IMPROVEMENTS ON
HUMANIZED NOD/SCID IL-2rg-/- MICE

LI, YAN(B.Sc., WUHAN UNIVERSITY)

A THESIS SUBMITTEDFOR THE DEGREE OF DOCTOR OF PHILOSOPHY
IN COMPUTATIONAL AND SYSTEM BIOLOGY(CSB)
SINGAPORE MIT ALLIANCE
NATIONAL UNIVERSITY OF SINGAPORE
2012

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In#Loving#Memory#of#my#Father#Acknowledgement
I would not have the courage to start my Ph.D if I knew the difficulties and themisfortunes I would confront in the past five years. It is impossible for me toaccomplish my thesis without the people who helped me in my research andsupported me in my tough times. Hence, I take this opportunity to express mygratitude to these people.
When I was graduating from Wuhan University, I received offers from Ph.D
programs abroad and companies in China. It was my father, who supported
my decision to embark on the route of academia. Throughout my Ph.D, helistened to my confusions, motivated me to challenge myself in research
topics, and encouraged me to pursue my happiness. We were both proud ofeach other and treat our relationship not just as father and son, rather the bestfriends. I was hoping he could witness the completion of my Ph.D, but hepassed away on March 8, 2011 in an unforeseen hemorrhage of theintracranial aneurysm. My deepest gratitude goes to my father.
My thesis work was carried out in Prof. Jianzhu Chen’s laboratory at the Koch
Institute at MIT, Cambridge, USA and at Infectious Disease Group, Singapore
MIT Alliance for Research and Technology, Singapore. I am extremely
grateful for his patience in my first three years when my progress was not up
to his expectation. The training from him turns me from a fearless newbie to acompetent researcher.

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Many thanks go to Prof. Bing Lim in Genome Institute of Singapore, who
agreed to support my work in Prof. Chen’s Lab and offered me many valuablesuggestions throughout my Ph.D.
My special gratitude goes to Prof. Harvey Lodish, who served as chairman of
my thesis committee and reviewed my thesis every year. Prof. Harvey Lodish
genuinely helps the growth of the young scientists. I greatly benefit from his
criticism of my midterm research proposal and will keep the high standard I
learned from him.
My knowledge of my thesis project began with the interaction with Adam
Drake in MIT, who helped me analyze the drawbacks of the humanized miceand teach me the basic lab techniques. After I started my own project toimprove the humanized mice, he will never miss a chance to ask me criticalquestions during my presentation at lab meetings. I also like to thank Eileen,Mimi, and Ilya in MIT for their helpful suggestions. When I came back to
Singapore, the laboratory of Infectious Disease Group is still underconstruction. Farzad, Lanhiong, Elias and Rashidi spent effort to set up the lab
and help me start experiments. Then Qingfeng, Dahai and Maroun joined theSingapore lab. They offered me the most valuable suggestions and helped me
overcome challenges when I was about to give up. I witness their growth frompostdoc to junior faculties, and I wish success in their academic career.
Many thanks also to George, Carol, Zhuyan, Hooi Linn, Salim, Gino, Nicola
in Chen lab, Siew Chin, Maggie, Li Liang, Yok Hian, Gino and all others in
Singapore MIT Alliance for Research and Technology for their endlesssupport and friendship.
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My deepest gratitude goes to my family (my mom, dad and my relatives) andfriends in China and abroad (Song Xu, Liu Tao, Yang Pengbo, Zhu Shiwen,
Wang Junjie, Yin Lu, Liu Yachao, Fang Fang, Yinzi, Shi Yuan, Zhai Boxuan,
Xu Shuoyu, Zhang Kuan, Porkodi, Dong Di, and Ke Wei) for their neverending faith in me and sharing my tears and joys in the past five years.
Without you guys, I would have never been able to accomplish this.

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Table of Contents
Declaration
Acknowledgement .......................................................................................... i
Table of Contents ......................................................................................... iv
Summary ..................................................................................................... viii
List of Tables ................................................................................................ xi
List of Figures .............................................................................................. xii
List of Abbreviations ................................................................................. xiv

CHAPTER 1
INTRODUCTION
1.1 Introduction to the humanized mice ......................................................... 2
1.1.1 The development of humanized mice .......................................................... 3
1.1.2 Remaining issues of humanized NSG mice ................................................. 6

1.2 Focus and objectives of this thesis .......................................................... 13

CHAPTER 2
MATERIALS AND METHODS
2.1 Stem cell purification .............................................................................. 16iv

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2.2 Feeder cell-free culture of cord blood CD34+CD133+ cells ................... 17
2.3 Angplt5-expressing MSCs ...................................................................... 17
2.4 Co-culture of cord blood CD34+CD133+ cells ....................................... 18
2.5 Mice and intracardiac injection ............................................................... 19
2.6 Serial reconstitution assays ..................................................................... 19
2.7 Single cell preparation ............................................................................ 19
2.8 Flow cytometry and cell sorting ............................................................. 20
2.9 Immunization and ELISPOT assay ......................................................... 21
2.10 Hydrodynamic injections ...................................................................... 22
2.11 In vitro differentiation assay and colony-forming unit assay ............... 22
2.12 RNA extraction and real-time PCR ...................................................... 22
2.13 Histology and Giemsa staining ............................................................. 23
2.14 LPS challenge, influenza virus A infection and Bacillus
Calmette-Guérin infection ............................................................................ 24
2.15 Statistical analysis ................................................................................. 25

CHAPTER 3
IN VITRO EXPANSION OF HSC FOR HUMANIZED MICERECONSTITUTION
3.1 Introduction ............................................................................................. 27
3.1.1 The demand for expansion of HSCs for clinical and preclinicalapplications.......................................................................................................... 27
3.1.2 Methods for HSC expansion ...................................................................... 28v

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3.1.3 Expansion of HSC in angptl5 expressing MSC co-culture ........................ 29

3.2 Results ..................................................................................................... 31
3.2.1 Combination of co-culture and selected growth factors enhance
expansion of cord blood CD34+CD133+ cells. .................................................... 31
3.2.2 Evaluation of contributions of feeder cells and soluble factors to theenhanced expansion of cord blood CD34+CD133+ cells..................................... 35
3.2.3 Expanded CD34+CD133+ cells give rise to multiple lineages ofblood cells in NSG mice...................................................................................... 36
3.2.4 Expanded cells are capable of stable long-term and efficientsecondary reconstitution ...................................................................................... 41
3.2.5 Human T cells are functional in the reconstituted mice ............................. 43

3.3 Discussion ............................................................................................... 45

CHAPTER 4
HUMAN M-CSF MOBILIZES PROMONOCYTES IN BM
AND RESTORES THE FUNCTIONS OF HUMAN TISSUE
MACROPHAGES IN HUMANIZED MICE
4.1 Introduction ............................................................................................. 49
4.1.1 The problem of human monocyte/macrophage development in NSG
mice ..................................................................................................................... 49
4.1.2 The role of human monocytes/macrophages in immune responses ........... 49
4.1.3 Cytokines and monocyte/macrophage development .................................. 51vi

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4.1.4 Past efforts to improve monocyte/macrophages in humanized mice ......... 52
4.1.5 Improvements of human macrophage reconstitution and immune
function in humanized mice ................................................................................ 53

4.2 Results ..................................................................................................... 55
4.2.1 Human monocyte and macrophage development is blocked in BMof humanized NSG mice ..................................................................................... 55
4.2.2 Human myeloid differentiation stops at monoblast to promonocyte
stage in the BM of humanized mice .................................................................... 57
4.2.3 Human monocyte/macrophage development is restored by cytokine
treatment .............................................................................................................. 60
4.2.4 Human tissue resident macrophages are generated in M-CSF treatedmice ..................................................................................................................... 65
4.2.5 Human inflammatory responses are enhanced in M-CSF treatedmice ..................................................................................................................... 67
4.2.6 M-CSF treated mice mount stronger immune response against
Influenza A virus ................................................................................................. 70
4.2.7 Human peritoneal macrophages are recruited during
Mycobacterium infection in M-CSF treated mice............................................... 75

4.3 Discussion .............................................................................................. .78

CHAPTER 5
EFFORTS TO IMPROVE ANTIGEN-SPECIFIC ANTIBODYRESPONSES IN HUMANIZED MICE
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5.1 Expression of human CD40 ligand on human T cells to promote
the maturation and proliferation of antigen experienced B cells. ................. 85
5.2 Expression of NSG mouse MHC class II (IA-g7) on reconstituted
human cells for proper T cell activation and antibody response .................. 89

CHAPTER 6
SUMMARY AND FUTURE PERSPECTIVES
6.1 Summary of thesis................................................................................... 94
6.2 Future perspectives ................................................................................. 97

BIBLIOGRAPHY .................................................................................. 99
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Summary
Humanized mice offer a great platform to study human diseases in vivo and totest new therapies. Although much progress has been made to improve humanhematopoietic cell engraftment by generating new strains of immunodeficient
mice, the limited number of hematopoietic stem cells (HSCs) in a single unitof cord blood, and the poor reconstitution and function of humanmonocytes/macrophages still remain as major limitations in current non-obese
diabetic (NOD) /scid IL-2rg-/- mice (NSG mice).
To overcome these two problems, we first developed a robust HSC co-culture
system wherein cord blood CD133+CD34+ cells were co-cultured withmesenchymal stem cell (MSC) engineered to express angiopoietin-like-5(Angptl5) in cytokine supplemented media. The number of double positive(DP) cells was expanded around ~60 fold after 11 days in this culture system,and expanded cells were capable of long-term engraftment in NSG mice.
Further, the expanded cells supported multilineage reconstitution of humanblood cells in NSG mice, including a more efficient T cell reconstitution. With
this expansion method, large-scale experiments with humanized micereconstituted with cord blood HSCs become possible, and the variation among
different donors could be minimized.
The poor engraftment and function of human macrophages in NSG mice werecorrected by a single injection of plasmid encoding human macrophagecolony-stimulating factor (M-CSF). As the mouse M-CSF does not work onix!
human cells, we identified that the human macrophage development was
blocked at promonocyte stage in the bone marrow (BM) of NSG mice. This is
unexpected because M-CSF is believed to play a role at a much earlier stage inmouse macrophage development. After the induction of human macrophages
by human M-CSF, we observed engraftment of human macrophages invarious organs, such as liver and lung. This is the first direct evidence forhuman tissue macrophage reconstitution in humanized mice. Moreover, thesehuman tissue macrophages responded to infections with several natural humanpathogens in vivo, and were able to slow down disease progression. With theengraftment of human tissue macrophages in NSG mice, we not only facilitate
the study of human macrophages in vivo but also make the humanized mice
one step closer to clinical and preclinical applications.

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List of Tables
Table 1. Umbilical Cord Blood Expansion Characteristics……………...34
Table 2. Comparison of reconstitution levels in NSG recipient miceengrafted with expanded cells from either the MSC-A5 co-cultures or the
FCF cultures………………………………...………………………………39
Table 3. Primers list for quantitative PCR………………………………..61

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List of Figures
Figure 1. Combination of co-culture and growth factors enhance
expansion of CD34+CD133+ cord blood cells. ……………………….……32
Figure 2. Fold increase of total cells and DP cells for 14 days coculture………………………………………….………………………….....34
Figure 3 Evaluation of contributions of feeder cells and soluble factors to
the enhanced expansion of CD34+CD133+ cord blood cells ………...……36
Figure 4. Expanded cells are capable of differentiating into multiple
lineages of blood cells in NSG mice……………………………..………….38
Figure 5. Comparison of spleen sections of NSG mice and NSG miceengrafted with expanded cells from MSC-A5 co-culture……………...…40
Figure 6. Expanded cells are capable of stable long-term and efficientsecondary reconstitution …………………………......………………….…42
Figure 7. T cell responses in mice engrafted with co-culture expanded
cells…………………………………………………………………………...44
Figure 8. Poor human monocyte/macrophage reconstitution inhumanized mice……………………………………...……………………..57
Figure 9. Unique human SSChigh myeloid cell population in BM of
humanized mice. ………………………………………………………...…58
Figure 10. Characterization of SSChigh population in BM of humanized
mice…………………………………………………………………………..60
Figure 11. Gene expression profile of SSChigh population in BM of
humanized
mice…………………………….……………………………………...……..61
Figure 12. in vitro differentiation of SSChigh populations from the BM of
humanized mice. …………………………………………....……………....62xii

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Figure 13. in vivo differentiation of SSChigh populations from the BM of
humanized mice. ……………………………………………………………63
Figure 14. Expression of GM-CSF or M-CSF stimulates humanmonocyte/macrophage development in vivo………………………………65
Figure 15. Tissue resident macrophages in M-CSF treated mice….….....66
Figure 16. Immunofluorescence staining of mouse frozen section..……..68
Figure 17. Enhanced human inflammatory response in M-CSF treatedmice after LPS challenge……………………………......………………….70
Figure 18. Influenza A virus infection with M-CSF treated mice........….73
Figure 19. Enhanced human inflammatory response in M-CSF treatedmice after influenza infection…………………………………..…….…….74
Figure 20. Q-PCR analysis of cytokine production after influenza A virus
infection………………………………………………....…………..……….76
Figure 21. BCG infection with M-CSF treated mice….……………..…....78
Figure 22. Expression of human CD40L on human cells…………….…..86
Figure 23. IA-g7 lentiviral vector and its expression in human cells…....90

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List of Abbreviations
Angptl5

Angiopoietin-like 5

BCG

Bacillus Calmette-Guérin

BFU-E

Erythroid burst-forming unit

BM

Bone marrow

BSA

Bovine serum albumin

CBA

Cytometric bead array

cDNA

Complementary deoxyribonucleic acid

CD40L

Cluster of differentiation 40 ligand

CSF-1

Colony stimulating factor 1

C/EBP

Ccaat-enhancer-binding protein

CFU

Colony-forming unit

CFU-E

Erythroid colony-forming unit

CFU-G

Granulocyte colony-forming unit

CFU-GEMM Granulocyte, erythrocyte, monocyte, megakaryocyte colonyforming unit
CFU-GM

