doi:10.1182/blood-2005-09-3621Prepublished online October 27, 2005;
Abby L Olsen, David L Stachura and Mitchell J Weiss cellsDesigner blood: creating hematopoietic lineages from embryonic stem
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Olsen et al. Producing blood lineages from ES cells 1
Designer Blood: Creating Hematopoietic Lineages from Embryonic Stem Cells
Abby L. Olsen1,2, David L. Stachura 1 and Mitchell J. Weiss3,4
Running head: Producing blood lineages from ES cells
1. Cell and Molecular Biology Graduate Program, The University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
2. Combined Degree Graduate Program, The University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
3. The Children’s Hospital of Philadelphia, Division of Hematology and the University of Pennsylvania, Philadelphia, PA 19104, USA
4. Corresponding Author: Mitchell J. Weiss, Division of Hematology, 3615 Civic Center Blvd., Abramson Research Center, Philadelphia, PA 19103, [email protected], Fax: (215) 590-4834, Phone: (215) 590-0565
Financial Support: This work was supported by NIH grants R01 DK064037 (M.J.W.), T32-HL007971-04 (D.S.).
Word counts: Abstract: 163 Text: 5574
SCIENTIFIC HEADING: Review Article
Blood First Edition Paper, prepublished online October 27, 2005; DOI 10.1182/blood-2005-09-3621
Copyright © 2005 American Society of Hematology
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Olsen et al. Producing blood lineages from ES cells 2
Abstract
Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any
differentiated tissue upon appropriate manipulation in culture, a property that has been
beneficial for studies of hematopoiesis. Until recently, the majority of this work utilized
murine ES cells for basic research to elucidate fundamental properties of blood cell
development and establish methods to derive specific mature lineages. Now, the advent
of human ES cells sets the stage for more applied pursuits to generate transplantable cells
for treating blood disorders. Current efforts are directed toward adapting in vitro
hematopoietic differentiation methods developed for murine ES cells to human lines,
identifying the key interspecies differences in biological properties of ES cells and
overcoming current obstacles to generating ES cell-derived hematopoietic stem cells that
are competent to repopulate adult hosts. The ultimate medical goal is to create patient-
specific and generic ES cell lines that can be expanded in vitro, genetically altered and
differentiated into cell types that can be used to treat hematopoietic diseases.
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Olsen et al. Producing blood lineages from ES cells 3
Introduction
Embryonic stem (ES) cells, first isolated in 1981, are primary cell lines originally derived
from the inner cell mass (ICM) of pre- or peri-implantation mouse blastocysts1,2. ES cells
are distinguished by their capacity to self-renew in vitro and differentiate into tissues
derived from all three embryonic germ cell layers. Following microinjection into host
blastocysts, donor ES cells contribute to all tissues of chimeric mice, including the germ
line3. This property, combined with the ability to easily manipulate the ES cell genome
via homologous recombination, provides the tools to generate new animal strains in
which genes of interest are knocked out or altered. In addition, ES cells can be induced
to form various differentiated cell types in vitro. Here we review the production of blood
lineages from ES cells.
The broad developmental capacity of ES cells and the relative ease by which resident
genes can be manipulated have been valuable for understanding the basic biology of
tissue development and offer tremendous potential for cell replacement therapies in
degenerative disorders, cancer and genetic diseases. The promise of clinical applications
for ES cells was stimulated recently by the development of human lines from blastocysts,
patients and normal adults4-6. Studies of murine hematopoiesis provide a paradigm for
utilizing ES cells as a model system to examine normal developmental processes and as a
source for tissue production. This work provides a launching point for efforts to
differentiate human ES cells into transplantable hematopoietic lineages that can be
employed therapeutically.
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Olsen et al. Producing blood lineages from ES cells 4
Culture of ES cells
The first requirement for successful propagation of ES cells is to maintain them in an
undifferentiated, pluripotent state by adherence to specific culture conditions.
Subsequently, differentiation into desired lineages can be initiated in a controlled and
synchronous fashion. ES cells are held in an undifferentiated state by numerous cell
intrinsic and environmental signaling pathways7,8.
In practice, murine ES cells are maintained in vitro by cocultivation on a stromal layer,
typically murine embryonic feeder (MEF) cells, and addition of leukemia inhibitory
factor (LIF), a cytokine that activates signal transduction through a cognate receptor
signaling complex9. Recent studies demonstrate that a combination of LIF and bone
morphogenic protein 4 (BMP4) can bypass serum and feeder requirements to allow self-
renewal of murine ES cells in defined medium10.
