenarios regarding their possible fates can be

envisaged (Fig. 7). Fertilized embryos that are “left over” from IVF

have three potential fates: disposal, generation of normal embryonic

stem cells or generation of a normal baby when implanted into the

womb. Similarly, the cloned embryo has three potential fates: it can

be destroyed or could be used to generate a normal ntES cell line

that has the same potential for therapy as an ES cell derived from a

fertilized embryo. In contrast to the fertilized embryo, the cloned

embryo has little if any potential to ever generate a normal baby.

An embryonic stem cell line derived by nuclear may, however, help

sustain existing life when used as a source for cell therapy that is

“tailored” to the need of the patient who served as its nuclear donor.
APPENDIX N. 401

If SCNT were accepted as a valid therapeutic option, a major

concern of its implementation as medical procedure would be the

problem of how to obtain sufficient numbers of human eggs that

could be used as recipients. Commercial interests may pressure

women into an unwanted role as egg donors. The recent

demonstration that embryonic stem cells can be coaxed into a

differentiation pathway that yields oocyte-like cells (Hubner et al.,

2003) may offer a solution to this dilemma. If indeed functional

oocytes could be generated from a generic human ES cell line,

sufficient eggs could be generated in culture and serve as recipients

for nuclear transfer without the need of a human egg donor. It seems

that technical issues, not fundamental biological barriers, need to

be overcome so that transplantation therapy can be carried out

without the use of human oocytes.
402 MONITORING STEM CELL RESEARCH

Fig 1: Reprogramming in normal development and nuclear

cloning.

a. The genome of primordial germ cells (PGCs) is

hypomethylated (“reset”, white boxes). Reprogramming and

establishment of parent specific epigenetic marks occurs over the

course of gametogenesis so that the genome of sperm and egg are

competent to express the genes that need to be activated in early

embryonic (box with wavy lines) and later (hatched box)

development. During cleavage and early postimplantation

development “embryonic” genes, such as Oct 3/4, become activated

(black box) and are repressed at later stages (stippled boxes) when

tissue specific genes (hatched boxes) are activated in adult tissues

(A, B, C). Epigenetic reprogramming of imprinted and non-imprinted

genes occurs during gametogenesis in contrast to X inactivation

and the readjustment of telomere length which take place

postzygotically.

b. Reprogramming of a somatic nucleus following nuclear

transfer may result in (i) no activation of “embryonic” genes and

early lethality, (ii) faulty activation of embryonic genes and an

abnormal phenotype, or (iii) in faithful activation of “embryonic”

and “adult” genes and normal development of the clone. The latter

outcome is the exception if it occurs at all.
APPENDIX N. 403

Fig. 2: The phenotypes are distributed over a wide range of

abnormalities. Most clones fail at two defined developmental

stages, implantation and birth. More subtle gene expression

abnormalities result in disease and death at later ages.
404 MONITORING STEM CELL RESEARCH

Fig 3: Parental epigenetic differences in normal and cloned

animals

A: The genomes of oocyte and sperm are differentially methylated

during gametogenesis and are different in the zygote when

combined at fertilization. Immediately after fertilization the paternal

genome (derived from the sperm) is actively demethylated whereas

the maternal genome is only partially demethylated during the next

few days of cleavage. This is because the oocyte genome is in a

different chromatin configuration and is resistant to the active

demethylation process imposed on the sperm genome by the egg

cytoplasm. Thus, the methylation of two parental genomes is

different at the end of cleavage and in the adult. Methylated

sequences are depicted as filled lollipops and unmethylated

sequences as empty lollipops.
APPENDIX N. 405

3B: In cloning a somatic nucleus is transferred into the enucleated

egg and both parental genomes are exposed to the active

demethylating activity of the egg cytoplasm. Therefore, the parent

specific epigenetic differences are equalized.
406 MONITORING STEM CELL RESEARCH

Fig. 4: Scheme for therapeutic cloning combined with gene and

cell therapy.

