[This working paper has been prepared by the staff of the President's
Council on Bioethics solely for the purpose of stimulating discussion at
the Council\'s January 2002 meeting.]{.smallcaps}

[Working Paper \# 2]{.underline}

[Scientific Aspects of Human and Animal Cloning]{.underline}

# Introduction

The following brief review of some scientific aspects of human and
animal cloning is based on scientific research published through the end
of December 2001. However, the scientific fields involved in cloning are
being very actively investigated, and significant new developments are
being published frequently. Publication of new results could change some
of the interpretations and emphases in this review.

Use of unfamiliar technical terms and jargon has been avoided wherever
possible. Scientific names and terms whose use was required are
described and defined in the Glossary of Terms below. In cases where
there is disagreement about definitions, this has been noted.

# Some Basic Facts about Human Cell Biology and Sexual Reproduction

These elementary facts about human cells, germ cells (egg and sperm),
and early embryogenesis will provide the background for understanding
the mechanism of cloning and the differences between sexual and asexual
reproduction.

Adult human cells with nuclei contain 46 chromosomes, 22 pairs plus two
X chromosomes if the adult is female, or 22 pairs plus one X and one Y
chromosome if the adult is male. These chromosomes contain the bulk of
the cell's DNA, and therefore the genes of the cell. During formation of
sperm cells, specialized cell division (meiosis) produces mature sperm
cells containing 23 chromosomes (22 chromosomes plus either X or Y).
During fertilization of eggs (or oocytes) by sperm, a pronucleus
containing half the egg chromosomes is ejected from the cell. Fusion of
egg and sperm cells and their nuclei (the defining acts of all sexual
reproduction) produces a zygote that again contains a nucleus with the
adult cell complement of 46 chromosomes, half from each parent \[See
Figure 1\]. The zygote then begins the gradual process of cell division,
growth, and differentiation. After several days in an appropriate
nutritive environment, the zygote attains the 100-200-cell (blastocyst)
stage. In normal reproduction, the blastocyst implants into the uterine
wall, where, suitably nourished, it continues the process of coordinated
cell, tissue, and organic differentiation that eventually produce the
organized, articulated, and integrated whole that is the new-born
infant.

Not quite all the genetic material of a cell resides in its nucleus.
Both egg and sperm cells contain small, energy-producing organelles
called mitochondria. Mitochondria contain a small piece of DNA that
contains the genetic information for making several essential
mitochondrial proteins. When additional mitochondria are produced in the
cell, the mitochondrial DNA is replicated, and a copy is passed along to
the new mitochondria that are formed. During fertilization, sperm
mitochondria are selectively tagged and subsequently degraded inside the
cell. Thus, the developing embryo inherits solely or principally
mitochondria (and mitochondrial DNA) from the egg.

Human reproduction, in cases hampered by one or another cause of
infertility, has been accomplished with the help of in vitro
fertilization of egg by sperm (IVF) and the subsequent transfer of the
early embryo to a woman for gestation and birth. Though such union of
egg and sperm requires laboratory assistance and takes place outside of
the body, IVF reproduction is still sexual in the biological sense: the
new human being arises from two biological parents through the union of
egg and sperm.

Egg and sperm cells combined in vitro have also been used to start the
processes of animal embryonic development. Implantation of the resulting
blastocysts into the uterus of a female of the appropriate animal
species is widely used in animal husbandry.

# "Reproductive" Cloning (Asexual Reproduction) of Mammals

The startling 1997 announcement that "Dolly" the sheep had been produced
by "reproductive" cloning (Wilmut et al, 1997) indicated that it was
possible to produce live mammalian offspring via asexual reproduction
through cloning with adult donor cell nuclei.[^1]

In outline form, the steps used to produce live offspring in the animal
species that have been cloned to date are (see Figure 1):

1.  Obtain an egg cell from a female of the animal species.

2.  Remove the nuclear DNA from the egg cell to produce an enucleated
    egg.

3.  Insert the nucleus of a donor adult cell into the enucleated egg to
    produce a reconstructed egg.

4.  Activate the reconstructed egg with chemicals or electric current in
    vitro to stimulate the reconstructed egg to commence cell division.

5.  Initiate development of the activated, reconstructed egg (zygote) to
    a suitable stage of early embryonic development in vitro, then
    transfer the embryo to the uterus of a female host that has been
    suitably prepared to receive it.

6.  A cloned animal is born that is genetically virtually identical to
    the animal that

> donated the nucleus. However, the mitochondria and mitochondrial DNA
> of such
>
> a cloned animal would most likely be derived from those of the egg.