Granulocyte, macrophage colony-forming unit

CFU-M

Macrophage colony-forming unit

DAPI

4', 6-diamidino-2-phenylindoleDC

Dendritic cell

DNP-KLH

2,4- Dinitrophenyl-keyhole limpet hemocyanin

DP

Double positive

EBV

Epstein-barr virus

EF1aElongation factor-1 alpha

EGR-1

Early growth response protein 1

ELISPOT

Enzyme-linked immunosorbent spot

EPO

Erythropoietin

FACS

Fluorescence-activated cell sorting

FCF

Feeder cell-free
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FCS

Fetal Calf Serum

Flt3

Fms-like tyrosine kinase receptor-3

FGF

Fibroblast growth factors

FSC

Forward scatter characteristics

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

GFP

Green fluorescent protein

GM-CSF

Granulocyte-macrophage colony-stimulating factor

HBSS

Hank's balanced salt solution

HIV

Human immunodeficiency virus

HLA

Human leukocyte antigen

HSC

Hematopoietic stem cell

H&E

Hematoxylin and eosin

IFN

Interferon

Ig

Immunoglobulin

IGFBP2Insulin-like growth factor binding protein 2

IL

Interleukin

LPS

Lipopolysaccharide

LZM

Lysozyme

M-CSF

Macrophage colony-stimulating factor

Min

MinuteMHC

Major histocompatibility complex

MLR

Mixed lymphocyte reaction

MMM

Mature macrophage marker

MMR

Macrophage mannose receptor

MNC

Mononuclear cell

MOI

Multiplicity of infection

MPO

Myeloperoxidase

mRNA

Messenger ribonucleic acid

MSC

Mesenchymal stem cell
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NK

Natural Killer

NOD

Non-obese diabetic

NSG

NOD/scid IL-2rg-/-

OD

Optical density

PBMC

Peripheral blood mononuclear cell

PBS

Phosphate buffered saline

PMA

Phorbol 12-myristate 13-acetate

PNAS

Proceedings of the National Academy of Sciences

RAG

Recombination-activating gene

RBC

Red blood cell

RT-PCR

Reverse transcription polymerase chain reaction

SCF

Stem cell factor

Scid

Severe combined immunodeficiency

SIRPa

Signal regulatory protein alpha

SRC

Scid repopulating cell

SSC

Side scatter characteristics

TLR

Toll-like receptors

TNF-a

Tumor necrosis factor-alpha

TPO

Thrombopoietin

TT

Tetanus toxoid

UCOE

Ubiquitous Chromatin Opening Element

VDR

Vitamin D receptor

VSVG

Vesicular stomatitis virus glycoprotein

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CHAPTER 1

INTRODUCTION

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1.1 Introduction to the humanized mice
The term ‘humanized mice’ is used to describe several experimental systems. In thisthesis, it refers to immunodeficient mice with components of the human immune
system. As a small animal model, humanized mice allow versatile experimentation
and are easily accessible to the research community. More importantly, theknowledge obtained from this model may be more relevant and applicable to humans.
Because of the differences between human and murine immune systems, the direct
extrapolation of data from studies of mouse models to the clinic becomescomplicated, sometime inappropriate. For example, TGN1412, a humanizedmonoclonal antibody binding to CD28 receptor of T cells, was originally developed
for treatment of B cell chronic lymphocytic leukemia and rheumatoid arthritis. In its
first clinical trial in 2006, it caused catastrophic systemic organ failure in the treatedsubjects at the administered dose, which was 500 times lower than the dose found safe
in animal tests (Hansen and Leslie, 2006; Kenter and Cohen, 2006; St Clair, 2008;
Stebbings et al., 2009; Suntharalingam et al., 2006). In a recent study from our lab,cytokine storm similar to what has been reported in clinical trials was observed in
improved humanized mice (manuscript in preparation). Hence, the humanized mice
offer a tool to bridge the bench and bed gap without putting the patient into risk.
Despite calls for humanized mice, the development of a mouse strain permissive forgood human cell engraftment has been slow. In the past 20 years, the increasing useof clinical hematopoietic cell transplantation in genetic malignant diseases andimmunodeficiencies, as well as the emergence of threatening pandemics caused by
infectious diseases, motivated many scientists to improve humanized mice from2!
extremely low human hematopoietic cell reconstitution to a functional immune
system. Thus, several major breakthroughs in this field are reviewed below.

1.1.1 The development of humanized mice
The history of humanized mice began with the discovery of a Prkdcscid mutation(proteinkinase,DNAactivated,catalytic

polypeptide;

severecombined

immunodeficiency) mutation in CB17 mice (Bosma et al., 1983). The Prkdcscid
mutation results in the absence of the catalytic subunit of a DNA-dependent protein
kinase (Araki et al., 1997; Blunt et al., 1996; Fried et al., 1996; Jeggo et al., 1996;
Kirchgessner et al., 1995), which plays an indispensable role in activation of a DNA
recombinase enzyme during V(D)J recombination of lymphocyte antigen receptors(Lieber et al., 1988; Malynn et al., 1988; Miller et al., 1995). Hence, this strain ofmice lacks mature and functional T lymphocytes. This scid mouse model allowed
only 0.5%-5% human cell reconstitution, but it was already a successful ‘proof ofconcept’. The extremely low levels of human reconstitution are due to the presence ofrelatively intact murine innate immunity (e.g. murine monocytes, macrophages, andnatural killer cells) and the leakiness of the mutation itself (Bosma et al., 1988; Shultz
et al., 1995).
In an effort to overcome the rejection of human graft caused by murine myeloid cells
and natural killer (NK) cells in CB17-scid mice, the scid mutation was introduced into
NOD mice, which reached 5-10 fold higher levels of human peripheral bloodmononuclear cell (PBMC) reconstitution (Hesselton et al., 1995). The improvement in
NOD/scid mice was thought to be due to additional defects in innate immunity of
NOD strain. For instance, NOD mice carry a polymorphism in signal regulatory
protein (SIRP) alpha, which serves as the inhibitory receptor on mouse macrophage to3

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prevent the phagocytosis of human cells (Takenaka et al., 2007). The hemolytic
complement pathways in NOD mice are also inactive due to the specific deficiency of
C5 complement (Baxter and Cooke, 1993). Moreover, the NOD-scid mice areresistant to the leakiness seen in CB17-scid mice, as only 10% of mice havedetectable immunoglobulin (Ig) in serum at 6 months of age compared to 90% of
CB17-scid mice at the same age (Shultz et al., 1995). Hence, the NOD/scid mice
became the gold standard for human cell engraftment studies and were widely used as
xenotransplant recipient mice since 1995. The usefulness of this model, however, islimited by the short life span caused by the development of fatal thymic lymphomasaround 8 months old (Prochazka et al., 1992; Shultz et al., 1995). In addition,although the engraftment of human cells is much improved in NOD/scid mice, highlevels of human cell engraftment are still not seen. This is likely due to remaining NK
cell activity in NOD/scid mice (Greiner et al., 1998).
To increase further the engraftment of human cells, several methods to genetically
block the development of NK cells were tested. One major success was achieved in
2005, when Shultz and colleagues back-crossed immunodeficient mice lacking thegene for the common cytokine-receptor  chain to the NOD/scid mice to create
NOD/scid IL-2rg-/- (NSG) mice (Ishikawa et al., 2005; Shultz et al., 2005). The
common  chain is a critical component for interleukins (IL) 2, 4, 7, 9, 15, 21
receptors (Asao et al., 2001; Sugamura et al., 1996). These cytokines play myriadroles in differentiation, maturation, activation and homeostasis of T cells (IL-2, IL-7
and IL-15), B cells (IL-7, IL-21), and NK cells (IL-2, IL-15) (Janeway, 2001;
Kovanen and Leonard, 2004). As a result of this deficiency, not only mouse T and B
cells, but also NK cells do not develop in NSG mice (Shultz et al., 2005). The average
human cell engraftment in this model is reported to be 6 times higher compared to4!
NOD/scid mice (Shultz et al., 2005). Furthermore, NSG mice are free of thymic
lymphomas, which increase the lifespan from 37 weeks in NOD/scid mice to 90
weeks in NSG mice (Shultz et al., 2005). This much longer life span makes long-term
experiments possible, especially for HIV studies. It is NSG mice reconstituted withhuman CD34+ cells that will be the focus of this thesis.

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1.1.2 Remaining issues of humanized NSG mice
The previous efforts to improve humanized mice specifically aimed for high levels ofhuman cell engraftment by the creation of various mouse strains with deficiencies inmouse immunity. This goal has been fulfilled by the creation of NSG mice. The
current focus of humanized mice switched to establishing a functional human immune
system and using this model for preclinical and clinical applications. Several criticalissues that need to be addressed in order to achieve this aim will be discussed below:
1.1.2.1 Stable reconstitution and the availability of human HSC
At least 1x105 human CD34+ cells are needed to achieve >10% reconstitution in
newborn pups or in adult NSG mice, and the reconstitution is only stable between 2 to
12 months. There are too few human cells before 2 months of reconstitution and one
type of human blood cells, usually human T cells, dominates after 10 months ofreconstitution. In contrast, single syngeneic mouse HSC could lead to more than 50%
hematopoietic chimerism for at least 10 months, whereas 10 fold greater numbers ofmouse HSCs are required for fully major histocompatibility complex (MHC)mismatched recipients. In addition, homeostatic expansion of human CD34+ cells is
not observed in humanized mice, and the frequency of scid repopulating cells (SRC)
and chimerism decline over time. This evidence suggests that human HSC are notmaintained in humanized mice (Manz, 2007). This could be due to the difficulty ofhuman HSC homing to mouse BM, or the insufficient support from mouse BM
niches, or the residual mouse immunity against human HSCs. To improve thissituation, co-transplantation of human MSC (Muguruma et al., 2006) or mouse MSC
engineered to express human cytokines with human HSCs may benefit the homing
and maintenance of human HSC. Genetically engineering human HSCs to express6!
mouse MHCs or to transiently express certain mouse chemokine receptors may also
help the homing and long-term reconstitution. Till now, not much progress has been
made on this issue of humanized mice.
The common sources for human CD34+ cells are human cord blood, human fetalliver, and less frequently, cytokine mobilized adult peripheral blood and adult BM.
Since the CD34+ cells isolated from adult humans are less effective to reconstitutehumanized mice, CD34+ cells from cord blood or fetal liver are typically used (Lepus
et al., 2009). As the availability of human fetal liver is uncertain, and the use of fetal
samples for research purpose is strictly regulated, many laboratories working withhumanized mice use cord blood as their routine source for human CD34+ cells.
However, the number of CD34+ cells isolated from a single unit of cord blood isaround 0.2-1x106, which is only enough to reconstitute 1-5 newborn pups. The limited
number of mice produced from one unit of cord blood makes it difficult to set up
experiments with proper controls. Humanized mice made from different batches ofcord blood or from pooled human CD34+ cells may be used to increase the number tomeet the requirement of experiments. However, the results obtained from different
units of cord blood often make it difficult to draw conclusions because thereconstitution kinetics and ability vary among batches (Drake et al., 2011). In
addition, the purchase of one unit of cord blood and the isolation process typically
cost several hundred dollars. It would be prohibitively expensive for a lab to generate
humanized mice routinely with this method. Thus, it would be desirable to expand a
population of long-term reconstituting human HSCs in vitro so that an entire study or
one set of experiments could be performed from a single unit of cord blood.

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1.1.2.2 Unbalanced human hematopoietic cell reconstitution
8 to 12 weeks after injection, human HSC and progenitor cells differentiate and form
a human hemato-lymphoid system in NSG mice. Almost all major cell populations of
the human immune system, including dendritic cells (DC), natural-interferon
producing cells, T cell, B and Ig-producing cells, could be found in organs andperipheral blood of NSG mice. However, the level of human cell reconstitution differs
significantly among different lineages.
B cells: Human B cell differentiation in BM of humanized mice seems to resemble
that in humans since several stages of B cell precursor populations exist, such as early
B cells, pro-B cells, pre-B cells and immature B cells (Watanabe et al., 2009).
However, the peripheral mature B cells in humanized mice express high levels ofCD5 (Matsumura et al., 2003), which is markedly different from human B cells foundin adult peripheral blood. CD5 was considered a marker for B-1 cells, a minor
fraction of B cells characterized by three functions: spontaneous IgM secretion,
efficient T cell stimulation, and tonic intracellular signaling (Griffin et al., 2011). The
origin and development of B-1 cells are still unclear but evidence suggests that they
may arise from precursors in fetal liver, and certain antigens are needed to shape therepertoire of the B-1 population (Bendelac et al., 2001). Recently, B1 population isdefined by CD20+CD27+CD43+CD70- in umbilical cord blood and adult peripheral
blood and the overlap of this population with CD20+CD5+ is only ~34% (Griffin etal., 2011). As the majority of B cells in humanized mice are CD5+ B cells, further
characterization of this population is needed to understand the problem of human B
cell development.
T cells: The major achievement in NSG mice is efficient intrathymic de novo human
T cell development (Gimeno et al., 2004; Ishikawa et al., 2005; Shultz et al., 2005). T
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cells in NSG mice, including CD4 and CD8 T cells, show a broad V distribution.
The ratio among CD4 T cells, CD8 T cells, Foxp3+ CD25+ regulatory T cells and T cells is quite close to physiological level. The problem for human T cells inhumanized mice is poor activation and function, not the development orreconstitution. That problem will be discussed separately.
NK cells: NK cells development could be detected in BM as early as 4 weeks after
HSC transfer in humanized mice. However, the number of peripheral NK cells is
generally low or undetectable (Chen et al., 2009). The poor reconstitution of humanNK cells possibly results from inappropriate cytokine cross-reactivity between mouseand human (Manz, 2007). In 2009, hydrodynamic injection was used to express
human IL-15 and Flt-3/flk2 required for NK cell development. This method
successfully improved the reconstitution and function of NK cells in NSG mice (Chenet al., 2009). In other studies, human NK cells were also induced by in vivo
supplementation of human IL-15/human IL-15R  complex or by transgenic
expression of human IL-2 in NOG mice (Huntington et al., 2009; Ito et al., 2012).
Monocytes/Macrophages and DCs: Although monocytes/macrophages and DCs
develop in humanized mice, their frequencies are usually very low. In a study fromour lab, expression of human granulocyte-macrophage colony-stimulating factor(GM-CSF) /IL-4 and human macrophage colony-stimulating factor (M-CSF) also
resulted in significantly increased numbers of human DCs and monocytes inperipheral blood of humanized mice, respectively (Chen et al., 2009). Similarly,
monocytes and alveolar macrophages were boosted in M-CSF and GM-CSF/IL-3
human cytokine knock-in NSG mice, respectively (Rathinam et al., 2011; Willinger etal., 2011). But to date, there is no report of human tissue macrophages in humanized9!
mice, except for alveolar macrophages in GM-CSF/IL-3 mice, and in vivo immune
functions in NSG mice with improved monocyte reconstitution have not beenreported.
Granulocytes and erythrocytes: Granulocytes and erythrocytes comprise the largest
fraction of human hematopoietic cells. These two cell types, however, are the lowest
reconstituted populations in humanized mice. Several groups attempted to improvethese two types of cells but without much success. A slight increase in CD15+
granulocytes was observed in the BM of stem cell factor (SCF), GM-CSF and IL-3transgenic NSG mice (Billerbeck et al., 2011). Some human CD66+ granulocytes also
were produced in the BM of Thrombopoietin (TPO) knock-in mice (Rongvaux et al.,2011). In a study from our lab, a low but significant level of human erythrocytes in
NSG mice could be induced by hydrodynamic injection of plasmid expressing
erythropoietin (EPO) and IL-3 (Chen et al., 2009). However, the induction took morethan one month. Collectively, the development of granulocytes and erythrocytes could
be partially improved by the addition of human cytokines.
1.1.2.3 Weak antigen specific T cell and B cell responses
Upon unspecific stimulation, these generated human T cells are capable ofproliferating and producing cytokines both in vitro and in vivo. However, human T
cells have a limited ability to respond to antigen stimulation. To understand thisproblem, we have to unravel what MHC is to be expected from human T cells
educated on a mouse background. As the positive selection of thymocytes
preferentially occurs on epithelial cells, and no evidence of human thymic epithelial
cells could be derived from reconstituted CD34+ cells, human thymocytes should bepositively selected on mouse MHC content. There has been a report of human DCs in
the mouse thymic DC compartment, and negative selection of thymocytes occurs on
10!
both epithelial cells and hematopoietic derived DCs. Hence, human thymocytes may
be negatively selected on both mouse and human MHC content.
This hypothesis of human thymocyte selection in a mouse thymus was supported by
the mixed lymphocyte reactions (MLRs), in which human T cells from NSG miceproliferated vigorously when stimulated with human allogeneic DCs, but proliferated
weakly when stimulated with autologous human DCs or mouse DCs (Traggiai et al.,2004). In addition, we used humanized mice to establish Epstein-Barr virus (EBV)and dengue infection models in our lab, but human T cells in mice were unable tocontrol the virus replication (unpublished data). There were also studies on influenza
virus infection in humanized mice. They reported that human T cells specific for viral
epitopes were only found in the context of mouse MHC (Legrand et al., 2006). Taken
together, the evidence suggests that human T cells in mice are tolerant for both
autologous human and mouse MHC. They frequently mount reactions towards
allogeneic MHC but produce, to some extent, specific responses only in the context ofautologous mouse MHC.
As a result of insufficient human T cell responses in vivo, antibody production inhumanized mice is far from optimal. Human IgG concentration in serum increases
over the course of reconstitution, and is on average 10-100 fold lower than in humanadults and wild-type mice (Baenziger et al., 2006; Traggiai et al., 2004). Specific IgG
responses against tetanus toxoid (TT), ovalbumin, and dinitrophenyl-keyhole limpet
haemocyanin (DNP-KLH) are very low or undetectable (Baenziger et al., 2006;
Ishikawa et al., 2005; Traggiai et al., 2004). To enhance the antibody response in
humanized mice, the key is to establish a robust T cell response to adequately help B
cells mature into antigen-specific antibody-producing cells. The obvious solution tothis problem is to replace the mouse MHC with human MHC components. Till now,11!
NSG mice with human MHC class I have been created. Cytotoxic human T
lymphocytes became functionally mature, and mounted human leukocyte antigen(HLA)-restricted cytotoxicity against EBV infected human B cells in NSG-HLA-A2
mice (Jaiswal et al., 2012). However, the antibody response in this mouse was notmuch improved. The NSG-HLA-DR mice and the NSG-HLA/A2-HLA/DR mice are
still

