lymphohematopoietic from - university of toronto t ......(eai3) cells (p. 40) figure 11....
TRANSCRIPT
LYMPHOHEMATOPOIETIC DIFFERENTIATION FROM
EMBRYONIC STEM CELLS IN VITRO
Samh K. Cho
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Graduate Department of immunology
University of Toronto
O Copyright by Sarah K. Cho 2001
National Library Bibliothèque nationale du Canada
. .. and Acquisitiorrsef = Services selvicas bibliographiques 395 Wangton Street 395, rue WeOingtm ûtrawaON K l A W OttawaON K 1 A W Canada Canada
The author has gmted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive ?ennettant à la National Library of Canada to Bibliothèque nationaie du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de
reprochiction sur papier ou sur format électronique.
The author retains ovmership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette dièse. thesis nor substantial extracts f h m it Ni la thése ni des extraits substantiels rnay be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son . . permission. ammsahnn_
Lymphohematopoietic differentiation h m embryonic stem cells in vitro. PhD. Thesis Abstract.
2001. Sarah K. Cho. Department of Irnmunology. University of Toronto.
The in viîro differentiation of embryonic stem (ES) cells provides a mode1 system for the
study of hematopoietic kvelopment. However, the extent to which ES cells can recapitulate in vivo
lymphopoiesis and facilitate the study of lymphohematopoietic differentiation remains to be
detennined ES cells cultwed on the bone marrow (BM)-derived stroma1 ce11 line, OP9 (ES/OP9
coculture), were reported to differentiate into B cells in vitro but it was not known whether these
phenotypic B cells were capable of functional responses. ES cell-derived B lymphocytes were
shown to be functionally analogous to normal fetd liver (TT)- or BMdenved B lineage cells at
different developmental stages, including response to the cytokine Flt-3 ligand at early stages, and
response to mitogen at later stages. Although B cells could be efficiently generated h m ES cells. it
remained unknown whether ES cells could give nse to T lymphocytes in vitro. ES cells,
unfractionated ES cell-derived precursors. and ES cell-derived CD49 hematopoietic precursoa
proved to be unsuitable progenitors for the generation of T lymphocytes in fetal thymic organ
cultures (FTOCs). In conh-ast, ES cell-derived Flkl+ CD45' precursors reproducibly gave rise to T
cells when trdnsferred into FïOCs. The molecular events regulating the onset of
iymphohematopoietic differentiation are not well characterized. Thus, the ES cell-derived FM*
population was M e r examined for the expression of ceil surface molecules that might serve to
identify the earliest lymphohematopoietic progenitors. CD LOS (endoglin), an accessory receptor of
the TGF-$ superfarnily , was expressed during early hematopoiesis h m Flkl+ precursoa (Flkl+
C D 6 to FM- CDS+). Members of the TGF-$ superfamily have been implicated in rnesoderm
speuficaiion and hematopoiesis. Thus, a functional role for CD 105 was assessed by examining the
lymphohernaiopoietic differentiation of CD 105-cie.ficient ES cells in ESIOP9 cocultures. In the
absence of CD 105, myeloerythropoiesis was severely irnpai~d in conûast to lymphopoiesis, which
appeared to be only mildly affected. These findings should help to further Our understanding of the
mechanisms by which the bone rnarrow and thymic microenvironments induce or support
1 ymphohematopoietic di fferen tiation from mesodemal precmors.
DEDICATION
To rny grandmother, the loving maûiarch:
Man Ho Choi
( lgO7-2OO 1 )
Thar 1 may srrive fo honour the rnemory of your srrengrh and sacrifice.
To my cousin:
Tommy Lee
(1968-2001)
Thar I may be reminded. each &y is God's gijk
ACKNOWLEDGEMENTS
1 am thanldul to my supervisor, Dr. Juan Carlos Zuniga-Pflücker, for always king
available, for his ceaseless energy. and for his exarnple of proficiency and expeditiousness. 1 have
learned a lot frorn him, and admire him for extending fnendship and generous effort to al1 his
students.
1 am grateful to the memben of my supervisory cornmittee ... Dr. Pamela Ohashi for her
insights and for prompting me to always "ask the next question" ... Dr. Tak Mak for showing me
that a great measure of humanity can be intertwined with a gnrat measure of intellect ... and
especially Dr. Noman Iscove for the infrequent but nourishing and memorable discussions of
science, for his example of integrity. and for his srniles and support.
Grad school was easier to endure+qecially on those difficult days that are inherent to
science-with the fnendship and patience of membea of the Ztiiiiga-Pflücker lab, past and present:
Alison Michie, Belma Ljutic, Quyen Fong, Sebastien Trop, Tom Schmitt, James Carlyle, Ross La
Motte-Mohs, Mark Konrad, and Maria Ciofani. 1 thank them for helpful discussions and the
quaiities that 1 admire in each of them. Of speciai mention, 1 hank Alison for the laughs, for her
willingness to help everyone. for her perspectives and genemsity , and for the comfy bed to crash
on... Belma for the refreshing tête-à-têtes about life ... and Quyen, for al1 her assistance and
contributions to the lab, and for her fiiendly and steadfast presence.
M y fondest acknow ledgements to my parents, for k i r love and encouragement ... and to
Jesus, whose love reminds me that "If 1 ..cm fathom al1 mysteries and ail knowledge ... but have not
love, I am nothing" (1 Corinthians 132).
Lastly, I wish to thank the Lady Tata Memonal Fund for financial support.
TABLE OF CONTENTS
. . ABSTRACT ......................................................................................................................... 11
....................................................................................... DEDICATION ........... ...........,,...... i v
................................................................................................... ACICNOWLEDGOMENTS v
TABLE OF CONTENTS ................................................................................................. vi
LIST OF FIGURES ............................................................................................................. x
............................................................................................................... LIST OF TABLES xiii
................................................................................................ LIST OF PUBLICATIONS xiv
............................................................................................................ AB B REVIATIONS.. xv
............................................................................... SELECTED CD NOMENCLATURE xvii
ERUDITION ........................................................................................................................ xviii
CHAITER 1. LNTRODUCTTON ........................................................................................ 1
. . Basic concepts of hematopoiesis ................................................................................... i
Embryonic and Fetal Hematopoiesis ............................................................................. 2
3 Origins of hematpoiesis from the induction of mesodemi ........................................ - Onset of hematopoiesis in the embryo ...................................................................... 5
Microenvironmental influences on early hematopoiesis ............................................ 10 . . . Hematopoiesis in the Bone Marrow ............................................................................. 1 1
The bone marrow microenvironment .............................. ...... ............................ 14
Hematopoietic regulation by cytokines .................. ... .......................................... 14
Defining stages of B ce11 development ................................................................... 16 . . ......................................................................................... Hematopoiesis in the Thymus 18
The thymic microenvironment ....................................... .. ...................................... 18
............................................................................ Early T cell progenitors ........ ... 20
..................................................................... Defining Stages of T ceIl Development 20
37 ............................................................... Mode1 systems to study hematopoiesis in vitro -- 37 In vitro culture of hematopoietic progenitors ............................................................. ,,
In vitro differentiation of embryonic stem cells ......................................................... 23 . . . B lymphopo~esis rn vitro ........................................................................................... 24
. . ........................................................................................... T lyrnphopoiesis in vitro 24
Thesis focus .................................................................................................................... 25
CHAPTER II . Functional characterization of B lymphocytes generated in vitro from
ernbryonic stem (ES) cells ............................................................................ 27
Summary ......................................................................................................................... 28
Introduction ............................................................................................................... 28
........................................................... Induction of hematopoiesis in ES/OP9 cocultures 31
RT-PCR Analysis of Cytokines Expressed by OP9 Cells .............................................. 34
Fît-3L enhances the generation of B lymphocytes h m ES/OP9 cocultures ................... 14
Direct generation of ES cell-derived A-MuLV-transformed pre-B @AB) cells in vitro ... 37
Genention of mature rnitogen-responsive Ig-secreting B lymphocytes ........................... 42
....................................................................................................................... Discussion 44
Acknow ledgments .......................................................................................................... 46
CHAPTER iII . In vitro generation of T lymphocytes from embryonic stem cellderived pre-
................................................................................. hernatopoietic precursors 47
Summary ......................................................................................................................... 48
Introduction .................................................................................................................... 48
............*.. ...............*.......................................*......................... Results and Discussion ,., 49
........................................................................................................... Acknow ledgments 58
CHAeTER IV . Expression and function of CD 105 during the onset of hematopoiesis
from Flk l+ precursors ........................................................................... 59
Sumrnary .............................................................................................................. 60
Introduction ............................................................................................................ 60
............................................. Characterization of ES ce11 derived hematopoietic lineages 63
............................................... Expression of Flkl dunng ES ce11 differentiation in vitro 65
CD 1 O5 expression during the onset of hemaiopoiesis frorn Rk 1 + cells .......................... 65
ES cells lacking CD 105 (eng") differentiate normally into Rklf mesodemal
precurson ........................................................................................................................ 69
............................... Impaired erythropoiesis and myelopoiesis in the absence of CD105 73
.......................................................... Definitive erythropoiesis is impaired in eng"' cells 73
Erythroid precursor frequencies are severely reduced in eng" ES/OP9 cocultures ......... 75
............................................... Lymphopoiesis in the absence of CD105 appears normal 77
Discussion ..................................................................................................................... 79
AcknowIedgments ........................................................................................................... 82
............................................................ CHAPTER V . SUMMARY AND DISCUSSION 83
........................................................... ES cell-derived B cells are functionally competent 83
........................................................................ Embryonic. fetal or adult hematopoiesis? 84
The genention of ES cellderived T cells in vitro ........................................................... 85
Optimal ES cell-derived T ce11 progenitors ...................................................................... 86
The rote of CD105 expression and hmction in hematopoiesis from Rkt+ precarsors .... 87
In search of the HSC ....................................................................................................... 88
Isolating early CD49 CD 1 05* hematopoietic progenitors ............................................. 90
Microenvimnmental influences on lineage-specific differentiation and cornmitment ....... 92
Distinct hematopoietic microenvironments: graduating to the HSC? .............................. 94
Dissecting the hematopoiehc microenvironment with stroma1 ce11 lines ........................... 95
,...,.**,..,, ....*..... ................................................................. Of mice and men , . . ........ 97
MATERLALS AND METHODS ............................... ., .................................... 100
Ce11 lines ......................................................................................................................... 100
ENOP9 coculture, in vitro generation of B and T cells. and A-MuLV infection .............. 100
4- CeII culture and differentiation of eng ES cells ............................................................. 102
Flow cytometric anaiysis and cell sorting .................................................................. 103
RT-PCR analysis ............................................................................................................. 104
........................................................... Fetal thymic organ culture (ROC) reconstitution 105
Limiting dilution analysis ................................................................................................ 105
REFERENCES ..................................................................................................................... 107
LIST OF FIGURES
Figure I Hematopoiesis originates h m mesoderm-derived precursors (p. 3)
Figure 2 Sites of hematopoiesis during ontogeny (p. 6)
Figure 3. Hematopoietic activity in embryonic tissues (p. 7)
Figure 3. Stages of B ceIl development (p. 17)
Figure 5. Stages of T ceIl deveIopment (p. 2 I )
Figure 6. Outline of the standard protoc01 used for the differentiation of ES cells into B lymphocytes (p. 32)
Figure 7. Temporal kinetics of hematopoie tic induction in ESlOP9 cocultures (p. 33)
Figure 8. Cytokine expression in the OP9 ce11 line (p. 35)
figure 9. Fit-3L enhances the in vitro generation of ES cell-derived B lymphocytes (p. 36)
Figure 10. Characterization of ES-deri ved Abelson murine leukemia virus-msfotmed pre-B (EAI3) cells (p. 40)
Figure 11. Recombination anaiysis in EAB cells (p. 41)
Figure 12. In vitro generation of ES-derived mature functional B cells (p. 43)
Figure 13. Schematic for the differentiatiion of ES celis intn B andT lymphocytes (p. 50)
Figure 14. Differentiated ES cells transferred into FTOCs (p. 53)
Figure 15. Lymphohernatopoietic potentid of ES cell-derived Flkl+ precursors (p. 51)
Figure 16. In vitro generation of T cells h m ES cell-derived pre-hematopoietic precunoa (P. 56)
Figure 17. Mu1 tiple hematopoietic Iineages generated h m in vitro-di fferentiated ES cells on OP9 stroma1 cells (p. 64)
Figure 18. Temporal analysis of Akl expression during differentiation of ES cells (p. 66)
Figure 19. Hematopoietic potential in RklC fractions fmm ES/OP9 cocultures (p. 67)
Figure 20. RT-PCR analysis of ES/OP9 cocultures (p. 70)
Figure 71. Early differentiation of CD1OS-deficient (eng4) ES cells in virro (p. 71)
Figure 12 Myeloid and erythroid defects in eng'- celis (p. 74)
Figure 23. Lymphopoiesis in eng" cells appears normal (p. 78)
Figure 24. Scherne depicting possible generation of HSCs from ES cells in viîro (p. 89)
Figure 25. CD105 expression on eady hematopoietic precursm (p. 91)
Figure 26. Mode1 of microenvironmental influences on lineage-specific differentiation and cornmitment (p. 93)
Figure 27. The education of an HSC in the PSp/AGM region (p. 96)
Figure 28. Lirnits of two-dimensional stromd ce11 cultures (p. 98)
Table 1. Transcription factors that regulate hematopoiesis (p. 12)
Table II. Ceil cycie analysis in ESIOP9 cocu l~es (p. 38)
Table m. Erythroid precursor potential of RklC cells (p. 76)
J.R. Carlyle. A.M. Michie, S.K. Cho, and J.C. Ztliiiga-Pflücker. 1998. Natural killer cell
development and function p ~ c e d e a@ T cell differentiation in mouse feral thymic ontogeny. J.
Imrnunol. 160: 744-753.
S.K. Cho, T.D. Webber. J.R. Carlyle. T. Nakano. S.M. Lewis. and J.C. ZuÏiiga-Pnlicker. 1999.
Functional characterizütion of B lymphocyes generated in vitro from ES cells. Proc. Natl. Acad
Sei. (USA) 96: 9797-9802.
N. Joza. S.A. Susin. E. Daugas. W.L. Stanford, S.K. Cho. T. Sasaki. A. Elia. M. Cheng. L.
Ravagnan. K.F. Ferri, A. Wakeham, T.W. Mak, J.C. Zïifiiga-Ptlücker, G. Kroerner, and J.M.
Penninger. 2001. Essentiai role of the rnitochondrial apoptosis inducing factor in cell death.
Nature. 4 10: 549-554.
S.K. Cho. A. Bourdeau, M. Letane. and J.C. Zuiiiga-PtlUcker. Expression and function of CD105
during the onset of hematopoiesis from nkL+ precurson. Blood. In press.
S.K. Cho, J.R. Carlyle, and J-C. Ztiiiiga-Pflücker. In vitro generation of T lymphocytes fiom
embryonic stem cell-denved pre-hematopoietic precmors. Submined
ABBREVIATIONS
AGM
A-MuLV
BCR
BM
CLP
dG
DN
DP
EA£3
ES
FACS
FL
Flt-3L
FT
FTOC
HSC
HSA
EL
Ig
LTR
rnAb
M-CSF
NK
P-Sp
aorta-gonad-mesone p hros region
Abelson murine leukemia virus
B cell receptor
bone m m w
comrnon 1 ymphoid progeni tor
2-deoxy guanosine
double-negative
double-positive
ES cell-derived Abelson-hansformed pre-B
embryonic stem
fluorescence-activated cell sorter
fetai liver
Fit-3 ligand
fetai thymus
fetd thymic organ culture
hematopoietic stem cell
heat stable antigen
interleukin
immunoglobulin
long-terrn reconstituting
monoclonal anti body
macrophage-colony stimulating factor
naturai killer
paraaortic splanchnopleura region
RAG
SCF
SP
TCR
TGF
TLP
TN
ncombination-acti vating gene
stem c d factor
single-posi tive
T cell receptor
transforming growth factor
thymic lymphoid progenitor
triple-negati ve
SELECTED CD NOMENCLATURE
T3 cornplex; y. 6, E, 5 (q) subunits; associates with TCR af3 and y6 subunits.
L3T4; binds c las II MHC.
Ly- 1 ; binds CD72.
Lyt-YLyt-3 (cd$ subunits); binds class 1 MHC.
Mac- l a chain, integrin a, chain: Mac- 1 (CD 1 1 b/CD 18) binds CD54 and iC3b.
B lineage signal innsduction molecule.
HSA: gpi-Iinked; binds CD62P.
IL-2Ra; binds IL2 associated with CD 12YCD 132.
platelet endotheliai ce11 adhesion molecule (PECAM- 1).
sidomucin; marker to e ~ c h for eariy hematopoietic progenitors.
S7, leukosialin, sidophorin; binds CDW.
Pgp- 1 ; binds components of the exiracellular matrix such as hyaluronate.
Ly-5. leukocyte cornmon antigen (LCA).
B220; CD45 isoforrn.
endoglin, accessory receptor of the TGF-p superfamily.
c-kt; Steel, stem£ell, mastcell p w t h factor receptor.
VEîadhenn; homotypic binding.
Here Itills and vales, the woodlund and the plain.
Here earth and warer seem ro srrive again;
Nor chos-like rogether crushed and bruised.
But, as the world. hannoniously confiued:
Where order in vu- we see,
And where, rhough all things differ. al1 agree.
- Aiexander Pope
CHAPTER 1: INTRODUCTION
The pmcess of differentiation allows for the generation of remarkably diverse cell types
from a stem cell, which acts as a continuous and self-renewing source for the generation of
multilineage progeny cells. Undentanding how molecular signals in developing tissues induce
commitment and differentiation of stem cells is a fundamental question of developmental biology.
In the context of blood cells, this question also has therapeutic implications in the treamient of
leukemia, which arises h m dysregulation during hematopoietic differentiation.
BASIC CONCEPTS OF IElEMATOPOIESIS
The existence of a blood stem ce11 was illustrated elegantly in observations that bone
marrow (BA4)derived cells could fom multilineage colonies in the spleens of irradiated recipients.
and that cells from these colonies could then give rise to more spleen colony-forming cells '?
Additional evidence for the existence of a hematopoietic stem ce11 (HSC) has ken provided by
experiments in which single stem cells or clones replenished the blood ce11 compartments of
lethally-irradiated adult recipients for long-term 5-7. Long-tem reconstitution (LTR) of a
lethally-imdiated adult recipient is the definitive assay for the presence of an HSC. Furthemore, if
HSCs are present, BM from reconstituted rnice should be able to confer LTR activity on secondary
recipients after senal transplantation in order for a single HSC to fully replenish the blood
compamnents of an adult mouse, it must be capable of self-renewal as it gives rise to differentiated
progeny cells. Differentiated HSCs give rise to a hierarchy of lineage-restncted multipotent,
oligopotent, and unipotent progeny cells with diffenng degrees of self-renewal capacities 5*7.'0.
Multilineage differentiation cm be exhibited by a population of cells containing multiple unipotent
precursorr or multipotent pursors or both. Thus, the presMce of dtiptent CU CUL SOLS can
only be estabüshed from the multilineage differentiation of a single ce11 or clone.
During differentiation. lineagecornmim~nt can be defined as an irrevocable determination
of a ce11 towards a panicular restricted fate. For example, o ce11 exhibiting characteristics associated
with the T ce11 lineage cm be desipated as committed to the T ce11 lineage when it is no longer able
to differentiate into other hematopoietic lineages. under normai conditions that are amenable for the
differentiation of other hematopoietic Iineages.
In the developing embryo, hernatopoiesis occurs in different embryonic tissues, and
differentiated blood cells and multipotent precursors are detected prior to the establishment of
HSCs, thus revening the hierarchy of adult hematopoiesis. Once genented. HSCs mignte from
embryonic and fetal sites of hematopoiesis to seed the BM and thymus. the major sites of
hematopoietic differentiation in the adult. The BM sustains a continual source of HSCs and also
supports myeloerythmid and non-T lymphoid differentiation, and the thymus is a speciaiized organ
required for the differentiation of T lymphocytes.
EMBRYONIC and FETAL HEMATOPOIESIS
Origios of hematopoiesis from the induction of mesoderm
Mesodenn is the intermediate genn tsyer formeà betweerr the ectodennat md enduciend
genn layers during early embryogenesis. The formation of mesoderm is an inductive process
&pendent on the expression of mesoderm-specific genes and influenced by secreted proteins.
Mesoderm-derived precursors give rise to various tissues including heart, musc le, bone, notochord,
blood vessels, and blood (Fig. 1). Membea of the transforming growth factor (TGF)-p
superfamil y, an extensive fami ly w hich includes TGF-B, activins and bone morphogenetic proteins
(BEIps), have been implicated in the induction of mesoderm and the initiation and regdation of
hem muscle bone notochord
0 - - /i lymphoid progenitor
/' - -
"Y. myeloery throid hemungiogenic progeni tor precursor
endothelid celld blood vessels
8 Cells NK Cells T Cells
erythroçytes megarküryoc y tes mast cells neutrophils monocytes/mucrophages
Figure 1. Hcmatopoicsis orienates from mesoderm-deriveû precursors. The mesoderm germ layer is fonned between the endodennul and ectodemül germ layers during gastrulation of the developing embryo. Mesoderm gives nse to various tissues, and also gives nse to a common hemangiogenic precursor for the endotheliai and b l d lineuges. In the established adult hem~topoietic system the hemûtopoietic stem cell (HSC) contiiiues to generate progenitors that give rise to all blooû cells.
hematopoiesis 11-14. Mice lacking BMP-4 die at around the time of gamlation showing linle or
no rnesoderm differentiation 15, which suggests that BMP4 is essential for normal gasû-ulation and
mesodemi formation. In addition, BMP4 was shown to mediate the formation of ventral
mesodenn and Activin A was shown to mediate the formation of dorsoanterior-like mesodenn
during the differentiation of mouse embryonic stem (ES) cells in chemically defined medium. In
this sarne assay. BMP4 was also show to mediate the formation of hematopoietic progenitors 16.
In vitro cultures of moue epiblast tissue revealed that addition of exogenous BMP-4 and activin A
induced hematopoiesis in an ectopic site 17. Furthemore, mice lac king TGF-p 1 or its receptor.
TGF-$M. exhibit early defects in hematopoietic and endotheliai development from extraembryonic
yolk sac mesodenn
In addition to d e s during mesodenn induction and hematopoiesis, TGF-ps are capable of
ngulating a vast array of cellular processes in a variety of tissues throughout development.
Regulation of TGF-fk involves a highly cornplex network. TGF-fk are newly synthesized as
zymogens, peptides w hich require prote01 ytic cleavage to generate the active form. Once cleaved,
the TGF-f3s are rnaintained in a biologically inactive state and are released by cells as latent
complexes with latency-associated proteins (LAPS). L M complexes can Further bind to latent
TGF-bbinding proteins (LTBPs) which allows association of the complexes to the extracellular
rnatrix and, presumabl y, storage of the TGF-fis 12-' '. Accessory recepton, suc h as CD 105
(endoglin), provide additional complexity of regulation at the receptor level. CD 105 is expressed
on uascular endotheliai celb anci subsets of hemtopoietic progenitors and associates with various
members of the TGF-f3 superiarnily in complex with their cognate receptors '*'@? CD105 can
bind TGF-$ I , TGF-$3. acti vin- A, BMP-2, and BMP-7 'O2'. However, CD 105 function and iü
potential regdation of signals transduced by these growth factors remains largely unelucidated.
Targeted deletion of CD 105 in mice resulted in embryonic lethality due to defects in angiogenesis
and cardiovascular development, indicating that CD 105 plays an important role in these processes
'5-27. In addition, studies have provided evidence that CD105 can antagonize or potentiate
TGF-prnediated signals 28-3 l . For example. addition of antisense CD 105 oiigonudeotides resulted
in a specific reduction of CD 105 mRNA and protein. and greater TGF-pl-mediated inhibition of
vascular tube formation and viability '. The close association of the de novo generation of blood cells and vessels during the
formation of yolk sac blood islands from mesodemal precumrs led to the hypothesis that
hematopoietic and endothelid lineages derive from a comrnon precursor. termed the hemangioblast
i g 1). Indeed, a number of genes are comrnonly expressed in hematopoietic and endothelial
lineages such as the transcription factor SCYTal-1 the sialomucin CD34 33, and the receptor
tyrosine kinase Tie-Yïek 3435. Gene-targeting ("knockout") experiments revenled the
requirement for the receptor tyrosine kinase Fikl (vascular endothelial growth factor receptor 2,
VEGFR-2) in the development of both the hematopoietic and endothelial lineages. which funher
suggested that these lineages may be denved from a common precursor 36*37. Moreover.
Nishikawa et al. showed that a single Flkl+ ce11 codd give rise to cells of the hematopoietic and
endothelial lineages 38. Choi et al. also demonstrated the existence of a clonal hemangiogenic
precursor 39. Although Flkl knockout mice displayed severe blocks in vasculogenesis and
hematopoiesis, residual levels of endothelid and hematopoietic differentiation in in vitro assays
revealed that Flkl is not absolutely required for cornmitment towards these lineages. It was
suggested, instead that Fikl may be required for proper horning and migration of early precursors
to appropriate sites during embryonic development 37.
Onset of hematopoiesis in the embryo
Embryonic hematopoiesis is a complex process in which varying degrees of hematopoietic
activity and multilineage potentiality occur sequentially in different anatomical sites (Figs. 2 and 3).
Rior to the establishment of circulation between the mouse yolk sac (YS) and embryo at E8.5,
hematopoiesis occurs de novo in two sites. the extraembryonic yolk sac and the intraembryonic
timeline
embryo adult
Figure 2. Sites of hematopoiesis during ontogeny. Hematopoiesis is first observed in the yolk sac (YS). Hernatopoietsis is independently initiated in the paraaortic spIanchnopleura/aorta-gonad-mesonephros (P-SplAGM) region shortly aftenvards. After circulation is estabiished, possible cellular interchange between the sites makes it difficult to determine whether hematopoietic precursors that seed the fetal liver (FL) were derived fmm the YS or P-SP/AGM. Nevertheless, the FL becomes the dominant site of fetd hematopoiesis and FL-denved precmors seed the thymus and bone marrow.