A piece of tail from a mouse homozygous for the recombination

activating gene 2 (Rag2) mutation was removed and cultured. After

fibroblast-like cells grew out, they were used as donors for nuclear

transfer by direct injection into enucleated MII oocytes using a

Piezoelectric driven micromanipulator. Embryonic stem (ES) cells

isolated from the NT-derived blastocysts were genetically repaired

by homologous recombination. After repair, the ntES cells were

differentiated in vitro into embryoid bodies (EBs), infected with the

HoxB4iGFP retrovirus, expanded, and injected into the tail vein of

irradiated, Rag2-deficient mice (after (Rideout et al., 2002)).
APPENDIX N. 407

Fig. 5: Blastocysts retain epigenetic memory of donor nucleus

Blastocysts can be derived from the fertilized egg or by nuclear

transfer. After implantation development of the embryo strictly

depends on the donor nucleus: Blastocysts derived from a fertilized

egg will develop with high efficiency to normal animals; blastocysts

derived by NT from an ES cell donor will develop with high efficiency

to abnormal animals; blastocysts derived by NT from a fibroblast or

cumulus cell donor will develop with low efficiency to abnormal

animals; blastocysts derived by NT from B or T donor cells will not

develop to newborns by direct transfer into the womb (only by a 2

step procedure, compare (Hochedlinger and Jaenisch, 2002a).
408 MONITORING STEM CELL RESEARCH

Fig. 6: The establishment of ES cells from blastocysts erases

epigenetic memory of donor nucleus

Most cells of the inner cell mass turn off Oct-4 like genes and die

after explantation of blastocysts into tissue culture. Only one or a

few cells turn on the Oct-4 like genes and proliferate. The surviving

cells will give rise to ES cells. During this highly selective outgrowth

of the surviving cells all epigenetic memory of the donor nucleus is

erased. Therefore, regardless of donor nucleus (fertilized egg or

somatic nucleus in cloned embryos), all ES cells have an equivalent

potency to generate functional differentiated cells.
APPENDIX N. 409

Fig. 7: Normal and cloned embryos have three possible fates

Embryos derived by IVF (“left over embryos”) have three fates: they

can be disposed, create normal babies if implanted or can generate

ES cells if explanted into tissue culture. Cloned embryos have also

three fates: they can be disposed, can generate abnormal babies if

any when implanted or can generate ES cells when explanted. The

ES cells derived from an IVF embryo or a cloned embryo are

indistinguishable (same potency, see figure 6)
410 MONITORING STEM CELL RESEARCH

Table 1

Development of normal embryos and embryos cloned from ES cell

and somatic donor nuclei. Note that normal and ES cell derived

blastocysts have a similar potency to develop to term if calculated

from the fraction of transplanted blastocysts.

1: (Eggan et al., 2001; Eggan et al., 2002; Rideout et al., 2000); 2

(Wakayama et al., 1998; Wakayama and Yanagimachi, 1999); 3

(Hochedlinger and Jaenisch, 2002a).

Acknowledgements

I thank my colleagues Bob Weinberg, Gerry Fink, George Daley and

Andy Chess for critical and constructive comments on this

manuscript.

ENDNOTES

1 Reprogramming: The genome of a somatic cell is in an epigenetic state that is

appropriate for the respective tissue and assures the expression of the tissue specific

genes (in mammary gland cells, for example, those genes important for mammary

gland function such as milk production). In cloning, the somatic nucleus must

activate those genes that are needed for embryonic development but which are

silent in the donor cell in order for the cloned embryo to survive. The egg cytoplasm

contains “reprogramming factors” that can convert the epigenetic state (see endnote

Donor nucleus Mice

(% of blastocysts) Phenotype Ref.

Fertilized zygote 30 - 50 % Normal

Nuclear transfer

from

ES cell 15 - 30 %

Most if not

all clones

are abnormal

1

Cumulus cell,

fibroblast 1 - 3 % 2

B, T cell < 1/3000 3
APPENDIX N. 411

3) characteristic of the somatic donor nucleus to one that is appropriate for an

embryonic cell. This process is very inefficient leading to inappropriate expression

of many genes and causes most clones to fail early.

2 Imprinted genes: For most genes, both copies, the one inherited from father and

the one inherited from mother, are expressed. In contrast, only one of the two copies

of an imprinted gene, either the maternal one or the paternal one, is active. The two

copies are distinguished by methylation marks (see endnote 4) that are imposed on

imprinted genes either during oogenesis (maternally imprinted genes) or during

spermatogenesis (paternally imprinted genes). Thus, the two copies of imprinted

genes are epigenetically different in the zygote and remain so in all somatic cells.