under

development

by

the

JacksonLaboratory(http://research.jax.org/collaboration/escell.html).

12!

1.2 Focus and objectives of this thesis
Despite high human cell engraftment in humanized NSG mice, those issues discussed
above greatly hampered its impact and application in basic and applied preclinicalhuman immunology research. In this thesis, we only focused on three criticalproblems of humanized mice:

Limited supply of human cord blood HSCs and variations from small
cohorts of cord blood HSCs in reconstituted humanized mice
Chapter 3 describes the efforts to develop a method for HSC expansion in vitro. Fourgoals were addressed: 1) Establish and optimize the conditions for human HSCs
expansion. 2) Evaluate and compare the differentiation potential of expanded HSCs
into hematopoietic lineages in NSG mice. 3) Evaluate the long-term reconstitution
potential of expanded HSCs. 4) Investigate the functions of human immune system inhumanized mice engrafted with expanded HSCs.

Poor understanding of human macrophage development and no tissue
macrophage reconstitution in humanized mice
Chapter 4 shows the efforts to generate human monocytes/macrophages in humanized
mice. Four goals were addressed 1) Investigate the problem of humanmonocyte/macrophage development in humanized mice. 2) Develop methods torestore the monocyte/macrophage population in peripheral blood and tissues ofhumanized mice. 3) Test the functions of induced monocytes/macrophages. 4)
Evaluatetheinnateimmunityinhumanized

mice

with

improved

monocyte/macrophage reconstitution.

13

!

Weak antigen specific antibody response in humanized mice
Chapter 5 shows the efforts to increase antigen specific antibodies in humanized mice.
Two approaches were designed: 1) Expression!of!human!CD40!ligand!(CD40L)!on!
human! T! cells! to! promote! the! maturation! and! proliferation! of! antigen=experienced! B! cells.! 2)! Expression! of! NSG! mouse! MHC! class! II! (IA=g7)! on!
reconstituted!human!cells!for!proper!T!cell!activation!and!antibody!response.!

14

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CHAPTER 2

MATERIALS AND METHODS15

!

2.1 Stem cell purification
Purified human CD133+ cord blood cells were purchased from AllCells (California).
Alternatively, umbilical cord blood was obtained from the Singapore Cord Blood
Bank. Red blood cells (RBCs) were removed by Ficoll-Hypaque density gradient.
Human fetal liver samples were obtained from aborted fetuses at 15-23 weeks ofgestation, in accordance with the institutional ethical guidelines of the National
University Hospital of Singapore. All women gave written informed consent for the
donation of their fetal tissue for research. Fetuses were collected within 2 h of thetermination of pregnancy. A single cell suspension was prepared. To isolate humanCD34+ cells, fetal liver samples were excised into small pieces and digested withcollagenase IV (2 mg/ml) at 37 °C for 15 min. A single cell suspension was preparedby passing the digested tissue through a 100 m cell strainer (BD Biosciences).
CD34+ cells were purified with the RosetteSep system using the CD34 positive
selection kit (Stem Cell Technologies, Vancouver, BC). The purity of purified cells
was 90 to 99% CD34+. On average, 95% of the purified CD34+ cord blood cells, and
30% of purified CD34+ fetal liver cells were also CD133+ (data not shown).
Following in vitro expansion, CD133+ cells were purified by staining cells with PEconjugated anti-CD133 (Ebiosciences) followed by a PE positive selection kit (StemCell Technologies).

16!

2.2 Feeder cell-free (FCF) culture of cord blood CD34+CD133+ cells
Cryopreserved or freshly isolated CD34+CD133+ cord blood cells were cultured invitro as described (Zhang et al., 2008). Briefly, StemSpan medium (Stem Cell
Technologies, Vancouver) were supplemented with 20 ng/ml SCF (R&D), 10 ng/ml
TPO (R&D), 10 ng/ml fibroblast growth factors-1 (FGF-1) (Gibco), 100 ng/ml
Insulin-like growth factor-binding protein 2 (IGFBP2) (R&D), 500 ng/ml
angiopoietin-like 5 (Angplt5) (Abnova, Taiwan), 500 µg/ml of heparin (Sigma), 1x
penicillin and streptomycin (Gibco) to obtain the expansion media. About 104
CD34+CD133+ cord blood cells were plated in a 96-well round bottomed plate, in 200
ml/well of the expansion media. Cells were transferred to a 6-well plate 4 days later,
and fresh media were added to keep cells at 200,000 /ml one day after the transfer.
Cells were supplemented with more fresh media 2-3 days later, in order to maintainthem at 700,000 cells/ml and then allowed to expand until the end of the 11-day
culture.

2.3 Angplt5-expressing MSCs
The human Angptl5 gene (DNA NM_178127.2) was excised from the pCMV6-XL5
vector (Origene, Rockville, USA) using Not I, and cloned into the pLB2 lentiviral
vector at the Not I site. The resulting vector encodes both Angplt5 and GFP under thesame EF1a promoter (provided by Dr. Patrick Stern of Massachusetts Institute ofTechnology). To produce lentiviruses, 293FT cells were co-transfected with lentiviral
vectors, the HIV-1 packaging vector Delta8.9 and the VSVG vector. Supernatants
containing lentiviruses were harvested on day 2 or 3, concentrated by17!
ultracentrifugation at 25,000g for 90 min, and frozen until use. Lentivirus titers were107-108 /mL based on GFP expression in 293FT cells.
Human MSCs from the BM of adult donors were purchased commercially (Lonza or
Stem Cell Technologies). MSCs were cultured in MesenCult MSC Basal Medium and
Mesenchymal Stem Cell Stimulatory Supplements (Stem Cell Technologies,
Vancouver) at a density of 5,000-6,000 cells per cm2 in a T175 flask, and passaged
before reaching 70% confluence. MSCs from 2nd to 4th passages were transducedwith a lentiviral vector (pLB2) expressing GFP alone or both GFP and Angplt5 at
MOI of 5. Four to five days post-transduction, 30-45% of cells were GFP+ by flow
cytometry analysis. The mixture of transduced and non-transduced MSCs wereexpanded and used for co-cultures.

2.4 Co-culture of cord blood CD34+CD133+ cells
MSC-A5 or MSC-GFP was plated in a 24-well plate at 5 x 104 cells/well overnight.
CD34+CD133+ cord blood cells were added to the culture at a cord blood cell-to-MSC
ratio of 1:5 (i.e., 104 cord blood cells for 5x104 of pre-plated MSCs per well). The
expansion media was used for co-culture except for the addition of Angptl5. The
initial volume of culture media was adjusted to 300 µl/well, and fresh media wereadded every 2 days starting on the 3rd day of culture. At different time points of theculture, hematopoietic cells were resuspended by carefully pipetting 5-7 times(avoiding the detachment of the MSCs), counted and analyzed by flow cytometry toobtain the total cell number and the number of DP cells. After 11 or 14 days ofculture, hematopoietic cells were resuspended thoroughly (to loosen strongly attachedcells to the feeder layer), analyzed for CD34 and CD133 expression.

18!

2.5 Mice and intracardiac injection
NSG mice were obtained from the Jackson Laboratory and maintained under specificpathogen-free conditions in the animal facilities at Massachusetts Institute ofTechnology, National University of Singapore and Nanyang Technological
University. Pups within 48 h of birth were sublethally irradiated (100 rad) using a
Cesium source, and engrafted with either expanded or unexpanded cells byintracardiac injection.
For engraftments, either 105 purified CD34+CD133+ unexpanded or expanded cells in
50 l were injected per recipient, or total expanded cell suspension containing 105
CD34+CD133+ cells in 50 l were injected per recipient. All research with humansamples and mice was performed in compliance with the institutional guidelines.

2.6 Serial reconstitution assays
Serial reconstitution was performed as follows: Fourteen weeks after thereconstitution, BM cells were harvested from both femurs and tibias of the primary
recipients. Human CD34+ cells were stained with PE-conjugated anti-CD34 followed
by a PE positive selection kit (Stem Cell Technologies). CD34+ cells were pooled
from different mice reconstituted with expanded cells from the same cord blood
donor, and then 104 cells were injected into sublethally irradiated newborn pups.

2.7 Single cell preparation
Single cell suspensions were prepared from the spleen and BM by standard
procedures. To isolate hepatic mononuclear cells (MNC) from humice, the liver was
19!
pressed through a 200-gauge stainless steel mesh, and debris was removed bycentrifugation at 50 g for 5 min. Supernatants containing MNCs were collected,
washed in PBS, and resuspended in 40% percoll (Sigma) in RPMI medium 1640. The
cell suspension was gently overlaid onto 70% percoll, and centrifuged at 750 g for 20
min. MNCs were collected from the interphase, washed twice in PBS. To isolate lung
MNCs, lungs were minced, suspended in medium containing 0.05% collagenase(Sigma) and 0.01% DNase I (Sigma), and incubated at 37 °C for 20 min. The lungsamples were passed through a 200-gauge stainless steel mesh, and MNCs wereisolated by percoll centrifugation as described above. To isolate MNCs fromhumanized mice brain, the brain was homogenized in 7 ml RPMI 1640 and added 3
ml stock isotonic percoll to make 30% percoll. 10 ml cell suspension was gently laid
onto 70% percoll, and centrifuged at 500 g for 30 min. MNCs were collected from theinterphase, washed with HBSS, and centrifuged for 7 min at 500 g.

2.8 Flow cytometry and cell sorting
MNCs were analyzed by flow cytometry using FACScalibur, FACS-Canto or LSR II
cytometers (Beckton Dickinson). Cell sorting of monocyte/macrophage progenitors in
BM was performed using FACS Aria cell sorter (BD Biosciences). The following
antibodies were used: CD45 (2D1), CD34 (581), CD38 (HIT2), CD209 (DCN46)
from BD Biosciences; CD14 (HCD14), CD45.1 (A20), HLA-DR (L243), CD33
(WM53), MMR (15-2), CD209 (9E9A8), CD56 (MEM-188), CD3 (HIT3a), CD19
(HIB19), CCR5 (HEK/1/85a), CD40 (5C3), CD7 (CD7-687), CD116 (4H1), CD114(LMM741), CD15 (HI98), TLR2 (TL2.1), CD11c (3.9), CD11b (ICRF44), CD36 (5271), CD80 (2D10), CD86 (IT2.2), CD16 (3G8), CD64 (10.1), CD163 (GHI61),
CD68 (Y1-82A) from Biolegend; CD13 (WM15), MMM (25F9), TLR4(HTA125)
20

!
from Ebiosciences. Dead cells were excluded from analysis by DAPI staining.
Analyses were performed with the FACS Diva (BD Biosciences) or FlowJo (TreeStarInc).