Figure 3. Hematopoietic activity in embryonic tissues. The fmt appearance of different hematopoietic activities in yolk sac (YS), and the paraaortic splanchuopleura/aorta-gonad-mesonephros (P-SpIAGM) region is depicted. Day of gestation is indicated. EM=erythroidbyeloid potential, L=lymphoid potential. CFü-S=potentiai to form colony units in the spleen in vivo, HSC=long-term reconstituting activity.
region designated as the para-aortic splanchnopleura @Sp) or later, as the aorta-gonad-
mesonephros (AGM) *"% Rimitive erythrocytes detected in YS blood isiands at E7.5 provide
the first observable evidence of hernatopoiesis in the developing embryo. However, erythroid and
granulocyte-macrophage potential can be detected as early as €7 by in vitro assays ".
Lymphoid potential was found in both the YS and P-Sp beginning at E8.5 ". However,
since circulation is established around this time, it could not be conclusiveIy determined whether
lymphoid potential arose indigenous to both sites, or whether lyrnphoid progeniton migrated h m
one site to the other. To resolve this issue, Curnano et al. assessed lymphoid potential by culturing
hematopoietic progenitors denved fmm YS and P-Sp tissues that were explanted prior to circulation
JO. Lpphoid potential was present in the P-Sp region at E7.5. pnor to the establishment of
circulation. Under the sarne culture conditions. lyrnphoid potential was not detected from the YS ".
These data dernonstrated that lymphoid potential originates in the P-Sp region, but not the YS, prior
to circulation M, and suggest that lymphoid potentid in the YS is derived from migrating P-Sp
precursors.
Hematopoietic progenitors capable of in vivo reconstitution can be detected by the formation
of colonies in the spleens of irradiated mice (colony forming unit-spleen, CN-S) '. Pmgenitoa
that exhibited CFü-S activity were detected from the YS and the P-Sp/AGM at E9. However,
CN-S activity was observed at a higher frequency in the AGM 43.
Evidence for the presence of HSCs is best determined by assays for LTR activity. Müller et
al. assayed for LTR activity in ex vivo ernbryonic tissues. At E 10, LTR activity was observed h m
the AGM but not the YS and fetal liver (FL), when cells were injected intravenously into recipient
mice. At El 1, LTR activity was also o b s e ~ e d in the YS and FL 9. The observation that LTR
activity appears first in the AGM suggests that HSCs originate in this site at EIO and subsequently
seed the YS and FL. To discount the possibility that the AGM was seeded by HSCs originating
fimm an alternative site, Medvinsky et al. isolated YS, AGM, FL, and body remnants beginning at
E9, cultured these explants for 2-3 days, and transferred cell suspensions h m these tissue explants
into irradiated adult rnice. Cornparison of various tissues at E9 and E IO showed that only El0
AGM exhibited LTR activity 8. These data provide strong evidence that LTR-HSC activity initiaies
in the AGA4 at E10.
Hematopoietic activi ty persists in the YS and AGM until about El2 to E13. At the same
tirne, the fetal liver becomes the dominant site of hematopoiesis until the BM is colonized prior to
birth '". Figure 1 depicts the various tissues that act as hematopoietic sites during ontogeny.
Although E9 YS cannot confer LTR activity when transferred directly into irradiated d u l t rnice, in
vivo transfer studies demonstrated that E9 YS can exhibit LTR activity when transferred into
sublethally conditioned neonatai mice, especially when injected k t l y into the neonate liver
8""*45. ûn the other hand, El0 AGM-derived cells can bypass a fetal or neonate liver
microenvironment and directly seed BM for long-term reconstitution 8*9"1. Thus, the FL is not
absolutely required for the development of definitive hematopoiesis. However, considering that
hematopoietic activity in the YS and AGM ceases by approximately EL3, the FL is required to
function as a reservoir before the establishment of BM hematopoiesis " . With respect to the
presence of muitipotent progenitors and definitive HSCs, fetal Iiver hematopoiesis resembles adult
BM de fini tive hematopoiesis. However, evidence of unique FL-derived 1 ymphoid su bsets 46-48
suggests that the FL microenvironment may pssess unique hinctions. in addition to s e ~ n g as a
reservoir.
Microenvironmental influences on early hernatopoiesis
Certain transcription factor knockout studies have highhghted microenvironmental
differences in ernbryonic and adult hematopoiesis, suggesting that tissue-specific induction and
expression of distinct sets of genes is cntical for hematopoietic ontogeny. For example, definitive
hematopoiesis from the FL stage onward was show to be critically dependent on AML-1. whereas
YS-derived erythropoiesis was normal in AML- 1 knockout mice. Inde«L viability of AML-1
knockout mice is likely to have ken dependent on the establishment of FL hematopoiesis, as
mutant embryos died around E12.5. Generation of chimenc rnice with AML-I-deficient ES cells
revealed ihat the hematopoietic defects were ceil-intrinsic 49. An inhiguing report by Wang et al.
revealed that establishment of hematopoiesis in the BM, but not the YS or FL, is dependent on the
transcription factor TEL. Anal ysis of chimenc mice reveded that TEL-deficient ES cells
contributed to non-hematopoietic tissues but not to BM hematopoiesis. Low levels of
hematopoietic progeni tors were reponed to be present in neonatal BM. indicating that TEL is not
required for definitive hematopoiesis or to colonize the BM, but is required for maintenance of
hematopoiesis in the BM 50.
Studying how distinct ontogenic microenvironrnents restnct, induce, or support varying
degrees of hernatopoiesis may help to detennine the regulatoty mechanisms underlying the self-
renewd and differentiation of multipotent progenitors and HSCs. Novel insights in this area will
have implications for leukemia diagnosis and therapy. and for hernatopoietic tissue-engineering and
reconstitution therapies.
HEMATOPOIESIS IN THE BONE MARROW
Throughout adulthooà, it is the unique and important function of the BM to replenish blood
cells and sustain the immune system by the continual differentiation and self-renewal of HSCs.
Transcriptional hierarchy of hematopoiesis
Multilineage differentiation is achieved by the timely and specific expression of select sets
of genes within the cell. A variety of facton act in combination to regulate transcription including
elements that directiy regulate sequence-specific binding to promoter regions, and elernents that
regulate accessibility to promoter regions by altering chromatin structure Knockout
experiments have highlighted the critical importance of a number of transcription factors during
lymphohematopoietic differentiation that support a hierarchical mode] of hematopoictic
development from the HSC (Table 1).
At the top of the hierarchy is the transcription factor SCyTal- 1, reported to be critical for
the development of al1 hematopoietic lineages 52"5. and also show to play an important role during
angiogenesis in Iater endothelial developrnent 56. Recent studies have provided evidence that SCL
is required for the development of al1 hematopoietic lineages because it functions to specify the
hematopoietic fate h m rnesoderm 5758. Other transcription facton do not affect al1 hematopoietic
lineages but play crirical roks in the differentiation and cornmitment of multiple or single lineages.
For example, myeloid and lymphoid cells were markedly reduced or absent in PU. 1 knockout rnice,
indicating that the development of these lineages is critically dependent on PU. 1 function 59.
Pervasive lyrnphopoietic deficiencies in Ikaros knockout or dominant-negative mutant mice ~veaied
the importance of this transcription factor in the establishment of lymphoid lineages **". Cenain
transcription factors appear to be critical only for the development of single lineages. For exarnple,
Table 1. Transcription factor regulation of hematopoiesis.
Transcription factor Fami l y Hematopoietic phenotype in knockout mice
Essenriul to establiulittm t 01 tttultiple lineuges:
PU. 1 13
Ets
1 karos Zn Finger
Essential to e.stablishnrt of o particulur lineuge:
GATA- 1 GATA (Zn finger)
GATA-3 GATA (Zn finger)
Pax-SIBSAP püired domain
Ets- l Ets
lack of erythroid, myeloid. and lymphoid lineuges
myeloid and lymphoid Iineages absent or severely reducec macrophages and B cells more severely affected chan neutrophils and T cells
nuIl mutant: lack of B and NK cells, fetal T cells dominant negative mutant: complete lack of B. T, and NK cells
early block in development of erythmcytes
block ut eürliest stage in thymic T cell development
lack of fetlil B cells. eurly block in adult B cell clevelopment
severe reduction in NK cells
GATA- 1 knockout mice exhibited an early block in the elythroid lineage, but not in other
hematopoietic lineages 62a. Pax4 is critical for B ce11 development 65*66, Ets-1 for NK cell
development 67, and GATA-3 for T ce11 development ". When multilineage defects are exhibited
in transcription factor knockout mice, it is often assumed that the defect occurs in a common
multipotent precursor. However, it is do possible that the defect occurs independently in each
lineage.
Novel insights to the mechanisms of transcriptional regulation have been gained from recent
studies on Ikaros, PU. 1, and PaxS. A more complex three-dimensional mechanism of
transcriptionai regulation has been proposed for Ikaros based on the observation that it localizes to
centmmeric heterochromatin in association with transcriptionally silent genes, which suggests that it
functions to repms genes by recruiting them to centmmeric foci 69*70. Studies revedinp that
ikaros can bind to chromatin remodelling complexes and histone deacetylases further support the
notion that it functions as a transcriptionai repressor "*". In a report by DeKoter and Singh, B
lyrnphopoiesis was shown to predominate from FL precursors expressing low levels of PU. 1.
whereas macrophages predorninated h m precunors expressing high levels of PU. 1, suggesting
that graded expression of the same transcription factor rnight contribute to the commitment or
differentiation of one lineage over another 73. Recent reports on the transcription factor Pax5 have
prornpted a reevaluation of B ce11 lineage commitment. In Pax5 knockout mice. cells expressing
surface markers and genes normaily indicative of B lineage commitment, displayed the surprising
ability to differentiate to other hematopoietic lineages. Thus, the data show that lincage-associated
events do not necessady indicate lineage cornmitment, and demonstrate that Pax5 is a detennining
66.73 factor of B ceil lineage cornmitment .
The bone.marrow mirr-vira-
The BM microenvironment, an inaicate network including reticular stromal cells,
macrophages, adipocytes, and extracellular matrix 75*76, mut allow for the maintenance and
self-renewal of HSCs while simultaneously supporting HSC differentiation. Conceptually, the
organization of distinct stromd cells into microenvironmental niches may be a possible means by
which these two seemingly opposite functions can be achieved In vitro evidence using transfonned
ce11 lines support the notion that stmmal cells can differ in their ability to efficiently support the
maintenance of stem cells 77*78. However, it is unclear how relevant these data are to stroma1 cells
in vivo. Distinct characteristics of stromd cells could be attributed to varied expression of
cytokines, signalling and adhesion molecules. and extracel lular matrix (ECM) components. The
ECM is established from secreted products of siromal cells. Cell adhesion to ECM components.
such as fibronectin and thrombospondin, differs during stages of myeloid and erythroid
development and suggesu that the ECM rnay contribute to regulating hematopoiesis 76.
75.76 Components of the ECM dso play a role in the presentation of cytokines .
Hematopoietic regulation by cytokines
Stroma1 cells of hematopoietic rnicroenvironments, including the BM and thymus,
contribute to the regulation of hematopoiesis, in part, by the production of cytokines which help to
regulate the survivat, profiferation and differentiation of hematopoietic progenitoa Some
cytokines, such as stem ce11 factor (SCF'), c m be found in secreted and membrane-associated
forms. The si gni ficance of these different f o m is not clear, however, there is evidence to support
that they play distinct d e s 8z83. Cytokine-induced signais are transduced upon binding of
cytokines to their cognate receptors on the ce11 surface. Cytokine receptors are categorized into two
main families 'O. Cytokines such as SCF, Ht3-L, and MCSF act through a farnily of tyrosine
b a s e receptors that possess intrinsic kinase activity. The second family of receptors, tenned the
cytokine receptor superfamily, are similar to tyrosine kinase receptors in the extracellular domain
but lack a kinase domain in the intracellular portion, and therefore do not possess intrinsic kinase
activity. Included in this second farnily are receptors for cytokines such as I D , erythropoietin,
GM-CSF, IL-7. and other interleukins Cytokines are characieristicaily pleiotmpic and c m
exhibit overlapping functions, which may be a reflection of the fact that many cytokine receptors
share one or more common subunits of a receptor complex 79*80. Although members of the
cytokine receptor superfamily lack intrinsic kinase activity, recent studies have shown that signals
are t~ansduced by binding intracellular kinases such as the JAK kinases, which can act on
downstrearn substrates called STATs (signal msducers and activators of transcription) or activate
the ras pathway by creating docking sites for SRcontaining pmteins 79*sl. CD45, a receptor
tyrosine phosphatase, serves as a useful hematopoietic marker because it is expressed on al1
hematopoietic cells except mature erythrocytes Recently, Irie-Sasaki et al. reported that
CD45 cm negatively regulate cytokine signalling by dephosphorylating IAKs 87. thus potentially
endowing functional purpose to CD45 that is congruent with its widespread expression on al1
nucleated hematopoietic cells.
The SI and W genetic loci in mice were shown to encode the cytokine, SCF. and its cognate
receptor. CD1 17 (c-kit), respectively. Studies revealed that hematopoietic defects resulting from
mutations in the W locus were intrinsic to hematopoietic cells, whereas those resulting h m
mutations in the SI locus were amibuted to the hematopoietic microenvironment 82. CD1 17 is
widely expressed on hematopoietic progenitors, including stem cells, multipotent progenitors, and
iineage-specific progenitois. However, most Lineage-specific progenitors lose expression of
CD1 17 upon further differentiation and maturation 6*83*8&9J. CD1 17 expression on these various
progenitors suggests that they should be responsive to SCF-induced signals. Indeed SCF has
suniival 96, synergize with other cytokines to promote growth, and in the case of mast cells, promote
effector functions ".
Similar to SCF, the cytokine Fit3-L can synergize with other cytokines to promote growth.
However, the effecü of Ht3-L and the expression of its receptor, nt-3. are more restricted than for
SCF. Furthemore, Flt-3L seems to have a more prominent effect on lymphopoiesis 83.9749
Veiby et al. reported that the addition of Nt-3L and IL-7 promoted growth of p m B cells, whereas
SCF and IL-7 generated mostly myeloid cells, during the culture of Lin- Sca-l+ mouse BM
progenitors98. In addition, growth of the earliest multipotent thymic lyrnphoid progenitors is also
more efficiently stimulated by Rt-3L than SCF '? Mice deficient for Flt-3. the receptor for FM-L,
83.lûû exhibit defects in hematopoiesis and lymphopoiesis .
Defming Stages of B ce11 Development
In the adult rnouse, B lymphocytes are generated from HSCs within the bone m m w
through a pmcess of sequential differentiation events. Stages of B ceIl differentiation have been
characterized by the expression of various ceIl surface molecules (Fig. 4). CD 1 17+ hematopoietic
progenitors with restricted B ce11 lineage potential cm be identified by expression of CD19 and
CD45R (B220) on the ce11 surface. Early B ce11 progenitors, termed pro-B cells. are M e r
characterized by the surface expression of CD43 in contrast to later progenitors, termed pre-B cells,
which lack expression of this marker 83v101*'". B lymphocytes and T lymphocytes are unique in
their expression of antigen receptors. An extraordinary degree of diversity is generated in the
antigen Rceptor repertoire by a pmcess termed V@)J recombination, during which recombination
of V, D, and J gene segments of the antigen receptor loci are mediated by the
Pro-B Pre-B Immature B Mature B
Figure 4. Stages of B c ~ l l development. A simplified scheme is shown that depicts changes in the expression of characteristic ceIl surfpce molecules dunng different stages of B cell <levelopment. Also shown is expression of CD1 17, Flt-3, and IL-7R (a chain), receptors for the cytokines SCF, Flt-3L, and IL-7. respectively. CeIl size is indicative of relative proliferüiive status.
recombination-actiu~g gene W G ) pmducts, RAG- 1 and M G - 3 B cell antigen receptor @CR)
gene remangement initiates at the immunoglobulin heavy ( igH) chah locus in pro-B cells, while
the light (IgL) chain locus remains in germline configuration. During the pre-B ce11 stage,
completed rearrangement of the IgH p n e loci and coupling of the peptide to a sunogate light chah
leads to the production of a pre-BCR, which sipals these cells to undergo prolifemtion and
promote differentiation to a late pre-B ce11 stage 10'*103. Pmductive rearrangements at the light
chah (K or A) loci results in the generation of immature surface I N B cells which undergo
selection. leading to the generation of mature B cells expressing IgM and IgD on the ce11 surface
1 O3
HEMATOPOIESIS IN THE THYMUS
The thymic microenvironment
The BM microenvironment is not sufficient for the differentiation of T lymphocytes.
Instead, mu1 tipotent progeniton begin to seed the thymus. at approximatel y E 1 1 -E L 1. in order to
complete their differentiation program into T cells. The thymus is forrned during embryonic
development h m an involution of the third pharyngeal pouch between ElO-El 1 104.105~ The
thymic anlage hclucks epithelid and mesmdiyrnd tissues, both of which are required for
functional thymopoiesis 10501". Interestingly, the El2 fetal thymus in culture does not appear to
mature into a pnuine thymic microenvironment capable of supporting complete T ce11
di fferentiation 107"08, suggesting that the fetai thymus itself requi~s further maturation
concomitant with the arriva1 of incoming precursots. However, addition of fibrobIastic ce11 tines to
the day 12 thymic nidiment results in the restoraaon of an optimal thymic microenvironment that
suppw the fuB diffcsren~iation of mature @T e e k 'O7. Ceus of hemrttqxktic Mgin dso
contribute to the thymic microenvironment, including dendritic cells and macrophages. which play
an important role in thymocyte selection 'O5. Evidence suggests that the architecture of a mature
and Functional thymic rnicmenvironrnent is dependent on reciprocal interactions between the thymc
stroma and developing thymocytes. The contml of thymic architectural organization by developing
T cells is termed "thymic crosstalk" 109-111
The architecture of the thymus is organized such that incorning hernatopoietic precursors
enter the outer/subcapsular cortex region. where the majority of early thymocyte differentiation
takes place, and subsequently migrate inwards 'O5*' lr ' 13. Early progeniton continue to
di fferentiate in the corticomedullary junc tion w here earl y selection events and commi tmen t towards
CD4+ or CD8+ T ce11 lineages occurs, progress further inward to the medulla where late selection
events take place, and finally, appmach the core of the thymus where they exit to the periphery as
mature and functional T cells ' 13. More recently, it has been demonstrated that BM-derived
precursors first enter the thymus near the corticomedullary junction and subsequently migrate to the
outer cortex ' ' '. The mechanisms by which unique aspects of the thymic microenvironment.
including archi tecniral organization and soluble factors. skew differentiation towards T lymphocytes
remain to be elucidated.
Notch receptors and their ligands are differentially expressed in siromal cells and
thymocytes during thymic development, and are involved in ceIl fate decisions, suggesting that they
rnay play a d e in T cell Lineage cornmitment l 1 '. Overexp~ssion of a constihitively active Notchl
intracelIular domain inhibited B cell development in the BM ' 14. In complementary experiments,
the induced inactivation of Notch 1 biocked T ce11 development at the most immature thymocyte
stage lS. in addition. BM cells expressing retrovirally-introduced activated Notch 1 gave rîse
exelusivdy te tumocus of immature l tell p h a t p e ' 16. Cdküvely, h e data p v i d e s m g
evidence that Notch 1 is important for T ce11 Lineage fate decisions.
Early T ce11 progenitors
hcursors that seed the thymus travel via the fetal circulation and origmate from sites of
hematopoiesis such as the AGM and FL during early development 8*9*1 17. and from neonatal and
adult BM later in development la. Both fetal and adult thymopoiesis depend on a continual source
of incorning hematopoietic precurson '". Once multipotent precurson enter the thymic
microenvironment. they rapidly commit to one of a few hematopoietic lineages 93*' "J 19. The most
immature hematopoietic precursors comrnon to the fetal and adult thymus are termed thymic
lyrnphoid progenitors (TLPs) because they are able to give rise to B. T, natural killer (Mo, and
lymphoid dendntic (LD) cells 93s112. Notably. TLPs do not give rise to myeloid, erythroid, and
megakaryocytic lineages in vivo 93*1 12120*121, and c m give rise to T lineage cells faster than HSCs
'". It remains unclear whether thymic immigrants are noncommitted hematopoietic precurson. or
a rnixed progenitor population including preîommitted T ce11 precunon 93~' 12*' 19.
During mouse ontogeny, thymopoiesis proceeds such that the discrete stages of
developrnent are observed sequentially on successive days of fetal üfe 93. In the adult thymus,
however, al1 stages of development are present simultaneously and a dynarnic equilibnum is
established between incorning precunors and emigrating T cells 93*112.
Defrning stages of T ce1 development
Stages of intrathymic T ce11 development have k e n weii-characterized (Fig. 5) by the
surface expression of the coreceptors CD4 and CD8, and progression of stages prior to the
TLP PrwT Early Pre-T Late Pre-T ISP DP SP
Figure 5. Sîages of T ce11 development. A simplified scheme is shown thai depicts phenotypic chwdcterization of different stages in T cell development; thymic lymphoid progenitor (TLP). immature single-positive (LSP). double- positive (DP), single-positive (SP). CD1 17 is the receptor for SCF. Also shown is the expression of Flt-3 and IL-7R (a chain), receptors for the cytokines Fit-3L, and IL-7, respectively. Cell size is indicative of relative proliferative siatus.
expression of a T ceIl antigen receptor UCR) are identifieci by four major subsets in the following
order: CD4 CD8- double-negative 0, CD& CD8+ double-positive OP), and CD4+ and CD8+
single-positive (SP) populations. The earliest DN thymic immigrants lacking TCR expression are
also referred to as triple-negative 0 thymocytes. The TN population can be further subdivided
into four discrete subsets based on the differentid expression of CD 117 and CD25 (IL-2Ra)
93.119.123
Multipotent TLPs are defined by a CD 117+ CD25- surface phenotype (Fig. 5). Following
the CD1 17+ CD25- stage, down-reguiation of CD 1 17 expression and induction of CD25
expression mark progression to the CD 1 17+ CD29 pro-T ce11 stage, and characterizes T lineage
cornmitment. Subsequently, pmliferation slows down and rearrangement of the TCR-$ gene locus
initiates in early pre-T cells, which are defined by a CD1 17- CD25+ phenotype. 121-124*1?5. The
transition h m early pre-T cells to CD 1 17- CD25- late pre-T cells is dependent on the cümpletion
of TCR-B rearrangement. This important developmental checkpoint is termed bselection and is
also dependent on the expression of a pre-T a chah which pairs with the K R - 6 chah to form a
pre-TCR. Thyrnocytes that have successfully rearranged their TCR-$ locus are selectively enabled
to expand, differentiate, and proceed to the CD4+ CDF DP stage of thymocyte differentiation Iz'
12'. DP thymocytes proceed with ~amingement of the TCR-a chain, and those expressing a
comptete afbTCWCD3 complex undergo positive and negative selection which results in the
generation of mature SP thymocytes 128-133.
MODEL SYSTEMS TO STUDY HEMATOPOLESIS IN VITRO
In vitro culture of hematopoietic progenitors
Various systems have been developed that support the differentiation of @id., rnyeloid,
and 1 ymphoid celis h m FL- or BM-dnived hemaîopoietic progenitors in vimo. in addition. LTR
assays have shown that HSC-activity can be rnaintained in vitro, however, maintenance of HSC
three-dimensional organization. and cellcell interactions of hematopoietic tissues in vivo. and are
therefore limited in the scope of questions that can be addressed in those contexts. However. the
simplicity and minimalistic aspects of in vitro systems can dlow for greater contml and ease of
manipulation in certain investigations such as studying the effects of different soluble factors,
dissecting the contributions of certain variables. expanding rare populations of cells, and defining
the kinetic progression of events during differentiation.
In vitro differentiation of embryonic stem ceiis
Embryonic stem (ES) cells are isolated from the inner ce11 mass of blastocysts. Established
ES ceIl lines can be maintained in an undifferentiated state in culture. exhibit pluriptency. and cm
contribute to al1 tissues of an animal. including germ cells, when transplanted back into a developing
blastocyst '35*136. Differentiation of ES cells in vitro provides a mode1 system to study the
development of various tissues and lineages 13'. The use of differentiated ES cells allows for the
study of genetic alterations such as the addition of hansgenes or targeted gene disruption. which
ad& a compounded advantage to those men tioned in the previous section regarding in vitro
systems. Mo~over, ES ce11 differentiation facilitates the snidy of certain gene products with criticai
or widespread d e s in development for which targeted deletion results in embryonic lethality.
Furthemore, the pluripotency of ES cells allows for the study of hematopoietic differentiation from
pre-hematopoietic precursors.
Differentiation of ES cells into hematopoietic lineages has been achieved by various
approaches including culture in meth ylcellulose or liquid medium in petti dishes, coculture on
stroma1 ceIl monolayers, and culture in liquid media suspended in hanging drops 13'. Particularly
well-characterized is the generation of myeloerythroid Lineages by the differentiation of ES cells
into embryoid bodies (EBs). Of distinctive importance are the findings that suggest that the course
of hematnpaietic diffetentiation h m E S tells in vina c i d y parallels the events of hematnpietic
development in vivo 138-140
B lymphopoiesis in v&o
In vitro hematopoietic differentiation cm be supported in the absence of stroma1 cells. In
particular, myelopoiesis and erythropoiesis occur readiiy in iiquid or semi-solid cultures with the
addition of various combinations of exogenous cytokines lY. B ce11 differentiation cm also occur
in the absence of stroma1 cells "".
The degree to which in vitro system can efficiently support the differentiation of B
lymphocytes fmrn ES cells remains uncertain. Reports demonstrateci that ES cells could give rise
to hematopoietic precmon with lymphoid ptential. However, lymphoid differentiation was
pnmarily dependent on transfer of hematopoietic progenitors into host anirnals 142-'u. Nakano et
al. desaibed a system in whicii ES cells were induced to differemtiate by coculture on the stroma1
ceIl line, OP9 (=/OP9 coculture). The OP9 ceIl line was denved h m M-CSF-deficient rnice
(op/op). Significantly, this study reported that IgM+ B lymphocytes were detectable after 40 days
of ESIûP9 coculture. The absence of M-CSF likely enhanced B ceIl potentiai by disallowing
macrophage differentiation and expansion from ovemwding the cocultures, and by increasing the
efficiency of ES ce11 diffe~ntiation and subsequent hematopoietic induction '".
T lymphopoiesis in vitro
In vitro T ce11 differentiation occurs efficiently in fetd thymic organ culture (FTOC), which
maintains the threedimensionai thymic microenWonment. FTOCs provide a powerful mode1
system to study T ce11 development. Moreover, the use of FTOCs that have been depleted of host
thymocytes allows for the study of thymic induction events by seeding these FïûCs with HSCs or
other p r e c ~ ~ 91*1&14. in v i m generation of T ~ y m p ~ ~ ~ y t e s hum ES celb wodd
facilitate the study of T ce11 lineage cornmitment and differentiation. particularly with respect to
genetically modified ES cells. However, to date it is unclear whether any in virro system can
support the generation of T lymphocytes from ES cells although. simila. to B cells, reports have
been made that ES cell-derived hematopoietic progenitors cm differentiate into T iymphocytes
when transfemed into host animals '42-1u.