Human ES cells can be maintained on stromal cells in the presence of basic fibroblast
growth factor (b-FGF)4. In contrast to its effects on murine ES cells, LIF does not
promote self-renewal of human ES cells11,12. Human ES cells are reported to have a
particular propensity for spontaneous differentiation in culture, perhaps due to species-
specific requirements for self-renewal that are incompletely understood13-15. In this
regard, further studies to optimize the propagation of human ES cells and eliminate their
requirements for animal feeder cells are important for future clinical applications. For
example, human ES cells can be maintained on human stroma16,17, and numerous
signaling pathways and transcription factors that participate in ES cell self-renewal have
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Olsen et al. Producing blood lineages from ES cells 5
been exploited for developing feeder free culture systems18-21. This includes the use of
basic fibroblast growth factor (b-FGF)18,22 and Wnt signaling agonists23,24. The nuclear
proteins Sox2, Oct3/4 and Nanog are critical for both human and murine ES cell
maintenance, and manipulation of their expression appears to bypass some cytokine and
stromal requirements for self-renewal8,25-27. It appears that these three transcription
factors are central components of a concerted autoregulatory network that promotes ES
cell self-renewal and pluripotency by activating and repressing numerous target genes,
predominantly those encoding other nuclear proteins28.
The expansion of ES cells and maintenance of their undifferentiated state in culture are
monitored by visual inspection, testing for developmental potential under various
differentiation conditions and gene expression analysis. In reference to the latter point,
an enormous amount of data on gene expression profiling of human and mouse ES cell
lines have been generated22,29-37. Distilling this information to produce a defined
molecular signature for ES cells and to identify all of the genes important for maintaining
their self-renewal and pluripotency has not yet been achieved, although as mentioned
above, Sox2, Nanog and Oct3/4 appear to be central to this process.
Creating blood lineages from ES cells
Major technological advances in deriving mature tissues from ES cells were established
through studies of the hematopoietic system using murine cells (for practical reviews, see
references 38-40). The first step in differentiating ES cells in vitro is to remove them from
the feeder cells and cytokines that maintain their pluripotency. Subsequently, if serum is
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present, a substantial fraction of ES cells differentiate into mesoderm, the germ cell layer
from which blood is derived. Under serum free conditions, murine ES cells can be
induced to form mesoderm and hematopoietic progenitors by added cytokines, most
importantly, BMP4 and vascular endothelial growth factor (VEGF)41,42. As mentioned
above, in other culture contexts, BMP4 participates in murine ES cell self-renewal10,43.
BMP-4 also stimulates mesoderm formation by human ES cells, although these
experiments were performed with serum present44. Mesoderm formation, which
represents an early step in transforming ES cells into blood, is regulated by numerous
positive and negative regulatory factors45,46. An ultimate goal is to develop defined
culture conditions that optimize mesoderm formation from ES cells; these conditions may
differ somewhat between human and mouse systems.
Two different experimental systems are used for the majority of experiments to generate
blood precursors from ES cells: formation of embryoid bodies (EBs)47,48 and induction of
hematopoietic differentiation on stromal cells49. Upon removal from stroma and
withdrawal of LIF, suspension cultures of ES cells form EBs, aggregates of differentiated
cells including mesodermal derivatives capable of hematopoietic differentiation. In 1985,
Doetschman et al noted that EBs in liquid culture form cystic structures that contain
blood islands analogous to those found in the embryonic yolk sac48. Subsequent studies
established that erythroid and myeloid lineages develop within EBs50,51. The emergence
of hematopoietic progenitors in this culture system can be tracked by the appearance of
lineage specific markers through gene expression analysis and
immunohistochemistry47,52-54. In addition, disruption of EBs by trypsin or collagenase
produces single cell suspensions from which hematopoietic progenitors can be analyzed
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and enumerated by flow cytometry or methylcellulose culture assays. In this fashion, the
effects of specific manipulations such as drugs, cytokines or gene targeting can be
analyzed. Cells within developing EBs synthesize various cytokines and consequently,
the initial stages of hematopoiesis occur when serum is the only source of added growth
factors39,47. However, addition of specific cytokine combinations optimizes the
development of specific mature lineages (see “In vitro production of specific
hematopoietic lineages from ES cells”).