2.9 Immunization and ELISPOT assay
Sixteen weeks after engraftment, mice were immunized intraperitoneally (i.p.) with10 l of TT vaccine adsorbed on hydrated aluminum hydroxide (Teabag, Sanofi
Pasteur, France) diluted in 90 l PBS, representing 1/50 of the recommended
vaccination dose for a human adult. Mice were boosted twice with the same dose at
three-week intervals. Two weeks after the third immunization, mice were sacrificed
and the frequencies of IFN- secreting cells in the spleen were measured by
ELISPOT assay (Ebiosciences, San Diego, CA). Briefly, single cell suspensions wereprepared from spleens, counted and analyzed by flow cytometry for the frequencyof human CD3+ T cells. 5x105 CD3+ cells were plated per well in a 96-well ﬂatbottomed plate (Multiscreen-IP, Millipore, MA, USA) that was coated overnight at 4
°C with anti-IFN- monoclonal antibody. For T cell stimulation, 10 ng/ml of PMA
or 0.5 mg/ml of the tetanus toxin peptide (830-843) (Genscript, USA) were added tothe culture. After 48 hours of incubation at 37 °C, 5% CO2, IFN- immunospots
were detected according to the manufacturer’s instructions. The spots were counted
using an ImmunoSpot S5 Versa Analyzer (Cellular Technology Ltd. Ohio, USA) andanalyzed with ImmunoCapture software (Analysis Software).

21!

2.10 Hydrodynamic injections
pcDNA3.1 (+) vector was used to clone human cytokine genes. Plasmid DNA wasprepared by Maxi-prep Kit (Promega) with endotoxin removal. For hydrodynamic
injection, 50 g plasmid of each cytokine gene in 2 ml PBS was injected into miceolder than 8 weeks within 7 seconds using a 27-gauge needle.

2.11 In vitro differentiation assay and colony-forming unit (CFU) assay
BM MNCs were flushed out from tibias of 12-week old humanized mice. Sorted
monocyte/macrophage progenitors from BM MNCs were cultured in RPMI 1640,
10% FCS, supplemented with 50 ng/ml M-CSF for macrophage differentiation or 50
ng/ml GM-CSF, 50 ng/ml IL-4 for DC differentiation. All cytokines were purchasedfrom R&D Systems.
Sorted monocyte/macrophage progenitors were plated at 2000, 5000, 10000 cells perdish in complete MethoCult media (MethoCult H4435 enriched; Stem Cell
Technologies) according to the product manual. Cells were incubated in 35 mm
culture dishes at 37 °C with 5% CO2. The numbers of colonies were counted under astereomicroscope after 14 days in culture.

2.12 RNA extraction and real-time PCR
Total RNA was extracted from lysed tissues or cells with the RNeasy Plus Mini kit(Qiagen) according to manufacturer’s instructions. 1 g of RNA was used for cDNA
synthesis with ImProm-II Reverse Transcription System (Promega). Quantitative RT22

!
PCR was performed with the CFX96 Real-Time System (Bio-rad) with self-designed
primers (supplementary Table 1). Expression values were calculated by thecomparative threshold cycle method, and normalized to mouse L32 or human
GAPDH.

2.13 Histology and Giemsa staining
For histology, each mouse was intracardiacally perfused with saline buffer for 10 min.
Organs were harvested, placed in disposable molds, immersed in OCT (theembedding compound) and flash frozen in liquid nitrogen. The blocks were stored at 80 °C and cut at 10 m per section in histology laboratory at National University ofSingapore.
Hematoxylin and eosin (H&E) staining was performed on formalin-fixed, frozen
spleen sections. Immunofluorescence staining was performed on 75% acetone/25%
ethanol fixed, frozen spleen sections. Monoclonal antibody to CD20 (L26, Abcam,
UK), CD68 antibody (KP1, Abcam), mouse CD68 antibody (FA-11, Abcam), andpolyclonal antibodies to CD3 (Abcam) were used as primary antibodies. Alexa
fluro647 donkey anti-mouse IgG (Invitrogen, USA) and Alexa fluro546 donkey antirabbit IgG (Invitrogen) were used as secondary antibodies. H&E stains andimmunofluorescence stains were visualized with the MIRAX MIDI Fluorescencemicroscope (Zeiss).
For Giemsa staining, 105 cells in 100 l 2% FCS-PBS were added to the cytospin
cassette, and centrifuged 1000 rpm for 5 min. The cell smears on polylysine-coatedslides were air-dried, fixed with methanol for 1 min, stained with Giemsa for 5 min23!
and washed with distilled water. The stained slides were examined under amicroscope.

2.14 LPS challenge, influenza virus A infection and bacillus calmetteguérin (BCG) infection
10 g LPS (Sigma) were injected i.p. into mice. Sera were collected 0 h, 2 h and 12
h after injection. Concentrations of human inflammatory cytokines in sera wereanalyzed by the Cytometric Bead Array System (CBA) (BD Biosciences) on LSRII
according to the manufacturer’s instructions, and data were analyzed by FCAP Arraysoftware (BD Biosciences).
300 pfu of influenza A/PR8 (H1N1) virus in 75 l PBS were administered toanesthetized mice via intratracheal route. Lungs were harvested at 0 h, 24 h, 48 h, and
72 h after infection. For each mouse, the left lobe was used for the single cell
preparation for flow cytometry analysis as described above. The superior lobe of rightlobes was excised to extract RNA for quantitative RT-PCR analysis as describedabove. The rest of the three right lobes were weighted and homogenized in 100 mg
tissue per 1 ml of ice-cold HBSS with 0.1% Bovine serum albumin (BSA). The lunghomogenates were centrifuged for 10000 g, 10 min at 4 °C to collect the supernatantsfor CBA assay to measure the cytokine concentration.
Mycobacterium bovis BCG Pasteur strain bacilli (ATCC, Manassas, VA, USA) was
grown in 7H9 culture medium at 37 °C in an 850 cm2 polystyrene roller bottle(Corning) at 10 rpm. The culture reached an optical density (OD600) of 0.6, at which
point the concentration of cells was ~5x107 /ml. Each mouse was injected i.p. with
5x106 CFU of BCG in 0.5 ml. The peritoneal lavage was flushed out with 5 ml PBS,24

!
and the spleen was homogenized in 10 ml PBS at 0 h, 24 h, and 72 h post-infection.
To determine the numbers of recovered viable bacilli in a culture or tissue
homogenate, colony counting was performed with serial dilutions of the samples in
7H9 with 100 µL of serial dilutions plated on 7H11 agar, and incubated at 37 °C with
5% CO2. Serial dilutions and 7H11 plates were performed in triplicate, and thenumbers of colonies on each plate counted after 4 weeks. The rest of the peritoneal
lavage was centrifuged down to obtain cells for flow cytometry analysis, and to
extract RNA for quantitative RT-PCR analysis as described above.

2.15 Statistical analysis
Data are presented as mean and standard error of the mean. The nonparametric MannWhitney U test was used to analyze the differences between two groups. A P-value of<0.05 was considered as statistically significant.

25

!

CHAPTER 3

IN VITRO EXPANSION OF HSC FOR HUMANIZEDMICE RECONSTITUTION
!
!
!
!
!
!
!
!
!
!
!

Work has been published as (Khoury et al., 2011) and LI Yan is the co-author with
Dr. Maroun Khoury on this paper. LI Yan contributed in all the experiments presented
in this chapter and independently designed and performed the experiments in Figure 5
and Figure 7.
26

!

3.1 Introduction
3.1.1 The demand for expansion of HSCs for clinical and preclinical applications
HSCs give rise to all blood lineage cells, which function in diverse biologicalprocesses from immune responses to oxygen transport. Because of this uniqueproperty, there has been great interest in clinical and preclinical applications of HSCs.
BM transplant is widely used to treat congenital immunodeficiencies, where thetransfer of HSCs from normal to deficient individuals is the basis of the treatment.
Transplantation of purified HSCs from BM, mobilized peripheral blood or umbilicalcord blood is also a well-established treatment for certain hematologic malignancies.
However, these treatments are often limited by the availability of HSCs in terms offinding suitable matching donors as well as obtaining sufficient numbers of cells.
In preclinical applications, there has been an enormous effort in developing mice withhuman blood lineage cells (humanized mice) for studying human blood cell diseases
and immune responses to pathogens. Construction of humanized mice requires
engraftment of human HSCs in scid mice. With the development of a new generationof scid mice, such as NSG mice, which also lack NK cells, it is now possible toroutinely construct humanized mice with high levels of human blood cellreconstitution. The easiest accessible source of human HSCs is cord blood. However,
only limited numbers (105~106) of CD34+ cells can be isolated from a single cord toreconstitute a small number (1~10) of mice. Although HSCs from different cords canbe used, to minimize variations due to differences in the genetic background of HSCs,
humanized mice reconstituted with HSCs from the same source are ideally used in thesame experiment. Large-scale application of humanized mice for research and27

!
preclinical studies requires access to sufficient numbers of human HSCs (Giassi et al.,2008).

3.1.2 Methods for HSC expansion
One way to overcome the shortage of HSCs is via the expansion in vitro. In general,
two approaches have been used: FCF culture in the presence of specific growth
factors and co-culture with feeder cells, such as MSCs. In FCF culture, the best in
vitro expansion of human HSCs achieved to date was reported recently by Delaney etal. 2010. Using a cocktail of growth factors, including human IL-3, IL-6, TPO, Flt-3
ligand, SCF and notch ligand, total and CD34+ cells were expanded ~790 and ~200
fold, respectively, after 17-21 days of culture, resulting in a ~16 fold expansion of
SRCs as assayed in NSG mice (Delaney et al. 2010). Using a different combination of
growth factors, including TPO, SCF, FGF-1, insulin growth factor binding protein 2(IGFBP2), and angiopoietin-like 5 (Angptl5), Zhang et al. 2010 reported a lower total
cell expansion (~200 fold) as cells undergo senescence after 14 days. Nevertheless, a20-fold expansion of SRCs was observed in NOD-scid mice following a 10-11 day
culture (Zhang et al., 2008; Zhang et al., 2006).
Another way to expand HSCs is co-culture using a feeder layer that mimics thephysiological microenvironment of HSCs. In particular, BM-derived MSCs have beenshown to support the growth of HSCs from cord blood through an increased cell
division and a better maintenance of the primitive phenotype (Walenda et al., 2009).
MSCs from other sources, including Wharton’s jelly of umbilical cord segment(Wang et al., 2004), cord blood (Lee et al., 2004), amnion-derived adherent cells (In 't
Anker et al., 2004; Kita et al., 2009), and other fetal tissues (Campagnoli et al., 2001),
have also been shown to support ex vivo expansion of HSCs (Bakhshi et al., 2008;28

!
Campagnoli et al., 2001; Huang et al., 2007; Jang et al., 2006; Lee et al., 2004;
Mizokami et al., 2009; Walenda et al., 2009; Wang et al., 2004; Wang et al., 2009).
Many of these reports suggest that co-culture is superior to the FCF culture in termsof both total cell expansion and the yield of CD34+ cells when compared under thesame culture conditions. Depending on the co-culture conditions, 80 to 800-foldincrease in total cell numbers, and 4 to 100 fold increase in the CD34+ cells werereported (Alakel et al., 2009; Briquet et al. 2010; da Silva et al. 2010). The highest
expansion was achieved by culturing HSCs with MSCs in a 3D configuration using a
woven mesh (96 fold expansion CD34+ cells in 10 days, (Zhang et al., 2006).
However, most studies with co-culture did not rigorously examine the scid
repopulating activity of expanded cells in mice using long-term reconstitution,
limiting dilution or competitive reconstitution assays. In the few cases where
expanded cells were tested in mice, only one dose of expanded progeny was injectedin NOD-scid mice, and reconstitution was assayed 6 weeks later in the BM (Briquet etal. 2010; Fei et al., 2007; Zhang et al., 2006). Another report showed a 3.4-foldincrease of SRCs when the progeny of CD34+ cells co-cultured with human brain
endothelial cells were injected into mice (Chute et al., 2004).

3.1.3 Expansion of HSC in angptl5 expressing MSC co-culture
One way to further enhance HSC expansion without promoting differentiation is tocombine co-culture with growth factors that are shown to promote HSC maintenanceand expansion. In this study, we co-cultured HSCs with engineered MSCs that
express Angptl5 plus the addition of SCF, TPO, FGF1 and IGFBP2 in the media.
Dramatically, we observed ~60 and ~150 fold expansion of CD34+ CD133+ DP cells
10 days and 14 days after co-culture, respectively. The expanded DP cells possess the29!
same repopulating and long-term HSC properties as uncultured cells using long-termand serial reconstitution. In addition, mice reconstituted with the expanded HSCs
from the co-culture have an enhanced ability to differentiate into T cells, leading to amore normal T cell to B cell ratio in the reconstituted mice. These results suggest thatco-culture in the presence of selected growth factors in the media is an efficientmethod to expand human HSCs in vitro.

30

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3.2 Results
3.2.1 Combination of co-culture and selected growth factors enhance expansionof cord blood CD34+CD133+ cells.
To investigate whether MSCs and recently identified growth factors, such as IGFBP2
and Angptl5, synergize in HSC expansion, we tested expansion of CD34+CD133+
cells on an MSC feeder layer in the presence of additional growth factors. Because
human Angptl5 was required at high concentrations (Zhang et al, 2010) and
expensive, we expressed it in human primary BM MSCs using a lentiviral vector. The
Angptl5 expressing MSCs are referred to as MSC-A5. As a control, GFP-expressing
MSCs (MSC-GFP) were constructed following the same approach. For expanding
HSCs, purified CD133+ cord blood cells which were also CD34+, were cultured on
top of a confluent MSC-A5 or MSC-GFP feeder layer at a ratio of 1:5 in the presenceof SCF, FGF, IGFBP2, and TPO in the culture media. For comparison, purified
CD133+ cells were also cultured in FCF culture, i.e., serum-free medium
supplemented with Anglt5, SCF, FGF, IGFBP2, and TPO. At the start of the coculture, hematopoietic cells were observed to attach to the MSC feeder layer (Figure1A). By day 5, the density of hematopoietic cells was noticeably increased. By day 7,
proliferation of hematopoietic cells was evidenced by cell density and cell counting(Figure 1A). By day 11, hematopoietic cells had completely covered the MSC-A5
feeder layer (Figure 1A) and were split into fresh feeder layers and medium. To
monitor HSC expansion, cultured cells were assayed for CD34 and CD133 expression
since we have shown previously that SRCs reside in the CD34+CD133+ DP fraction,and some of the DP cells possess the long-term HSC activity (Drake et al, submitted).
At the beginning of the co-culture, more than 95% of the input cells were CD34+ and
31

!