THESIS FOCUS
Tissue-specific events and molecular mechanisms that control hematopoietic differentiation.
From the induction of mesoderrnal precursors to the generation of mature and functional biood cells,
are poorly undentood The in vitro differentiation of ES cells has provided a powerful mode1
system to study myeloerythroid differentiation. However, ES cell-derived lyrnphopoiesis has not
been wellestablished, lirniting the snidy of lymphohematopoietic differentiation and the
àevelopment of potential therapeutic applications. Thus, the work presented in this thesis aims to
establish the efficient generation of lymphocytes h m ES cells, and to m e r o w understanding of
lymphohematopoietic differentiation.
An in vitro system in which B lymphocytes couid be generated h m ES cells had k e n
pviously established by differentiating ES cells on OP9 stroma1 ceIl monolayers. However, littie
was known about the functional capabilities of these ES cell-derived B ceiis, and whether the
efficiency of B ce11 differentiation could be enhanced To address these questions, B ce11
differentiation in ES/OP9 cocuInires was further characterized, exarnining whether exogenous
cytokines could enhance B lymphopoiesis and determining whether B lymphocytes generated in
vitru are functionaily andogous to their cornterparts Rt vivo. To detexmine whether ES cells cm
give rise to T c e h in vitro, various precursor populations derived fmmESlûP9 coculhires were
assessed for their abiliiy to diffe~ntiate into T cells in FTOCs. To siudy lyrnphohematopoietic
differentiation events, the ce11 surface expression of Fikl and CD 105 was and yzed to potentidl y
isolate the earliest hernatopoietic precursors generated duing ES/OP9 coculture. Finally. a possible
d e for CD105 in early hematopoiesis was determined by examining the expression and function
of CD105 during the course of lyrnphohematopoietic diffe~ntiation in ES/OP9 cocultures.
Functional characterization of B lymphocytes generated in vibo
€rom embryonic stem cells
Sarah K. ho*. Travis D. ~ebber ' , lames R. ~ a r l ~ l e * . Tom ~akano'. Susanna M. ~ewis? and
Juan Carlos ~ ~ f i i ~ a - ~ n ü c ker'
'~e~artment of imrnunology, University of Toronto. Toronto, Ontario M5S lA8, Canada.
t Department of Molecular Ce11 Biology, Research Institute for Microbial Diseases. Osaka
University, Yunada-Oka 3- 1, Suita, Osaka 565, Japan. in Genetics and Genomic
Biology , The Hospital for Sick Children Research Institute. Toronto. Ontario MSG 1x8. Canada
(A-MuLV infections and extrachromosomal recombination assays were performed by
T.D. Webber and S.M. Lewis. Al1 other data shown was performed by S.K. Cho)
Published in The Proceedings of the National Academy of Sciences (USA)
August 1999, Volume 96, pp. 9797-9802
SUMMARY
To study molecular events involved in B lymphocyte development and V(D)J
rearrartgement, we have established an efficient system for the differentiation of embryonic stem
(ES) cells into mature immunoglobulin (Ig)-semting B lymphocytes. Here. we show that B
lineage cells generated from ES cells are hinctionally analogous to normal fetal liver (FL)-derived
or bone marrow (BM)-derived B lineage cells at three important developmental stages: fint. they
respond to FIt-3 ligand during an early lymphopoietic progenitor stage; second. they becorne targets
for Abelson murine leukernia virus (A-MuLV) infection at a pre-B ce11 stage: third, they secrete Ig
upon stimulation with lipopolysaccharide (LPS) at a mature mitogen-responsive stage. Moreovet.
the ES cell-derived A-MuLV-tmsformed pre-B (EAB) cells are phenotypicall y and func tional l y
indistinguishable from standard A-MuLV-transformed pre-B cells derived from infection of mouse
R or BM. Notably, EAB cells possess functional V@)J recombinase activity. Thus, in particular.
the genention of A-MuLV-transformants h m ES cells will provide an advantageous system to
investigate genetic modifications that will help to elucidate molecular mechanisrns in V@)J
recombination and A-MuLV-mediated transformation.
During development, B lymphocytes undergo a process of stage-specific differentiation
'49*1S0. CD1 I7+ (c-kit) hematopoietic progenitors with a restricted B ce11 lineage potential can
be identified by surface expression of CD45R (B220). CD43, and AA4.1 molecules 149*150.
Recentiy. it has been shown that the cytokine Fît-3 ligand (Fit-3L) enhances B ce11 lineage
also synergizes with interleukin-7 (IL-7) to induce the proliferation of primitive CD43+ CW~R'O
CD24- (heat stable antigen) B ce11 pmgenitors '".
Following cornmitment to the B ce11 lineage. upregulation of CD19 and CD24 surface
expression characterizes differentiation to the pro-B ce11 stage. B ce11 progeniton begin to undergo
DNA rearrangement of their V. D, and J loci to generate a diverse repertoire of antigen-specific
surface Ig 901149*150. Experiments with Abelson murine leukemia virus (A-MuLV)-transformed
pre-B ce11 lines have provided key insights into the regdation and mechanism of V@)J
rearrangement, and have been instrumental in establishing the phenotype of B ce11 precursoa lS3-
15'. Re-gement at the Ig heavy (IgH) chah locus begins in pro-B cells and productive
rearrangements result in the expression of 1 heavy chah during the pre-B ce11 stagc '4929.15Q. The p
heavy chah pairs with a s m g a t e light chain to form a pre-B ce11 receptor (BCR) complex which
signals these cells to pmliferate and promotes their differentiation to the CD4f CD2r late pre-B
ce11 stage ( c D ~ ~ R ~ C D lg+C~%+) 'O3. During this stage, productive rearrangement at the light
chah loci (K or A) results in the generation of immature surface IgW B cells that undergo selection
and give nse to mahue IN IgD' B cells 'O3. Functionally. B cells that have differentiated tu a
mame s t a g are denoteci by their ability to secrete Ig upon mitogen activation '".
Several methods have ken described for the generaiion of lymphohematopoietic
pmgenitors fiom embryonic stem (ES) cells in vitro 142*143*145. In a method described by Nakano
et al., undifferentiated ES cells were cocultured on the macrophage colony-stimulating factor
(M-CSF)-deficient BM stroma1 ce11 line, OP9, resulting in the appearance of hematopoietic ce11
clusters eight days later 14'. The advantages of this system over others for the generation of
small fraction of ES cells eventually gave rise to I N B cells afier long terni culture (40 days) on
the OP9 strornal cell line 14? In light of these findings, we sought to determine whether the
differentiation of ES cells in vitro resulted in progenitor and mature B lymphocytes that am
functiondly equivdent to those genented in vivo. To this end, we chose to investigate
representative events from early, rniddle. and late stages of B cell developrnent: response of eariy
progenitors to nt-3L. susceptibility of pre-B cells to A-MuLV infection. and mitogenic activation
of mature B cells by lipopolysaccharide (LPS).
Our findings show that the addition of nt-3L dramatically enhanced B lymphopoiesis in the
ESIOP9 cocultures. and pmmoted the pmliferation of a progenitor B ce11 subset. Furthemore, we
were able to generate permanent ES cellderived A-MuLV-transfomed pre-B (EAB) cell lines.
These EAB cell lines were phenotypically and functionally equivalent to standard mouse FL-derived
A-MuLV-transformed pre-B ceIl lines. Finally, our results show that ES/OP9 coculturederived
progenitor B cells develop into mature mitogen-responsive B cells, sec~ting Ig upon stimulation
with LPS. Thus, our results demonstrate that the OP9 stroma1 ce11 line, with the addition of Fit-3L,
is suscient for the differentiation of ES cells into lymphohematopoietic progenitors, and efficient
for the development of these progeniton into mature functionai B cells. Collectively, our findings
suggest that the developmenrai program of B cells derived in vitro from ES cells closely parallels
the intrinsic development of B cells in vivo.
T'herefore, the coculture system alone or in combination with A-MuLV infection, using
differentiated genetically-manipulated ES ceiis in vitro, should prove valuable in the elucidation of
molecular mechanisms controlling differentiation and Ig gene rearrangement during B cell
development
Induction of hemasopoiesis in ESOP9 coculntres
An NI vitro system developed by Nakano et al. allows for the differentiation of ES cells into
lymphohematopoietic precursors upon coculture with the M-CSF-deficient BM-derived stroma1 ce11
line. OP9 "'. Figure 6 provides a schematic outline of the ESiOP9 coculture system (see
Materials and Methods for details). As show in Figure 7, flow cytomeaic analyses of cells
harvested at different time points after initiation of the WOP9 coculture revealed that CD49 cells
'57-159 were first observed by day 5 of coculture. By day 8. the CD45+ cells also expressed
CD1 17 and Sca-1 on their surface, thus displaying a phenotype analogous to that of early
hematopoietic stem cells (Fig. 7) 15'* '? Typicall y, a signifiant portion of earl y hematopoiesis
occurring in the coculture system gave rise to cells of the erythroid lineage 16'. This was evident by
the large fraction of CD24+ cells staining positive for TER-1 19 (days 8 & 12; Fig. 7). Although
the majority of cocultured &y 12 cells belonged to the erythroid lineage ( ~ ~ 2 4 ~ C D 6
TER4 19+), Figure 7 also shows that the CD4Sç cells expressed low to high levels of CD45R.
This phenotype indicated that B-lineage cells emerged h m the coculture between day 8 and 13
'49*'M. Although this B lineage phenotype was clearly apparent by day 12, long-tem cultures (>
20 days) seldom resulted in the generation of CD19+ I N B cells (data not shown). This result is
in keeping with that previousl y observed by Nakano et al. '". To address one possible rnechanisrn
for the inefficient B lyrnphopoiesis observed, we charactrrized the cytokines expressed by OP9
ceiis.
Infection S tart First Second + A-MuLV cocu lm e passagea passageb * 1 day 15
I I 1 1 1 I I 1 I 1 1 1 I 1 1 1 1 I I I 1 I I I I 1 I I
Day O 5 8 12 15 Mesodenn colony Hcmmpdeitic blasts Rolifcrative burst of diffcnntiation cobble stoae colonies hematopoetic colonies
a Includes treatment with aypsin to disrupt the coculture prior to passaging. Cells were replated onto confluent OP9 cells, with the addition of Fit-3L.
b Loosely sdherent cells were rransfemd onto confluent OP9 cells (see text). * Media changes. with the addition of cytokines as indiwted (see text).
Figure 6. Outhe of the standard protocol used For the dif'ferentiation of ES ceUs hto B üneage lymphocytes. ES cells were induced to differentiate into hernatopoietic cells on the bone-marrow derived stromal cell line, OP9. To generate mature. functional B cells, hematopoietic cells were maintained on OP9 cells. To generate ES-denved Abelson- transfomed pre-B (EAB) cells. cocultures were infected with A-MuLV at 2 day 15. Flt-3L was added for dl media changes after day 5.
Day 5 Day 8 Day 12
Figure 7. Temporal kinetics of hematopoietic induction in ES/OP9 crniultures. Representative flow cytometric analysis of various surface markers as indicated are shown for days S,8, and 12 of coculture. These results represent cocuitures without exogenous addition of Flt-3L.
RT-PCR analysis ulcytokines expressed by OP9 ceUs
Several groups have demonstrated a criticai role for certain cytokines in eariy B ce11
developrnent 83. Stem ce11 factor (SCF), Flt-3L, and IL-7 have ken shown to pmmote and support
the growth of earl y B ce11 progeniton 83~97198*15'*162*'63. TO characterize the potential role these
cytokines may play in the induction of ES ce11 differentiation upon cocultu~ with OP9 cells, we
performed an RT-PCR analysis for cytokines expressed by OP9 cells. As shown in Figure &OP9
cells expressed high levels of S C F and moderate levels of TL-7. but almost undetectable levels of
Fit-3L. The low level of Flt-3L suggested a possible explanation for the inefficient B
lyrnphopoiesis in ES/OP9 cocultures. This is in accord with recent findings demonstrating that
Fit-3L synergizes with IL-7 to enhance B ce11 differentiation from uncommitted BM progenitor
cells 98. The importance of Flt-3L for the ES/OP9 in vitro system is underscored by the
observation that. whereas Flt-3L + IL-7 selectively stimulate the production of pro-B cells. SCF +
IL-7 predominantly support the production of mature granulocytes 98. Thus. the emerging ES
cell-derived hematopoietic progenitors may be adversely affected by the low level of Flt-3L relative
to SCF.
Flt-3L enhances the generation of B l~mphocytes from ES/OP9 cocultures
In light of the above findings, we exarnined whether hernatopoietic progenitors generated
hwn ESIOP9 cctcul~~.es wouM respond io exogenous addifion of RE-3L, resulting in an enhanced
induction of B lineage cornmitment. To this end, Flt-3L was added at &y 5 of the ES/OP9
coculture, when hematopoietic cells were first observed (Fig. 7). Analysis of &y 19 cocultures
revealed thaî the addition of nt-3L dramatically enhanced the generation of B lymphocytes from the
ES/OP9 cocultures (60% venus 6% CD45R+ cells, with or without Flt-3L. respectively) (Fig. 9).
Thus, the addition of Fit-3L to the ES/OP9 coculture at day 5 increased the recovery of B lineage
1- Flt-3 Ligand
Figure 8. Cytokine expression in the OP9 ceil Iine. Total RNA from OP9 cells was analyzed for the expression of several cytokines by RT-PCR. cDNAs were prepared from 1 pg of total RNA. OP9 cDNAs were diluted in a 1 5 senes and then amplified simultaneously by PCR using gene specific primer pairs. as indicated.
F i 9. Flt-3L enhances the in vitro generation of ES ceii-àerived B lymphocytes. Fiow cytometric analysis for various ce11 surface markers expresseci on ES celiderived B iineage cells h m &y 19 ES/OP9 cocultures. The addition of Flt-3L to the cocultures at &y 5 resulted in a ciramatic increase in the generation of B lymphocytes. Flt-3L was added each the the culture media was replace& as indicated in Figure 6.
(Mac-1). and erythroid, TER- 1 19+, cells was diminished in the Fit-3L-treated cultures (Fig. 9).
Evidence for T lymphocyte differentiation was not observed in these cultures (CD3 v. CD8; Fig. 9).
The phenotype of day 19 ES/OP9 coculture cells clearly showed that Flt-3L addition resulted in a
specific increase in the generation of CD 19+ CD45R+ AA4.1+ CD24+ IgM? cells (Fig. 9). although
only s slight increase in the total ce11 number was observed (-30%). With the addition of Fit-3L at
&y 5. B lymphopoiesis in the =/OP9 coculture system occured with high efficiency in d l
independent trials (>25) to date.
To directly Iissess which stage of differentiation was predominantly affected by the addition
of exogenous FIt-3L. we analyzed the ce11 cycle status of different subseü of ES/OP9 coculture
cells by flow cytometry. Table Ii shows that the AA4.1+ CD45R+ CD 19' population of early pro-
B cells is most dramatically affected by the addition of exogenous Fit-3L, with an alrnost two-fold
increase in the percentage of cells in the S-GZM phase of the ce11 cycle. Our analysis also revealed
a similar increase in the percentage of hematopoietic progenitor cells, ~ ~ 4 5 ' ~ ~ ~ 3 4 " , proliferating
in the presence of exogenous Fit-3L (Table II). Taken together, these results are consistent with
previous findings showing that Flt-3L enhances B lineage cornmitment and differentiation h m
early lymphoid progenitors 83-98. Thus, the Flt-3L response of early B ce11 progenitors derived
h m ES/OP9 cocultures is akin to the response of in vivo B ceIl progenitors.
Direct generation of ES-derived A-MuL V-transfomed pre- B (W) tells
It is likely that during the course of ES ce11 differentiation, fmt into lymphoid progenitors
and then into B cells Flg. 9), cells should transit through a developmental stage comsponding to
that of FL-derived pre-B cells. Because FL is a rich source of targets for A-MuLV transfomation
' y it seemed likely that A-MuLV pre-B tmsformants might be directiy obtainable h m this in
Table ïI. CeU cycle anaiysis in ESIOP9 cocultures.*
* Cells from day 19 ES/OP9 cocultures, with and without exogenous addition of Fît-3L, wexe analyzed for surface phenotype and DNA content by flow cytom~try. Percentage of cells in S + G2/M within CD19 by AA4.1 and CD45 by CD34 subpopulations.
vitm system As outlincd in Figure 6. differentiating ES cells were infected a k r day 15 when a
significant population displayed a phenotype consistent with B iineage cornmitment. i.e. CD 19+
CD45R+ AA4.1+ (Fig. 10A). After the addition of viniscontaining supernatants, cells were
cultured for an additional 2 4 week pend until transformed cells became apparent (see Materiais
and Methods). EAB ce11 cultures were then cloned by limiting dilution.
Fiow cytomeaic analysis of a representative EAB ce11 clone is showi? in Figure IOB. The
phenotype of these cells was virtually identical to that of Abelson lines derived by in virro
transformation of FL or adult BM cells lS3*'". In panicular, EAB cells displayed a CD45R'
CD lgC CD24' A A ~ . 1+ s1wflo Lin' phenotype (Fig. lOB), correspondhg to that of pre-B cells
'49*'50. Moreover, PCR andysis of DNA '" extracted from these cells revealed that they had
undergone DI and M)J rearrangement of their IgHchain DNA loci (data not shown).
A signahue feature of A-MuLV pre-B transformants is their ability to exhibi t continued
V(D)J recombination activity in culture This feature can be assessed using extrachromosomal
V@)J recombination substrates '66. Recombination activity is scored by the ability of the ceil to
site-specificaily recombine a plasrnid containing V@)l joining signals, after which the plasrnid will
confer chlorarnphenicol resistance when introduced into E. coli '? Al1 of the EAB clones tested
were p s i tive with this assay . In cornparison to a standard A-MuLV pre-B ce11 line, 204- 1 -8 16',
EAB clones showed recombination ac tivi ty of equd or higher levels (Fig. 1 1 A). The site-specific
nature of the recombinants was confirmed by testing plasrnid DNA recovered from
chloramphenicol-resistant bacterial colonies for the presence of a diagnostic ApaLl site 166*'". AS
show in Figure 1 IB? most plasmids possessed the expected ApaLl site, indicating that an
authentic "signai joint" had been generated. Exceptional cases were due to imprecise joining, as
Figure 10. Characteriution of ESderived Abelson marine Ieukemia virus-transformed pre-B @AB) ceh. A) Row cytometric analysis of day 15 ES/OP9 coculture for surface expression of CD 19 vs.CD45R. and CD45 vs AA4.1. The generation of ES cell-derived B lineage cells is clearly evident after 15 &y of coculture. B) Flow cytomeaic analysis of a representative EAB ce11 line shows nearly 100% homogeneity of pre-B ce11 associated phenotype. Conirol staining with T and NK lineage mAbs is shown in the top left panel. The other panels show a composite phenotype, CD45R+ CD19+ CD24+ AA4.1+, consistent with the EAB cells designation as transformed pre-B ceils.
a The number of colonies was corrected for plasmid and transformation dilutions.
r, Site-specific recombinants fiom EABs
' New ApaL 1 site
Figure II. Recornbütation aaalysis in EAB ceiis. Site-specific recombination of the transfected V(D)J- containing plasmid, pWTSJA, results in ûamcrîption of the chloramphenicol-resistance gene, and mates a new ApaL1 restriction endonuclease site. A) Recombination index (RI) of two EAB ce11 lines versus a standard Abelson line, 204-1-8. %RI is the ratio of double resistant Amp%CatR colonies over AmpR colonies. B) Inverted EtBr gel image of site-specific recombination products in the extrachromosomal V(D)J recombination assay, showing the two bands generated by a new ApaLl site.
typically obsewedat low levels for A-MuLV lincs ued with the V(DU recnmhinahm assay
155*'68*169. In addition, alternative recombination products, in which a cryptic joining signal in the
plasrnid is targeted '67. occurred at nonnd low fiequencies (100 recombinants were mened by
colony filter hybridization; data not shown) 167. Thus, the quantity and quality of the recombinant
products scored by the extrachromosomal assay was in agreement with previous observations made
on typical A-MuLV-transformed pre-B ce11 lines.
Generaiion of mani re mitogen -responiive Ig -secreting B lymphocytes
Analysis of cells harvested from later time points of W O P 9 cocultures with Flt-3L showed
a large increase in the percentage of cells positive for B-lineage marken. Following a four week
culture p e n d nearly al1 (90%) of the cells present in the coculture were B lineage CD45Rf
CD l9+ lymphocytes (Figure 12B). These ES-derived B lymphocytes displayed a CD 11 b"'
phenotype, with a small subset (2-3%) of CDS+ B cells (Fig. 9 and data not shown). suggesting that
CD9 B cells a were not readily generated in the W O P 9 cocultures.
To further demonstrate the functional capabilities of the in vitro-generated B cells. &y 38
cocultures were treated with LPS 156. Following the addition of LPS. mature surface IgM+ CDl9+
B cells increased in size and proliferated extensively (Fig. l2A. and data not shown). Following
nritogcn advation we d y z e d for the expression of CD80 (B7.1). e costirnutatory molecule
normdly upregulated on mature B cells following activation "O. Figure 12C shows the increased
surface expression of CD80 upon stimulation with LPS. indicating that ES-denved B cells behave
in a similar manner to nomal B cells. Furthemore, culture supernatants h m LPS-stimulated cells
were positive for the presence of IgM by ELISA anaiysis ( 16.4 11.4 pg/rnl), revealing that these
cells were capable of robust levels of Ig secretion. In conmt to previous reports '""", our
m + LPS
Figure 12. Ia vitro generation of ESderived mature huictional B c e k Flow cytometric analysis for A) forward-scatter (FSC) by si&-scatter (SSC) and B) CD 19 by CMSR of ES/OP9 coculture with aod without LPS treatment for 4 days- ELISA analysis of the culture supernatant collected 4 days after LPS stimulation showed high titer for secreted IgM (-16 j@ni). C) Flow cytometnc analysis for CD80 (B7-1) with and without LPS matment for 48 h. Mean flourescence intensities: 10.25 for -LPS, and 31.81 for +LPS.
findings pmvide the first evidence for the differentiation of ES cells into mature rnitogen-respnsive
Ig-secreting B cells in vitro.
DISCUSSION
The addition of exogenous Fït-3L to the ES/OP9 coculture system was found to be a key
element in the development of an efficient and practical mode1 system for the generation of mature
functional B lymphocytes from ES cells in vitro. The present findings support the notion that nt-
3L is an important factor in early B lymphopoiesis in vitro 98. Moreover. it also elucidates how the
addition of Flt-3L to the ESlOP9 cocultures facilitates the generation of B lymphocytes. Fiow
cytometric analyses of ES/OP9 cocultures revealed that the differentiating ES-denved precunors
approximated the temporal kinetics and phenotypic progression of known developmentai stages that
occur during B ce11 differentiation in vivo (Figs 7,9, 10A, 12, and data not shown). Thus. the fact
that ES-denved B cells follow a normal developmentai pathway, and are Functionaily analogous to
progenitor and mature B cells in vivo, provides strong evidence that this system will prove vaiuable
in dissecting the molecular events that govern B cell differentiation.
Additional applications will follow from the abiiity to obtain A-MuLV-transformed
differentiated stable ceil lines h m a genetically-modified ES cell, entireIy in vitro. Because A-
MuLV msfomiants a~ simple to generate and mainiain, with a rapid doubling tirne, the derivation
of EAB ceil iines will add to the armory of possible approaches in studying lineage-specific gene-
targeted mutations. For exarnple, nul1 mutations in certain genes involved in V@)J recombination
c m be embryonic lethal 17'. In such cases. one rnight still be able to assess their d e in V@)J
recombination program by exarnining EAB cells derived dirraly from homozygous mutant ES cells
172
Togehr wih ihe meni c h t e ~ r a t i o n of human ES cells 'TJv'74, OU^ findings make ii
possible io envisage a system for the genention of hurnan B ce11 progenitoa a d o r B lymphocytes
directly h m ES cells in vitro. Such a system would provide a limitless souce of
geneticaily-defined ES ceil-derived B cells, with potentiai therapeutic applications for individuals
suffering frorn agarnrnaglobulineriuas or specific B ce11 dysfunctions.
ACKNOWLEDGEMENTS
We th& A. M. Michie for cntical ~ a d i n g of the manuscript. We also thank C. Furlonger and N.
Rosenberg for reagents md advice regarding A-MuLV infection. A. h b b é for assistance with the
ELISA analysis, S. Trop for help with the ce11 cycle analysis. and G. Caruana for help in setting up
the OP9 cocuInire system. This work was supported by gants from the National Cancer Institute
of Canada (NCIC) with fun& from the Terry Fox Run (JCZ-P and SML). and the Medical
Research Council (MRC) of Canada (JCZ-P). SKC is supported by an award from the Lady Tata
Mernorial Trust. JRC is supported by a studentship from the MRC. SML is a Research Scientist
of the NCIC. JCZ-P is supported by a Scholarship from the MRC.
In vitro generation of T lymphocytes from embryonic
stem cell-derived pre-hematopoietic progenitors
Sarah K. Cho, James R Carlyle, and Juan Carlos ZiiRiga-Pfiücker
Department of Irnmunology. University of Toronto, Toronto, Ontario M5S lA8. Canada.
(Experiments represented in Figure 2 were performed by J.R. Carlyle.
Ail other data shown was perforrned by S.K. Cho)
Embryonic stem (ES) cells cm differentiate into most blood cells in vitro, providing a
powerfbl mode1 system to study hematopoiesis. However, the generation of T lymphocytes has
rernained elusive, suggesting that T ce11 potential is either absent or difficult to isolate during ES ce11
differentiation in vitro. Attempts to generate T lymphocytes in vitro h m ES cellderived
hematopoietic progenitors have been largely unsuccessful. In order to circumvent the p s i bi lity
that ES cell-derived hematopoietic progeniton may rapidly commit to non-T ce11 lineage fates, ES
cell-derived pre-hematopoietic precursors were used to reconstitute fetd thymic organ cultures.
However, when used unfractionated, these precursors were deleterious to the thymic architecm
and failed to give rise to thymocytes. In contrast. a transient subset (Rkl+ CD45') of these
precursors was able to eficiently generate T lymphocytes Nt vitro . These findings reveal that T ce11
potential is restricted to an early stage of ES ce11 differentiation, and support the notion that the
thymic microenvironment is capable of inducing T ceIl differentiation h m a subset of pre-
hematopoietic progenitors. The ability to generate T cells in vifro h m a defined source of stem
cells should contribute io the development of future therapeutic approaches.