Differentiation of ES cells on stromal lines provides a second means for inducing
hematopoiesis. The most commonly used system is OP9, a line established from calvaria
of newborn op/op mice, which lack functional macrophage colony-stimulating factor (M-
CSF)49,55. OP9 cells enhance hematopoietic development by providing a supportive
microenvironment for differentiation. The absence of M-CSF prevents the overgrowth of
monocyte-macrophage cells, which tend to outgrow other lineages in culture systems
using wild-type stroma. ES cells plated onto OP9 stroma form mesodermal colonies after
approximately 5 days, and cells within these colonies differentiate into multipotential
hematopoietic progenitors after an additional 3 days. Similar to what is observed in EBs,
ES cell-derived erythroid, myeloid and B lineage cells are generated on OP9 stroma
without exogenous growth factors, although cytokine supplementation influences lineage
output (see “In vitro production of specific hematopoietic lineages from ES cells”). The
EB and OP9 systems exhibit similar kinetics and efficiencies for many aspects of
hematopoietic development, although each approach may offer unique advantages56. For
example, empiric studies indicate that one specific ES cell differentiation system (EB or
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Olsen et al. Producing blood lineages from ES cells 8
OP9) may optimize the production of different hematopoietic lineages, although this has
not been examined rigorously.
Hematopoietic progenitor and stem cells develop optimally in vivo through cellular
interactions and paracrine effects provided by stromal niches57,58. Recapitulating these
microenvironments in vitro could enhance cell production. Toward this goal, three
dimensional culture systems incorporating extracellular matrix (ECM) proteins have been
used to provide a scaffold for EB development59-61. It is likely that refining this approach
to more closely mimic the in vivo niches that support cell specification and
organogenesis will further optimize tissue production from ES cells. For example,
mKirre is a type Ia membrane protein that is expressed by OP9 cells and contributes to
their ability to support hematopoiesis62. Incorporation of recombinant mKirre into
artificial matrix systems could, therefore, enhance hematopoietic culture of ES cells
under more defined conditions.
Examining early hematopoietic ontogeny through studies of ES cells
During embryogenesis, the hematopoietic system is established spatiotemporally through
waves of distinct progenitors arising in different tissues63-66. The first recognizable blood
cells in the embryo are yolk sac-derived primitive erythrocytes that differ from adult-type
definitive erythrocytes in many respects including gene expression, morphology and
cytokine requirements. Hematopoietic stem cells (HSCs) capable of reconstituting adult
hosts are found initially in the murine yolk sac and in an intraembryonic region termed
the aorta-gonad-mesonephros (AGM)67,68. Around midgestation, the major site of HSC
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activity and production of mature blood cells shifts to the fetal liver where definitive
erythrocytes and other adult-type lineages are produced69. Numerous studies in avian and
murine species suggested that the earliest embryonic hematopoietic progenitors and
endothelial cells derive from a common progenitor termed the hemangioblast70-72. As
discussed below, identification and analysis of this previously elusive progenitor was
greatly facilitated through the use of ES cells.
Remarkably, hematopoiesis in cultured ES cells largely recapitulates embryonic events.
For example, distinctly timed waves of primitive and definitive erythropoiesis develop in
vitro from ES cells in a fashion that parallels the onset of these developmental programs
in early embryos47. Moreover, Keller and colleagues discovered a transient progenitor in
early EBs, termed blast-colony forming cell (BL-CFC), which gives rise to primitive
erythrocytes, definitive lineages and endothelial cells73,74. Nishikawa et al extended these
studies, using cell surface markers to identify a developmental hierarchy in ES cell
differentiation in which hematopoietic cells arise from a precursor expressing the
endothelial markers Flk-1 and VE cadherin. They also identified single progenitor cells
capable of hematopoietic and endothelial differentiation75. Together, these studies helped
to establish lineage relationships between the first murine hematopoietic precursors and
verify the existence of the hemangioblast (Figure 1). These and subsequent studies are
beginning to define the characteristics of hemangioblasts, including cytokine and
transcription factor requirements. For example, BL-CFCs express the cytokine receptor
Flk-1 and are dependent on its ligand, vascular endothelial growth factor (VEGF). Gene
targeting studies have identified numerous transcription factors and signaling molecules
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important for the generation or maintenance of murine ES-cell derived hemangioblasts 76-
81.
Recently, Huber et al identified hemangioblasts from the primitive streak of the early
mouse embryo82. Importantly, these progenitors have similar properties to ES cell
derived BL-CFCs but are an extremely rare and transient population in vivo. Most
embryos examined contained less than 10 hemangioblasts that could be identified by
current culture conditions. This study further illustrates the parallels between
developmental hematopoiesis in early embryos and ES cell differentiation cultures,
validating the biological relevance of the latter. Using ES cells, hemangioblasts can now
be generated and purified on a scale much greater than what could be achieved from
embryos. This, in turn, allows for further studies into the basic biology and therapeutic
potential of this progenitor.