32

!
CD133+ (data not shown). After culture for 7 days, the percentage of DP cells
decreased to ~50% in MSC-A5 or MSC-GFP co-culture, and ~30% in FCF culture(Figure 1B).
The percentages continued to decrease and by day 14, they were ~15% in co-culturesand ~10% in FCF culture. Despite the decrease of the percentages of DP cells in thecultures, because the total cell numbers increased dramatically (Figure 2A), the actual
numbers of DP cells increased ~60 fold in MSC-A5 co-culture, ~29 fold in MSC-GFP
co-culture, and ~20 fold FCF culture at day 11 (Figure 2B). By day 14, while thenumber of DP cells did not increase significantly in the FCF culture, the number of DP
cells increased ~150 and ~60 fold in MSC-A5 and MSC-GFP co-cultures, respectively.
When CD34+ cells isolated from fetal liver and mobilized peripheral blood were
cultured using the same conditions, expansion of DP cells in MSC-A5 co-culture wasalso significantly higher than that in the FCF culture (data not shown). These results
show that the combination of MSCs and the different growth factors can support amuch more robust CD34+ CD133+ HSC expansion.

33

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!

34

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3.2.2 Evaluation of contributions of feeder cells and soluble factors to theenhanced expansion of cord blood CD34+CD133+ cells
To further assess the role of each of the five growth factors in the expansion of
CD34+CD133+ cord blood cells, we performed the co-cultures without adding specificgrowth factors. For easy comparison, the expansion of DP cells in the different
cultures was normalized to that under the standard condition where CD34+ CD133+
cord blood cells were co-cultured with MSC-A5 in the presence of SCF, TPO, FGF
and IGFBP2 in the media. When CD34+CD133+ cord blood cells were co-cultured
with MSC-A5 without the addition of any of the growth factors, they did not expand atall (Figure 3). Addition of SCF and TPO, which are well-established HSC growth
factors and broadly used together (Liu et al., 1999; Ohmizono et al., 1997), into theabove co-culture led to expansion of DP cells, reaching 19% of that obtained under the
standard condition. With further addition of FGF or IGFBP2, the relative expansion
was 36% and 61%, respectively. Without exogenously added Angplt5 (co-culture with
MSC-GFP in the presence of SCF, TPO, FGF, and IGFBP2), the relative expansion
was 43%. These results suggest that all five growth factors contribute to the expansionof CD34+CD133+ cord blood cells, with IGFBP2 and Angplt5 having the biggest
impact.

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3.2.3 Expanded CD34+CD133+ cells give rise to multiple lineages of blood cells in
NSG mice
We tested the ability of expanded cells from the co-cultures to reconstitute different
lineages of human blood cells in NSG mice. CD34+CD133+ cord blood cells were
either expanded in the MSC-A5 co-culture or the FCF culture for 11 days. Total
expanded cells, containing 105 DP cells, were engrafted into sublethally irradiated
NSG newborn pups. Fourteen weeks later, the presence of different human blood
lineage cells in the peripheral blood, spleen and BM were analyzed by flow cytometry.
Among various human blood lineage cells analyzed, T cells (CD3+), B-lineage cells(CD19+ or CD19+IgM+), macrophages (CD14+CD33+), myeloid precursor cells(CD33+), hematopoietic stem/precursor cells (CD34+ or CD34+CD133+), DCs(CD11c+ HLA-DR+), neutrophils (CD15+), and NK cells (CD56+) were detected in the
blood, spleen or BM (Figure 4 and data not shown). Importantly, the levels of various
human cell reconstitution, both in terms of median percentage and the range among
different recipient mice, were similar regardless of the conditions used to expand
CD34+CD133+ cord blood cells (Table 1), except significantly higher percentages of
CD3+ T cells were observed in the blood and spleen of recipient mice that were
injected with cells from MSC-A5 co-culture than the FCF culture (p<0.05). H&E
staining revealed a restoration of the splenic architecture with the appearance of thewhite pulp in mice reconstituted with co-cultured cells, but not in unreconstituted NSG
mice (Figure 5). Furthermore, T cells and B cells were found in the same follicles, with
CD3+ T cells residing predominantly in the inner region of the follicles, whereas
CD20+ B cells resided in the outer region of the follicle. These results suggest that
expanded cells from the co-cultures are capable of differentiating into multiple
lineages of blood cells in NSG mice.
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3.2.4 Expanded cells are capable of stable long-term and efficient secondary
reconstitution!
The expanded cells were tested for their ability to give rise to stable long-termreconstitution in NSG recipients. Following 11-day expansion in either MSC-A5 coculture or FCF culture, expanded cells were transferred into sublethally irradiatednewborn NSG pups. By 12 weeks following engraftment, the percentages of humanleukocytes in the blood were less than 50% (Figure 6A). These percentages increasedslightly or were maintained at 24 weeks after engraftment (Figure 6A, B). No
difference in reconstitution was observed between expanded cells from MSC-A5 coculture or FCF culture in the blood and spleen (Figure 6A). The level of T cellreconstitution in both blood and spleen was higher with expanded cells from co-culture
than FCF culture (data not shown). These results suggest that expanded cells from bothprotocols possess similar levels of long-term reconstitution activity.
We also assessed the presence of long-term HSC activity in the expanded cell
population by serial transfer. Expanded cells from day 11 MSC-A5 co-culture weretransferred into irradiated NSG pups. Fourteen weeks after engraftment, humanCD34+ cells were magnetically enriched from the BM of primary recipient mice, andinjected into sublethally irradiated adult NSG mice. Twelve to 16 weeks after, BM
cells of the secondary recipient mice were analyzed by flow cytometry for human andmouse CD45+ cells. Among the 14 secondary recipient mice, 11 had 0.1-2% of
human CD45+ cells in the BM, one had 11% and the

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other two had ~0.03%. A representative mouse is shown in Figure C. These data showthat the expanded CD34+CD133+ cells from MSC-A5 co-culture retain a secondary
reconstitution potential, suggesting the presence of long-term HSC activity.
3.2.5 Human T cells are functional in the reconstituted mice
The increased T cell development with co-culture expanded DP cells prompted us totest if the T cells are functional. Similarly reconstituted mice with expanded cells fromeither co-culture or FCF culture were immunized with TT and boosted twice at aninterval of 3 weeks. Two weeks after the last immunization, splenocytes were assayedfor IFN- expression by ELISPOT assay. Without immunization, significant numbersof IFN- immunospot were detected only if splenocytes were stimulated with PMA
during the assay, regardless of whether the cells were from mice reconstituted withexpanded cells from the co-culture or the FCF culture (Figure 7A). Similarly,
significant numbers of spots were detected in PMA-stimulated splenocytes from
immunized mice, regardless whether reconstituted with expanded cells from the coculture or the FCF culture. As expected, with TT stimulation of splenocytes in theculture, significant numbers of spots were detected only in TT immunized mice.
However, the average numbers of spots were significantly higher in mice reconstituted
with cells from the co-culture than the FCF culture (104 ± 37 vs. 22 ± 14, P < 0.005).
These results suggest that enhanced T cell development leads to an enhanced T cell
response in mice reconstituted with co-culture expanded cord blood cells.

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3.3 Discussion
Here we report an improved method to expand HSCs from umbilical cord blood by
combining the HSC growth factors recently identified by Zhang et al. 2010 with a
BM-derived MSC feeder layer. We reasoned that by adding the growth factors into
the co-cultures it might be possible to further enhance HSC expansion while
minimizing differentiation. By all measures, this approach worked significantly better
than the FCF culture or co-culture without the selected growth factors. In our
experiments, CD34+ CD133+ cells from the same cord blood donor were expanded
using the three different conditions, and the fold expansion of DP cells was monitored
because we have shown previously that the SRCs reside in the DP cell fraction.
Compared to FCF culture, which expanded DP cells 20-25 fold during 11-14 days,
co-culture with MSC-A5 expanded DP cells 60-150 fold, whereas co-culture without
Angptl5 (MSC-GFP) expanded DP cells 28-60 fold during the same period. Theseobservations were further supported by results from experiments where different
growth factors were added into the MSC-GFP co-culture. It is noticeable that the coculture system expanded the SRCs, as measured by CD34 and CD133 expression,
faster than FCF cultures and retained a higher proportion of CD34+ (47% vs. 20% on
day 14) and CD34+CD133+ cells (15% vs. 9% on day 14), producing 150 fold ofexpansion of DP cells by day 14. These findings suggest that both MSCs and theselected growth factors, especially Angptl5 and IGFBP2, contribute significantly tothe expansion of CD34+ CD133+ cord blood cells in the co-culture.
Different from previous co-culture studies, we rigorously evaluated the reconstitution
activity of expanded cells from the co-culture in long-term and serial reconstitution.
We show that the expanded cells were able to support stable long-term (6 months)

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reconstitution. When human CD34+ cells were isolated from the BM of primary
recipient mice and transferred into second recipient mice, 12 of 14 recipients had
more than 0.1% human cells in the BM 12-16 weeks later. These results suggest thatsome of the expanded cells still possess the long-term HSC activity.
MSCs have been shown to stimulate differentiation of HSCs. With the recent
development of the NSG mouse model that supports robust lymphoid engraftment, we
examined the ability of the co-culture expanded cells for T and B cell reconstitution.
While unexpanded or expanded CD34+CD133+ cells from the FCF culture give robust
B cell but poor T cell reconstitution initially, we show that the co-culture expanded
cells gave significantly enhanced T cell reconstitution, reaching the normal T to B cellratio as in humans and mice. The observed T cell-bias was opposite to the effectreported by Giassi et al. (Giassi et al., 2008), further suggesting a critical role of MSC
in regulating differentiation in the co-culture. Given that we did not observe any
reduction in long-term repopulation capacity, the T cell-biased reconstitution suggeststhat MSCs also support progenitor cell expansion. Furthermore, the expanded T cell
population significantly enhanced the antigen-specific T cell response in micereconstituted with co-culture expanded cord blood cells as assayed by ELISPOT.
However, whether the improvement in cytotoxic T cell response is due to theincreased T cell number or improved functionality is still not known.
In both clinic and laboratory, the supply of HSCs is limiting with little prospect offinding significant new sources of cells. Strategies to make the existing sources ofcells go further are essential for both improving treatments of hematologic
malignancies, and our understanding of human hematology and immunology. While
progress is being made with strategies to improve delivery of stem cells, ex vivo
expansion of HSCs is the key to improving clinical outcomes. In the first clinical trial,46!
transfer of unexpanded and expanded cells from different donors together
significantly reduced the period of neturopenia following transplantation (Delaney etal. 2010). However, over time the graft was universally dominated by unexpanded
cells in all patients. In another clinical trial, HSCs from umbilical cord blood wereexpanded by approximately 12-fold on donor-related MSCs and injected in 6 cancer
patients. Five out of the six patients were in complete remission after one year of theengraftment (Kelly et al., 2009). The improved approach for expanding HSCs
reported here not only solves one bottleneck for the humanized mice research field togenerate large numbers of humanized mice from the same HSC source for large-scalepreclinical studies, but also provides a promising method to expand HSCs fortransplantation.

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CHAPTER 4

HUMAN M-CSF MOBILIZES PROMONOCYTES IN BM
AND RESTORES THE FUNCTIONS OF HUMAN TISSUE
MACROPHAGES IN HUMANIZED MICE!
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A manuscript is being prepared for this work and LI Yan is the co-first author with
Dr. Qingfeng Chen on this paper. LI Yan designed and performed all the experiments,
analyzed and interpreted the data, and wrote the manuscript.!

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4.1 Introduction
4.1.1 The problem of human monocyte/macrophage development in NSG mice
A major avenue for improvement of current humanized mice is to generate mice witha functional immune system of human origin. To achieve this, a complete
hematopoietic system is a prerequisite. In NSG mice, human T and B cells can stably
reconstitute the mice for more than one year while NK cell reconstitution, which wasvery poor in NSG mice, is also improved by expression of human IL-15 and Flt3 (orflk-2) (Chen et al., 2009; Hiramatsu et al., 2003; Ishikawa et al., 2005; Shultz et al.,2005; Yahata et al., 2002). Nevertheless, myeloid lineage cells, such as
monocytes/macrophages, granulocytes, megakaryocytes and erythrocytes, are poorly
developed and maintained in humanized mice (Ishikawa et al., 2005; Legrand et al.,2006; Manz, 2007; Shultz et al., 2007). The defect of human myeloid development in
NSG mice has been speculated to be the result of an inefficient myelopoiesis, and a
relatively intact mouse myeloid compartment (Macchiarini et al., 2005; Manz, 2007;
Shultz et al., 2007). In view of the fact that myeloid cells play a vital role in oxygen
transport (erythrocyte), blood coagulation (megakaryocytes) and development ofefficient immunity against infectious pathogens (granulocyte and macrophages),
generation of humanized mice with efficient human myelopoiesis is of paramountimportance.

4.1.2 The role of human monocytes/macrophages in immune responses
In this part of the thesis, we will focus on correcting the defect of humanmonocyte/macrophage development in humanized mice. Monocytes/macrophages,
along with DCs, form the mononuclear phagocytic system (Murray and Wynn, 2011).
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The mononuclear system is derived from common myeloid precursors in BM,
circulates in the blood as monocytes, and populates tissues as macrophages in thesteady state and during inflammation. Monocytes function as immune effector cells,
and do not proliferate in a steady state (Auffray et al., 2009a; Auffray et al., 2009b).
The expression of chemokine receptors and adhesion receptors on monocytes
facilitate their migration from blood circulation to sites of inflammation. In the courseof migration to tissues, they produce inflammatory cytokines and differentiate into
inflammatory DCs or macrophages. Even in the steady state, monocytes are believedto serve as a pool to replenish tissue macrophages and DCs (Serbina et al., 2008).
Macrophages are resident phagocytic cells in both lymphoid and non-lymphoidorgans. They participate not only in the initiation and resolution of immune response
during tissue injury or infection, but also play a critical role in steady-state tissue
homeostasis, via the clearance of apoptotic cells, and the production of growth factors(Geissmann et al., 2010). In addition, there is accumulating evidence that many
diseases may be caused by dysregulated macrophage function, such as adipose tissue
macrophages in metabolic disorders, M1-like macrophages in atherosclerosis, andKupffer cells in liver fibrosis (Murray and Wynn, 2011). The study of macrophage
biology would greatly help to develop therapies for the above diseases. Nevertheless,the developmental origin and the function of tissue macrophage subsets, such as
microglia, dermal macrophages, alveolar macrophages, Kupffer cells, splenicmarginal zone and metallophilic macrophages remain insufficiently understood(Geissmann et al., 2010).