INTRODUCTION
Embryonic stem (ES) cells are tofïpotent cells isolated h m the inner ce11 mass of
blastocysts and cm contribute to al1 tissues of an animal, including germ cells, when transplanted
back into a developing blastocyst 135*136. The in vitro differentiation of ES cells provides a mode1
system to study the development of various tissues and lineages 13'. In particdar, ES cells can
difierentiate into most blood ceil lineages. facîlitating the study of hernatopoietic differentiation
137,144
In the thymus, T cells differentiate from hematopoietic stem ce11 (HSC)-derived lymphoid
progeni ton ' Thus. the findings that B and natural killer (NK) lymphocytes can be
generated from ES cells in vitro suggested that ES cell-derived developmental
intermediates should also possess the ability to become T lymphocytes. There is some evidence to
suggest that ES celldenved precursors exhibit T ce11 potential in vivo 14*10*178. However, the ES
cell-derived precursor population responsible for the generation of T cells in vivo was undefined.
and it also remains unhown whether ES cells cm differentiate into T lymphocytes in vitro.
To address these questions, a culture system was employed that efficiently supports in vitro
lymphopoiesis from ES cell-derived progenitors by differentiating ES cells on the stmmal ce11 line
OP9 (ES/OP9 coculture) 1459176. Using this ESIOP9 coculture system, we report that ES
cell-derived Flklt CD45 pre-hematopoietic progenitors cm effsciently give nse to T lymphocytes
when transferred into fetai thyrnic organ cultures (FïOCs). The in vitro generation of T cells from
this defined pre-hematopoietic subset of differentiaied ES cells should help to elucidate the
thymic-specific mechanisms which allow for the induction and support of T ce11 lineage
cornmitment and differentiation. In addition, these findings establish a foundation for tissue
engineering applications and the development of future therapeutic approaches, which may requi~ a
geneticaily-defined source of T lymphocytes.
RESULTS AND DISCUSSION
ES cells cocultured on O B ce11 monolayers (ES/OP9 cocultures) give rise to hematopoietic
clustea, and subsequently differentiate into B lymphocytes (Fig. 13) 145v176*'79. We previously
Rcsccding onto OP9
6 - 9 &YS
O B cells
'4 s d i n g of feral thymic lobes by hanging drop
Figure 13. Schematic for the differentiation of ES c e h into B and T lymphocytes. ES cells were induced to differentiate on the bone marrowderived stromai ce11 line, OP9. Intitial seeding of ES cells ont0 OP9 cells is designated as &y 0. After 5 to 6 days of coculture, mesodemi-like colonies were observed and were disaggregated by treatment with 0.25% Trypsin. Reseeding onto fresh OP9 ce11 monolayers dlows for the generation of B lymphocytes. For the generation of T lymphocytes. cells were seeded into fetal thymic lobes by "hanging drop" for 1 to 2 days, and then transferred to fetal thymic organ cdttues (FTOCs) for approximately 2 weeks. Flow cytometric d y s i s of B ce11 lineage markers (CD 19 and CD45) and T ce1 lineage markers (CD4 and CD8) are shown for a &y I9 EWOD c o c u l ~ ~ and a &y 14 FTOC, respectively . Using this protocol, the generation of B lymphocytes is routinely observed (6% CD 19+ CD45+). while T lymphocytes are not generated (>97% CD4- 0%).
phenotypicall y nsernble multipotent hematopoietic progenitors IN. There fore, we reasoned that
these progenitors might exhibit T ceil potential when transferred to host thymaiytedepleted
FTOCs TO test this notion, CD49 CD 1 17+ cells were isolated from ES/OP9
cocultures on various days and transfemd into FTOC. Reconstitution of JTOCs with ES
cell-derived hematopoietic precursors was rare (c 5% of independent experiments gave rise to ES
cell-derived thymocytes. n>20: data not shown). This low frequency of reconstitution may be due
to a loss of hematopoietic multipotency during ES ce11 diflerentiation. or other as yet undefined
limitations of in vitro ES cell cultures. Indeed, it remains unclear whether tme HSC activity,
including full-lineage multipotency and self-renewal, is possible during in vitro ES ce11
differentiation 1?J*137. Thus, in the absence of a thymic microenvironment, ES cell-derived
hematopoietic precursors may be npidly induced to commit to non-T ce11 lineages. We reasoned
that these problems rnight be circumvented by the isolation and transfer of ES cell-denved
pre-hematopoietic precursors into FTOCs, where induction towards the hematopoietic lineage
would occur de novo within a thymic microenvironment, therefore favoring T ce11 differentiation.
During embryogenesis. the hematopoietic lineage is established from mesodemal
precursors ". Mesodem-like colonies are observed by &y 5 of W O P 9 coculture, and when
reseeded ont0 OP9 cells give rise to B lymphocytes (Fig. 13) '45*179. Thus, we tested whether
these precursors from day 5 ES/OP9 cocultures could give rise to T lymphocytes when transferred
into FTOCs (Fig. 13). After 14 days in FTOC, analysis by flow cytometry showed no evidence of
donor-derived T ce11 differentiation (Fig. 13). Additionally, the FTOCs resernbled arnorphous ce11
aggregates, instead of the well-smictured thyrnic lobes that are normally observed in FTOCs,
suggesting that the thymic architecture was alterrd
The three-dimensional architecture of the thymus is critical for normal T ce11 development
'". Tedeiemum- whether differentiated EScells adversely affect& Ihe thymic rnicroenvir~cunen~,
cells From a day 5 ES/r)P9 coculture were serially titrated and added to FTOCs in conjunction with
a known source of HSCs (13 dpc fetal liver, FL, cells). As shown in Figure 14, reconstitution of
CD4 and CD8 thymocyte populations was observed in lobes that were seeded with FL-denved
progenitors (fig. 14. upper right panel), compared to conml lobes that received only media (Fig.
14. upper left panel). Again. ES cell-derived thyrnocytes were not detected (Fig. 14 bottom panels,
and data not shown). Moreover. Figure 14 shows that thymocyte reconstitution from 5 x 1 0 ~
FL-derived progenitors was disrupted by the presence of differentiated ES cells, such that the
addition of 560 differentiated ES cells completely abolished reconstitution. Collectively, these
results suggest that ES cell-derived progenitors from day 5 cocuInires inhibit FTOC reconstitution
because they are detrimental io the three-dimensional thymic architecture. In contrast, we
previously showed that day 5 ES/OP9 coculture cells efficiently gave rise to lymphohematopoietic
cells when reseeded back ont0 OP9 ceIl monolayen ''! Thus, only a subset of differentiated ES
cells may be amenable to the thymic microenvironment.
Flkl (vwular endothelial growth factor receptor-2, VEGFR-2) is a receptor tyrosine kinase
that is expressed on subsets of rnesoderm and on the earliest endothelial and hematopoietic
precursors 38J9~i39*i8 ' . Specificdly, Kabrun et al. suggested that a transient population of Fl k l +
precursors may represent the onset of embryonic hernatopoiesis '39. We hypothesized that
isolating FIklf cells from day 5 to day 6 ESIOP9 cocultures might exclude cells that are deleterious
to the thymic architecture (Fig. 14). Moreover, Rkl+ CD45 precursors are pre-hematopoietic and
would therefore be uncornrnitted to particdar hematopoietic lineages, and thus permissive to T ce11
cornmitment and difierenbation. Ce11 surface expression of Rkl dming the course of ES ce11
differentiation was analyzed by flow cytometry (Fig. 15A). The resuits in Fiam 15A indicate that
the greatest percentage of Rklf cells was observed at approximately day 5 of ES/OP9 coculture.
Figure 14. Deleterious efkct of dierenüated ES ce@ on FTOCs. Fetal thymic lobes (14- 15 dpc) from Swiss (S w) mice were depleted of host thymocytes by irradiation (3000 cGy). Media was added to lobes as a negative control (top le fi). Host thymic lobes were reconstituted with 5000 Sw fetal liver (FL) cells (top right). In addition, some tobes received 60, 180,560 or 5000 differentiad ES c e h (day 5 ESlOP9 coculture). Lobes were analyzed by flow cytornetry for ce11 surface expression of CD4 and CD8 on day 13 of FTOC. Day O FïOC corresponds to the day of initial seeding of the thymic lobes by hanging drop.
# of days differentiation
Figure 15. Lymphopoietic potenaal of ES ce1I-àerived F M + precursors. A) 104 ES cells were seeded onto OP9 ce11 monolayers in 6-well piates. Cells were hmested from ESOP9 coculn~es on various days and andyzed by fiow cytometry for Nr 1 surface expression. The percent of cells expressing FIkl is shown. Cocultures were disaggregated with aypsin on day 6. B) 2 x 104 ES cell- derived Flkl+ cells were isolated by flow cytomeüic ce11 sorting (day 5 ES/OP9 coculture) and reseeded ont0 fresh OP9 ce11 monolayers. Evidence of B lymphopoiesis on day 14 was detennined by flow cytometnc analysis for the surface expression of CD45 and CD19. The total number of cells obtained from the coculture is indicated in parentheses.
were sorted and reseeded back ont0 OP9 cells (Fig. 193). Figure 15B shows that Rkl+
progenitoa were able to give rise to B lymphocytes with continued coculture on OP9 cells.
To determine whether Hkl+ CD45' progenitors from &y 5 to day 6 ESIOP9 cocultures can
generate T lymphocytes in vitro. Flkl+ CD45' cells were isolated by fiow cytometric ce11 sorting
(Fig. 16A) and seeded into FTOCs that were derived from RAG-2"' '82w'83 CD45.1 congenic
mice. After 13-16 days in FTOC. donor-derived thymocyte reconstitution was detnmined by flow
cytometric analysis for the surface expression of CD45.2. Control host FTOCs receiving only
media did not contain cells expressing CD452 (Fig. 16B). Detection of donor-detived CD45.2+
cells indicated that thyrnic reconstitution was successf'ul in FTOCs that were seeded with ES
cell-derived FIklf CD4S pre-hematopoietic precurson (Fig. 16B). These ES cell-denved
thymocytes differentiated into ail the normal CD4 and CD8 thymic subsets (Fig. 16C). The in
vitro generation of T lymphocytes from ES cells for three independent experiments is shown in
Figure 16C. Furthemore, ES cell-denved FlklC CD4S precursors gave rise to T lymphocytes in 5
of 11 independent experiments. Our finding that T Iymphocytes cm be generated in R O C from
pre-hematopoietic precursors is consistent with the results reponed by Nishikawa et al., in which
CD45 endothelid-like cells derived from E9.5 to El0 embryonic tissues gave rise to T cells in
thymic organ cultures 38.
ES cell-derived T cells were generated mote frequently and efficiently (1 10-fold) h m
Rkl* CD45' as opposed to CD49 CD1 l? progeniton. This supports the notion that during ES
cell differentiation, newly generated precursors may rapidly lose theû T ceIl potential as
they are induced to differentiate towards other hematopoietic tineages. It is interesting to speculate
Expt. 1 (135 x 103 soned ml+ ctlwlobc: 4 l o k )
Figare 16. In Pibo genemtion of T lymphocytes h m ES ceil-derived Flkit precarsors. A) Cells were ~ e s î e d f r o m d a y 5 a & y 6ESIQP9 cocuîtures and Flk t + CD45- ceiis were isolated by flow cytometric ceii sorting. B) Sotted ml+ cells were seeded into host ceiidepleted fetd thymic lobes ( 14- 16 dpc) from RAG-Ldeficient CD45 1 congenic mice by hanging drop, culnired overaight, and then transferred to FfOC. Two weeks later, donordenved reconstitution was determined by flow cytometric andysis for CD45.2 surface expression. Negative control (no donor) lobes received only media In two additional experimcnts CD45.2+ cells were undetettable in control lobes (no donor), and represented 91% and 89% in lobes that received ES cellderived Ftk 1 + precursors. C) Evidence of donor- derived reconstitution was determined by flow cytometry in three independent experiments. Anaiysis of CD4 and CD8 surface expression is shown for CD45.2+ gaced cells. Individual lobes were pooled for analysis. Experiments 1 and 2 were analyzed on day 14 FTOC, aad expriment 3 was analyzed on &y 16 FTOC. The total number of cells is indicated in parentheses.
Expt 2 (7.8 x 103 s o d Fut[+ celMok 2 lobes)
rhat the difficulties in isolatirtg CD49 purstxs witf, T cell p e ~ t i d in ESOP9 cocultures
amount to the sarne difficulties that have peaisted in isolating HSCs from ES cells diffe~ntiated in
vi t ro '34v137. In this regard, it can be postulated that tnie HSCs might be generated from ES cells
in vitro. however. these HSCs may rapidly becorne diffe~ntiated into restricted muftipotent
progeniton. Characterization of the Rkl' CD45* precurson generated in ESfOP9 cocultures rnay
help to further define ES cell-derived T ceIl progeniton, as welI as to identify inducible factors that
could conaibute to T ce11 lineage cornmitment andor maintenance of HSC-like multipotency.
Two key findings have contributed to the ability to repducibly generate T cells h m ES
cells in vitro. First. Flkl' pre-hematopoietic precursors weR non-deleterious and responsive to the
differentiation cues provided by the thymic microenvironment. Second, ES cellierived Flklf
progenitors exhibited T ce11 potential at a higher incidence than ES celi-derived CD49
hematopoietic progenitors, which suggests that the Flkl+ population is eruiched for precursors that
have not committed to other hematopoietic fates. These findmgs explain why the differentiation of
ES cells into T cells has been diffîcult to achieve compared to other blood cell lineages, and should
help to advance the study of thymic T ce11 differentiation and lineage cornmitment. In conjunction
with the ment derivation of human ES ceIl lines ".'. and establishment of mamrnalian ES cell lines
after nuclear transfer from somatic cells 18J*'85, these findings Rpresent r fundamentally important
k t step towards the development of hurnan T ce11 reconstitution therapies from a
geneticaily-defined source of stem cells.
We thank Drs. Alison Michie and Ross La Motte-Mohs for Mticd reading of the manuscript.
This research was supported by the National Cancer hstitute of Canada with funds from the
Canadian Cancer Society. SKC was supported by a studentship from the Lady Tata Memorid
Fund (UK). JCZP is supported by a Scientist Award from the Canadian Institute of Health
Researc h.
Expression and function of CD105 during the omet of hematopoiesis
from FUCI* precursors
Sarah K. ho*, Annie ~ourdeau*~, Michelle ~etarte*~, and
Juan Carlos zufiiga-Pflücker*
' ~e~ar tment of Irnmunology, University of Toronto. Toronto. Ontario MSS lA8. Cana&. *cancer
and B l d Research Program, The Hospital for Sick Children Research Institute, Taronto. Ontario
MSG 1x8, Canada,
(Ail data shown was performed by S.K. Cho)
B lood. In press.
During ontogeny. the hematopoietic system is established from mesodennderived
precursoa. however molecular evenü regulating the omet of hematopoiesis are not well
characterized. Several members of the transforming growth factor (TGF)-$ superfamily have ken
implicated to play a role during mesodenn specification and hernatopoiesis. CD 105 (endoglin) is
an accessory receptor for memben of the TGF-fil superfarnily. Here we report that during the
differentiation of rnwine embryonic stem (ES) cells in vitro, hematopoietic cornmitment within
Flkl' mesodemal precursor populations is characterized by CD105 expression. In particular,
CD 105 is expressed during the progression from the Fik l+ CD4Y to Flkl- CD49 stage. The
developrnentiilly regulated expression of CD105 suggests that it may play a rûle during early
hematopoiesis from Flklf precunon. To determine whether CD105 plays a functional d e during
early hematopoietic development, we assessed the potential of CD 105deficient ES cells to
differentiate into various hematopoietic lineages in vitro. In the absence of CD105, myelopoiesis
and definitive erythropoiesis were severely impaireci. In contrast. lymphopoiesis appeared to be
only mildly affected. Thus, our findings suggest that the regulated expression of CD105 functions
to support lineage-specific hematopoietic development h m Flkl+ precursors.
INTRODUCTION
During vertebrate embryogenesis. the onset of hematopoiesis and vasculogenesis occurs in
the extraembryonic yolk sac with the formation of blood islands from aggregates of mesodemal
precursors. CeUs within these clustes differentiate into primitive erythrocytes while those at the
periphery differentiate into endothelial cells. The temporal and spatial coupling in the appearance of
hernatopoietic and endothelial cells led to the hypothesis that these lineages are derived from a
common progenitor '". Recent snidies have shown that the mis-like receptor tyrosine kinase Flkl
(also known as vascular endothelial growth factor receptor-2, VEGFR-2), whch is expressed on
subsets of mesoderm is critical for the normal development of both hernatopoietic and
endothelial lineages. Fikl-deficient vklJ-) mice die in utero ai E8.5 due to defects in blood island
and vasculature formation 36. Further investigations revealed thatflklJ' embryonic stem (ES) cells
failed to contribute to hematopoietic or endotheliai cells in chimenc rnice, although residual
hematopoietic md endothelial activities were observed during differentiation in vitro 37,186.1 87
Moreover, in vitro clonal assays provided direct evidence that hematopoietic and e~idothelial ceIl
lineages are derived from a cornmon precursor 38-39 that expresses Hkl 38. Thus, Flkl expression
may serve to identify the hemangioblast, a putative bipotent progenitor for the hematopoietic and
endothelial lineages. The molecular events contributing to hematopoietic and endothelial
development immediately following the Flkl+ stage remain to be elucidated
Members of the TGF-fi superfamily enert multiple effects during developrnent, including
d e s in mesoderm paneming and hematopoietic differentiation 13. CD105 (endoglin) is an
accessory receptor for severai memben of the TGF-$ superfamiIy 2a2'. CD105 was fint
identified on human leukemic cells of the pre-B phenotype and was then shown to be
transiently expressed on subsets of normal human hematopoietic iineages such as proerythroblasts,
macrophages, and fetal marrow early B cells U-24*189. It is expressed on ail types of vessels and is
implicated in endothelial ce11 func tion '? CD 1 OS-deficient (en&) mice exhi bi t normal
vasculogenesis but die in utero of impaired vascular and cardiac development, suggesting that
CDlOS M critical fur angiogenesis 25-27. A ,le f a CD105 in hematopoiesis is suggested hy its
expression on hematopoietic subsets and its potentiai involvement in signalling by memben of the
TGF-p superfamily. However, hematopoietic developrnent beyond yolk sac erythropoiesis was not
assessed in eng-'- mice due to embryonic lethality at E1O.O- 10.5.
Differentiation of ES cells in vitro provides a powerfd mode1 system to study
hernatopoietic development 13? The progression of events appears to parallel that of the developing
embryo, and various hematopoietic lineages can be generated, including erythroid and myeloid cells
i 3 7 ~ i 3 8 ~ i ~ * ' 6 i . In particular, ES cells differentiated on the macrophage colony stimulating factor
(M-CSF)&ficient bone mamw stromal cell line, OP9. are aiso able to generate lymphocytes.
allowing for the characterization of rnyeloerythroid and lymphoid development within the sarne
system ''j*145*176. Therefore, we used this approach to elucidate the expression and function of
CD 105 during lymphohematopoietic development. We demonstrate that during ES ce11
differentiation in vitro. CD 105 is coexpressed on Rkl + precurson with hematopoietic potential,
and furthemore, expression is maintained at intermediate levels on the earliest detectable CD45+
cells. These data suggest that CD 105 may be a useful marker to M e r investigate early
hematopoietic development from Hkl+ precursors. In addition, our findings suggest that CD 105
plays an important functional role in hematopoietic differentiation h m Flkl+ mesodemal cells, as
we observed severely diminished myeIoelythropoiesis in the absence of CD 105. However, CD 105
does not appear to play a prominent role in lymphopoiesis. Thus, our data implicate CD105 as a
lineage-specific regulatory molecule during the onset of hematopoiesis frorn FIk14 precursors.
Characterization of ES cell-de rived hematopoietic lineages
ES cells were differentiated on OP9 cells (ES/OP9 coculture) as p~viously described
1"m176, and the generation of multiple hematopoietic ce11 lineages was maiyzed by fîow cytomeuy
(Fig. 17). A small fraction of cells expressing the receptor tyrosine phosphatase. CD45 (leukocyte
cornmon antigen, LCA), which is present on all hematopoietic cells except mature erythrocytes
86*'n? is detectable as early as &y 5 of coculture 176. Some variation is observed in the temporal
kinetics of the different hematopoietic lineages in independent ES/OP9 cocultures. Typicaily.
erythropoiesis in =/OP9 cocultures peaks between &y 13 to day 14, and rnyelopoiesis peaks
between day 12 to day 16. Although significant lymphoid populations cm be obsewed by day 14.
1 ymphopoiesis generall y peaks after day 16, when myeloerythropoiesis subsides. such that beyond
day 19 the cocultures usually consist primarily of lymphocytes. Analysis by flow cytometry
(representative of day 14 to &y 16 cocultures) identified four distinct populations. labelled a d (Fig.
17A), as determined by ce11 surface expression of CD45 and CD24 (heat stable antigen, HSA)
191g192. Figure 17B shows further analysis of these populations with lineage-specific markers as
follows: CD45- CD24+ cells (Fig. 17A, a) corresponded to TER1 19+ " erythrocytes (Fig. 178. a);
CM'"' cetls (Fig. I7A. b) comsponded to CD45R (8220)+ B Iymphocytes L93w'9J which
also coexpressed CD 19 (Fig. 17B, b; and data not shown); CD49 CD24+ cells (Fig. 17A. c)
comsponded to CD L l b (Mac- l)+ myeloid cells (Fig. 17B, c); and ~ ~ 4 5 ~ cells (Fig. 17A, d)
comsponded to NK lymphocytes c haracteris ticdl y lac king CD24 expression (Fig. 17A, d) and
94,196 expressing NK ceIl markea DX5 and CD90 (Thy 1) (Fig. 1 A, d) .
Figure 17. Multiple hematopoietic lineages generateà from ÙJ viao-differentiated ES ceb on OP9 -mal ceiis (EUOP9 coculture). Hematopoietic iineages were characterized fmm a dl4 ES/OP9 cocultures by ceIl surface expression of lineage specific markers. A) Staining with control mAbs is s h o w in the left panel. In the right panel, four distinct populations, kbelled ad, were detected by flow cytometry on the basis of CD45 and CD24 surface expression. B) Populations a d were W e r characterized by lineage-specific markers for erythtocytes (a, TER1 IF), myeloid ceIIs (c, CD1 lb+), B lymphocytes (b, CD45R+), and NK lymphocytes (à, DX5+ CD90+).
Expression of Flkl dunng ES ce11 dzxerentiatr'on ni vitro
To study events during the onset of hematopoiesis. we sou@ to characterire the
hematopoietic potential from FlklC precurson. A transient wave of Flkl expression was observed
during the in vitro differentiation of ES cells into embryoid bodies (EBs) '39. The rnajority of cells
with hematopoietic potential were shown to be Rkl+ during the early stages of differentiation. and
Flkl- at later stages 139. ES cells differentiated on collagen IVsoated plates were able to give rise
to A k l + hemangioblasu, some of which expressed the vascular endothelid cadhenn. CD144 38.
We assessed the temporal appearance of Flkl' precursors during ES/OP9 coculture and
determined that Flkl expression peaked between &y 4 to day 6 (Fig. 18), with subsets of cells
coexpressing CD 144. We further determined that hematopoietic potential was pedominantly
contained within the Flkl+ fractions at &y 5 and day 6 (Fig. 19 and data not shown). These
findings are consistent with previous reports in the EB differentiation system 139. demonstrating the
transient nature of Flkl+ expression by a population of cells containing the earliest hematopoietic
precursors.
CD IO5 e-rpression during the onset of hernatopoiesisjiom Flkl+ cells
In order to funher define the population of m l + precursors with hematopoietic potential.
we characterized the R k l + subset based on the expression of CDIOS and CD3 1 (platelet
endothelial ce11 adhesion mlecuie, PECAM- 1 ), w hich have been reported to be expressed on
subsets of hematopoietic cells, including early progenitors t"4-'97-'99. Flow cytornetric analy sis
of day 5 ES/OP9 cocuInires revealed that CD105 and CD3 I expression subdivided the Akl+
haction into discrete populations (Fig. 19B). OP9 cells did not express any of the markers
Figure 18. Temporal analysis of Flkl surîace expression during differentiation of ES celis. Flow cytometnc analygis of Flk 1 and CD144 surface expression from days 3-6 ESlOP9 cocultures is shown. Undifferentiated ES cells (left panel) did not express Flk 1 or CD 144.
(3.8 x ld celis)
sorted F k l + CD lOS+CD3l+
Figure 19. Hematopoietic potential in F M + fractions €mm ES/OP9 cocultures. A) Day 5 ESIOP9 cocultures were andyzed for FIkl surface expression (solid Iine). Staining with control mAb (dotted line) is also shown. B) Flkl+-gated ceIIs were analyzed for the surface expression of CD IO5 and CD3 1 C) Flk 1 - and Fkl+ cells were isolated fkom day 5 ESIOP9 cocultures. FM+ cells were further fiactionated into CDlOS+ CD3 1+, CD105+ CD3 I -, and CD t 05- CD3 1- subsets. An equai number of cells was sorted from each subset, reseeded ont0 OP9 cells, and analyzed by flow cytometry on subsequent days (with day O corresponding to the start of the coculture). The CD49 CD105- quadrants include OP9 stroma1 cells ( d u 1 l), and erythrocytes (dl 1). The totai number of cells obtained fiom each coculture is indicated in parentheses.
subsets was isolated by flow cytometric cell sorting at day 5, reseeded ont0 OP9 cells. and andyzed
by flow cytornetry for hematopoietic activity on various days, with the initial seeding of ES cells
designated as &y 0. Andysis for the surface expression of CD45 (Fig. 19C) and TER1 19 (data
not shown) revealed that hematopoietic potentid was Iargely contained withm the Flkl' ftactions
(Fig. 19C, bottom three rows), compared to residual levels within the Fikl' fraction (Fig. 19C. top
row). Notably, CD 105+ subsets accounted for the majonty of hematopoietic potential within the
Flkl+ fraction (Fig. 19C. rows 2 and 3). compared to CD 105- subsets (Fig. 19C. row 4 and data not
shown: see Materials and Methods). In contrast, similar levels of hematopoietic activity were
observed in CD3 l+ and CD3 1- cocultures (Fig. 19C. rows 2 and 3). It was suggested previously
by Kabrun et al. that Fikl expression defines early hematopoietic precVmon which could represent
the onset of embryonic hematopoiesis 139. Our data support this notion. In addition. Our findings
that CD109 cells accounted for the majority of hematopoietic potential within Flkl+ fractions
suggest that early hematopoietic pmuaors coexpress Flkl and CD 105. Moreover. inducth of
CD105 expression was observed in cells that had been sorted CD105- (Fig. 19C, fourth row, days
6 and 8), and at &y 6 a population of cells exp~ssed CD105 prior to the detection of CD45+
hematopoietic cells at day 8. Furthemore, CD49 cells did not coexp~ss Rk l (data not shown),
and the majority of CD45+cells at &y 6 and day 8 were CD3 1' (data not shown), but intemediate
levels of CD105 expression were maintained on emerging CD49 cells (Fig. 19C, day 8). W e
c m o t exclude the possibility that some CD49 celis obsemed at day I l were generated directly
h m CD 105' precurson. Nonetheless, taken together. our data suggest that CD 105 should serve
as a useful marker to further dissect events during the progression of developmental stages h m
appeared to be transient. as coexpression on some CD45+ cells was diminished afier day 8 (Fig .