These epigenetic marks distinguish the two copies and cause only one copy to be

expressed whereas the other copy remains silent. It is estimated that between 100

and 200 genes (of the total of 30,000 genes) are imprinted. Disturbances of normal

imprinted gene expression lead to growth abnormalities during fetal life and can

be the cause of major diseases such as Beckwith-Wiedeman or Prader-Willi

syndrome.

3 Epigenetic changes: Cells of a multicellular organism are genetically identical but

express, depending on the particular cell type, different sets of genes (“tissue

specific genes”). These differences in gene expression arise during development

and must be retained through subsequent cell divisions. Stable alterations of this

kind are said to be “epigenetic”, as they are heritable in the short term (during cell

divisions) but do not involve mutations of the DNA itself.

4 DNA methylation: Reversible modification of DNA (methylation of the base cytosine)

that affects the “readability” of genes: usually, methylated genes are silent and

unmethylated genes are expressed. DNA methylation represents an important

determinant of the “epigenetic state” of genes and affects the state of the chromatin:

methylated regions of the genome are in a “silent” state and unmethylated regions

are in an “open” configuration that causes genes to be active.

References

Bortvin, A., Eggan, K., Skaletsky, H., Akutsu, H., Berry, D. L., Yanagimachi, R.,

Page, D. C., and Jaenisch, R. (2003). Incomplete reactivation of Oct4-related genes

in mouse embryos cloned from somatic nuclei. Development 130, 1673-1680.

Buehr, M., Nichols, J., Stenhouse, F., Mountford, P., Greenhalgh, C. J.,

Kantachuvesiri, S., Brooker, G., Mullins, J., and Smith, A. G. (2003). Rapid loss of

oct-4 and pluripotency in cultured rodent blastocysts and derivative cell lines. Biol

Reprod 68, 222-229.

Byrne, J. A., and Gurdon, J. B. (2002). Commentary on human cloning.

Differentiation 69, 154-157.

Cibelli, J. B., Campbell, K. H., Seidel, G. E., West, M. D., and Lanza, R. P. (2002).

The health profile of cloned animals. Nat Biotechnol 20, 13-14.

Coalition, and Ethics, A. f. R. (2003). Do no harm - Reality check: proof of

“therapeutic cloning”? wwwstemcellresearchorg/pr/pr_2003-03-10htm.
412 MONITORING STEM CELL RESEARCH

Eggan, K., Akutsu, H., Loring, J., Jackson-Grusby, L., Klemm, M., Rideout, W.

M., 3rd, Yanagimachi, R., and Jaenisch, R. (2001). Hybrid vigor, fetal overgrowth,

and viability of mice derived by nuclear cloning and tetraploid embryo

complementation. Proc Natl Acad Sci U S A 98, 6209-6214.

Eggan, K., Rode, A., Jentsch, I., Samuel, C., Hennek, T., Tintrup, H., Zevnik, B.,

Erwin, J., Loring, J., Jackson-Grusby, L., et al. (2002). Male and female mice derived

from the same embryonic stem cell clone by tetraploid embryo complementation.

Nat Biotechnol 20, 455-459.

Ferguson-Smith, A. C., and Surani, M. A. (2001). Imprinting and the epigenetic

asymmetry between parental genomes. Science 293, 1086-1089.

Giles, J., and Knight, J. (2003). Dolly’s death leaves researchers woolly on clone

ageing issue. Nature 421, 776.

Gurdon, J. B. (1999). Genetic reprogramming following nuclear transplantation

in Amphibia. Semin Cell Dev Biol 10, p239-243.

Hochedlinger, K., and Jaenisch, R. (2002a). Monoclonal mice generated by nuclear

transfer from mature B and T donor cells. Nature 415, 1035-1038.

Hochedlinger, K., and Jaenisch, R. (2002b). Nuclear transplantation: Lessons

from frogs and mice. Curr Opin Cell Biol 14, 741-748.

Hochedlinger, K., and Jaenisch, R. (2003). Nuclear transplantation, embryonic

stem cells, and the potential for cell therapy. New England Journal of Medicine 349,

in press (July 17, 2003).

Hubner, K., Fuhrmann, G., Christenson, L. K., Kehler, J., Reinbold, R., De La

Fuente, R., Wood, J., Strauss, I. J., Boiani, M., and Scholer, H. R. (2003). Derivation of

Oocytes from Mouse Embryonic Stem Cells. Science.