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4.1.3 Cytokines and monocyte/macrophage development
The development of a mononuclear phagocyte system is governed by cytokines: small
secreted proteins that promote cell-cell communication, and can act as growth anddifferentiation factors. The generation of monocytes and macrophages, and to someextent DCs, is dependent on cytokine M-CSF (or CSF-1), which was originallydefined by its ability to generate macrophage colonies when added to cultures of BM
progenitor cells in vitro (Stanley et al., 1975). Subsequent discovery of
CSF1op/CSF1op mouse, which carried a spontaneous mutant lacking a functional Csf1
gene, showed the indispensable role of M-CSF in the generation and maintenance ofmonocytes and tissue macrophages (Cecchini et al., 1994; Yoshida et al., 1990). At
which point the M-CSF is critical in macrophage development is still debated (Lenzo
et al., 2011). In the op/op mice, there is severe depletion of monocytes in circulation.
The production of macrophage colony-forming cells and monocytic cells(monoblasts, promonocytes and monocytes) is also defective in BM, and various
tissue macrophages are deficient (Felix et al., 1990a; Felix et al., 1990b; WiktorJedrzejczak et al., 1992; Wiktor-Jedrzejczak et al., 1991; Wiktor-Jedrzejczak et al.,1982). This evidence together suggests that the absence of M-CSF causes themacrophage development to be blocked at an early myeloid progenitor stage.
However, a few immature macrophages are present in op/op mice, which are believed
to be M-CSF independent macrophages derived from granulocyte/macrophagecolony-forming cells or earlier hematopoietic cells with the help from GM-CSF and
IL-34 (Naito et al., 1991; Wei et al., 2010; Wiktor-Jedrzejczak et al., 1992). Anotherimportant cytokine for monocyte/macrophage development is GM-CSF, which was
also found in the in vitro BM cell culture to generate both granulocyte andmacrophage colonies. However, unlike M-CSF, the deficiency in GM-CSF does not51!
compromise the steady-state myelopoiesis and most tissue macrophage populations.
The major phenomenon observed in GM-CSF deficient mice is the pulmonary
alveolar proteinosis because of a defect in alveolar macrophage maturation (Stanley etal., 1994). Taken together, this evidence supports the argument that M-CSF plays amajor role in monocyte/macrophage development and acts at the early stages of
myeloid development, whereas the role of M-CSF, GM-CSF and IL-34 in humanmacrophage development is still unclear due to the lack of a proper model to study.

4.1.4 Past efforts to improve monocytes/macrophages in humanized mice
Of note, the understanding of mouse tissue resident macrophages does not often apply
to human macrophages because homotropic pathogens are predominantly or solely
found in humans, and different sets of molecules are employed by humanmacrophages to control infections (Murray and Wynn, 2011). To translate knowledge
obtained from animal models to better understand human macrophages and their rolein associated diseases, humanized mice may offer an important in vivo readout if theengraftment and function of human macrophages reach a comparable level to humans.
Moreover, the “human” innate and adaptive immune responses in current humanized
mice, which are measured without the participation of human macrophages, are not anatural response as it is in humans and may hinder the application of humanized micefor preclinical testing. Hence, there is a great need to improve the reconstitution oftissue resident macrophages in humanized mice.
As certain murine cytokines critical for development of monocytes/macrophages,
such as M-CSF, GM-CSF and IL-34, do not act on human counterparts (Fixe andPraloran, 1997; Ginhoux and Merad, 2010; Manz, 2007; Metcalf, 1986; Stevenson
and Jones, 1994; Wei et al., 2010), we used hydrodynamic tail vein injection to52!
introduce human GM-CSF or M-CSF into humanized mice and significantly
improved the reconstitution of human DCs and monocytes in peripheral blood andvarious organs, respectively (Chen et al., 2009). However, to what extent thistreatment would restore the population and function of human tissue macrophages arestill uncharacterized. Billerbeck et al. 2011 created NSG mice transgenically
expressing human SCF, GM-CSF and IL-3 (NSG-SGM3), and then humanized themwith CD34+ human fetal liver (FL) cells (Billerbeck et al., 2011). Although thefrequencies were elevated, human myeloid cells were still mostly found in the BM,
but few in the peripheral organs. Subsequently, several other groups created human
GM-CSF/IL-3 or M-CSF gene knock-in humanized mice to improve the engraftmentof monocytes/macrophages (Rathinam et al., 2011; Willinger et al., 2011). Despitemuch progress that has been made in improving the engraftment of human monocytes
in peripheral blood of these cytokine-supplemented mice, there is lack of direct
evidence of improved reconstitution and function of human tissue macrophages.
Moreover, a clear explanation of the problem of monocytes/macrophage developmentin humanized mice is still lacking.

4.1.5 Improvements of human macrophage reconstitution and immune function
in humanized mice
Based on the hypothesis that human monocyte/macrophage development is impairedby cytokine cross-reactivity, we analyzed the BM of humanized mice and identified a
unique large population of SSChighCD14lowCD45+ human monocyte precursors. Thesecells could be differentiated in vitro and in vivo to monocytes and macrophages when
cultured with human GM-CSF or M-CSF. By hydrodynamic injection of plasmids
encoding human M-CSF, we were able to generate significant levels of tissue resident

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macrophages in various organs in humanized mice compared to untreated mice.
Furthermore, enhanced engraftment and functionality of monocytes/macrophages
leads to substantial improvement in human immune responses in M-CSF-treated micecompared to untreated mice.

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4.2 Results
4.2.1 Human monocyte and macrophage development is blocked in BM of
humanized NSG mice
To generate humanized mice, sublethally irradiated newborn pups were injected via
intracardiac route with CD34+ HSCs isolated from human FL. Twelve weeks after
injection, human monocyte and macrophage reconstitution was examined inperipheral blood and various organs. Among human CD45+ MNCs, the levels of
CD14+CD11b+ monocytes ranged from 0% to 6% in blood. Although monocytes weredetected in blood and organs, their frequency was relatively lower than that incorresponding human tissues and murine tissues (Figure 8A) (Fluks, 1981). To
determine the frequency of tissue resident macrophages, CD14+CD68+ cells were
examined in spleen, BM, lung and liver (Figure 8B). Most CD14+ cells did not
express the macrophage marker CD68 in the spleen, lung and liver. This suggests thathuman monocytes cannot efficiently differentiate into macrophage in humanizedmice.
One important observation is that a large CD14low population was present only in BM
but not in other organs or blood. Along with this observation, we also found a uniquepopulation with high side scatter (SSC) in BM (Figure 9A). The average number ofthis population per mouse BM (one tibia) was ~3 million, which is ~10% of humancells in BM. To find out the relationship between these two populations, the CD14low
population was gated, and traced back to forward and side scatter characteristics(FSC/SSC) gating. As a result, >80% of CD14low cells are SSChigh cells (Figure 9B).
Since both the low expression of CD14 and high SSC are the characteristics of55

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promonocytes (van Lochem et al., 2004), we hypothesized that the monocyte
development in BM stopped at the promonocyte stage and hence monocyte could not
be released into the peripheral.

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4.2.2 Human myeloid differentiation stops at monoblast to promonocyte stage inthe BM of humanized mice
To further confirm the developmental stage of this unique population in BM, SSChighcells were purified (>90%) to check their morphology by Giemsa staining (Figure
10A). The majority of these cells had an oval or indented nucleus with several
nucleoli (Figure 10B). This is in agreement with the high SSC and suggests they arepromonocytes. In addition, the phenotype of these cells was CD45+CD34CD13+CD33+CD15+CD11b+CD14low, which resembled the surface expression aspromonocytes (van Lochem et al., 2004) (Figure 10C). Quantitative RT-PCR analysisof these cells also showed that they expressed myeloid genes (e.g. EGR-1, PU.1,
VDR and LZM), and the levels of expression were lower than the levels in CD14+
cord blood monocytes (Figure 11). This suggests that these BM CD14low cells belong
to the myeloid lineage. Their low expression of the early myeloid commitment genec-myb, and the expression of the differentiation gene C/EBP further restrained thedevelopmental progression to post-monoblast stage (Valledor et al., 1998). As
myeloid cells are known to down-regulate the expression of MPO after maturing tomonocytes (Lubbert et al., 1996), the relatively high expression of MPO suggests
these cells are still at an immature stage. Based on these results, we conclude theseimmature cells in BM are CD14lowSSChigh promonocytes. Hence, the accumulation ofpromonocytes and lack of mature human macrophages in peripheral blood strongly
suggests that human monocyte/macrophage development in humanized mice may beblocked at the promonocyte stage.

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found.

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4.2.3 Human monocyte/macrophage development is restored by cytokine
treatment
Human monocyte/macrophage lineage commitment and maturation rely on serial setsof cytokines. GM-CSF, M-CSF and IL-34 are one set playing a critical role in thedevelopment of granulocyte and monocyte precursor (CFU-GM) (Valledor et al.,1998). However, mouse GM-CSF, M-CSF and IL-34 are not cross-species active onhuman cells (Manz, 2007). Hence, we also performed the colony-forming cell assay
to obtain information on the frequency and types of progenitors present in the SSChighpopulation, and their ability to proliferate and differentiate when supplemented withhuman cytokines. Cells were plated in enriched MethoCult, which supports the
optimal growth of erythroid progenitors, including colony-forming-unit-erythroid(CFU-E) and burst-forming-unit-erythroid (BFU-E);

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granulocyte/macrophage progenitors, including colony-forming-unit-granulocyte
macrophage (CFU-GM); colony-forming-unit-granulocyte (CFU-G) and colonyforming-unit-macrophage(CFU-M);andmultipotential-colony-forming-unit-

granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM). Two weeks later,
only CFU-M was detected under the microscopy with a frequency of 1/220 (Figure
12B). Many small CFU-M like colonies with less than 20 cells were observed but notcounted. These colonies may be derive from monoblasts and promonocytes, whichhave limited proliferation potential. Hence, it is likely that SSChigh population is
predominantly promonocytes, but may also contain a few monoblasts.

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The finding that SSChigh population comprised monocyte/macrophage progenitors,
and that human monocytes developed in vitro in the presence of M-CSF and GMCSF/IL-4 suggests that these human cytokines may also drive the differentiation ofSSChigh populations to mature monocytes in vivo. To introduce human cytokines into
mice, we used hydrodynamic delivery of cytokine-encoding plasmids. 7 days after asingle injection of 50 µg human M-CSF or GM-CSF encoding plasmids,
promonocytes disappeared and abundant human CD11b+ monocytes were presented
in blood, while promonocytes and blood monocytes remained unchanged in untreatedcontrol mice and in mice injected with human IL-4 encoding plasmids (Figure 13).
The percentages of human CD11b+ monocytes also significantly increased in the
spleen, BM, liver and lung in M-CSF or GM-CSF treated mice, but not in IL-4 treatedmice (Figure 14A). Human monocytes/macrophages induced by M-CSF are known toexpress high CD14, whilst GM-CSF or IL-4 stimulated monocytes down-regulate theexpression CD14 and exhibit accessory cell functions like DCs (Liu et al., 2001;
Ruppert et al., 1991; Shalhoub et al., 2011). The same phenomenon was observed inour cytokine-treated humanized mice. To further explore whether the increased
monocytes in M-CSF and GM-CSF treated mice were macrophages, we purified
CD45+CD11b+ cells from splenocytes of GM-CSF treated mice and M-CSF treatedmice, and stained these cells with Giemsa. These sorted cells from both GM-CSF
treated mice and M-CSF treated mice exhibited typical macrophage morphology, i.e.
large in size, round shape and heavily vacuolated (Figure 14B). Based on the results
obtained, we found promonocytes could be mobilized by GM-CSF or M-CSF
treatments in the human monocyte/macrophage development in humanized mice.

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4.2.4 Human tissue resident macrophages are generated in M-CSF treated mice
Since CD14+CD11b+ monocytes/macrophages were significantly increased in M-CSF
treated mice, and the sorted CD11b+ cells exhibited macrophage morphology in both
GM-CSF treated mice and M-CSF treated mice, we further analyzed the M-CSF
treated mice for tissue resident macrophages. M-CSF treated mice were perfused withsaline, and their organs including brain, spleen, lung and liver were harvested. Single
cell suspensions were prepared, and stained with tissue resident macrophage marker
CD163 and CD68. Interestingly, untreated humanized mice did not reconstitute wellwith CD14+ cells, and also most of CD14+ cells did not express the macrophage
marker CD68 and CD163. These results suggest that monocytes/macrophages
naturally developed in humanized mice cannot mature into tissue resident
macrophages. As expected, the frequencies of human CD14+CD68+ andCD14+CD163+ cells were significantly increased in all organs of M-CSF treated mice
(Figure 15).
To further support the findings that tissue resident macrophages can develop in MCSF treated mice, histology analysis of liver, lung and kidney was performed on
saline-perfused M-CSF treated mice. Consistent with the flow cytometry data,
untreated humanized mice showed no human CD68+ cells in the examined organs, but
a considerable number of mouse CD68+ cells were observed in the lung and liver(Figure 16A). In contrast, the lung and liver of M-CSF treated mice were substantiallyreconstituted with human CD68+ macrophages (Figure 16B). Of note, these M-CSF
induced macrophages selectively reconstituted in the liver and lung, but not in thekidney. Their frequency and distribution patterns were similar to their mousecounterparts. Taken together, these data demonstrate that tissue resident macrophages

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successfully reconstitute M-CSF treated mice and were strategically located inselected target organs.

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4.2.5 Human inflammatory responses are enhanced in M-CSF treated mice
To investigate whether the induced monocytes and tissue macrophages in M-CSF
treated mice were functional, we injected LPS i.p. to induce systemic inflammation. 2hours and 12 hours after injection, mice were bled and the serum levels of humanproinflammatory cytokines were determined by CBA assay. Consistent with theincreased frequencies of monocytes and macrophages in M-CSF treated mice,significantly elevated levels of IL-6, TNF-, IL-10, IL-1 and IL-8 were detected in
the M-CSF treated mice (Figure 17). Of note, the basal levels of these cytokines were
the same between M-CSF treated mice and untreated mice, which indicated thequiescent status of newly generated monocytes and macrophages in M-CSF treatedmice. Overall, human inflammatory responses mediated by human monocytes andmacrophages were enhanced with M-CSF treated mice.