19C). Interestingly, expansion of C ~ 4 5 + CD 105 cells by day 1 1 (Fig. 19C) corresponds to the
approximate time when lineage-specific differentiation is observed in ESIOP9 cocultures. Thus, the
developmentally regulated expression of CD 105 on CD45+ cells may serve to identify the earliest
hematopoietic cells, and fuither, suggests that CD105 may play an important role during the onset
of hematopoiesis.
The expression of CD 105 during ES/OP9 coculture was confirmed by RT-PCR (Fig. 20).
Consistent with the flow cytometric analysis, CD105 transcripts were present at day 5 of coculture
and diminished by day 12. In addition, we observed the expression of TPR-II (type II TGF-$
receptor) hanscripts. indicating the presence of this receptor for TGF-p that cm interact with
CD105 'O. CD105 and TpR-II mRNAs were not expressed in OP9 cells but were present at low
levels in El5 fetal liver cells (Fig. 20). Since CD105 and ALKl (a type 1 TGF-p receptor) are
mutated in hereditary hemorrhagic telangiectasia type I and type 2, respectively '6*200, expression of
ALKl was examined. Figure 20 shows that ALKl was expressed in OP9 cells and in =/OP9
cocultures at days 5,8, and 12, but not in El5 fetal liver cells. Control RT-PCRs were perfonned in
the absence of cDNA templates (dH,O), and using &actin pnmers. -
ES cells lacking CDIOS (eng") dzrerentiare nonnully into Hkl+ mesodeml precursors
To determine whether CD105 is fùnctionally important for hematopoietic development, we
assessed the differentiation potential of mg4' ES cells in vitro. The eng'- ES cells were generated
h m heterozygous eng+'- ES cells (clone 4A-36; '9 following selection in high concentration
G418, and confirmed by multiplex PCR as previously described 26 (Fig. 21A). Figure 21B shows
Figure 20. RT-PCR analysis of ES/OP9 cocultures. Expression of transcripts for CD 105. TBR Il. ALK 1 and p-actin was analyzed by RT- PCR. cDNAs for ûndysis were prepared directiy fiom OP9 cells, El5 FL cells, and ES/OP9 cocultures at days 5,8, and 12 (total unhctionated cells were passaged and reseeded at day 5). Control RT-PCRs were performed in the absence of cDNA templates (dH20), and using Pactin primen.
Figure 21. Early differeatiatiotr of CDl05-defitient feirg-f-) ES tells in vitro. A) Genotyping of en& and en@/- ES cells was detemùned by multiplex PCR. DNA was isolated fiom ES ce11 clones and gene-specific primers were used to amplify wüd type (300bp) or targeted (476bp) exon 1, and wild type exon 2 (383 bp). B) Flow cytometric analysis of Rkl ce11 surface expression from &y 6 eng-/- and en&+ ES/OP9 cocultures is shown. C) Expression of rie-2, brachyury. and /%actin transcripts was analyzed by RT- PCR from &y 6 and day 9 ESlOP9 cocultures. cDNAs for day 6 analysis were prepared directly h m sorted Flk l+ cells, and cDNAs for day 9 analysis were prepared after sorted FM+ cells were cocultured for an additionai 3 days. Control RT-PCR reactions were performed using RNA from OP9 ceIis and E 13 feral liver (FL). D) Flk l+ cells were sorted from day 6 cocultures, reseeded onto OP9 cells, and analyzed by flow cytometry for the surface expression of CD105 and CD45 at day 9. CD IO5 expression is not detected in eng/- coculnires. The total number of cells obtained from each coculture is indicated in parentheses.
and eng+'+ cocultures were sorted and reseeded onto new OP9 cells for flow cytometnc and
RT-PCR analyses on various days. Figure Z1C shows the results of RT-PCR analysis from Flkl+
CD4Y cells directly sorted at day 6, or after coculm for an additional 3 &ys (coculhue &y 9).
Anal y sis of eng*'-. mg'", and eng+/+ cocultures revealed simi lar expression levels of brachvury. a
rnesdem-specific transcription factor , and rie-2, a receptor tyrosine kinase associated with
endothelial ce11 differentiation and reportedly expressed in fetal liver HSCs Y*35203v204. Thus,
expression analysis of Rkl. rie-2. and brachyry suggest that the early differentiation potential of
eng" ES cells is normal. In contrast. hematopoietic differentiation appears to be impaired (Fig.
21D). Fiow cytometric analysis at &y 9 revealed that C W 5 + hematopoietic cells were severely
diminished in eng" as compared to eng+'+ cocultures (Fig. 2 ID). This was observed in four
independent experiments. However, the presence of a small fraction of CD 105- CD45+ cells in
eng"- cocultures indicated that CD105 function, albeit important, was not absolutely required for
hematopoietic cornmitment and m e r differentiation. Although we previously determined that
hematopoietic activity is predominantly contained in Flkl+ fractions h m &y 5 and 6 ES/OP9
cocultures (Fig. 19 and data not shown), we addressed the possibility that hematopoietic potential
couid be shified to the Flkl- fraction in the absence of CD 105. Consistent with previous
observations, the Flkl- sorted fiaction contained minimal hematopoietic activity in eng" coculaires
(data not shown).
Lmpaired eqthropoiesis and myelopoiesis in the absence of CDLQ.5
To assess the hematopoietic precursor potential of eng-'- ES cells, Rkl+ cells were sorted
h m day 5 or day 6 ES/OP9 cocultures, reseeded ont0 OP9 cells, and analyzed by flow cytometry
on various days for ce11 surface expression of erythroid and myeloid lineage marken. Figue 22A
shows that erythroid and myeloid cells were efficiently generated from sorted Rkl' precwors
derived from control (+/+) cocultures. In contrast, Flkl+ precurson from eng'" cocultures
exhibited severely diminished myeloerythroid potential (Fig. 22A). Although eng'" ES cells could
differentiate into erythruid (CD45' TER 1 192 and myeloid (CD49 CD 1 1 b') cells, erythropoiesis
was diminished by approximately 1 5-fold (Fig. 22B; &y 12). and myelopoiesis by 5- to 8-fold
(Fig. 22B; day 9 and &y I I ) in eng-'' cocultures. as compared to eng+'+ cocultures. Simila. results
wece observed using two different eng-" ES ceIl clones (Fig. 22B and C), and in four independent
experiments. The eng+'* ES cells were not impaired in their ability to generate erythroid and
myeloid cells (Fig. 12C). The elevated ce11 numbers for eng+'- ES cells in Figure 22C are not
deemed to be significmt as this was not consistently observed. Eyrthropoiesis and myelopoiesis in
eng'' cocultures, aibeit at much reduced levels, followed the same time course and duration as for
eng+'+ and eng+'' control cocultures (Fig. 22C). This suggests that the temporal kinetics of
hematopoietic differentiation were not a l t e ~ d by the targeted deletion of the eng gene.
Definitive erythropoiesis is impaired in eng'- cells
Our findings indicated that sorted Rkl+ precursors h m day 5-6 ES/OP9 cocu1 tures
typically gave rise to TE31 19+ erythrocytes by day 12 (Fig. 22). Nakano et al. previously reportai
that two waves of erythropoiesis are observed in ES/OP9 coculhues, similar to erythropoiesis
Figure 22. Myeloid and erythroid defects in eng-/- cells. A) Sorted Flkl+ cells from day 6 ESIOP9 cocultures were reseeded ont0 OP9 cells, and analyzed at day 9 and day 12 for cell surface expression of CD45 TER1 19, and CD1 Ib by flow cytometry. B) Toul cellularity fiom cocultures, as indicated, was determined for erythroid and myeloid Iincages for the expriment represented in A). C) Total cellularity from separate experimnts with an erig+/- JS clone and a second mg-/- ES clone are shown. D) Expression of embryonic-type C-globin and üdult-type p-globin transcripfs, identifying primitive or definitive erythropoiesis, respectively, was analyzed from dny 12 cocultures, OP9 cells, and El3 by RT-PCR.
during mouse oniogeny. with k henrst aaosient wave gaemhg p.rimitive erythmcytes expresshg c- globin at day 6, and the second wave generating definitive erythrocytes expressing adult pglobin
beginning at day 10 '". Consistent with the observations of Nakano et al. 16' (-globin transcripts
were not detected in any &y 12 cocultures denved h m sorted Flkl' precursors (Fig. 22D). To
determine whether definitive erythropoiesis was affected by the absence of CD 105, we examined
the expression of fbglobin from &y 12 cocultures. RT-PCR analysis clearly indicated that Pglobin
expression was severely diminished in eng'- cocultures. as compared to eng+/+ cocultures (Fig
22D). This finding, taken together with our flow cytometric analysis, demonstrates that definitive
erythropoiesis is impaired in the absence of CD 105. Two groups observed signficant leveis of
erythrocytes in the yolk sac. suggesting that primitive erythropoiesis occurred efficiently x26.
However, another group " reported severe anernia in eng" yolk sacs. As Rkl+ precursors are
isolated from day 5-6 cocultures, a cornparison of primitive erythpoiesis in eng" and eng+'+
ESIOP9 cocultures would be difficult to interpret due to the fact that primitive erythropoiesis is
o c c h n g concomitantiy or soon after the reseeding of nkl+ celis ont0 OP9 celis. Thus, the extent
to which primitive erythropoiesis is dso dependent on CD105 function remains unclear.
Erythroid precursor frequencies are severely reduced in eng-'- ESlOP9 cocuItures
Potmtid differencff in erymid progeiiitor freqtteney rUKL c d m y stze were determined, by
lirniting dilution analysis, in engJ' and eng"+ cocultures flable III). Day 5 sorted Flkl' cells were
titrated by send dilution, reseeded ont0 OP9 cells, and analyzed by flow cytometry at day 14.
Rogenitor frequency was estimated by the statistical method of maximum likelihood 205 h m the
anaiysis of individual cocultures that were scored for the presence of CD45-TER119+ eiythrocytes.
From this analysis. erythroid progenitor frequency from nkl+ precursors was estimated to be
Table LII. Erythroid precursor potential of F M + cells*
Rogenitor Frequency - 1 (95% confidence limits)
+/+ 4-
* Flkl+ cells were isolated from ci5 W O P 9 coculturcs, reseeded ont0 new OP9 cells in senal dilution, and analyzed by flow cytornetry on &y 14 for surface expression of the erythroid lineage marker, TERl 19.
f Statistical analysis was performed using the methoci of maximum likelihood applied to the Poisson model, with 95% confidence lirnits shown in parentheses.
appmimately L&fdd lower in eng-'- cocultwes, w i L a freqüency of ln843 (4-) as compared to
11463 (+/+) (Table DI). This difference was statistically significant @ c 0.025) and is consistent
with the data (Fig. 22) showing severe erythropoietic defects in eng'" cocultures. Flow cytometric
analysis of positive cocultures at iimiting dilution, and examination under a microscope, revealed no
obvious differences in colony size from eng" and eng+'+ cocultures.
Lymphopoiesis in the absence of CDlû.5 appears normal
We previously reported that efficient lymphopoiesis occurs in ES/OP9 cocultures (Fig. 23)
''! However, B lymphocytes ( ~ ~ 4 5 ' " CD 19+) were not consistently generated from eng-" ES
cell-denved Flkl+ precursors. In these experiments. low numbers of sorted Rkl+ cells (7 - 8 x
id) were seeded per well ont0 OP9 cells. At this number of input cells, even Flklf precurson
derived h m control ES cells failed to give rise to B cells in a consistent manner. Tiierefore, we
considered that B lymphopoiesis might be inefficient due to low progenitor frequency in the n k l C
subset. Thus, we perfonned sepamte experiments in which 8 x 10" sorted Rklf cells were seeded
per well. This approach revealed that B 1 ymphopoiesis ( ~ ~ 4 5 ' ~ ' CD l9+ CD 1 1 b', Fig. 23) and NK
lymphopoiesis ( ~ ~ 4 5 ~ ' CD 19- CD1 lb-, Fig. 23) were similar in eng"- and control eng+"
cocultures compared to the severe defects observed in myeloerythropoiesis (CD45+ CD 19-
CD1 lb+, Fig. 23; and data not shown) which were still evident in mg" cocultures, as in previous
experiments (Fig. 22). However, data from Figure 23 and Iimiting dilution analysis indicate that a
possible rnild defect may be exhibited in lymphopoiesis h m eng"' ES cells ( Fig. 23, day 19; Cho
et al-, unpublished observations). The extent to which lymphopoiesis may be dependent on CD 105
hinction remains to be determineci. Nonetheless, these data suggest that lymphoid and
(8 x l@ cells) -1-
(9 x 10s cells)
(30 x ld cells) (21 x 1 6 cells)
I
Figure 23. Lymphopoiesis in mg-'- eells appears normal. Soned Fik 1 +
cells tiom day 6 +/- and -/- ESlOP9 cocuitures were reseeded onto OP9 cells and analyzed by flow cytometry on &y 16 and day 19 for surface expression of CD45, CD 19, and CD 1 1 b. The total number of celis obtained h m each coculture is indicated in parentheses.
non-lymphoid hemampoiesis may be distinguhhak an the basis of their developmental
requirement for CD LOS in that myeloerythropoiesis is stmngly dependent on CD 105 function.
DISCUSSION
Early hematopoietic and endothelial precursors, which may include hemangioblasts, have
been reported to express Flkl 38J9*139*'81. Our results show that CD 105 is coexpressed on Flk l+
early hematopoietic precursors. and CD 105 expression can be induced on Flkl+ CD 105' cells. The
Flkl+ CD los+ population of earl y hematopoietic precurson may also include cornmitted
endothelial precurson and hernangiogenic cells. Indeed, CDIOS has been reported to be expressed
on cells of both hematopoietic and endothelial lineages "240189*'90. Nonetheless, we show that
CD105 expression is rnaintained on CD49 cells that have cornmitted to the hematopoietic lineage.
Furthemore, our data revealed that CD 105 is not required for the differentiation of CD45+ cells but
rather appears to play an important role in early myeloerythropoietic progenitors, suggesting that
CD105 plays a role in hematopoietic developrnent after specification to the hematopoietic lineage.
The initiai reports that examined endothelial development in CDiO5-deficient mice showed that
while vasculogenesis appeared normal, subsequent stages in angiogenesis appeared to be defective
3-27. Taken together. these &ta support the notion that CD105 hinctions after differentiation is
specified to either the hematopoietic or endothetid lineage. Moreover, Flkl+ mesodemal
precursors were efficiently generated h m eng'/- ES cells. This finding M e r suggests that
CD105 could play a role in hematopoietic development following the Rkl+ stage. This is
supported by the more severe and eartier defects in Rkl-deficient mice compared to
is dependent on Rkl signalling in a comrnon differentiation pathway.
The most siriking phenotype we observed in CD1OSdeficient ES cells was the profound
reduction in myeloid and erythroid cells, which suggests that the survival, self-renewal, or
proliferation of a common myeloerythroid progenitor may be smngiy dependent on CD IO5
function. Normally, the microenvironment mated by OP9 stmmai cells allows for the efficient
differentiation of erythroid, myeloid, and lyrnphoid lineages Lymphopoiesis did not appear to
be significantiy altered in eng-'- cells. However, in the absence of CDlO5, it appears that either
inhibitory cues were not being sufficiendy antagonized, or stimulatory cues were not king
sufficiently arnplified during myeloerythropoiesis. In addition, the balaice between differentiation
and self-renewal signals may have been dysregulated Our observations are consistent with a roie
for CD LOS in regulating differemtiaton at early stages of myeloerythropoiesis, rather than later
stages. First, CD LOS expression was highest at day 8 and was downregulated by day 12 in nonnal
ES cells, suggesting that defects due to the absence of CD 105 would have a direct impact on early
myeloid and erythroid pmgeni tors. Thus, in the absence of CD 105, it appears that a smaller pool of
myeloerythroid precursors are generated, which are. albeil able to differentiate normally. This is
supported by the observations that eng*'' cells exhibited a lower progenitor frequency but displayed
kinetics and colony size similar to control cells.
The absence of CD105 appertn todempen early hernatopoietic differentiation fmm ES cells,
but other factors likeiy determine the extent of this effect. CD 105 is an accessory receptor for
members of the TGF-$ superîamily and can bind TGF-pl, TGF-$3, activin-A, BMP-3, and BMP-7
'OJ' in complex with their cognate receptors. When present in the receptor complex, CD 105 can
modulate cellular responses to TGF-$1 and is capable of acting as an antagonist of inhibitory and
stimulatory signals 289051. However, it c m also potentiate effects of TGF-$1 '9*206 and is
therefore b a t describai as a regulatary cornpanent af the receptur cornpleh The phenotype of
eng4 embryos is reminiscent of that observed for TGF-BI~' and TGF-f3 receptor II" embryos
and therefore suggests an important role for CD105 in conjunction with this growth factor
and its receptor complex in angiogenesis and hematopoiesis. In general, TGF-fll exerts a negative
control on the ce11 cycle of primitive murine hematopoietic cells and shows a preferentiai p w t h
inhibitory effect on early progenitors '4*18'07. For example, TGF-f3 I was shown to inhibit the
expression of stem ceIl factor (SCF) and its receptor CD 1 17 (c-kit) 'O8, and was also shown to be
P major regulator of erythropoiesis, inhibiting early stages but stimulating later stages ".
Interestingly, Pierelli et al. nported that CD105 is expressed on primitive HSCs. and suggested that
autocrine TGF-$1 helps to maintain the resting state and self-renewal capacity of these cells 19'.
Other members of the TGF-B superfamily, such as activin-A. BMP-2, BMP4, and BMP-7, have
also been irnplicated as important regdators of mesodemal specification to the hematopoietic fate.
or of early hematopoietic differentiation 1"172m. Mechanistically, TGF-$ signals are tmnsduced
by Smad proteins l3 that can synergize with components of the Wnt signalling cascade ' which have also been show to regulate hematopoietic differentiation """' . Thus, dthough the
precise mechanism remains to be detemiined, it is Iikely that CD105 functions as a regulator of
microenvironmen ta1 cues delivered through TGF-p cytokines.
Our studies have identified a potentid role for CD105 during the onset of hematopoiesis
from FIkl+ precursors. With the multiple effects exerted by members of the TGF-b superfamily
throughout developrnent 12", and the potentiai ability of CD105 to regulate responses to several of
these factors. it will be important to &termine which pathways are regulated in specific tineages
during hematopoietic development.
We thank Dr. Norman Iscove for helpful discussion and Cheryl Smith for technical assistance with
cell sorting. We would also like to th& Dr. Daniel I. Dumont for advice regarding the derivation
of CD105-deficient ES cells. SKC was supported by a studentship from the Lady Tata Mernorial
Fund (UK). AB was supported by a Studentship from the Medical Research Council of Canada
(MRC). ML is a Teny Fox Research Scientist of the National Cancer Institute of Canada. JCZP
is supported by a Scientist Award from the Canadian Institute of Health Research.
Zn vitro systems provide unique models for the study of development. Several culture
systems exist that ailow for the generation of various hematopoietic lineages. which will aid the
study of hernatopoieiic differentiation and lineage commitment. The use of an in ritm culture
systern that employs the differentiation of embryonic stem (ES) cells confen additional advantages
to the study of hematopoietic development in conjunction with the greater conml and manipulations
that an in virro system allows. For example, ES cell-derived pre-hematopoietic precurçors cm be
isolated to study the affects of various rnicrwnvironrnents and conditions on naive hematopoietic
differentiation. that is, pnor to the exposw of any cues which rnay signal through hematopoietic
receptors such as CD45 In addition, hematopoietic differentiation can be studied h m ES cells
with genetic alterations such as targeted gene disruption or the addition of transgenes, especially in
the case of targeted deletions that result in embryonic lethality.
Differentiation of rnyeloid and erythroid lineages from ES cells has k e n well established.
However, it remained controversial whether lymphocytes could be efficiently generated from ES
cells in vitro. It was suggested that M-CSF contributed to replete myelopoiesis during the
differentiation of ES cells on the stromal ce11 lines ST2, PA6, and W. 10 IJ5. Thus, Nakano et
al. induced ES ceils to differentiate on the stromal ce11 line, OP9, which was derived from
M-CSF-deficient mice (opfop). Pmnising Bndings indicaied that IgM+ B lymphocytes could be
generated by differentiating ES cells on OP9 ce11 monolayers (ES/OP9 coculture) lJ5.
ES cehderived B ceils are huictiondly competent
It remained unknown whether B cells generated in ESlOP9 cocultures possessed functionai
capabiliries andogous to their in vivo counterparts. The findings shown in Chapter II indicate that
=/OP9 cocultures cm serve as a mode1 system for the study of functional aspects of B ce11
developrnent such as cytokine ~esponse, retraviral-mediatedtransfhon, V@)J recombinaâion,
and rnitogen response. The exogenous addition of Fit-3L greatiy enhanced the efficiency of B
lymphopoiesis in ESIOP9 cocultures by augmenting B ce11 differentiation in individual cultures.
and also by increasing the consistency of cocultures that exhibited significant levels of B ce11
differentiation. A prolifentive ce11 cycle response to Fit-3L was exhibited by ex1y B ce11
progenitors with a CD19+ A A ~ . 1+ phenotype. ES cell-derived B lymphocytes also served as
targets for Abelson murine leukernia virus (A-MuLV) infection at a pre-B ce11 stage, similar to FL
or BM pre-El cells. Furthemore, the ES cell-derived A-MuLV-transformed pre-B (EAB) cells were
shown to possess functional V@)J recombinase activity and thus, should help to elucidate
molecular mechanisms in V@)J recombination and A-MuLV-mediated transformation. Finally, ES
cell-cienved B cells were show to upregulate the expression of the costirnulatory molecule, CD80
(B7-1). and semete Ig in response to the mitogen lipopolysacchaxide (LPS).
Embryonic, fetal o r adult hematopoiesis?
Some aspects of embryonic and fetal hematopoiesis are distinct from adult hematopoiesis
*. For example, it has ken proposed that CD5+ B cells represent a unique B ce11 lineage that is
derived from FL *. Ce11 transfer experiments showed that neonatal liver but not adult BM could
effectively reconstitute CD5+ B ce11 populations. Furthemore, FL-derived pro-B cells transferred
into irnrnunecompmm~sed (SCID) mice generated predorninantly CDT B cens whereas
BM-denved pro43 cells generated predorninantly 0 5 - B cells As indicated in Chapter II.
B cells generated h m ES/OP9 cocultures are predominantly CDS B cells, indicating that
fetd-type B lyrnphopoiesis does not occur and that BM-derived OP9 cells rnay efficiently support
on1 y aduit-type hematopoiesis.
However, primitive and definitive eryttuocytes are generated in ES/OP9 cocuitures and their
embryogenesis, primitive erythropoiesis occurs in the yolk sac and is msient. while definitive
erythropoiesis becomes established in the FL and persists throughout adult hematopoiesis ".
Thus, aithough the OP9 cells are BMderived, embryonic-type erythropoiesis occurs in ESlOP9
c o c u l t ~ s . It is possible that the generation of primitive erythrocytes is not dependent on a unique
embryonic microenvironment. Altematively, other ce11 types that concornitantly differentiate from
ES cells may provide an embryonic-like microenvironment that supports primitive erythropoiesis.
However, it appears that ES ce11 differentiation does not compensate for the lack of a FL-like
microenvironment that supports significant CD9 B lymphopoiesis. The extent to which ESlOP9
cocuitures Rpresent embryonic, fetal and adult hematopoiesis remains unclear.
The generation of ES cell-derived T celis in vitro
The thymus is seeàed by multipotent hematopoietic progenitors that undergo a
differentiation program which gives rise to T lymphocytes 93*'12*119. However. the early stages of
differentiation and lineage cornmitment, and the nature of the tissue-specific inductive events during
thyrnic T ce11 development are poorly understood. In vitro techniques such as fetal thyrnic organ
cultures O C s ) have allowed for a greater understanding of T ce11 development. These FTOCs
have been seeded with FL- or BM-derived hematopoietic progenitors 91*'61". The ability to
generate T lymphocytes in FTOCs from ES cell-derived progenitors would facilitate the study of
early T ce11 development by allowing a greater repertoire of feasible genetic modifications and in
vitro manipulations. The findings presented in Chapter II that eficient B lymphopoiesis occurs in
EUOP9 cocuitures led to the hypothesis that T ce11 progeniton codd aiso be generated This
hypothesis was based on the assurnption that dong the differentiation pathway to B cells,
uncommitted lymphoid progenitors should be generated. The ability of ES cefls to generate T
Lymphocytes in fetal thymic organ cultures (FL'OCs) was assesseb The e x p i m a t s presented in
Chapter IIi show that undiffe~ntiated ES cells and mesoderm-like differentiated ES cells did not
give rise to T cells in JTOC, and hirthermore, were detrimental to the integrity of the thymic
architecture. This suggested that only a subset of differentiated ES cells might be suitable for
seeding into FTOCs. Therefore, FT'OCs were seeded with defined CD49 hematopoietic
progenitors, but generation of T cells from these ES cell-derived progenitors proved to be extremely
rare. The OP9 microenvironment may not be adequate to support the maintenance of hematopoietic
stem cells (HSCs), and once specified to the hematopoietic lineage, progeniton may rapidly commit
to non-T ce11 fates. Thus, in order to ennch for multipotent progenitors, and possibly
pre-hematopoietic progeniton, isolated Flkl+ CD4S precursoa were seeded into FTOCs to allow
for hematopoietic induction or differentiation to occur within the thymic mirnoenvironment. As
show in Chapter m. ES cellderived Flkl+ CD4S precursors could give nse to T cells in a
reproduci ble manner.