Humpherys, D., Eggan, K., Akutsu, H., Friedman, A., Hochedlinger, K.,

Yanagimachi, R., Lander, E., Golub, T. R., and Jaenisch, R. (2002). Abnormal gene

expression in cloned mice derived from ES cell and cumulus cell nuclei. Proc Natl

Acad Sci U S A 99, 12889-12894.

Humpherys, D., Eggan, K., Akutsu, H., Hochedlinger, K., Rideout, W.,

Biniszkiewicz, D., Yanagimachi, R., and Jaenisch, R. (2001). Epigenetic instability

in ES cells and cloned mice. Science 293, 95-97.

Jaenisch, R., and Wilmut, I. (2001). Developmental biology. Don’t clone humans!

Science 291, 2552.
APPENDIX N. 413

Kyba, M., Perlingeiro, R. C., and Daley, G. Q. (2002). HoxB4 confers definitive

lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac

hematopoietic progenitors. Cell 109, 29-37.

Lanza, R. P., Cibelli, J. B., Faber, D., Sweeney, R. W., Henderson, B., Nevala, W.,

West, M. D., and Wettstein, P. J. (2001). Cloned cattle can be healthy and normal.

Science 294, 1893-1894.

Mayer, W., Niveleau, A., Walter, J., Fundele, R., and Haaf, T. (2000). Demethylation

of the zygotic paternal genome. Nature 403, 501-502.

Oback, B., and Wells, D. (2002). Donor cells for cloning-many are called but few

are chosen. Cloning Stem Cells 4, 147-168.

Ogonuki, N., Inoue, K., Yamamoto, Y., Noguchi, Y., Tanemura, K., Suzuki, O.,

Nakayama, H., Doi, K., Ohtomo, Y., Satoh, M., et al. (2002). Early death of mice cloned

from somatic cells. Nat Genet 30, 253-254.

Prentice, D. (2002). Why the “Successful” Mouse “Therapeutic” Cloning Really

Didn’t work. wwwcloninginformationorg/info/unsuccessful_mouse_therapyhtm.

Reik, W., Dean, W., and Walter, J. (2001). Epigenetic reprogramming in mammalian

development. Science 293, 1089-1093.

Rideout, W. M., 3rd, Hochedlinger, K., Kyba, M., Daley, G. Q., and Jaenisch, R.

(2002). Correction of a genetic defect by nuclear transplantation and combined cell

and gene therapy. Cell 109, 17-27.

Rideout, W. M., Eggan, K., and Jaenisch, R. (2001). Nuclear cloning and epigenetic

reprogramming of the genome. Science 293, 1093-1098.

Rideout, W. M., Wakayama, T., Wutz, A., Eggan, K., Jackson-Grusby, L., Dausman,

J., Yanagimachi, R., and Jaenisch, R. (2000). Generation of mice from wild-type and

targeted ES cells by nuclear cloning. Nat Genet 24, 109-110.

Tamashiro, K. L., Wakayama, T., Akutsu, H., Yamazaki, Y., Lachey, J. L., Wortman,

M. D., Seeley, R. J., D’Alessio, D. A., Woods, S. C., Yanagimachi, R., and Sakai, R. R.

(2002). Cloned mice have an obese phenotype not transmitted to their offspring.

Nat Med 8, 262-267.

Wakayama, T., Tabar, V., Rodriguez, I., Perry, A. C., Studer, L., and Mombaerts, P.

(2001). Differentiation of embryonic stem cell lines generated from adult somatic

cells by nuclear transfer. Science 292, 740-743.
414 MONITORING STEM CELL RESEARCH

Wakayama, T., Whittingham, D. G., and Yanagimachi, R. (1998). Production of

normal offspring from mouse oocytes injected with spermatozoa cryopreserved with

or without cryoprotection. J Reprod Fertil 112, 11-17.

Wakayama, T., and Yanagimachi, R. (1999). Cloning of male mice from adult tailtip cells. Nat Genet 22, 127-128.

Wakayama, T., and Yanagimachi, R. (2001). Mouse cloning with nucleus donor

cells of different age and type. Mol Reprod Dev 58, 376-383.

Wilmut, I. (2001). How safe is cloning? Cloning 3, 39-40.

Young, L. E., Sinclair, K. D., and Wilmut, I. (1998). Large offspring syndrome in

cattle and sheep. Rev Reprod 3, p155-163.