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4.2.6 M-CSF treated mice mount enhanced immune response against influenza A
virus
After we have shown the presence of tissue resident macrophages and their responses
to LPS, we further used influenza A virus infection as a model to test whether thehuman alveolar macrophages can provide innate immunity to a natural pathogen in
M-CSF treated mice. We infected mice intratracheally with 300 pfu influenza A/PR8(H1N1) virus, and sacrificed them at 24 h, 48 h, and 72 h post-infection for various
tests. The influenza virus RNA was examined at each time-point by quantitative PCR
of lung homogenates and normalized to mouse gene L32 expression. The viral levels
were significantly lower in M-CSF treated mice than untreated mice during the courseof infection (Figure 18A). However, the difference of virus load between M-CSF
treated mice and untreated mice became smaller as the infection progressed. To
investigate if the decreased virus load was the results of improved human macrophage
reconstitution in the lung, we carried out several experiments to assay humanmacrophage response to influenza infection. First, the human alveolar macrophages
obtained from M-CSF treated mice displayed enhanced activation as assessed by the
upregulation of CD80 costimulatory molecules at 24 h post-infection (Figure 18B).
Second, in addition to the increased frequency of human alveolar macrophages (asdemonstrated in Figure 15 and Figure 16), their absolute numbers were significantly
higher throughout the course of infection (Figure 18C). Third, consistent with theincreased number and functionality of alveolar macrophages in M-CSF treated mice,significant levels of proinflammatory cytokines, such as IL-6, TNF- and IL-1,were detected both at protein and RNA levels in lung homogenates between 24 h and
72 h post-infection in M-CSF treated mice (Figure 19). In contrast, untreated mice
showed no significant inductions of proinflammatory cytokine during the course of70

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influenza A infection. Similarly, as in LPS condition, the fact that only basal levels of
proinflammatory cytokines were detected before infection supported that alveolar
macrophages were in resting status in M-CSF treated mice. Taken together, these data
show that M-CSF treated mice can exhibit enhanced immune response against
influenza virus due to improved human macrophage chimerism and functions in the
lung.

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Other interesting results were observed in influenza infected M-CSF treated mice.
Unlike LPS stimulated human macrophages that secreted IL-10 into serum, human
IL-10 protein was not detected both in lung homogenate and serum samples of MCSF treated and untreated mice after influenza infection (data not shown). However,mRNA expression of human IL-10 showed a higher level and an increasing trend in
M-CSF treated mice compared to untreated mice (Figure 20A). Activated antigen
specific T cells and tissue resident macrophages are the main producers of IL-10
during influenza virus infection to suppress the inflammation and prevent injury totissue. Since the specific role of IL-10 in the acute influenza infection is still unclear,
M-CSF treated mice may offer a tool to study the regulation and function of thiscytokine. Furthermore, since alveolar macrophages are the main type-I IFN producer
cells after infection with pulmonary viruses, we also measured the IFN- mRNA inlung homogenate. M-CSF treated mice had a steadily increasing trend of human IFN mRNA expression during the course of infection compared to untreated mice
(Figure 20B) (Kumagai et al., 2007). During influenza A virus infection, macrophages
function also as accessory cells by producing chemokines and cytokines to promote T
cell responses to clear the viruses. In M-CSF treated mice, an increasing trend of
human IL-12 mRNA expression correlated with the increased production of IFN-
mRNA most likely because macrophages secrete IL-12 to stimulate the production of
IFN- by NK cells and cytotoxic T cells (Desmedt et al., 1998; Murray and Wynn,2011) (Figure 20C). In untreated mice, both IL-12 and IFN- production decreased
from 24 h post-infection.

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4.2.7 Human peritoneal macrophages are recruited during Mycobacterium
infection in M-CSF treated mice
Macrophages are critical components of the innate and adaptive immune responses tobacterial pathogens. Infection of mice with the intracellular bacterium BCG via thei.p. route has served as a classic system for the study of macrophage activation(Hamerman and Aderem, 2001). We have demonstrated the systemic responses ofmonocytes and macrophages to LPS challenge, and the local response of tissue
resident macrophages to influenza A virus infection. Here, we took the advantage of
BCG infection as a model to test the ability of monocytes to migrate to inflammatory
sites and to differentiate into activated macrophages. 5 million CFUs of BCG were
injected into mice. Mice were sacrificed at 24 h and 72 h post-infection. Peritoneal
lavage and spleen homogenates were collected for BCG colony assays. Significantly
lower numbers of BCG colony counts found in the spleen and peritoneal lavage
samples of M-CSF treated mice (Figure 21A). Moreover, human CD14+CD11b+
macrophages were present only in M-CSF treated mice at 24 h post-infection (Figure
21B). These macrophages may be derived from activated monocytes recruited fromthe peripheral blood in response to BCG stimulation. However, the majority of
peritoneal cells were still of mouse origin (>80%) during the course of infection. We
also extracted RNA from peritoneal cells and examined the expression of human
inflammatory cytokines (Figure 21C). Only M-CSF treated mice showed increased
expression of IL-6 and IL-1 at 24 h post-infection. Taken together, a proportion ofhuman monocytes in M-CSF treated mice can be recruited to inflammatory sites todifferentiate into macrophages to control the growth of BCG.

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4.3 Discussion
The ability to study human hematopoiesis with in vivo settings is one of theadvantages of humanized mice. Given that cytokines are important for humanmonocyte/macrophage development (e.g. M-CSF, GM-CSF and IL-34), and are
species-specific (Manz, 2007; Wei et al., 2010), humanized mice may be regarded asa ‘human knockout model’ to study the impact of these cytokines. GM-CSF induces
proliferation and differentiation of intermediate myeloid precursors, while M-CSF
induces not only the proliferation and differentiation of late myeloid precursors but
also the maturation of monocytes into macrophages (Ogawa, 1993). IL-34 increases
monocytes viability, induces macrophage proliferation, and synergized with othercytokines to generate macrophages and osteoclasts from cultured progenitors. Basedon the functions of these three cytokines, human myelogenesis would stop at CFUGM or CFU-M stages. However, as described in our results, the characterization ofhumanized mice revealed that there was a unique human cell population in BM, andhuman monocyte/macrophage development was blocked mainly at the promonocyte
stage. Hence, it would be necessary to reevaluate our knowledge obtained frommouse experiments and in vitro data, and human monocyte/macrophage progenitors
might exploit other cytokines other than GM-CSF and M-CSF for differentiation topromonocytes in vivo.
Studies on op/op mice, which are deficient for mouse M-CSF, reveal that thehematopoietic system has the capacity to use alternative mechanisms to compensate
for the absence of M-CSF probably by the effect of GM-CSF and IL-34 (Begg et al.,
1993; Nilsson and Bertoncello, 1994; Wei et al., 2010). Hence, at least one of three78

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cytokines has to be presented for monocyte/macrophage development. With
supplementation of human GM-CSF or M-CSF by hydrodynamic injection, theblocked development could be restored and monocytes/macrophages could be
efficiently generated. Human monocytes/macrophages induced by M-CSF are known
to express high CD14, whilst GM-CSF or IL-4 differentiated monocytes downregulate the expression of CD14 and exhibit accessory cell functions like DCs(Ruppert et al., 1991; Shalhoub et al., 2011). The same phenomenon was observed inour cytokine treated humanized mice. Absence of both M-CSF and GM-CSF would
hinder human monocyte/macrophage development in later stages rather than earlystages of hematopoiesis, and the ratio of expression of these 2 cytokines affects thehomeostasis of monocytes, macrophages and DCs.
In agreement with the indispensable role of M-CSF in generation of mouse tissue
resident macrophages in organs from studies on op/op mice (Cecchini et al., 1994),
various human tissue macrophages were significantly induced after M-CSF treatment.
Previous efforts to improve human monocyte/macrophages, such as human GMCSF/IL-3 or M-CSF knock-in Balb/c Rag2-/-c-/- mice, have increased levels ofalveolar macrophages and circulating monocytes compared to control mice,
respectively (Rathinam et al., 2011; Willinger et al., 2011). However, tissue
macrophages other than alveolar macrophages, and circulating monocytes in GMCSF/IL-3 knock-in mice were not significantly improved. Although high percentagesof circulating monocytes were achieved in M-CSF knock-in mice, the highest level oftissue monocyte/macrophage reconstitution was still under 5% of human CD45+ cells.
There could be several reasons for the limited improvements seen in these knock-in
mice. First, these mice were created on Balb/c Rag2-/-c-/- mice, which do not have
the SIRP- polymorphism like NSG mice. The functional mouse macrophage would79

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prevent high levels of reconstitution of human cells in tissues. Hence, the developing
tissue macrophages and monocytes could be phagocytosed by mouse macrophages in
M-CSF and GM-CSF knock-in mice. Second, M-CSF is not only produced byendothelial cells and fibroblasts, but also by monocytes, granulocytes. Moreover,mouse cells also consume human M-CSF. Poor tissue macrophage reconstitution in
M-CSF knock-in mice may be the result of competition between human and mouse
macrophages for the less than optimal amount of human M-CSF produced frommouse nonhematopoietic cells. Hence, a useful control to compare the human M-CSF
levels in serum of knock-in mice would be the endogenous concentration of mouse
M-CSF in serum of wild type mice rather than immunodeficient mice. As we usedhydrodynamic injection to deliver cytokines into NSG mice, we circumvented theabove problems confronted by knock-in mice. Although hydrodynamic injection
provides short-term and variable levels of expression in vivo, the levels of expression
are much higher in hydrodynamic condition compared to the levels in knock-in
condition. This excessive amount of cytokines guarantees that both human and mousemonocyte and macrophages could be efficiently generated. Hence, both high levels of
circulating monocytes and different types of tissue macrophages were induced after asingle hydrodynamic injection of M-CSF plasmids. This method renders researchers arapid approach to test the effect of one or several secreted proteins in mice.
Furthermore, the level and duration of the expression of gene expression can be
optimized in hydrodynamic injection by the amount of plasmids, choice of thepromoters, and number of injections. Therefore, hydrodynamic injection of M-CSF
encoding plasmids into NSG mice leads to high levels of human tissue resident
macrophage reconstitution, and the tissue macrophage in NSG mice offers a uniqueand powerful tool to study the development of human monocyte/macrophage80!
development and compare the human macrophage with their cognate mouse model
systems. This is an area with great potential, as the chasm between understanding
human and mouse macrophages is wide.
Along with the improved reconstitution of human monocytes and tissue macrophages,
M-CSF treated humanized mice also exhibit significantly stronger proinflammatory
response to LPS and enhanced protection against influenza virus and BCG infection.
The levels of proinflammatory cytokine induced in M-CSF treated mice are ~46 foldand ~34 fold higher than untreated mice for IL-6 and TNF- at 2 hour post-injection,
respectively. The LPS stimulation in current knock-in mice results in only 2-3 foldhigher level of IL-6 than control humanized mice (Rathinam et al., 2011; Willinger etal., 2011). In addition, we first reported the enhanced immune response against
influenza virus and BCG infection in humanized mice. No difference of viral load in
the course of infection was seen between GM-CSF/IL-3 knock-in mice and control
mice, whereas significant lower level of viral load was seen between M-CSF treatedmice and untreated mice. In BCG infection, the numbers of BCG colonies weresignificantly lower in spleen and peritoneal of M-CSF treated mice than untreatedcontrol during the period measured. These improvements should be attributed tohigher engraftment and better function of monocytes and tissue macrophages.
Despite the presence of tissue resident macrophages and enhanced responses against
infections, the virus burden and bacteria were still significant in M-CSF treated miceover the course of infection. This could imply that resident macrophages alone areinsufficient to control the infection, and that subsequent T and B cell responses areneeded to clear the infection (McGill et al., 2009). In M-CSF treated mice, morehuman T cells could be found in lung after 3 days of infection (data not shown). This81

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suggests functional macrophages may secrete chemokines and cytokines to attract
more T cells and induce their differentiation into effector cells. To prove thesehypothesis, in vitro experiments, such as mixed leukocyte reaction, need to be
conducted to test the function of human T cells and macrophages from M-CSF treatedmice in the future. In vivo experiments, such as immunization of tetanus toxoid andinfection of influenza virus, also need to be carried out to test human B cell and T cell
response in M-CSF treated mice. With information from these experiments, not only
the functionality of M-CSF induced macrophages could be proved, the puzzle aroundhuman T cell activation in humanized mice may also be answered. If humanmacrophages in M-CSF treated mice were capable of stimulating human T cells in
vitro but not in vivo, it would be necessary to investigate the impact of missing lymph
nodes in humanized mice on efficient human immune responses. In addition, mostcells in the peritoneal lavage after BCG infection were of mouse origin and mouse
tissue resident macrophages were coexisting with human ones by histologicalanalysis. Further improvement of this model by temporary depleting mouse
macrophages before hydrodynamic injection may remove the undesired mouse
macrophages and make humanized mice a promising tool to model humantuberculosis.
Another limitation of this approach to improve human tissue macrophage inhumanized mice is that several types of macrophages are M-CSF independent. The
results have shown that macrophages in brain and kidney of humanized miceincreased to a much less extent than macrophages in other organs. This suggests MCSF do not have a critical impact on the development or proliferation of these typesof macrophages. However, this limitation could be easily overcome by injecting othercytokines or combining other cytokines with M-CSF. GM-CSF and IL-34 might be82!
interesting candidates to test. A more challenging and significant study would be
improving human macrophage populations, which originate from non-hematopoietic
progenitors, such as embryonic progenitors. Current humanized mice permit highlevels of engraftment of human hematopoietic cells but the engraftment of cells
derived from human tissues or embryos are generally poor. To improve theengraftment of macrophages, such as Langerhans cells, a subpopulation of microglia
and Kuffer cells, which develop in the embryo before the appearance of definitive
HSCs, humanized mice have to be further improved to achieve stable and high levels
engraftment of nonhematopoietic cells of human origin (Ginhoux et al., 2010;
Ginhoux and Merad, 2010; Hoeffel et al., 2012; Schulz et al., 2012).
In summary, the findings in this study may answer a long-lasting question of
monocyte/macrophage development in humanized mice. Hydrodynamic injection ofhuman M-CSF encoding plasmids into NSG mice greatly improved the reconstitutionof tissue resident macrophages and the related innate immune responses against
selected human pathogens. Apart from being a model for human hematopoiesis andimmune responses to human pathogens, the M-CSF treated mice may facilitatestudies on the role of human macrophages in the course of tumorigenesis,
autoimmune diseases and tissue homeostasis.

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CHAPTER 5

EFFORTS TO IMPROVEANTIGEN-SPECIFIC ANTIBODY RESPONSESIN HUMANIZED MICEThis work is still in progress. LI Yan designed and performed experiments, analyzed
and interpreted data.