Optimal ES cell-deriveà T ce11 progenitors
The precise phenotype and precursor potential of the thymus-colonizing ce11 has not been
well-characterized. However, thyrnic lymphoid progeniton (TLPs) serve as a fint approximation
because they are the most immature thymocytes comrnon to the fetal and adult thymus and
therefore, may represent cells that newly immigrated to the thymus. TLPs are lymphoid-restricted
CD49 hematopoietic progenitoa that efficiendy differentiate into cells of the lymphoid lineages
but not myeloid and erythroid lineages 93*'12*1"12'. Thus, Flkl+ CD45- precurson are not
identical to TLPs because they do not express CD45 and can eficientiy give rise to myeloid and
erythroid cells. Thus, Rk l+ CD45- cells may not be optimal precursors for seeding of the thymus,
which might only support efficient T ce11 development h m CD49 TLP-Like or FL-derived
not disrupt the integrity of the thymic architecture, CD45- precursors may generate
non-hematopoietic lineages or non-T cell lineages that inhibit T ce11 differentiation. Further
characterization of the Rklf population may reveal a subset of precursors that is mom optimal for
T ce11 differentiation in the thymus.
The role of CD105 expression and function in hematopoiesis
Several memben of the transforming growth factor (TGF)-f! superfamil y have ken
implicated in mesoderm specification and hematopoiesis. However molecular events regulating the
onset of hematopoiesis From mesodemi-derived pre-hematopoietic precurson remain poorly
undentood. In Chapter IV, characterization of the Hkl+ population revealed that subsets of Flkl+
cells coexpressed CD 105 (endoglin), an accessory receptor of the TGF-$ super f ' l y . The
majority of hematopoietic potential in the Flkl+ population could be isolated from the CD 105+
subset. Moreover, ce11 surface expression of CD105 was maintained during the transition from
FIklC C D 6 pre-hematopoietic cells to Flkl* CD45+ hematopoietic cells. The developmentally
regulated expression of CD105 suggested that it might play a role during early hematopoiesis from
Flkl+ precursors. Furthemore, CD LOS expression on subsets of human hematopoietic cells
suggested that it could play a role in multiple lineages, including erythroid, myeloid, and lymphoid
cells 2-24188. Thus, to determine whether CD 105 plays a functionai role during early
hematopoietic development, the potential of CDlOSdeficient ES cells was examined during
lymphohematopoietic differentiation in ES/OP9 cocdnires. As indicated in Chapter TV,
myelopoiesis and definitive erythropoiesis were severely impaired in the absence of CD105 In
contrast. lymphopoiesis did not appear to be significantly affected Althougb it is possible that
subde defects were undetected, the results suggest nonetheless that CD 105 serves an ancilIary
In search of the HSC
It is unclear whether ES cells are capable of giving rise to HSCs in vitro. This remains the
prirnary unresolved question regarding in vitro ES ce11 differentiation systems. The in ritru
generation of ES cellderived myeloerythroid cells, and B and NK lymphocytes had been
previously established '37*138*1JO-'JJ*'JS~'6i*176J77. However, the in vitro generation of T cells
had not been achieved If HSCs are generated h m ES cells, full multilineage potential should be
exhibited under appropriate conditions. Thus, it would be unlikely for ES cells to be able to
generate HSCs but not T cells. Results in Chapter III now describe that ES cells can dso give nse
to T lymphocytes in vitro, consistent with the possibility that ES cellitrived HSCs might aiso be
generated. However, multipotent progenitors capable of giving rise to mycloerythroid, and B, NK.
and T lineages also exist in the eady embryo pnor to the establishment of HSCs w2. Indeed.
during embryonic development. a "reverse cascade" to BM hematopoiesis is observed, as
di fferentiated prirni tive erythrocytes are detec ted pnor to mu1 ti potent precursors, prior to the HSC.
Therefore, evidence of hernatopoietic differentiation and multipotency are not indications of HSC
activity. The definitive hinctiond assay for HSC activity is long-term reconstitution (LTR) of a
lethally-imdiated host IW. Lack of evidence for LTR-HSC activity suggests that HSCs are not
generated from ES cells in vitro. However, ES cells are known to give rise to al1 tissues when
injected into blastocysts to generate chimeric adult rnice '35. indicating that ES cells are not
intrinsicdly lirnited in generating HSCs. Thus, the inability of ES cells to generate LTR-HSCs is
likely due to limitations of the in vitro microenvironment.
A number of possibilities c m explain the lack of LTR-HSC activity from in vitro ES ce11
differentiation systems (Fig. 24). First, HSCs might not be generated ïnstead due to the unique
nature of in vitro differentiation, multiple hematopoietic lineages may be derived h m restricted
O - P ~ $f = HSC-g
@ = HSC
= m-HP =BC
Figure 24. Scheme depicting possible generation of HSCs h m ES c e b Ut vilro. Evidence for HSCs with long-term reconstituting (LTR) capabilities have not been found in ES ce11 differentiation systems. A) HSCs may no< be generated h m pre-hematopoietic precurson (ÿHP). which may instead give rise dkctly to muftipotent hematopoietic precursors (m-HP) that differentiate into various blood cells (BCs). B) HSCs with grafting capabilities (HSC-g) may be generated h m ES cells but at fkequencies below detection levels. Dashed boxes indicate cells prexnt at lower frequencies than depicted. C) Even if HSC-g are generated, they may exist for only a short period of the . D) HSCs may be generated and maintained at low frequencies, however they may not possess grafnng capabilities.
89
multipatentar uni- progenitos thaiarise direc:tly fkmpre-hemsrnpoietic precurso~s A
second hypothesis is that ES cell-derived HSCs are generated so rarely that it would be technically
infeasible to inject, for LTR assays. the possibly enormous nurnber of cells necessary to ensure the
presence of one HSC. Even if HSCs are generated, they rnay only exist in a shon temporal window
before rapidly differentiating into restricted multipotent or unipotent progenitorr. making their
isolation and detection even more dificult. Thus, although HSCs rnight be generated, the in vitro
microenvironment rnay be unable to support HSC survival or self-renewal. However rare and
fleeting, once an HSC is generated, subsequent rounds of extensive self-renewal and prolifention
of restricted mu1 ti poten t progeni tors rnay alIo w for si gni ficant degrees of hematopoietic
differentiation. Lady. just as in the BM '". a rare but sustainable population of HSCs rnay exist
in vitro. In this scenario, the in vitro microenvironment rnay be able to support HSC self-renewal,
but rnay be deficient in inducing the expression of horning receptors and adhesion molecules
necessary for engraftment. In the ESIOP9 caculture system. multilineage hematopoiesis is not
sustained, which is more consistent with the possibility that HSCs are not generated or are rare and
fleeting. The enurneration of differentiated progeny h m early hematopoietic precursors in
ES/OP9 cocultures may help to address these possibilities.
Isolating early CD45+ CDlOg hematopoietic progenitors
As discussed if HSCs are generated fiam ES cells in vitro. they rnay be rare and fleeting.
Thus, studies of lymphohematopoietic differentiation h m the HSC, and isolation of multipotent
hematopoietic precursors that maintain T ce11 potential. rnay be dependent on defining the earliest
hematopoietic progenitors during ES/OP9 cocultures. One approach would be to characterize the
induction of pre-hematopoietic cells towards the hematopoietic lineage. The mlf CD45'
population likely includes pre-hematopietic cells. CD105 was expressed on these cells during the
transition to Rkl- CD45+ hematopoietic cells (Fig. 25). Thus, CD49 CD lO5+ expression could
Figure 25. CD105 expression on eariy hematopoietic precursors. The expression of CD 105 during the transition of pn-hematopoietic H k l+ CD45- precursors to Flkl- CD4+ hematopoietic cells suggests that CD 105 would be a usefuI marker to characterize the earliest hematopoietic cells during ESOP9 coculture, and to potentially isolate ES cell-derived HSCs.
define a population of cek th& represent the onset of hematopaiesis in ESIOP9 coculturesultures
Interestingiy, autocrine regulation by TGFB was implicated to enhance long-term culture-initiating
ce11 (LTC-IC) activity of human CD34+ primitive hematopoietic precursors that coexpressed
CD 105 '97*"7. CD 105 potentially regulates signais msduced by vanous rnemben of the TGFP
superfarnily. which play multiple d e s during mesoderm specification and hematopoiesis. Thus. it
will be important to analyze the expression of TGFB during ES/OP9 coculture to determine how
the pmsence or absence of these cytokines may contribute to the generation and maintenance of T
ce11 progenitors and HSCs.
Microenvironmental influences on lineage-specifie differentiation and commitment
Figure 26 depicts a mode1 of the ways in which distinct rnicroenvironments could influence
hernatopoietic differentiation and lineage commitment. Fini, the lineage potential of multipotent
progenitors might not be affected in one microenvironment compared to another (Fig. 26A).
Generai growth and survival factors comrnon to many rnicroenvironments might passively support
the differentiation of hematopoietic lineages. Secondly, the microenvironment could actively
support or inhibit the survivai or proliferation of lineagecommitted precursors but not have any
direct influence on lineage cornmitment (Tig. 26B). Cytokines are a common supportive or
inhibitory component of microenvironments. For example, the addition of IL-7 and Flt-3L
enhanced B lymphopoiesis, as discussed in Chapter II, and TGFfJ-1 has been reported to inhibit
eariy erythropoiesis 14. as discussed in Chapter IV. Thirdl y, the microenvironment rnay act to
instruct hematopoietic lineage commitment by induction or suppression.
Although there is a distinct conceptual difference between supportive/inhibitory
microenvironments and instnic ti ve rnicroenvironments (Fig. 26B and C), experimental approaches
in most studies do not distinguish hem with respect to lineage cornmitment. For example, Ht-3L
enhances B lymphopoiesis but it is unclear whether it does so by inducing commi tment towards the
A) PeFmiSsive Microenvironment I
sumival
@
swival proli feration
B) Supportivdnhibitory
Microenvironment I
Microenvironment II
Microenvironment U
P p Q proliferation
C) Instructive
Microenvironment 1 Microenvironment II
Figure 26. Mode1 of microuivironmentil influences on Iiwsgcspedfic dinerentiation and cornmitment. A) The microenvironment could be permissive and not exen any influence on mul tipotent progenitors (MPs) to c o d t to one üneage (LI) or another (L-II). B) A particular microenvironment may influence the slnvivd or proiiferation of MPs in a supportive or inhibitory fashion, subsequent to limage commitmen~ or C) may be necessary to actively induce or supprcss iineage cornmitment.
B ce11 lioeage or by supporthg the sumival and proliferation af e d y B lineage progenitors. or
perhaps functions in both capacities. In addition, the observation that T ce11 differentiation occurs in
the thymus but not in the BM invites the notion that the thymus induces T ce11 lineage cornmitrnent.
However, active support of T ce11 differentiation does not indicate that the thymus instructs T ce11
lineage cornmitment The thymic microenvironment rnay be unique in supporthg the survival and
proliferation of T lineage cornmitted cells once they differentiate from multipotent progeniton, and
inhibit or fail to support the differentiaùon of other lineages.
Phenotypic characterization of the earliest lineage-cornmitted precursors, and molecules
involved in the mechanisrns of Iineage cornmitment will help to identify instructive capacities of
mimnvironments. In particular, the study of differentiation events during ontogeny will be
helpful because changes in progeniton and microenvironmenu cm be examined as they initiate de
novo in various tissues. For exarnplr, potentiai instructive elements of a hematopoietic
microenvironment that induce commitrnent to the hematopoietic tineage should be easier to identify
in the AGM when hematopoiesis ini tiates de novo.
Distinct hematopoietic microenvironments: graduating to the HSC?
During embryonic hematopoiesis, the conventionai hierarchy of BM hematopoietic
differentiation is confounded by the observation that mature blood celis exist prior to the emergence
of HSCs. In addition, the point has ken raised that hematopoietic activity arises from mesodemai
g n m tayer cells which are fOtrned during gastntlation at E6.5. Thus, it seems implausible that
primitive erythrocytes observed one &y later at E7.5 were generated from a BM-iike HSC when it
takes weeks to generate erythrocytes from an HSC in an adult mouse 41. This puzzling observation
may reflect that embryonic- and addt-type hematopoiesis are derived h m distinct
pre-hematopoietic precursors. However, it is possible that primitive erythropoiesis &ses h m
mesoderm at E6.5 via the same pre-hematopoietic precursor that gives nse to definitive HSCs, but
the YS micrcenu- may cause or permit accekateddifferentiation, In addition, it is
interesting to note that multipotent hematopoietic progenitors and precursors capable of in vivo
grafting (CRI-S) also appear in the embryo pnor to the establishment of definitive HSCs '".
These confounding observations may refiect that embryonic tissues must acquire the ability to
support the "education" of "pre-HSCs." Thus, it is possible that pre-hematopoietic precursors
sequentially acquire the functional capabilities of the definitive HSC (ïig. 27). Mechanistically. one
could envision that these capabilities might depend on the expression of gene products that function
to cwrdinate and Iink components such as ceil cycle machinery. transcriptional regulation
machinery, chromatin remodelling complexes, and srnichiral and organizational proteins. It is
interesting to speculate that this sequential "education" of HSCs cm only be completed in the
AGM (after EIO), the site where definitive hematopoiesis is initiated and where the fint LTR-HSC
activity is observed (Fig. 27).
Regardless, because definitive hematopoiesis has been shown to autonornously initiate in
the AGM, this region is a critical hematopoietic site to study the mechanisrns involved in HSC
generation, maintenance, and differentiation. The finding that definitive hematopoiesis in the AGM
precedes that in the FL has pmmpted new studies on factors that were shown to be critical in FL
hematopoiesis. For example, defects in definitive hematopoiesis in AML- 1 and c-myb hockout
mice were attributed to defects in FL hematopoiesis but more recently, in vitm cultures of AGM
from the knockout mice demonstrated that severe hematopoietic defects occur in the AGM region
8.9.218119 . Thus, the observed hematopoietic defects in the FL rnay be consequent to defects that
originated in the AGM.
Dissecting the hematopoietic microenvironment with s t m d ceii lines
In addition to studying hernatopoietic development, investigating lymphohematopoietic
differentiation h m ES cells in vimo may help to dissect the varying components of distinct
pre-HP
HSC
/ pre-HSC HSC
pre-HP \ pre-HSC HSC
Figure 27. The education of an HSC in the PSplAGM region. A) Mesodemderived pre- hernatopoietic precursors (pre-HPs) may directly generate the distinct hematopoietic progenitors observed in the P-Sp/AGM region, Le. multipotent progenitors (MPs) at E7.5, MPs capable of in vivo engraftment (gr&) at E9, and definitive HSCs fully capable of these functions and also exhibiting long-term reconstitution (LTR) acîivity , at E 10. B) Aitematively , the P-SP/AGM region could provide educational cues thal allow for hematopoietic precunors to acquire specific functions, such as in vivo grafûng capabilities, and LTR activity. In particular, some MPs may be hypothetical pre-HSCs that sequentidy acquire functional characteristics to becorne HSCs.
microenvitanments that contribute to lineage-specifc differentiation and cornmitment. For exarnple,
it is intriguing to speculate that HSCs may not be generated in ES/OP9 cocultures because pre-
HSCs never encounter the necessary education provided by the AGM microenvironment (Fig. 27).
Thus, the lyrnphohematopoietic lineages generated in ES/OP9 cocultures may be derived from a
multipotent progenitor that is comparable to the multipotent progenitor of E7.5 AGM. prior to the
establishment of grafang and HSC-LTR activities. Analysis of genes that are differentially
expressed in an AGM-derived ce11 line "O as compared to the OP9 ceil line may yield
microenvimnmental components, such as soluble factors, that will enhance the ability of OP9 cells
to induce or support HSCs. In Chapter II, for exarnple. OP9 cells were shown to express minimal
levels of Flt3-L and exogenous addition of this cytokine greatiy enhanced B lymphopoiesis in
ES/OP9 cocultures. Thus, factors present in the AGM microenvironment and ahent in OP9 cells
could be added to ES/OP9 cocultwes to determine their effects.
The use of AGM-derived ceIl lines may facili tate the generation of HSCs from ES cells.
However, it is possible that a two-dimensional culture system will not suffice because the
microenvironment proMded by three-dimensional niches may not be effectively recapinilated fFig.
28). Thus. the generation of HSCs from ES cells, which may also serve as optimal T ce11
progeniton, could require the isolation of pre-hematopoietic ES cell-derived precursors combined
with organ culture of AGM tissue explants '**.
Of mice and men
A fundamental aim of biomedical research in animal mode1 systems should be to gain and
apply new understanding towards therapeutic goals. Dysregulation of hematopoietic differentiation
and Iineage commiûnent mechanisrns cm give rise to leukemia In essence. the study of
hematopoietic differentiation is also a study of Ieukemogenesis. Therefore, an understanding of
these processes is prerequisite to developing selective therapies. The more we understand about the
other stromal ce11
Figure 28. Limits of twoaimensional s t r o d ceIl cultures. A) Stromal cells organized in three-dimensionai microniches may be required to fully recapitulate an in vivo hematopoietic microenvironment. B) Rare HSC-supporting stromal cetis may become diluted in a hetemgeneous two-dimensional cuiture. Even with the isolation of HSC-supporting smmal ce11 lines, surface contact area and the formation of microniches is limited in the two- dimensionai culture.
specinc stepwise events dunng differentiatim the heüer we d i be able ta chamcie&e anomaiies
that cause leukemia, and to targe t speci fic therapies accordingl y. Although similari ties exist in
moue and human hematopoiesis, the ment derivation of human ES ce11 lines should allow for a
p a t e r undentandhg of human lymphohematopoietic differentiation with the development of
hurnan in vitro models ''! Another fundamental aim of animal models, in particular ES ce11 differentiation systems, is
to lay a groundwork for genetic and tissue engineering. Recently, mammalian ES ce11 lines have
been established ;ifter nuc tear transfer from somatic ceI1s '"*185. In conjunction with the derivation
of human ES ce11 lines "". these advances should facilitate groundb~aking progress in tissue
engineering for the purpose of thenpeutic goals involving lymphohematopoietic reconstitution.
The work presented in this thesis will hopefully facilitate the progress of these aims. In
addition. this work may facilitate the isolation and characterization of the still-elusive ES cell-derived
HSC.
They are il1 discoverers t l m think there is no land, when they can see norhing but sen.
- Francis Bacon
Mice
RAG-2" CD45.1 congenic mice were bred and maintained in our animal facility.
CeIl Iines
The bone marrow stroma1 ce11 line, OP9 "', was cultured as a monolayer in (rMEM supplemented
with 2.2 g/L sodium bicarbonate and 20% FCS (Hyclone, Logan, UT; ES grade and lot tested).
OP9 media was also used for ESlOP9 cocultures. The ES ce11 line RI, obtained from Dr. G.
Canima (Mt. Sinai Hospital, Toronto. Ontario). was cultured on a confluent monolayer of
mitomycin-C treated embryonic fibroblasts (EF) with 1 nghl leukemia inhibi tory factor (R&D
Systems, Minneapolis, MN). ES and EF cells were maintained in DMEM supplemented with 15%
FCS. 2 mM glutamine. 1 10 &ml sodium pynivate, 50 2-mercaptoethanol, and 10 rnM Hepes,
pH 7.4. The A-MuLV producer ceIl line, 54 clone-2, was obtained from Dr. G. Wu (Ontario
Cancer Institute, Toronto, Ontario). 54 clone-2 is an A-MuLV-PI60 transformed NM 3T3
nonptoducer ce11 line superinfected with Moloney-MuLV-Clone-2, denved by Rosenberg and
Witte The 54 clone-2 ceil line and all EAB ce11 lines were maintained in RPMI, 106 FCS, and
50 pM 2-mercaptoethanol. Al1 cocultures were incubated at 37% in a hurnidified incubator
containing 5% CO, in air. Periodic testing indicated that al! ceil lines were maintained as - rnycoplasma-Free cultures.
ESIOP9 coculture, in vitro generation of B and T ce&, and A-MuLV infection
For hematopoietic induction, a singlecell suspension of 101 R1 ES cells was seeded ont0 confluent
OP9 monolayen in 6-well plates. The media was changed ai &y 3, and by day 5 or &y 6 nearly
100% of the ES colonies differentiated into mesodem-like colonies. The cocultures were
aypuaized (025%; Gibco-BR& Gaitherssburg, MD) at day 5 or &y 6. the singieceii suspension
was preplated for 30 min., and non-adherent cells (1-2 x 106) were reseeded onto new confluent
OP9 layen in 10 cm dishes. At &y 6 or day 7 small clusten of hematopoietic-like smooth round
cells began to appear. At &y 8, loosely adherent cells were gently washed off and placed ont0 new
OP9 layers (without ûypsin). This treatrnent enriched for cells with hematopoietic porential. while
Ieaving behind differen tiated rnesodenn and undifferen tiated ES colonies. Following this passage.
hematopoietic colonies expanded wi th noticeable pmliferation between day 10 and day 12, and
thereafter. By day 19, the total number of CD49 cells recovered from the ES/OP9 cocultures was
in the order of 10' cells. Figure 6 provides a schematic outline of the ESIOP9 coculture protocol
for the generation of B-lineage cells, including the addition of cytokines as indicated. Human Flt-
3L was used at a final concentration of 5 ng/ml, or mouse Flt-3L was used at a final concenmation
of 20 nglml (R&D systems). Cells were cultured in the prescnce of exogenous At-3L fmm day 5.
The addition of Flt-3L at day 5 appeared to represent a cntical temporal window for the
enhancement of B lymphopoiesis, as the enhancement was not observed when Flt-3L was added at
later time points (2 day 8). Media was changed andior the cells were passaged without trypsin
(made into single ceIl suspension and filtered, 70prn) between day 8 and &y 15.
To generate S I N B cells, the lymphohematopoietic cells were harvested at day 15, and
replated ont0 a fresh OP9 monolayer. At day 28, cells were stimulated with LPS (10 &ml) for 4
&YS. CeHs mci culture supernetant wett then harvested for flow cytometry and ELISA analysis,
respectively. In a separate experiment, cells were stimulated with LPS (100 pg/mi) for 48 h. and
analyzed for the upregulation of CD80 (B7- 1).
To generate EAB ce11 lines, IL-7 (5 ngmil) (R&D systems) was added to Fit-3Lsontaining
ES/OP9 cocultures at day 8 to maintain immature preB cells. Cocultwes were infected by adding
an undiluted virus stock harvested from a 4 &y confluent plate of the A-MuLV producer ce11 h e ,
54 clone-2. Cocultures from a 10 cm dish were infected by replacing the media with 3 ml of virus-
stock cantaining 4 &mi of piybrene (Sigma St Louis. MO). d I L - 7 . The plate was rocked
periodically at 37OC for 2 4 h. After this penod, 5 ml of fresh OP9 media containing IL-7 was
added to the plate. The media was changed 5 days later to EAB media with IL-7, but without Flt-
3L. Subsequent media changes lacked IL-7. Three separate experiments gave rise to EAB ce11
lines. Infections were performed on either day 15.28. or 49. Flow cytometry analyses showed that
al1 EAB lines displayed the same phenotype. Although the day of infection was varied, in each
experiment a significant population of CD45R+ CD24+ IN immature pre-B cells, containing
+known A-MuLV targets. were pment Is3. However, we noted that tranformants arose 33 days
alter the day 15 infection and only 13 days afler the day 49 infection. Infected cells were grown in
bulk, then subsequently cloned by iimiting dilution. Abelson-tmnsformed clones showed a
doubling time of about 18 h, and the ptesence of integrated copies of the viral genome was
confirmed by Southern blot analysis. EAB cells or the Abelson line. 204-1-8 . were transfected
with the recombination-reporter plasmid (pWTSJA) as previously descn bed 166-168
To generate T cells, unsorted differentiated cells from day 5 WOP9 cocultures, isolated
CD&+ cells from &y 5-14 cocultures, or isolated Rkl+ cells from day 5-6 ocultures were
harvested or sorted and seeded into fetal thymic organ cultures (FïOCs).
Cell culture and differentiation of eng-'' ES cells
Experiments in Figures 17 to 19 were performed with R1 ES cells. eng+'- ES cells (clone 4A-36)
were previously generated '6 by gene targeting the parental wild type 129/0la-derived E l 4 ES ce11
lines, deleting 609 bp including eng exon 1 and its initiation codon, and leaving the endoglin
promoter intact. These Girl1 8-ganc yclovir resistant clones were able to give gedine transmission
in mice ". G418-resistant ES cells that had randomly integrated the gene-targeting consmict were
used as conml (+/+) cells, and results with these cells were similar to those obtained with El4
concentration G4l8 (2.4 mg/ml) ln. Screening of resistant colonies for eng"' was performed by
multiplex PCR as previously described 26. Two independently-derived eng'" clones were used for
ES differentiation experiments.
ES/OP9 di Eerenti ation cocu1 tures were performed as descri bed above '"? 104 ES ceils
were seeded ont0 OP9 monolayers in 6-well plates. or 5 x 10' ES cells were seeded ont0 OP9
monoiayen in 10 cm dishes. Flkl+ celis were sorted on day 5 or 6. reseeded ont0 new OP9 ce11
monolayers. and harvested on various days for analysis by fiow cytometry andor RT-PCR
analyses. For Flkl subset analysis shown in Fig. 19C. 2.5 x IO' cells were sorted on day 5.
reseeded onto new OP9 ce11 monolayers and anaiyzed on various days. The Hkl+ CD 105' CD3 1+
subset (Fig. 19B) represented a minor population which was not usually detectable and therefore
not included in the progenitor analysis shown in Fig. L9C. However, hematopoietic potential h m
this population was lower than that obsewed for the Flklf CD105' CD3 1' subset.
Flow cytometric analysis and ce11 sorting
SingleceII suspensions were stained for surface expression of various markers using ETI'C-, , or
APCconjugated mAbs in staining buffer (Hank's baianced salt solution W S S ) with 18 BSA
and 0.051 N a 3 ) . Conjugated mAbs were purchased fmm Pharmingen (San Diego, CA). except
for CD105 (clone U17/18) which was purchased from Pharmingen in purifed form, and
biotinylated Cells were stained in 100 pl for 20 to 30 min. on ice and washed twice pnor to
analysis. Stained cells were analyzed with a FacsCalibur flow cytometer using CeilQuest software
(Becton Dickinson. Mountain View, CA); data was live-gated by forwardfside Light scatter and lack
of propidium iodide uprake (except PI was omined for intracelldar stains). For ce11 sorting,
singk-cell susptmions were prepared and stained for FACS as desaibed above, except that no
N a . was added to staining buffer. Cells were sorted using a Coulter Elite cytometer (Hialeah.