The holy grail: hematopoietic stem cells from ES cells
By definition, transplantation of a single HSC into a suitable adult host can repopulate the
entire hematopoietic system for an extended period of time. While cultured ES cells give
rise to multipotent hematopoietic progenitors with extensive proliferative capacity, these
repopulate animals only weakly and inconsistently in most reported studies83,84. Burt et al
recently reported more robust in vivo reconstitution from purified EB-derived CD45+
cKit+ progenitors85. It is now important to further characterize these progenitors with
HSC activity, refine the conditions for their derivation and reproduce these encouraging
findings in other laboratories.
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Most ES cell-derived blood progenitors studied may exhibit relatively limited HSC
activity because they approximate an immature embryonic lineage that cannot effectively
survive and proliferate in adult hematopoietic niches86,87. Accordingly, “pre-HSCs” are
found in the yolk sac and para aortic splanchnopleura region of early embryos88-92.
Presumably, during fetal maturation, pre-HSCs are somehow made competent to
repopulate adult hosts, either though interactions with specific embryonic/fetal
microenvironments, execution of latent cell-autonomous genetic programs, or both
(Figure 1).
Recent work suggests that pre-HSCs from ES cells and embryos can be coaxed to
become adult repopulating cells through genetic manipulation93,94. The homeobox (Hox)
family of transcription factors participates in normal hematopoiesis and
leukemogenesis95-100. Work by Humphries’ group showed that ectopic expression of
HoxB4 can enhance the self-renewal capacity of hematopoietic progenitors and HSCs
from mice and ES cells101-105. Daley and colleagues extended these findings by showing
that enforced expression of HoxB4 in immature murine yolk sac or ES cell-derived
hematopoietic progenitors rendered them competent for long-term multilineage
engraftment of irradiated adult hosts106. In these experiments, a doxycycline-inducible
HoxB4 transgene was introduced into ES cells and expressed during a narrow window of
hematopoietic differentiation. Hence, ectopic HoxB4 can facilitate the maturation of pre-
HSCs but is not required subsequently. This result leads to several interesting questions
and areas for further study. First, for unknown reasons, lymphoid repopulation appeared
to be less robust than myeloid repopulation in the HoxB4-transduced cells, a finding
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warranting further examination106,107. Second, 2 million cells were used to reconstitute
mice in these studies. It is now important to fractionate this mixture, identify the
repopulating cells and demonstrate that a single cell is capable of generating all
hematopoietic lineages. Third, basic mechanistic studies are essential to understand how
HoxB4 might educate pre-HSCs. For example, structure-function studies identified a
HoxB4 mutant with more potent regenerating activity108. In addition, it is important to
define the HoxB4 transcriptional program associated with pre-HSC maturation109. It is
possible that this line of research will identify functionally important cell signaling
interactions that can be modulated by drugs.
Additional strategies to generate HSCs from ES cells will be facilitated by ongoing and
future studies to better understand how pre-HSCs are conditioned in the embryo. For
example, stromal interactions almost certainly contribute to HSC development and
maturation as hematopoietic populations shift during embryogenesis110. In support of
this, multilineage progenitors and HSCs develop autonomously in ex vivo organ cultures
of the paraaortic splanchnopleura and AGM regions89,91,92,111. More recent work by
several groups suggests that HSC activity is induced and supported by stromal lines from
various embryonic sources112-114. It will be interesting to determine whether such stromal
lines can support the maturation of HSCs from ES cell differentiation cultures. In this
case, the next step will be to identify the specific molecules that influence HSC
development by either direct stromal interactions or paracrine effects115. Ultimately, the
goal is to reconstruct these inductive events using recombinant molecules.
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In vitro production of specific hematopoietic lineages from ES cells
Early studies showed that mixtures of different hematopoietic cell types develop in ES
cell differentiation cultures47-50,52,55,116. Subsequently, methods were refined to generate
relatively pure populations of specific lineages. Important variables that affect lineage
output include duration and mode of differentiation and supplemental cytokines or
chemicals. Conditions for generating erythroid cells, megakaryocytes, mast cells,
granulocytes, eosinophils, osteoclasts and lymphoid cells have been established (Table
1). These methods are useful for studying the development and physiology of specific
mature blood lineages generated from both wild type and gene targeted ES cells.