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To improve antibody responses in humanized mice has been an intense topic ofresearch on humanized mice for many years. A humanized mouse with a proper
humoral immune response holds promise for developing effective vaccines or evengenerating neutralizing antibodies against human pathogens for which we do not havegood prophylaxis and therapeutics. Since de novo human T cell development andstable reconstitution were achieved in NSG mice, it was expected that humanantibody response would also be greatly improved. However, the human Ig detected
in sera of humanized mice is still mainly IgM, while very low levels of antigenspecific IgG could be generated. As reviewed in the Introduction, the cause of thisproblem is attributed to insufficient human T cell help. Jackson Laboratory tries tomodify the NSG mice to express one specific human HLA-DR so that human CD4
helper T cells will be selected on human HLA-DR, and be properly activated whenstimulated with antigen. This effort is still undergoing. Even if this approach was
successful, the difficulty to find matched HSC would still limit the application ofhumanized mice. Hence, we tried to overcome this problem via two approaches
detailed below:

5.1 Expression of human CD40 ligand (CD40L) on human T cells to
promote the maturation and proliferation of antigen-experienced B
cells.
The problems caused by the T cell activation may be partially remedied by expressing
CD40L on human CD4 T cells. Human CD40 is constitutively expressed on B cells
and other antigen-presenting cells. The CD40-CD40L interaction is essential for
humoral responses. Upon the recognition of antigen by B cell receptor, the CD40CD40L interaction provides the second signal to induce B cell activation and promote85!
B cell proliferation, germinal center formation, and Ig Isotype-switching (Grewal and
Flavell, 1998).

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However, CD40L expression is tightly controlled in T cells. It is only transiently
expressed for less than 24 hours on the surface of activated T cells (Castle et al.,
1993; Grewal and Flavell, 1998). Studies have shown that CD40L on activated helper
T cells is internalized upon encountering CD40-expressing B cells (Yellin et al.,1994). Since human T cells are educated on mouse MHC and are difficult to be
activated by human DCs, genetic engineering of human T cells in humanized mice toexpress CD40L may circumvent the need of T cell activation. Hence, antigenexperienced B cells could be directly transformed into antibody-secreting cells withthe help of CD40L expressing T cells.
In order to express CD40L on human T cells, we constructed a lentiviral vector withhuman CD40L under the human CD4 promoter and regulatory sequences (Figure
22A, B). We plan to infect HSCs with this lentivirus and inject infected HSCs into
NSG mice. When mice are reconstituted with human cells, the human helper T cells
would express CD40L to enhance the antibody production. With a proper
immunization protocol, we could amplify the antigen-specific antibody producing B
cells for further isolation of neutralizing antibody. Because murine CD40L, similar tohuman CD40L, has shown that the removal of the intracellular domain increases thetime of surface expression of CD40L (Higham et al., 2010), we also constructed
several truncations to remove the first 13-22 amino acids of the intracellular domainof human CD40L. These constructs successfully expressed human CD40L both on acultured human T cell line (Figure 22C) and on primary human T cells isolated by
CD3 selection from cord blood (Figure 22D). However, two issues prevented thisproject from moving forward. First, the CD4 promoter and regulatory elements did
not restrict the expression of CD40L on CD4 T cells (Figure 22E). During in vitro
culture, lentiviral-infected 293FT cells and human B cell line (Raji cell line) also87

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weakly expressed CD40L. We attempted to add more regulatory elements andredesign the promoter, but the results were not satisfactory. Second, the expression of
CD40L was turned off after reconstitution of lentiviral-infected HSCs into NSG mice(data not shown). After 2 to 3 months reconstitution with CD40L-expressing HSCs
infected with lentivirus, we could detect human cells but without the expression of
CD40L. The antibody isotype in these mice was still mainly IgM and low levels ofantigen specific IgG to TT were detected after immunization. The reason for theapparent silencing of CD40L expression is unknown, but it may be due to the poor
engraftment property of CD40L-expressing HSCs or the tight transcriptional
regulation of CD40L expression in cells. To solve this problem, it is critical to obtaina promoter to restrict the expression of CD40L to a specific cell type or to use an
inducible promoter to control the timing of CD40L expression.
The limitation of this approach is the unspecific activation of B cells. Given that the
CD40L would be forced to express on human T cells, all antigen-experienced human
B cells could transform into antibody-producing cells. With an inducible promoter tocontrol the expression of CD40L along with an immunization protocol, the targeted
antigen-specific B cells might be able to proliferate and outgrow those unspecificactivated B cells.

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5.2 Expression of NSG mouse MHC class II (IA-g7) on reconstituted
human cells for proper T cell activation and antibody response
The most obvious approach to correct the problem of human T cell selection in NSG
mice is to replace NSG murine MHC class II gene with human HLA-DR gene. The
human cells will thus be positively selected on mouse thymic epithelial cells
expressing HLA-DR. However, this approach requires gene knock-in on the NSG
background, and HLA-DR matched human HSCs to avoid graft-versus host response.
The embryonic stem cell line for the creation of knock-in NSG mice is currently
under development at the Jackson Laboratory. The requirement of HLA-DR matched
HSCs also limit a wider application of these mice. An alternative approach is toexpress NSG mouse MHC class II, termed as IA-g7, on reconstituted human cells bylentiviral transduction of the IA-g7 gene into HSCs. In this approach, the human T
cells are still educated on mouse MHC content. However, since human antigenpresenting cells utilize the NSG murine MHC molecule IA-g7 to present antigen,human T cells would have no difficulty to recognize the signal for activation. In
addition, no HLA mapping and matching is necessary before each injection of HSCs
into NSG mice, and expression of the mouse IA-g7 may increase the engraftment of
human HSCs in NSG mice due to the MHC compatibility.

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We constructed a lentiviral vector to express IA-g7 under the ubiquitous chromatin
opening element (UCOE) promoter (Figure 23A). This promoter drives stable andreproducible expression of IA-g7 alpha and beta chains. We infected the human Rajicell line with this lentiviral construct to verify the expression of IA-g7 on human B
cells in vitro by RT-PCR (Figure 23B). Initially, we failed to detect the expression of
IA-g7 by an antibody specific for IA-d, which shares the same alpha chain as IA-g7
and detects IA-g7 on NSG mouse cells. Since there was no specific antibody
produced against IA-g7 (Rinderknecht et al., 2010), we suspected that IA-d antibody
might not recognize IA-g7 molecule expressed by human cells. Hence, we tested
other antibodies specific for mouse and rat MHC. Finally, we identified a clone (RT-1,
targeted for rat MHC class I) that could recognize IA-g7 both on NSG mouse cells
and the lentiviral-infected human B cell line (Figure 23C, D). However, this projectencountered another problem. First, the expression of IA-g7 was very weak on HSCs
after infection (Figure 23E). HSCs usually do not express MHC class II molecules,
and associated accessory proteins, and this could explain the low expression of IA-g7
on the surface of human HSCs. Hence, we proceeded to reconstitute infected HSCs
into NSG mice. 2 to 3 months later, we could not detect any human cells expressing
IA-g7 in these mice (data not shown). To solve this problem, we will have to verify
the expression of IA-g7 in infected HSCs by RT-PCR. If the IA-g7 is inserted into thehuman genome but the expression is still not detected in reconstituted human cells, it
may be the possible that the RT-1 antibody fails to recognize the IA-g7 expressed on
primary human cells, or the expression of IA-g7 is switched off. Currently, a newbatch of mice with IA-g7 infected HSCs is being generated for this experiment.
Although these two projects are experiencing difficulties, efforts to find solutions tothose problems will continue. Besides these two approaches, there are also attempts to91

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increase the antibody response by boosting the DCs with human GM-CSF and IL-4 in
NSG mice (manuscript in preparation). This approach may enhance the antigen
delivery to human T cells and B cells. However, as GM-CSF and IL-4 induced DCs
are proinflammatory DCs, the massive production of proinflammatory cytokines andunspecific activation of human T cells may also pose potential problems.

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CHAPTER 6

SUMMARY AND FUTURE PERSPECTIVES

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6.1 Summary of thesis
Humanized mice offer a great platform to study human diseases in vivo and to test
new therapies. Although much progress has been made on the generation of new
mouse strains permissive for high human cell engraftment, many critical issues still
remain to be solved in order to bring the application of humanized mice to clinicaland preclinical use.
Focused on the widely used immunodeficient NSG mice, we addressed three urgent
problems as a priority: 1) Low HSC numbers in a single unit of cord blood caused
sample-to-sample variations, and restricted the application of humanized mice tolarge-scale preclinical studies. 2) Imbalanced human blood lineage reconstitution,
especially monocytes/macrophages, caused poor immune responses against pathogens
and reduced the predictive value of the human immune responses observed inhumanized mice. 3) Human T cell selection in the mouse thymus environment
rendered antigen-specific T cell activation difficult, and consequently poor B cell
response and antibody production. In this thesis, we targeted these three problems,and found partial solutions to the first two.
For the first issue of limited HSCs in a single unit of cord blood, we developed an
improved culture system that combines MSCs engineered to express Angptl5 withother growth factors recently identified to support HSC expansion. Using this method,
CD34+ CD133+ cord blood cells were expanded 60 and 150 fold in 11 and 14-day
cultures, respectively. The expanded CD34+ CD133+ cells efficiently reconstituted
secondary recipients. Furthermore, the expanded cells gave rise to multilineage
reconstitution of human blood cells in NSG mice, and a significantly higher level of T94

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cell reconstitution. These results demonstrate that expanded cells possess both shortterm and long-term HSC activity, and the improved approach should provide a more
efficient method for expanding human HSCs for both clinical and preclinicalapplications.
For the second problem of poor reconstitution and function of humanmonocytes/macrophages, we analyzed various organs of humanized mice tounderstand the root of the problem, and discovered that human monocyte/macrophage
progenitors accumulated in the promonocyte stage. To restore the normal
monocyte/macrophage development in vivo, we used hydrodynamic injection toexpress human cytokines in humanized mice. Mice with human M-CSF or GM-CSF
expression significantly improved engraftment of monocytes/macrophages. In
particular, M-CSF treated humanized mice supported the development of tissue
resident macrophages in various organs, such as liver and lung. Moreover, M-CSF
treated humanized mice had enhanced responses against influenza virus and BCG
infection. Hence, the humanized mice with human tissue resident macrophages offer aapproach for the study of human macrophages and their roles in disease progression
in a small animal model.
Although these two improvements in humanized NSG mice partially solved the above
problems in principle, it is acknowledged that several key issues still exist. For
instance, expansion of HSCs eliminates the variation among cord blood samples, but
in vitro culture is still expensive because of the relatively high concentration and costfor IGFBP2. Nonetheless, if the method developed here is widely applied and ismarketed, mass production may reduce the cost. Furthermore, the improvements ofhuman tissue macrophage reconstitution do not change the fact that many other95!
human myeloid compartments are still poorly reconstituted in humanized mice, suchas neutrophils, erythrocytes and megakaryocytes.
In this thesis, I also made two attempts at improving human IgG antibody response in
humanized mice. In the first attempt, several lentivectors were constructed to express
human CD40L on reconstituted human CD4 T cells. This approach planned to utilize
the costimulatory ability of CD40L to activate antigen experienced human B cells tosecret antibodies. However, the expression of CD40L was not restricted on T cells
despite that the CD40L gene expression was driven by a CD4 promoter. Moreover,the expression of CD40L was switched off after 2-3 months reconstitution. In anotherattempt, several lentivectors were cloned to express mouse MHC class II moleculeIA-g7 on reconstituted human cells. This approach targeted the problems of human T
cell selection on murine MHC content and hoped that matched MHC-TCR interaction
would enhance the human B cell activation in humanized mice. However, thisapproach encountered the similar problem of target gene silencing like CD40L
project. These problems are currently under investigations.
Despite room for further improvement on these problems in NSG mice, the current
achievements on HSC expansion and macrophage reconstitution have alreadybroadened the application of humanized mice to many new research areas. The bigger
group of humanized mice created from a single donor’s HSCs would makehumanized mice applicable to large-scale studies or preclinical test without concerns
about the variations within the group. The tissue macrophages in NSG mice offer aunique and powerful tool to study the development of human monocyte/macrophagedevelopment, and to compare the human macrophages with their cognate mousemodel systems. This is an area with great potential, as the chasm between our
understanding human and murine macrophages is wide.
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6.2 Future perspectives
This thesis focused on three problems in current humanized mice, and partially solved
two of them. For future investigations, work should continue to solve the antigenspecific antibody response. As discussed in Chapter 5, the expression of CD40L andIA-g7 was successful in vitro, but could not detected in the human cells in humanized
mice reconstituted with infected HSCs. We need to isolate human cells from thesemice, and examine if CD40L or IA-g7 is still within the human genome afterreconstitution. This result will guide the direction for the next steps. Once thesetechnical issues are solved, and CD40L or IA-g7 strategies indeed improve theantigen-specific antibody response, this model may have potential for generating
monoclonal antibodies against various exogenous substances, such as viral orbacterial target proteins. Such monoclonal antibodies may act as therapeutic agentsfor prevention of infections or allergies.
Another direction to improve humanized mice is to increase the reconstitution of
granulocytes and erythrocytes. Unlike macrophages and NK cells, the problem of
granulocyte and erythrocyte engraftment may not be simply the result of lacking
growth factors. Since granulocytes can only be found in the BM of humanized mice
after cytokine treatment (Billerbeck et al., 2011; Rongvaux et al., 2011), they
probably need serial sets of cytokines to help them further mature and migrate into theperiphery. Moreover, transient depletion of mouse granulocytes by antibody or
chemical treatment before adding human cytokines may provide space for humangranulocytes to grow. For erythrocyte development, more than one month is required
to detect the population of human erythrocytes in peripheral blood after EPO/IL-3
treatment (Chen et al., 2009). This result indicates that human erythrocyte97!
development may be blocked at very early stages. If human EPO/IL-3 knock-in mice
could be developed, the human erythrocyte reconstitution may be improved, and it
will facilitate the study of malaria in humanized mice. Currently, human erythrocytes
from human blood must be successively injected into the mice i.p. in order to obtain
persistent circulating human erythrocytes in blood for Plasmodium parasites to infect.
Recently, progress in the field of regenerative medicine has drawn the attention of thehumanized mouse research community. Following the establishment of humanembryonic stem cells, inducible pluripotent stem cells, and various identified adulttissue stem cells, artificial human organs or HSCs developed from embryonic stem
cells or inducible pluripotent stem cells may be available. Although these techniques
have not yet impacted the field of humanized mice, the future focus on improvements
in humanized mice could shift from purely hematopoietic cell engraftment to artificial
human organs and HSC-integrated systems.
After all, a mouse is not “human”. No matter how many improvements we make, theresults obtained from humanized mice still need to be verified by clinical studies.
However, this does not mean our efforts are meaningless. On the contrary, the morealike humanized mice and humans are, the fewer resources will be wasted on failed
drug in clinical trials, and increasing knowledge will be gained from the direct studyof human cells in vivo.

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