FL); sorted cells were 2 95% pure, as detemined by pst-sort analysis. Staining was not altered in
the presence of blocking FqRII/CII antibody (2.462).
RT-PCR analysis
Total RNA From single-cell suspensions was isolated using the Trizol RNA isolation protocol
(Gibco BRL, Gaitheaburg, MD). RNA was muspended in 25 4 diethylpyrocarbonate
@EPC)-treated (O. 1%) dH,O. cDNA was prepared from 1 pg of RNA using random hexamer - primers and the cDNA Cycle kit (Invitmgen, San Diego, CA). Subsequent PCR analysis in
Chapter 1 was performed using titrations of cDNA in a 1:s dilution senes in dH,O. - cH,O - and RT
reactions done in the absence of AMV revene transcriptase were included as negative contmls.
Total RNA h m OP9 cells and El3 fetal liver were used as contmls for al1 primer sets in Chapter
III. Wherever possible, primers were designed to span intronic regions. Ail PCR reactions were
performed using the sarne cDNA batches as shown for fbactin, and d l K R products comsponded
to the expected molecular sizes. Gene-specific pnmers used for PCR are as follows (5'->3'): f!-
actin 5'. GAT GAC GAT ATC GCT GCG CTG; fkactin 3', GTA CGA CCA GAG GCA TAC
AGG; SCF Sv, TCT TCA ACT GCT CCT A ï T T; SCF 3'. ACT GCT ACT GCT GTC AIT C;
FIt-3L 5' . ACA CCT GAC TGT TAC TTC AGC; FIt-3L 3', CCT GGG CCG AGG CTC TGG;
IL-7 S', ACT ACA CCC ACC TCC CGC A; IL-7 3'. TCT CAG TAG TC' CIT TAG G; CD105
5'. GGT GTT CCT GGT CCT CGT Ti'; CD105 3', CAA AGG AGG TGA CAA TGC TGG; T
BR-II 5'. T m TCT ACT C X X ACC GTG TCC A; T BR-II 3', CGT AAT C C ' TCA CTT CTC
CCA; ALKlS, GAA CAC GGC TCC CTC TAT GA; ALKl3'. ACT TTG GGC TTC TCT
GGA TTG; brachyury 5'. TAC TCï TTC TïG CTG GAC TT; brachyury 3', ATC TIT GTG
GTC GTT TCT TT; bglobin S, CAC AAC CCC AGA AAC AGA CA; pgiobin 3', CCA CTC
CAG CCA CCA CCT TC; c-globin 5'. ATG TGG GAG AAG ATG GCT GCT; c-giobin 3'. CAA
TAA AGG GGA GGA GAG GGA; tie-2 S', GTC CIT CCT ACC TGC TAC TT; tie-2 3',TTC
CAC TGT I T A CTT CAA TG. PCR was performed on an automated GeneAmp 9600
thermocycler (Perkin Elmer, Norwalk, CI') using 20-30 seconds denaturation at 94OC. 30-45
seconds annealing, and 30-60 seconds extension at 72°C for 32-35 cycles, with a hot start at 94'C
for 2 minutes and a final extension at 72OC for 6 minutes, using annealing temperatures specific for
primer pairs as determined using the OLIGû program (NB1 Software). Roducts were separated
by agarose gel electrophoresis on a 1.0 to 1.6% gel, and Msualized by ethidium brornide staining;
reverse photo images are shown.
Fetal thymic organ culture (FTOC) reconstitution
Lymphocyte-depleted thymic lobes were pnpared by culhlnng &y 14 to day 16 fetal thyrnic lobes
from timed-pregnant mice in iTOC medium containing 1 .ûû- 1.35 rnM 2-deoxyguanosine (dG), as
previously described 91*1Jb'Q. Briefly, host dG-treated FTOCs were cultured for 5-6 days.
dG-containing medium was replaced with FTOC medium for one day, then lobes were rinsed,
resuspended in 10 pi medium. and placed in Terasaki plates at one or two lobes per well. Sorted
donor cells were washed with medium, resuspended in 20 pl medium. and added to dG-treated
alymphoid fetal thyrnic lobes in Terasaki plates. After adding donor cells or medium done, the
plates were inve~ed ("hanging drop") and cufrms were incubated at 37°C in a humidified
incubator containing 5% CO, in air for 24-48 hrs. Lobes were then transferred to standard R O C - for 12- 14 days, except where otherwise noted
Limiting DUution Analysis
A 1:4 serial dilution fmm 1.92 x io4 to 3 x ld sorted Flkl+ cells from eng+'+ and eng" ES/OP9
cwulnnes (&y 5) were reseeded ont0 new OP9 ce11 monolayers in 24-well plates, with 6 replicate
weiis (n4) for each dilution. AU the cells h m each well were harvested, and analyzed individually
by flow cytometq at &y 14 for ce11 surface expression of CD45 and TERl 19 (data not shown).
The presence of CD45 TERl 19* erythrocytes was scored and the prugenitor frequency was
derermined by the method of maximum Iikelihood applied to the Poisson model 'O5. 2 analyses of
CD45' TER1 19+ erythrocytes indicated satisfactory conformance of the experimental data to the
Poisson model: X2131 = 1.598 (+/+) and SLs, = 3.332 (4). Cocultures were observed under an
inverted microscope, and anal yzed by flow c ytometry ai Limiang dilution to detemine approxirnate
clone size.
1.
2.
3.
4.
5 .
6.
7.
8.
9.
IO.
Till JE, McCulloch EA. A direct measurement of the radiation sensitivity of normal mouse
bone marrow cells. Radiat Res. 196 1; 14:z 13-222.
Becker AJ, McCulloch EA, Till JE. Cytological demonstration of the clonal nature of spleen
colonies denved from ümsplanted mouse marrow cells. Nature. 1963; l97:452454.
Sirninovitch L, McCulloch EA, Till JE. The distribution of colony-fomiing cells among
spleen colonies. I. Ceil. Comp. Physiol. 1963;62:327-36.
Wu AM, Till JE, Sirninovitch L, McCulloch EA. Cytological evidence for a relationship
between normal hematopoietic colony-forming cells and cells of the lymphoid system. J.
Exp. Med. 1967; 127:455-467.
Jordan CT. Lemischka IR. Clonal and systemic analysis of long-term hernatopoiesis in the
mouse. Genes Dev. 1 !WO;4:EO-32.
Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lyrnphohematopoietic
reconstitution by a single CD34- lowinegative hematopoietic stem cell. Science.
l996;273 :242-5.
Morrison SJ, Shah NM, Anderson DJ. Regulatory mechanisms in stem ceIl biology. Cell.
1997;88:287-98.
Medvinsky A, Dzieaak E. Definitive hematopoiesis is autonomousl y initiated by the AGM
region. CeIl. t 9%;86:8W906.
Muller AM. Medvinsky A, Strouboulis J, Grosveld F, Dzierzak E. Development of
hematopoietic stem ce11 activity in the mouse embryo. Immunity. 1994; 129 1-30 1.
Billia F, Barbara M, McEwen J, Trevisan LM, Iscove W. Resolution of p1uripotentia.i
intemediates in murine hematopoietic differentiation by global complementary DNA
amplification from single cells: confinnation of assignrnents by expression profiling of
cytokine receptor transcripts. Blood 2001;97:2257-68.
S m d F , Ryan K. Gurdon IB. Markers af vertebrate mesodenn induction, CLLU Opin
Genet Dev. 1997;7:620-7.
Moses HL, Serra R. Regulation of differentiation by TGF-beta. Curr Opin Genet Dev.
1996;6:58 1-6.
Massague J. TGF-beta signal transduction. Annu Rev Biochem. 1998:67:753-9 1.
Fortune1 NO, Hatzfeld A, Hatzfeld JA. Transforming p w t h factor-beta: pleiotropic role in
the regulation of hematopoiesis. Blood. 2000;96:2021-36.
Winnier G, Blessing M, Labosky PA. Hogan BL. Bone morphogenetic protein-4 is
required for mesoderm formation and patteming in the mouse. Genes Dev. 1995;9:2105-
16.
Johansson BM, Wiles MV. Evidence for involvement of activin A and bone morphogenetic
protein 4 in mamrnaiian mesoderm and hernatopoietic development. Mol Ce11 Biol.
1995;15:141-51.
Kanatsu M, Nishikawa SI. In vitro analysis of epiblast tissue potency for hematopoietic cell
differentiation. Development. 1996; lZ2:823-30.
Dickson MC, Martin JS, Cousins FM, Kulkarni AB, Karlsson S, Akhurst RJ. Defective
haematopoiesis and vasculogenesis in transfomiing growth factor-beta 1 knock out mice.
Development. 1995;121: 1845-54.
Oshima M, Oshima H. Taketo MM. TGF-beta receptor type II deficiency results in defects
of yolk sac ternatopopoiesis and vrtsculogenesis. Dev Biol. 19%: 179297-W.
Barbara NP, Wrana IL, Leme M. Endoglin is an accessory protein that interacts with the
signahg receptor complex of multiple members of the transforming growth factor-beta
superfamily. I Bi01 Chem. 1999:274:584-94.
Cheifetz S, Belton T, Cales C. Vera S, Bernabeu C, Massague J, Letarte M. Endoglin is a
component of the transforming growth factor-beta receptor system in human endothelid
cells. J Bi01 Chem. 1992;267: 19027-30.
Buheng HI. Muller CA. Letarte M. Gouges Saalmiiller A. van Agthoven AJ, Busch FW.
Endoglin is expressed on a subpopulation of immanire erythroid cells of normal human
bone rnarrow. Leukernia 1991;S:Ml-7.
Lastres P, Bellon T, Cabanas C, Sanchez-Madrid F, Acevedo A, Gougos A, Letarte M.
Bernabeu C. Regulated expression on human macrophages of endoglin. an Arg-Gly-Asp-
containhg surface antigen. Eur J Immunol. 1992;23:393-7.
Rokhlin OW, Cohen MB, Kubagawa H, Letarte M. Cooper MD. Differentiai expression of
endoglin on fetal and adult hematopoietic cells in human bone marrow. J Imrnunol.
1995; f54:4456-65.
Li DY, Sorensen LK, Brooke BS, Umess LD, Davis EC, Taylor DG, Boak BB, Wendel
DP. Defecti ve angiogenesis in mice Iacking endoglin. Science. l999;284: 1534-7.
Bourdeau A, Dumont DJ, Letarte M. A murine mode1 of hereditay hernonhagic
telangiectasia. J Clin lnvest. 1999; 104: 1343-5 1.
Arthur HM, Ure J, Smith AJ, Renforth G, Wilson DI, Torsney E, Charlton EtT Patums DV,
Jowett T, Marchuk DA, B um J, Diarnond AG. Endoglin, an ancillary TGFbeta receptor, is
required for extraembryonic angiogenesis and plays a key role in heart development. Dev
Biol. 2000;2 17:42-53.
Letamendia A, Lastres P, Botella LM, Raab U, h g a C, Velasco B. Attisano L, Bernabeu C.
Role of endoglin in cellular responses to transforming growth factor-beta. A comparative
study wiEh betagtycari. 3 Bi01 Cttern. t 99827 S:S30L 1-9.
Caniggia 1, Taylor CV, Ritchie JW, Lye SI, Letane M. Endoglin regulates trophoblast
differentiation dong the invasive pathway in human placental villous explants.
Endocrinology. 1997; 138:4977-88.
Lastres P. Letamendia A. Zhang H, Rius C, Aimendm N, Raab U, Lopez LA, Langa C,
Fabra A, Letarte M, Bemabeu C. Endoglin modulates cellular responses to TGF-beta 1. I
Ce11 Biol. l9%;133: 1109-21.
L i ~ H a m p s M ~ ~ H a m p s O n L I < u m a r P , B e r r r a b e 1 ~ ~ ~ S . ( 1 D 1 0 5 antagnnius the
inhibitory signaling of transforming growth factor betal on human vascular endothelid
cells. Faseb J. 2000;14:55-64.
Kallianpur AR, Jordan E, Brandt SJ. The SCLJTAL-1 gene is expressed in progeniton of
both the hematopoietic and vascular systems during embryogenesis. B I d 1994;83: 1200-
8.
Young PE, Baumhueter S, Lasky LA. The sialomucin CD34 is expressed on hematopoietic
celis and blood vessels during murine development B l d . 1995:85:96-105.
Dumont DJ, Yamaguchi TF. Conlon RA. Rossant J, Breitman ML. tek, a novel tyrosine
kinase gene locaied on mouse chromosome 4, is expressed in endotheüal cells and their
presumptive precunors. Oncogene. 1992;7: 147 1-80.
Hsu HC, Ema H, Osawa M, Nakamura Y, Suda T, Nakauchi K. Hematopoietic stem cells
express tie-2 receptor in the murine fetal liver. Blood 2000;96:3757-62.
Shalaby F, Rossant J. Yamaguchi TP, Gertsenstein M. Wu XF, Breitman ML, Schuh AC.
Friilure of blood-island formation and vasculogenesis in Fik-ldeficient mice. Nature.
l995;376:62-6.
Shalaby F. Ho J. Sianford WL, Fischer KD, Schuh AC, Schwartz L, Bernstein A. Rossant J.
A requirement for Flkl in primitive and definitive hematopoiesis and vascuIogenesis. Cell.
l997;89:98 1-90.
Nisltikawa S-1, Nistrikawa S. Hirashima M, Matsayoshi N, Kodama B. Rogressive heage
analysis by ce11 sorting and culture identifies FLKlfVEcadhennf cells at a diverging point
of endothelid and hemopoietic lineages. Development. 1998; 125: 1747- 1757.
Choi K, Kennedy MT Kazarov AT Papadimitriou JC, Keller G. A common precursor for
hematopoietic and endothelid cells. Development. 1998;125:725-32.
Cumano A, Dieteden-Lievre F, Godin 1. Lymphoid potential, probed before circulation in
k Rsaicted ta caridal intraembryoaic -km Cell1996:861907-16.
Dzierrak E, Medvinsky A. de Bmijn M. Qualitative and quantitative aspects of
haematopoietic ce11 development in the rnammalim embryo. Immun01 Toàay. 1998; l9:?38-
36.
Godin 1. Dieterlen-Liewe F, Cumano A. Emergence of multipotent hemopoietic cells in the
yolk sac and paraaortic splanchnopleura in mouse embryos, beginning at 8.5 days
postcoitus (published enatum appears in Roc Natl Acad Sci U S A 1995 Nov
7;92(23): 108 151. Froc Nad Acad Sci U S A. 1995;92:773-7.
Medvinsky AL, Samoylina NL, Muller AM. Dzierzak EA. An early pre-liver intraembtyonic
source of C N - S in the developing mouse. Nature. 1993;364:64-7.
Yoder MC, Hian K, Dun P, Mukhe jee P, Bodine DM. Orlic D. Characterizaiion of
definitive lymphohematopoietic stem cells in the &y 9 murine yolk sac. Immunity.
1997;7:335-44.
Yoder MC, Hiatt K. Mukhejee P. In vivo repopulating hematopoietic stem cells are present
in the murine yolk sac at day 9.0 postcoiw. Roc Natl Acad Sci U S A. 1997;94:6776-80.
Bonifer C. Faust N. Geiger H. Muller AM. Developmental changes in the differentiation
capacity of haematopoietic stem cells. Immun01 Today. 1998; 19:236-4 1.
ikuta K, Kina T, MacNeil 1, Uchida N, Peault B. Chien YH, Weissman IL. A developmental
swiich in thymic lymphocyte maturation potentiai occurs at the level of hematopoietic stem
ceth. Cetf. t 990;62:863-74.
Hardy RR, Li YSI Hayakawa K. Distinctive &velopmentd origins and specificities of the
CDS+ B-ceil subset. Semin. ImrnunoI. 19%;8:37-44.
Okuda T, van Deursen I, Hiebert SW, Grosveld G, Downing IR. AMLl, the target of
multiple chromosomal translocation in human leukernia, is essentiai for nomal fetal Liver
hematopoiesis. Cell. l9%;84:32 1-330.
Wang LC. Swat W, Fujiwara Y. Davidson L, Visvaàer J, Kuo F, Ait FW, Gihland DG,
Golub TR Orkui SH. The ï E E T V 6 g ~ e is required spedïcaiiy for hematopoiesis in the
bone m m w . Genes Dev. 1998;12:2392-402.
Ernst P. Smale ST. Combinatonai replation of transcription. 1: Gened aspects of
transcriptional control. Immunity. 19% $:3 1 1-9.
Robb L, Lyons 1, Li R, Hanley L. Kontgen F, Harvey RP. Metcalf D. Begley CG. Absence
of yolk sac hematopoiesis from rnice with a targeted disruption of the xl gene. Proc Nat1
Acad Sci U S A. 1995;92:7075-9.
Shivdasani RA, Mayer EL. Orkin SH. Absence of blood formation in mice lacking the T-
ce11 leukaemia oncoprotein tal-l/SCL. Nature. 1995;373:432-4.
Robb L, Elwood NJ, Elefanty AG, Kontgen F, Li R, Bamett LD, Begley CG. The scl gene
product is required for the generation of al1 hematopoietic lineages in the adult mouse.
Embo J. 1996; 15:4 123-9.
Porcher C, Swat W, Rockwell K, Fujiwara Y, Alt FW, Orkin SH. The T cell leukemia
oncoprotein SCUtal- 1 is essential for development of ail hematopoietic Iineages. Cell.
1996;86:47-57.
Visvader JE, Fujiwara Y, Orkin S R Unsuspected role for the T-ce11 leukemia protein
SCUtal- 1 in vascular development. Genes Dev. 1998; 12:473-9.
Gering MT Rodaway AR, Gottgens B. Patient EX, Green AR. The SCL gene specifies
haemangioblast development from early mesoderm. Embo J. 1998; 17:4029-45.
Robertson SM, Kennedy M, Shannon M. Keller G. A transitional stage in the cornmiment
of mesodenn to hematopoiesis requiring the transcription factor SCZltai- 1. De velopment.
2000; l27:%47-59.
Scott EW, Simon MC, Anastasi J, Singh H. Requirement of transcription factor PU.1 in the
development of multiple hematopoietic lineages. Science. 1994265: 1573-7.
Wang .J-H, Nichogiannopoulou A, Wu L, Sun L, Sharpe AH, Bigby M, Georgopoulos K.
Selective def~ecü in the development of the fetal and adult lymphoid system in rnice with an
Ilcaros nu l miitation, Immunity, i.9965537-549,
Georgopoulos K. Bigby M. Wang J-H, Molnar A, Wu P, Winandy S, Sharpe A. The karos
gene is required for the development of d l lyrnphoid lineages. Cell. 1994:79: 143- 156.
Pevny L. Simon MC, Robertson E, Klein WH, Tsai SF, DtAgati V, Orkin SH, Costantini F.
Erythroid differentiation in chimaeric rnice blocked by a targeted mutation in the gene for
transcription factor GATA- 1. Nature. 199 1 ;349:357-60.
Weiss MJ, Keller G, Orkin S. Novel insights into erythroid development revealed through
in vitro differentiation of GATA-1' embryonic stem cells. Genes Dev. 1994;8: 1184-1 197.
Pevny L, Lin CS. DtAgati V, Simon MC, Orkin SH, Costantini F. Development of
hematopoietic cells lacking transcription factor GATA- 1. Development. 1995; 12 1 : 163-72.
Urbanek P, Wang ZQ, Fetka 1, Wagner EF, Busslinger M. Complete block of early B cell
differentiation and altered patteming of the postenor midbrain in mice lacking PaxYBSAP.
Cell. 1994;79:901-12.
Nutt SL, Heavey B. Rolink AG, Busslinger M. Cornmitment to the B-lymphoid lineage
depends on the transcription factor PaxS. Nature. l999;4O 1 556-62.
Barton K, Muthusarny NT Fischer C, Ting CN, Walunas TL, Lanier LL, Leiden JM. The
Eu-1 transcription factor is required for the development of n a d killer cells in mice.
Imrnuni ty. l998;9:555-63.
Ting C-N, Olson MC, Barton KP, Leiden M. Transcription factor GATA-3 is requid for
development of the T-cell lineage. Nature. 19963 84:474-478.
Brown KE, Guest SS, Smale ST, Hahm KT Merkenschlager M, Fisher AG. Association of
transcriptionally silent genes with Ikaros complexes at cenmmenc heterochromatin. Cell.
1997;9 1 : 845-54.
Cobb BS, Morales-Alcelay S. Kleiger G. Brown EE, Fisher AG, Smale ST. Targeting of
ïkaros to pericentromeric heterochromatin by d k c t DNA binding. Genes Dev.
2000;14rU46-60-
Koipally J, Renold A, Kim J, Georgopoulos K. Repression by Ikaros and Aiolos is
mediated through histone deacety lase complexes. Embo J. 1999; 1 8:3OW- 1 ûû.
Kim J, Sif S, Jones B, Jackson A, Koipally J, Heller E, Winandy S, Vie1 A, Sawyer A, Ikeda
T, Kingston R, Georgopoulos K. ikms DNA-binding proteins direct formation of
chromatin rernodeling complexes in lymphocytes. Irnrnunity. 1999; 10:345-55.
DeKoter RP, Singh H. Regulation of B lymphocyte and macrophage development by
graded expression of PU. 1. Science. 2000;288: 1439-4 1.
Rolink AG, Nun SL, Melchers F, Busslinger M. Long-term in vivo reconstitution of T-ce11
development by Pax5 deficient Bcell progeniton. Nature. l999;M 1 :603-6.
Roberts R, Gallagher J, Spooncer E, Allen TD, Bloomfield F, Dexter TM. Heparan sulphate
bound growth factors: a mec hanism for stromd ceIl mediated haemopoiesis. Nature.
1988;332:376-8.
Klein G. The extracellular matrix of the hematopoietic rnicroenvironment. Experientia.
1995;s 1:914-26.
Muller-Sieburg CE, Deryugma E. The stroma1 cells' guide to the stem ce11 universe. Stem
Cells. 1995; l3:477-86.
Wineman J. Moore K, Lemischka 1, Muller-Sieburg C. Functionai heterogeneity of the
hematopoietic rnicroenvironment: rare stroma1 elements maintain long-terni repopulating
stem cetts. B l d . 1996;87:j082-90.
Watowich SS, Wu H, Socolovsky M, Klingmuller U, Constantinescu SN, Lodish HF.
Cytokine receptor signal transduction and the control of hematopoietic ce11 developrnent.
Annu Rev Ce11 Dev Biol. N96; l2:9Ll28.
Metcalf D. Hematopoietic regulators: redundancy or subtiety? Blood. 1993;82:35 15-23.
Dorshkind K. Regulation of hemopoiesis by bone m m w stmmd cells and their products.
Annu Rev hmunol. 1990;8: 1 1 1-37.
Bmudy VC. Stem ceii factor and hematopoiesis. Blood. 1997;90: 1345-64.
Lyman SD, Jacobsen SEW. c-kit Ligand and Flt3 Ligand: stemlprogeni tor ce11 factors wi th
overlapping yet distinct activities. Blood. l998;gl: 1101-1 134.
Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K, Silvennoinen O. Signding through the
hematopoietic cytokine receptors. Annu Rev hmunol. 1995; 13:369-98.
Trowbndge IS, Thomas ML. CD45: an emerging role as a pmtein tyrosine phosphatase
required for lymphocyte activation and development. Annu Rev Immunol. 1994;12:85-116.
Trowbndge IS, Ostergaard HL, Johnson P. CD45 a leukocyte-specific member of the
protein tyrosine phosphatase family. Biochim Biophys Acta. 199 1; 1095:46-56.
hie-Sasaki J, Sasaki T, Matsumoto W. Opavsky A, Cheng M. Welstead G, Griffith E,
Krawczyk C, Richardson CD, Aitken K, Iscove N, Koretzky G, Johnson P, Liu P, Rothstein
DM, Penninger IM. CD45 is a JAIS phosphatase and negativel y regulates cytokine receptor
signalling. Nature. 200 1 ;409:349-54.
Sanchez MJ, Holrnes A, Miles C, Dzierzak E. Characterization of the first definitive
hematopoietic stem cells in the AGM and liver of the mouse embryo. Irnrnunity.
l996;5:5 13-25.
Osawa M, Nakarnun K, Nishi N, Takahasi N, Tokuomoto Y, houe H, Nakauchi H. In vivo
self-renewal of c-Kit+ Sca-l+ Lin(low1-) hemopoietic stem cells. J Irnrnunol.
1996; lS6:32O7- 14.
Huns BE, Capone M. Zlomik A, Rennick D, Mowe TA. Acquisition of CD24 expression
by ~ i n - ~ ~ 4 3 + ~ 2 X f ~ ~ c k i t ' cells coincides with commiünent to the B ce11 Iineage. Eur. I.
Irnmunol. 1998;28:3850-3856.
Godfky DI, Zlotnik A. Suda T. Phenotypic and functiond characterization of c-kit
expression during intrathymic T ceIl development. J. Immunol, 1993; 1.
Honimi K, Koburi A, Sato T, Nozaki H, Nishikawa S-1, Nishimura T, Habu S. b T cells
in fetai thymus express c-kit and RAG-2 but do nat reamge the gene encading the T celi
receptor p chah Eur. J. Immunol. 1994;24:1339-1344.
Zfifiiga-Pfliicker JC, Lenardo UT. Regulation of thymocyte development fmm immature
progenitors. Curr. Opin. Irnmunol. 1996;8:2 15-224.
Carlyle JR. Michie AM. Cho SK. Zuniga-Pflucker JC. Naniral killer ce11 development and
function precede alpha beta T ce11 differentiation in m o w fetal thymic ontogeny. J
Immunol. 1998; 160:744-53.
Leary AG, Zeng HQ, Clark SC. Ogawa M. Growth factor requirements for survival in GO
and entry into the ce11 cycle of primitive human hemopoietic progeniton. Roc Natl Acad
Sci U S A. l993;89:4û 13-7.
Keller JR, Oniz M, Ruscetti FW. Steel factor (c-kit ligand) promotes the survival of
hernatopoietic stedprogenitor cells in the absence of ce11 division. Blood 1995;86: 1757-
64.
Hirayama F, Lyman SD, Clark SC, Ogawa M. The flt3 ligand supports proliferation of
lymphohematopoietic progenitors and early B-lymphoid progenitors. Blood. 1995;85: 1762-
1768.
Veiby OP, Lyman SD, Jacobsen SEW. Combined signaling through interleukin-7 receptors
and flt3 but no c-ki t potently and selectively promotes Bcell cornmitment and
di fferentiation from uncomitted murine bone marrow progenitor cells. B lood.
l9%*,88: 1 256-1 265.