"Reproductive" cloning carries with it several possibilities not
available through sexual reproduction. Because the number of presumably
identical donor cells is very large, a very large number of genetically
virtually identical individuals could be produced by this process,
limited only by the supply of eggs and female animals that could bear
the young. In principle, any animal, male or female, newborn or adult,
could be cloned, and in any quantity. Because mammalian cells can be
frozen and stored for prolonged periods at low temperature and grown
again for use as donor cells in cloning, one may even clone individuals
who have died. In theory, a clone could be cloned again, on and on,
without limit.

Since the report by Wilmut et al. (1997), attempts have been made to
clone several other mammalian species. As described in more detail
below, live offspring have been produced in a low percentage of cloning
attempts with sheep, cattle, goats, mice and pigs. According to a recent
press report (Kolata, 2001, see Appendix A), attempts to clone rabbits,
rats, cats, dogs and primates using adult cell DNA have not yet yielded
live offspring. While a variety of health problems have been reported in
some cloned animals, surviving cloned cattle appear physiologically
similar to their uncloned counterparts, and two cloned cows have given
birth to their own offspring (Lanza et al., 2001). Several aspects of
the data from the five species that have been cloned are compared and
summarized in Table 1.

# Human "Reproductive" Cloning

At this time it is unknown whether any experiments involving human
cloning for reproductive purposes have been carried out. Although
statements have appeared in the press that this was underway (Weiss,
2001), no published scientific report of any such experiments has been
made as of the end of December 2001. However, the steps in such an
experiment would probably be similar to those described for animal
"reproductive" cloning \[see References to Table 1\]. A recently
reported first attempt at human "research" cloning has followed the
first four steps (Cibelli, et al, 2001, see Appendix B).

# Human "Research" Cloning

In vitro production of human embryos through cloning could also be used
by scientists interested in studying the early embryonic forms of human
life, including those produced by cloning. Cloning would make available
embryos that are genetically virtually identical; this would greatly
facilitate comparison of results between different experiments. Specific
genes could be introduced into developing cloned embryos to study the
role(s) of these genes on early human development. Research on cloned
embryos is also being considered as a source of stem cells potentially
useful in developing treatments for degenerative diseases.

One such attempt at human "research" cloning has been published in the
scientific literature (Cibelli, et al., 2001) as of the end of December
2001. It involved the following steps (see Figure 1):

1.  Obtaining human eggs from informed and consenting female volunteers.

2.  Removing the nuclear DNA from the egg cell to produce an enucleated
    egg.

3.  Inserting the nucleus of a cell from an informed and consenting
    adult donor into the enucleated egg produce a reconstructed egg.

4.  Activating the reconstructed egg with chemicals to stimulate the
    reconstructed egg to commence cell division in vitro.

5.  Using a microscope to follow the early cell divisions of the
    reconstructed egg.

6.  In this experiment, 3 of 19 reconstructed embryos underwent cell
    division, but none progressed beyond the six-cell stage.

> In the "research" cloning experiments described by Cibelli et al
> (2001) the stated

intent was to create embryos that would progress to the 100-200 cell
(blastocyst) stage, at which point the embryo would be taken apart, stem
cells from the inner cell mass would be isolated, and an attempt would
be made to grow and preserve "individualized" human stem cells (see
Figure 2) for the possible future medical benefit of the donor (NRC/IOM
Report). Since the embryos stopped dividing at the six-cell stage, no
stem cells were isolated in these experiments. Although the steps
Cibelli et al (2001) followed up to the blastocyst stage were the same
as those that would be used by those attempting human "reproductive"
cloning, Cibelli et al (2001) distinguished their intent from
"reproductive" cloning by stating:

> Strict guidelines for the conduct of this research have been
> established by Advanced Cell Technology's independent Ethics Advisory
> Board (EAB). In order to prevent any possibility of reproductive
> cloning, the EAB requires careful accounting of all eggs and embryos
> used in the research. No embryo created by means of NT \[nuclear
> transfer\] technology may be maintained beyond 14 days of development.

[Parthenogenesis]{.underline}

It is also possible, using chemical or electrical stimuli, to stimulate
human eggs to undergo several rounds of cell division, as if they had
been fertilized (see Figure 1). In this case, the egg retains all 46 egg
cell chromosomes and egg cell mitochondria. In some animal species this
asexual reproduction process, known as parthenogenesis, has produced
live offspring that contain the same nuclear DNA as the egg. These
offspring are all necessarily female.

Cibelli et al (2001) activated eggs obtained from informed and
consenting human donors by parthenogenesis, and obtained multiple cell
divisions up to the early blastocyst stage in 6 out of 22 attempts.
Although there was no report that stem cells were isolated in these
experiments, it is possible that parthenogenesis of human eggs could
induce them to develop to a stage where embryonic stem cells could be
isolated.

# Epigenetic Modification and Reprogramming

During differentiation of embryonic cells to produce the cells of the
adult, specific chromosomal DNA segments are selectively repressed
through processes called epigenetic modification (see Glossary). In
adult cells, genes whose expression is required only during early
embryonic development are shut down. During the formation of egg and
sperm cells, "epigenetic reprogramming" of genes whose products will be
needed during early embryonic development is required to make these
genes active once more. In cloning using donor nuclei from adult cells,
a similar "epigenetic reprogramming" is required.

Based on the data on "reproductive" cloning experiments in animals in
Table 1 and the results of Cibelli et al (2001), most cloned embryos die
either in vitro or after transfer to the uterus. Why is production of
live cloned mammalian offspring a relatively rare event? Several factors
may play a role. Enucleation of the egg may (variably from one attempt
to the next) remove or damage its "epigenetic reprogramming"
capabilities. An optimal in vitro nutritive environment for the
development of cloned zygotes may not yet have been determined. One
interpretation (Rideout et al, 2001) attributes the early death of many
cloned embryos to complete failure or incompleteness of "epigenetic
reprogramming".

At this early stage of experimental work with cloned human embryos, it
is perhaps not surprising that 16 of 19 of the reconstructed eggs did
not undergo cell division and none of the other three reconstructed eggs
divided beyond the six-cell stage (Cibelli et al, 2001). A more detailed
discussion of epigenetic modification and reprogramming as they relate
to cloning will be developed as a component of a broader analysis of
"research" cloning.

\* \* \*

REFERENCES

Cibelli, J.B. et al., "Somatic Cell Nuclear Transfer in Humans:
Pronuclear and Early Embryonic Development", e-biomed: The Journal of
Regenerative Medicine, 2: 25-31 (2001) \[see Appendix B\]

Kolata, G. "In Cloning, Failure Far Exceeds Success", New York Times,
December 11, 2001, page D1 \[see Appendix A\]

Lanza, R.P. et al., "Cloned Cattle Can Be Healthy and Normal", Science,
294: 1893-4 (2001)

NRC/IOM Report - "Stem Cells and the Future of Regenerative Medicine"

Rideout III, W.M. et al., "Nuclear Cloning and Epigenetic Reprogramming
of the Genome", Science, 293, 1093-1098 (2001)

Weiss, R. "Human cloning bid stirs experts' anger; problems in animal
cases noted", Washington Post, April 11, 2001, page A1

Wilmut, I. et al., "Viable offspring derived from fetal and adult
mammalian cells" Nature, 385: 810-813 (1997)

# GLOSSARY OF TERMS

*In writing Working Papers 1-4 for the January 2002 Council meeting,
members of the Council staff have attempted to use terms consistently as
they are defined below in this glossary.*

### Asexual reproduction

The term "asexual reproduction" means reproduction not initiated by the
union of oocyte and sperm.

Blastocyst

> An early stage in the development of mammalian embryos, when the
> embryo is a spherical body comprising an inner cell mass that will
> become the fetus and an outer ring of cells that will become part of
> the placenta.

Biologically related children

> Children are "biologically related" to the individuals who are the
> sources of their genetic endowment, in the case of sexual
> reproduction, to one man and one woman who are the sources of sperm
> and egg (the "biological" parents), in the case of cloning, to the
> source of the donor nucleus.