Moore TA. Ziotnik A. Differential effects of Rk-2mt-3 ligand and stem ce11 factor on
murine thyrnic progenitor cells. J. Immunol. 1997; lS8:4187-4 192.
Mackarehtschian K, Hardin ID, Moore KA, Boast S, Goff SP, Lemischka IR. Targeted
disruption of the flkUflt3 gene leads to deficiencies in primitive hematopoietic progenitors.
Immunity. 1995;3: 147-61.
Henderson A, Calame K. Transcriptionai regdation during B cell development. Annu Rev
Immtinol. l998;l6: 163-200,
Tudor KS. Payne EU, Yamashita Y, Kincade PW. Functional assessrnent of precursoa from
murine bone marrow suggests a sequence of early B lineage differentiation events.
Irnrnunity. 2000; 1233535.
Melchers F, Rolink A. Grawunder W. Winkler TH. Karasuyama H. Ghia P. Andenson J.
Positive and negative selection events during B lymphopoiesis. Curr. Opin. Immunol.
l995;7:2 14-27.
Owen JJT, Jenkinson U. Early events in T lymphocyte genesis in the fetal thymus. Am. I.
Anat. 1984; l7O:3O 1-3 10.
Anderson G. Moore NC, Owen JJ, Jenkinson W. Cellular interactions in thymocyte
development. Annu. Rev. Immunol. 1996; 14:73-99.
Anderson G, Jenkinson EJ, Moore NC, Owen JJT. MHC class iI-positive epithelium and
mesenchyme cells are both required for T-ceIl development in the thymus. Nature.
l993;362:70-73.
Itoi M. Arnagai T. Inductive role of fibroblastic ce11 lines in developrnent of the mouse
thymus anlage in organ culture. Ceil. Immunol. 1998; lO:32-4 1.
Amagai T. Itoi M. Kondo Y. Limited developrnent capacity of the earliest embryonic murine
thymus. Eur. J. Irnmunol. 1995;25:757-762.
Shores ETK, Van Ewijk W, Singer A. Disorganization and restoration of thymic medullary
epihlial tells in T ceil reeeptor-negative scid micc evidmce that receptor-beanng
lymphocytes influence maturation of the thymic microenvironment. Eur J Immunol.
lggl;?l: 1657-61.
van Ewijk W. Wang B, HoIlander G, Kawamoto H. Spanopoulou ET Itoi M, Amagai T, Jiang
YF, Germeraad WT. Chen W. Katsura Y. Thymic microenvironments, 3-D versus 2-D?
Sernin ImmunoL 1999:11:57-64.
Rockop S. Petrie HT. Ce11 migration and the anatomic conml of thymocyte precursor
differentiatio~ Semin Immiind 200QrI3.4-3.
Shortman K, Wu L. Early T lymphocyte progenitors. Am. Rev. Immunol. 1996;14:29-47.
Boyd EU, Tucek CL, GodfRy DI, Izon DJ, Wilson TJ, Davidson NJ, Bean AG, Ladyman
HM, Ritter MA, Hugo P. The thymic microenvironment. Immunol. Today. 1993; 14:445-
459.
Pui JC, Allman D, Xu L, DeRocco S, Karnell FG, Bakkour S, Lee JY, Kadesch T, Hardy
EtR, Aster JC, Pear WS. Notchl expression in early lymphopoiesis influences B versus T
lineage determination. Immunity. 1999; 1 1299-308.
Radtke F, Wilson A, Stark G, Bauer M, Meerwijk Jv, MacDonald KR. Aguet M. Deficient
T ce11 fate specification in rnice with an induced inactivation of Notchl. Immunity.
1999; lO:S47-558.
Pear WS, Aster JC, Scott ML, Hasse jian RP, Soffer B, Sklar J, Baltimore D. Exclusive
development of T ceIl neoplasms in mice transplanted with bone marrow expressing
activated Notch alleles. J Exp Med 1996; l83:2283-229 1.
Delassus S, Cumano A. Circulation of hematopoietic progenitoa in the mouse embryo.
Irnmunity. l996;4:97- lO6.
Antica M, Wu L, Shortman KT Scollay R. Thyrnic stem cells in mouse bone marrow. Blood.
l994;84: 1 11-1 17.
Godfrey DI, Zlotnik A. Conml points in early T-ce11 development. Immunol. Today.
k 993; f 4547-553.
Moore TA, Zlotnik A. T-cell Lineage cornmitment and cytokine response of thymic
progeniton. Blood. lWS;86: 1850- 1860.
ZUniga-Pflücker JC, Jiang D, Lenardo M. Requirement for TNF-a and IL-la in fetal
thyrnocyte cornmitment and differentiation. Science. 1995;268: lW6- l9O9.
Wu L, Scollay R, Egerton M, Pearse M, Spangrude GJ, Shortman K. CD4 expressed on
earliest T-lineage precursor cells in the aduit murine thymus. Nature. 199 l;349:7 1-74.
Di Santo JI?, Rodewald tIIl In vivo mles of recep~ar tyrnunt kinases and cytokine
receptors in early thymocyte development. C u r ~ Opin Immunol. 1998; 10: 196-207.
Godfrey DI, Kennedy J, Mombaerts P, Tonegawa S, Zlotnik A. Onset of TCR-$
reanangement and role of TCR-$ expression during CD3-CD4'CDK thymocyte
differentiation. J. Immunol. 1994; 152:4783-4793.
Dudley EC, Petrie HT, Shah LM, Owen MJ, Hayday AC. T cell receptor $ chain gene
rearrangement and selection during thymocyte development in adult mice. immunity.
1994;1:83-93.
von Boehmer H, Fehling HI. Structure and function of the pre-T ceIl receptor. Annu. Rev.
Immunol. 1997: l5:433+52.
Levelt CN, Eichmann K. Receptors and Signais in Early Thymic Selection. Imrnunity.
1995;3:667-672.
Wilson A, Held W. MacDonald HR. Two waves of recombinase gene expression in
developing thymocytes. J. Exp. Med. 1994; 179: 1355- 1360.
Kisielow P, von Boehmer H. Negative and positive selection on immature thymocytes:
timing and the role of ligand for cdfl T cell receptor. S e m . Immunol. 1990;2:35-44.
Ziiiïiga-Pflücker JC, Jones LA. Chin LT, Kniisbeek AM. CD4 and CD8 act as CO-receptors
during thymic selection of the T cell repertoire. Semin. Immunol. 1990;3: 167-172.
Hugo P, Kappler JW, Marrack PC. Positive selection of TcR alpha beta thymocytes: is
cortical thymic epithelium an obligatory participant in the presentation of major
histocompatibility complex protein? Immunol. Rev. 1993; 135: 133- 155.
Fowlkes BJ, Schweighoffer E. Positive selection of T cells. C m Opin. Immunol.
l995;7: 188-195.
Ohashi PS. T ce11 selection and autoimmunity: flexibility and tuning. Curr .Opin. Immun01 .
1996:8:808-814.
Mamson SI, Uchida N, Weiumaa IL The hidogy of hematopoietic stem ceb. h u Rev
Ce11 Dev Biol. 1995; 11:35-71.
Bradley A, Evans M, Kauhan MH, Robertson E. Formation of germ-line chimaem fmm
em bry O-denved teratocarcinorna ce11 lines. Nature. 1 984;309:255-6.
Beddingîon RS, Robertson EJ. .ei assessrnent of the developmental potential of embryonic
stem cells in the midgestation mouse embryo. Development. 1989: 105:733-7.
Keller GM. In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol.
1995;7:862-9.
Keller G, Kennedy M, Papayannopwlou T, Wiles MV. Hematopoietic cornmitment during
embryonic stem ceil differentiation in culture. Mol Cell Biol. 1993;13:473-86.
Kabnin N, Buhring HJ, Choi K. Ullrich A, Risau W, Keller G. Rk-1 expression defines a
population of early embryonic hematopoietic precursors. Development. 1997; 124:2039-48.
Kennedy M. Firpo M, Choi K, Wall C, Robertson S, Kabrun N, Keller G. A common
precursor for primitive erythropoiesis and definitive haematopoiesis. N a m . 1997;386:488-
93.
Kee BL, Cumano A, Iscove NN, Paige CI. Strornal ceil independent growth of bipotent B
cell-macrophage precursors from murine fetal liver. In t [mmunol. l994;6:4O 1-7.
Potocnik AJ, Nielsen PJ, Eichmann K. In vitro generation of lymphoid precursors from
embryonic stem cells. EMBO J. 1994; l3:5274-5283.
Gutierrez-Rmos JC, Pdafios R. In vitro differentiation of ernbryonic stem cetls into
lymphocyte precursors able to generate T and B lymphocytes in vivo. Proc. Natl. Acad. Sci.
1992;89:9171-9175.
Nakano T. Lymphohematopoietic developrnent h m embryonic stem ceils in vitro. Semin.
Irnrnunol. 1995;7: 197-203.
Nakano T, Kodama H, Honjo T. Generation of lymphohematopoietic cells h m embryonic
stem cells in culture. Science. 1994;265: 1098- 1 101,
JenkinsanrnFranchiLL, KhgsmnR.+ OwenJI,Effect af deoxyguanosine on
lymphopoiesis in the developing thymus nidiment in vitro: application in the production of
chimeric thymus rudiments. Eur J Immunol. 1982;12:583-7.
Kingston R, Jenkinson U, Owen JJ. A single stem ce11 can recolonize an embryonic
thymus, producing phenotypicdly distinct T-cell populations. Nature. 1985;3 17: 8 1 1-3.
Anderson G, Jenkinson EJ. The role of the thymus during T-lymphocyte developrnent in
vitro. Semin. Immunol. 1995;7: 177- 183.
Hardy RR. Carmack CE. Shinton SA, Kemp ID, Hayakawa K. Resolution and
characterization of pro-B and pre-pro-B ce11 stages in normai mouse bone marrow. J. Exp.
Med. l99l;l73: l?l3-1225.
Li YS, Wassennan R, Hayakawa KT Hardy RR. Identification of the earliest B lineage stage
in rnouse bone marrow. h u n i t y . 19965527-535.
h t a K, Uchida NT Friedman I, Weissman IL. Lymphocyte devel~prnent h m stem celis.
Annu. Rev. Imrnunol. 1992; 10:759-783.
Hunte BE, Hudak S, Campbell D, Xu Y, Rennick D.jiWfl3 Ligand is a potent cofactor for
the growth of primitive B cell pmgenitors. J. Imrnunol. 1996; 156:489-96.
Rosenberg N. A bi-mediated transformation, immunoglobulin gene reamgernents and
arrest of B lymphocyte differentiation. Semin. Cancer Biol. l994;5:95- 102.
Rosenberg N, Kincade PW. B-lineage differentiation in normal and transfonned cells and
the microenvironment ihat supports it. C m ûptn. fmunot. f Wt;6:203-3 1 1.
Lewis SM. The mechanism of V(D)J joining: lessons frorn molecular, immunological, and
comparative analyses. Adv. hmunol. l9%*56:27- 150.
Curnano A, Dorshkind K, Gillis S, Paige CI. The influence of S17 strornai cells and
interleukin 7 on B ce11 development. Eur. J. Immunol. lgW30:2 183-2 189.
Ledbener JA, Hemberg LA. Xenogeneic monoclonal antibodies to mouse lymphoid
di fferentiation antigens. Immunol. Rev. l979;47:63-90.
Miller B A ADtognetti G, SpMger TA. Identifcation of ce11 suface antigens present on
murine hematopoietic stem cells. J. Immunol. 1985; 134:3386-3290.
Huang H, Auerbach R. Identification and charactenzation of hematopoietic stem cells from
the yolk sac of the early mouse embryo. Proc. Nail. Acad. Sci. 1993;90: 101 10-10114.
S p m p d e GJ. Heimfeld S, Weissman IL. Purification and characterization of mouse
hematopoietic stem cells. Science. 1988;241:58-62.
Nakano T, Kodama H, Honjo T. In vitro development of primitive and definitive
erythrocytes from different precursors. Science. 1996;272:722-724.
Hudak S, Hunte B, Culpepper J, Menon S. Hannum C, Thompson-Snipes L. Rennick D.
FLT3/FLK2 ligand promotes the growth of murine stem cells and the expansion of colony-
fonning cells and spleen colony-forming units. Blood. 1995;85:2747-2755.
Jacobsen SE, Okkenhaug C. Myklebust J, Veiby OP, Lyman SD. The E T 3 ligand potently
and directly stimulates the growth and expansion of primitive murine bone rnarrow
progenitor cells in vitro: synergistic interactions with interleukin (IL) 11, IL-12, and other
hernatopoietic growth factors. J. Exp. Med. 19%; 18 1 : 1357- 1363.
Rosenberg N, Baltimore D. A quantititative assay for transformation of bone marrow cells
by Abelson murine leukernia virus. J. Exp. Med. 1976; 143: 1453- 1463.
Pennycook JL, Marshall AJ, Wu GE: PCR assays for endogenous Ig gene rearrangement.,
in Lefkovits 1 (ed): Immunology Methods Manual. Toronto, Academic Ress, 1997. p
2237-257
Hesse JE, Lieber MR, Gellen M. Mizuuchi K. Exnachromosornai DNA substrates in pre-B
cells undergo inversion or deletion at imrnunoglobulin V-@)-J joining signais. Cell.
1987;49:775-783.
Lewis SM, Agard E, Suh S, Czyzyk L. Cryptic signals and the fidelity of V(D)J joining.
Mol. Cell. Biol. 1997; 17:3 125-3 136.
Lieber MR, Hesse JE, Mizuuchi K, Gellert M. Lymphoid V@)J recombination: nucleotide
insertion at signal joints as well as codingjoints, Roc NatL Acad S& 1988:85:8588-8593
Meier JT, Lewis SM. P nucleotides in V@)J recombination: A fine-structure analysis. Mol.
Cell. Biol. 1993;13: 1078-1092.
Hathcock KS, Lasdo G, Pucillo C, Linsley P, Hodes RI. Comparative analysis of B7- 1 and
B7-1 costirnulatory ligands: expression and function. J. Exp. Med 1994; 180:63 1-40.
Frank KM, Sekiguchi JM, Seidl KJ, Swat W, Rathbun GA, Cheng HL, Davidson L.
Kangaloo L. Alt FW. Late embryonic lethality and impaired V(D)J recombination in rnice
lacking DNA ligase IV. Nature. 1998;396: 173-7.
Mortensen RM, Conner DA, Chao S, Geisterfer-Lowrance AAT, Seidman JG. Production
of homozygous mutant ES cells with a single targeting consmict. Mol. Cell. Biol.
1992; l2:EN 1-2395.
Sharnblon MJ, Axelman J, Wang S. Bugg EM, Littlefield W. Donovan PJ, Blumenthd PD,
Huggins GR, Gearhart JD. Derivation of pluripotent stem cellr fmm culnired human
primordial germ cells. Roc. Natl. Acad. Sci. USA. 1998;95: 13726-3 1.
Thomson SA, Itskovitz-Eldor J, Shapiro SS, Waknia MA, Swiergiel JJ, Marshdl VS, Jones
M. Embryonic stem ce11 lines derived hom human blastocysts. Science. 1998;282: 1145-7.
Kondo M, Weissman LL, Akashi K. Identification of clonopnic common lyrnphoid
progeniton in mouse bone marrow. Cell. l997;9 1 :66 1-72.
Cho SK, Webber TD, Carlyle JR, Nakano T, Lewis SM, Zuniga-Pflucker JC. Functional
chancterizatim of B tymphocytes gniented in vitro h m mbryornc stem cetts. Roc Nad
Acad Sci U S A. 1999;96:9797-802.
Nakayarna NT Fang 1, Elliott G. Natural killer and B-lymphoid potential in CD34+ cells
derived h m embryonic stem ceiis differectiated in the presence of vascular endothelial
growth factor. B l d 1998;9 12283-95.
Chen U, Kosco M, Staen U. Establishment and characterization of lymphoid and myeloid
mixed-cell populations h m mouse late embryoid bodies. "embryonic-stemceil fenises".
Proc Nat1 Acad Sci U S A, L992;89%41-S,
Cho SK, Bourdeau A, Letarte M, Ziiiiiga-Pflücker JC. Expression and function of CD LOS
during the onset of hematopoiesis from Flk 1 + precursors. Blood. 200 1 ;in press.
Hare KT, Jenkinson U, Anderson G. In vitro models of T ce11 development. Semin
Irnmunol. 1999; 1 1 :3- 12.
Yamaguchi TP, Dumont DJ, Codon RA, Breitman ML, Rossant J. flk-1, an flt-related
receptor tyrosine kinase is an early marker for endothelial ce11 precursors. Development.
1993; f 18~489-98.
Shinkai Y, Rathbun G, Lam K-P, Oltz EM, Stewart V, Mendelsohn M, Charron J, Datta M,
Young F, Stail AM, Alt W. RAG-2-deficient mice lack mature lymphocytes owing to
inability to initiate V@)J rearrangement. Cell. 1992;68:855-867.
Mombaerts P, Iacornini J, Johnson RS, Hemp K, Tonegawa S, Papaioannou VE. M G - 1-
deficient rnice have no mature B and T lymphocytes. Cell. 1992;68:869-877.
Munsie MJ, Michalska AE, O'Brien CM, Tmunson AO, Pen MF, Mountford PS. Isolation
of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei.
Cun Biol. 2000; lO:989-9L
Wakayama T, Tabar V, Rodnguez 1, Perry AC, Studer L, Mombaerts P. Differentiation of
embryonic stem ce11 lines generated From adult somatic cells by nuclear transfer. Science.
200 1 ;292:740-3.
Schuh AC, Faloon P, Hu QL, Bhimani M, Choi K. In vitro hematopoietic and endothelial
potential of flk-l(-/-) embryonic stem cells and embryos. Roc Nat1 Acad Sci U S A.
l999;96: 2 159-64.
Hidaka M. Stanford WL, Bernstein A. Conditional requirement for the Fik- l receptor in the
in vitro generation of earl y hematopoietic cells. Proc. Natl. Acad Sci. U.S.A.
1999;96:7370-5.
Quaekenbush U. Leme M. IdentificaÉion of seved ceB surface proteins of non-T, mn-B
acute !yniphoblastic leukernia by using monoclonal antibodies. l hmunol. 1985; 134: 1276-
85.
O'Comell PJ, McKenzie A, Fisicm N. Rockman SP, Pearse MJ, dApice M. Endoglin: a
180-kD endothelial ce11 and macrophage restricted differentiation molecule. Clin Exp
Immunol. 1992;90: 154-9.
Gougos A, Letarte M. Identification of a human endothelial ceII antigen with monoclonal
antibody 4464 produced against a pre-B leukernic cell line. J Imrnunol. 1988; 141:1925-33.
Springer T, Galfre G, Secher DS, Milstein C. Monoclonal xenogeneic antibodies to murine
ce11 surface antigens: identification of novel leukocyte differentiation antigens. Eur J
Imrnunol. 1978;8:539-5 1.
Alterman LA, Crispe IN, Kinnon C. Characterization of the muriiie heat-stable antigen: an
hematolyrnphoid differentiation antigen defined by the J 1 Id, Ml169 and B2A2 antibodies.
Eur J Immunol, 1990;20: 1597-602.
Coffman RL. Sunace antigen expression and immunoglobulin gene rearrangement during
mouse pre-B ceIl development. hmunol Rev. 1982:69:5-23.
Krop 1, Shaffer AL. Fearon DT, Schlissel MS. The signaiing activity of munne CD19 is
regulated during ce11 development. 1 Immunol. 1996;157:48-56.
Springer T, Galfre G, Secher DS, Milstein C. Mac4 : a macrophage differentiation antigen
identified by monoclonal antibody. Eur 3 Immunol. l!lW$k3O t -6.
Moore TA, von Freeden-Jeffry U, M m y RT Zlotnik A. Inhibition of gamma delta T ceIl
development and early thymocyte maturation in IL-7 -1- mice. J ïmmunol. 1996;157:2366-
73.
Pierelli L, Scambia G, Bonanno G, Rutella S, Puggioni P, Banaglia A, Moveni S. Marone
M, Menichella G, Rumi C, Mancuso S, Leone G. CD34+/CD 105+ cells are enriched in
primitive circulating progenitors residing in the GO phase of the ceIl cycle and contain al1
bone maunw and cord blocxi (lD3LkLCD381~wf- ~ U I S O L S Br I be.maioL
ZOûû; lO8:6 10-20.
Ling V, Luxenberg D, Wang J, Nickbarg E, kenen PJ, Neben S. Kobayashi M. Structural
identification of the hematopoietic progenitor antigen ER-MP 12 as the vascular endothelial
adhesion moiecule PECAM- 1 (CD3 1). Eur J h u n o l . lgW;Y:509- 14.
Watt SM, WilIimon J, Genevier H, Fawcett J, Simmons DL, Hatzfeld A, Nesbitt SA,
Coombe DR. The heparin binding PECAM-1 adhesion molecule is expressed by CD34+
hematopoietic precursor ceUs with early myeloid and B-lymphoid cell phenotypes. Blood.
1993:82:2649-63.
Johnson DW, Berg IN, Baldwin MA. Gallione Ci, Marondel 1, Yoon SJ. Steruel TT, Speer
M, Pericak-Vance MA, Diamond A, Guttmacher AE, Jackson CE, Attisano L, Kucherlapati
R, Porteous ME, Marchuk DA. Mutations in the activin receptor-like kinase 1 gene in
herediiary haemorrhagic ~ ; d + t a s i a type 2. Nat Genet. 1996; 13: 189-95.
Hemann BG, Kispert A. The T genes in embryogenesis. Trends Genet. 1994; 10280-6.
Wilkinson DG, Bhan S. Hemnann BG. Expression pattern of the mouse T gene and its role
in mesoderrn formation. Nature. 1990;343:657-9.
Sato TN, Tozawa Y, Deutsch U, Wolburg-Buchholz K, Fujiwara Y, Genhn-Maguire M,
Gridley T, Wolburg H, Risau W, Qin Y. Distinct roles of the receptor tyrosine kinases Tie-
1 and Tie-2 in blood vesse! formation. Nature. 1995;376:70-4.
Dumont V3, Gndwoht G, Fong GH, Puri MC. Gertsenstekr M. A u d a c h A, Breitman ML.
Dominant-negative and targeted nu11 mutations in the endothelial receptor tyrosine kinase,
tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev. 19948: 1897-9O9.
Fazekas de St. Groth S. The evaluation of limiting dilution assays. Journal of
Immuno1ogica.I Methods. 1982;49:R 1 1-22.
Ma X Labinaz M, Goldstein J, Miller H, Keon WJ, Letarte M, O'Brim E. Endoglin is
overexpressed after artenal injury and 1s required for transfonning growth factor-ss-
indlireriinhibition of smooth muscie di migzaiinn. Artenosder T h r d Vasc BiaL
2000:20:2546-52.
Goey H, Keller IR, Back T, Longo DL, Ruscetti FW, Wiltrout RH. Inhibition of early
murine hemopoietic progenitor ce11 proliferation after in vivo locoregional administration of
msforming p w t h factor-beta 1. J Immunol. 1989; l43:877-80.
Heinnch MC, Dooley DC, Keeble WW. Transforming growth factor beta 1 inhibits
expression of the gene products for steel factor and its receptor (c-ki t). B lood.
l!BS;85: 1769-80.
Bhatia M, Bonnet D, Wu D, Mudoch B, Wrana J, Gallacher L, Dick JE. Sone
morphogenetic proteins regulate the developmental program of human hematopoietic stem
cells. J Exp Med. 1999; 1 89: 1 139-48.
Nishita M, Hashimoto MK, Ogata S, Laurent MN, Ueno N, Shibuya H, Cho KW.
Interaction between Wnt and TGF-kta signalling pathways during formation of Spemann's
organizer. Nature. 7000;403:78 1-5.
Labbe E, Letamendia A, Attisano L. Association of Smads with lymphoid enhancer binding
factor l/T cell- specific factor mediates cmperative signaling by the transfoming growth
factor-beta and wnt pathways. Proc Nat1 Acad Sci U S A. 2000;97:8358-63.
Austin TW. Solar GP. Ziegler FC, Liem L, Matthews W. A roIe for the Wnt gene family in
hematopoiesis: expansion of multilineage progenitor cells. Blood 1997:89:3624-35.
Brandon C, Eisenberg tM, Eisenberg CA. WM signaimg modulates the divemfrcation of
hematopoietic cells. Blood 2ûûû;96:4 132-4 I .
Reya T. O'Riordan M. Okarnura R, Devaney E, W i k t K, Nusse R, Grosschedl R. Wnt
signaiing regdates B 1 y mp hoc yte proli feration through a LEF- 1 dependent mec hanism.
Immunity. 2000; 13: 15-24.
Van Den Berg DJ, S h m a AK. Bruno E, HoMnan R. Role of members of the Wnt gene
family in human hematopoiesis. B l d 1998;92:3 189-202.
216 Hayakawa K, Hardy RR, LA, Progenitors foE Ly- 1 B cells are distinct from
progenitors for other B cells. J Exp Med 1985; 16 1 : 1554-68.
217. Pierelli L, Marone M. Bonanno G, Mozzetti S, Rutella S, Morosetti RT Rumi C, Mancuso S.
Leone G, Scambia G. Modulation of bcl-2 and p27 in human primitive proliferating
hernatopoietic progeni tors b y autocrine TGF-beta l is a ce11 cycle-independent e ffect and
influences their hematopoietic potential. B l o d 2000;95:3001-9.
2 18. Mukouyama Y, Chiba N, Ham TT Okada H, Ito Y, Kanainam R, Miyajima A, Satake M,
Watmabe T. The AMLI transcription factor functioos to develop and maintain hematogenic
precursor cells in the embryonic aorta-gonad-mesonephros region. Dev Biol. 2000;220:27-
36.
2 19. Mukouyama Y, Chiba N. Mucenski MLT Satake M. Miyajima A, Han T, Watanabe T.
Hematopoietic cells in cultures of the murine embryonic aorta-gonad- mesonephros region
are induced by c-Myb. Curr Biol. 1999;9:833-6.
220. Ohneda O, Fennie C, Zheng 2, Donahue C, La H. Villacorta R Cairns B, Lasky LA.
Hematopoietic stem ceil maintenance and differentiation are supported by embryonic aorta-
gonad-mesonephros region-derived endothelitm. Blood. f998;9?:W8- 19.
221. Rosenberg NT Witte ON. Abelson murine leukemia virus mutants with alterations in the
virus-specific pl20 molecule. J. Virol. 1980;33:340-348.