Cloning

> *"Reproductive" cloning* -- 1) Obtaining a human or animal egg cell
> and removing its DNA. 2) Inserting a nucleus or a cell from a donor
> human or animal in order to produce a reconstructed egg that is
> genetically very similar to the donor. 3) Implanting the reconstructed
> egg in a uterus and delivering the resulting baby or newborn animal.
>
> *"Research" cloning* -- 1) Obtaining a human or animal egg cell and
> removing its DNA. 2) Inserting a nucleus or a cell from a donor human
> or animal to produce a reconstructed egg that is genetically very
> similar to the donor. 3a) In some cases, termed "therapeutic" cloning
> or "cell replacement through nuclear transfer" (CRNT), the next steps
> include growing the reconstructed egg to the 100-200 cell embryo
> (blastocyst) stage, then taking the embryo apart to isolate and
> preserve "individualized" stem cells for immediate or future use in
> cell transplantation therapies. 3b) In other cases, the next steps
> comprise various scientific experiments designed to better understand
> the earliest stages of human embryonic development.
>
> *Gene (molecular) cloning* -- Using carrier pieces of DNA (called
> vectors) to isolate and characterize DNA segments coding for proteins.
>
> *"Human cloning"* -- The term 'human cloning' means the asexual
> reproduction of a new human organism that is genetically virtually
> identical to an already existing, or previously existing, human being.
> Operationally, it is currently accomplished by introducing the nuclear
> material of a human somatic cell into an oocyte whose own nuclear
> material has been removed or inactivated, to produce a living organism
> -- at whatever stage of development -- that has a human (or
> predominantly human) genetic constitution.

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 cell
division. Chromosomes thus transmit the genes of the organism from one
generation to the next.

Embryo

1.  An organism in the early stages of development.

2.  In humans, the developing organism from conception until
    approximately the end of the second month; developing stages from
    this time to birth are commonly designated as fetal.

Epigenetic modification

> Turning genes encoded by chromosomal DNA on and off during cell
> differentiation through changes in a) DNA methylation, b) the assembly
> of histone proteins into nucleosomes, and c) remodeling of
> chromosome-associated proteins such as linker histones.

Epigenetic reprogramming

> The process of removing epigenetic modifications of chromosomal DNA,
> so that genes whose expression was turned off during embryonic
> development and cell differentiation become active again. Epigenetic
> reprogramming of the donor cell DNA is believed to be an essential
> process in generating live offspring through "reproductive" cloning
> using adult donor cells and nuclei.

Eugenics

Activity seeking to alter (with the aim of improving) the genetic
constitution of future generations.

Infertility

The inability to conceive a child through sexual intercourse.

Mitochondria

> Small energy-producing organelles inside of cells. Mitochondria give
> rise to other mitochondria by copying their small piece of
> mitochondrial DNA and passing one copy of the DNA along to each of the
> two resulting mitochondria.

Nuclear transfer

> Transferring the nucleus with its chromosomal DNA from one (donor)
> cell to another (recipient) cell. In cloning, the recipient is a human
> egg cell and the donor cell can be any one of a number of different
> adult tissue cells.

Oocyte

As used here, the term oocyte is synonymous with egg.

Parthenogenesis

> A form of nonsexual reproduction...in which the female reproduces its
> kind without fecundation by the male.

Parthenogenote

An embryo that has been produced by parthenogenesis.

Somatic cell (human)

> The term "somatic cell" means a diploid cell (having a complete set of
> 46 chromosomes) obtained or derived from a living or deceased human
> body at any stage of development.

Stem cells

> "The definition of 'stem cells' is still a debated issue (Morrison et
> al., 1997; Watt and Hogan, 2000). According to the currently
> prevailing view (Watt and Hogan, 2000) a stem cell can be defined on
> the basis of the following two features: (1) it has an unlimited or
> prolonged self-renewal capacity (i.e. the capability to maintain a
> pool of undifferentiated stem cells besides giving rise to
> differentiated daughter cells); (2) it has uni/multipotency, i.e. the
> potential to produce one or more differentiated descendent cell types.
> ....
>
> Uncertainty that can derive from the terminological ambivalence of the
> term 'stem cell' is increased by the misuse of this term, which is
> often inappropriately employed (or perhaps voluntarily 'manipulated')
> in the popular press to sustain scientific and/or ethical positions
> for political ends." (Guena, S., et al, 2001)

In this paper, we use the Watt and Hogan (2000) definition given above.

Zygote

The diploid cell that results from the fusion of a sperm cell and an egg
cell.

## GLOSSARY REFERENCES

Guena, S., et al., "Adult Stem Cells and Neurogenesis: Historical Roots
and State of the Art"

The Anatomical Record (New Anat.), 265: 132-141 (2001)

Morrison, S.J., et al., "Regulatory mechanisms in stem cell biology"
Cell, 88: 287-298 (1997)

Watt F.M. and Hogan B.L.M. "Out of Eden: Stem cells and their niches"
Science, 287: 1441-1446 (2000)

[^1]: Previous experiments dating from the 1950s had shown that it was
    possible to clone amphibians. Earlier experiments had also produced
    clones of animals using embryonic donor cells. What made the report
    of Dolly's birth stand out was the use of adult donor cells and the
    fact that a mammal had been cloned.