Analysis of hematopoiesis from genetically altered ES cells is particularly useful when
the mutation produces a complex phenotype with early embryonic lethality that interferes
with examination of definitive hematopoiesis. This application has been utilized
extensively; a few illustrative examples are discussed below. The methods summarized
in Table 1 were established using murine ES cells, but these are now being adapted for
use with human ES cells.
Erythroid cells.
When 7-8 day old EBs are disaggregated and plated into methylcellulose cultures with
erythropoietin (Epo) and Kit ligand (KL), the majority of resultant colonies are
erythroid47. We used this strategy to show that ES cells with a disrupted Gata1 gene,
which encodes an essential hematopoietic transcription factor, produce normal appearing
proerythroblasts that subsequently undergo maturation arrest and apoptosis117,118. In
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these experiments, use of ES cells allowed us to easily generate a synchronous cohort of
erythroid precursors to pinpoint the developmental stage at which GATA-1 function
becomes essential. We used the same system to generate an immortalized GATA-1–
proerythroblast line that undergoes GATA-1-dependent terminal erythroid maturation119.
This line, termed G1E, for GATA-1– Erythroid, has been a useful tool for various
applications including erythroid gene discovery and examining mechanistic aspects of
GATA-1 function in erythroid development120-128. Zheng et al generated ES cells in
which the endogenous Gata1 gene was ablated and a tetracycline regulated Gata1 cDNA
expression cassette was inserted, permitting conditional rescue of the gene defect129.
Using this system the authors identified unique functions for GATA-1 at various stages
of erythroid development. Together, these studies illustrate how gene functions can be
studied through genetic manipulation and in vitro differentiation of ES cells.
Methods to derive definitive erythrocytes from ES cells were advanced by Beug’s group,
who developed culture conditions in which EB-derived immature erythroid progenitors
can be expanded in a relatively pure state roughly 107-fold over the course of several
months130. This approach relies on prior findings that dexamethasone promotes erythroid
progenitor self-renewal131-133. When dexamethasone is removed and the cytokine
mixture is adjusted, terminal maturation ensues. Carotta et al used this approach to
demonstrate an unrecognized function for the cytokine receptor Flk-1 in erythroid
development130. This role was not appreciated from in vivo studies, as Flk1–/– embryos
die early with complex, cell non-autonomous defects in hematopoiesis134.
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Megakaryocytes.
Culturing ES cells on OP9 stroma with thrombopoietin (Tpo) generates populations
enriched for megakaryocytes with characteristic functional features including proplatelet
production and fibrinogen binding after exposure to platelet agonists135-139. Eto et al used
this system to show that CalDAG-GEF1, a guanine nucleotide exchange factor that is
deficient in megakaryocytes lacking the transcription factor NFE2, promotes integrin-
mediated megakaryocyte signaling138. Fujimoto et al reported similar methods to derive
functional megakaryocytes and platelets from ES cells135. Our laboratory adapted these
culture methods to show that Gata1 gene-disrupted ES cells produce a unique self-
renewing bipotential progenitor with the capacity to undergo GATA-1-dependent
megakaryocyte and erythroid maturation140. A defined role at this stage of hematopoietic
development is relevant to recent findings that mutations in the GATA1 gene contribute to
Down’s Syndrome associated acute megakaryoblastic leukemia (AMKL), which exhibits
both erythroid and megakaryocytic features, suggesting that malignant transformation
may occur within a megakaryocyte-erythroid progenitor (MEP)141.
Granulocytes.
Lieber et al generated > 75% pure neutrophil cultures from ES cells142,143. This approach
involves inducing the maturation of EB-derived hematopoietic progenitors on OP9 cells
with added cytokines. The resultant neutrophils exhibit signature markers, chemotactic
response and superoxide production. The authors used this system to demonstrate
migration defects in neutrophils lacking mitogen-activated protein (MAP)/extracellular
signal-related kinase (MEKK1) through targeted mutagenesis.
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Mast cells.
Culturing EB-derived hematopoietic progenitors in suspension with the cytokines IL-3
and KL generates pure populations of mast cells that can be expanded significantly in
vitro47,50,144,145. Upon transplantation into mast cell-deficient mice (KitW/KitW-v), ES cell-
derived mast cells survive, proliferate, mature and mediate IgE-dependent immune
responses. This provides a powerful strategy to analyze the effects of genetic and
chemical alterations on mast cell development and function in vitro and in vivo. For
example, IgE-induced crosslinking of its high affinity receptor triggers mast cell
activation with release of inflammatory mediators, contributing to the pathophysiology of
allergic response. Several groups are dissecting this pathway by creating ES cells with
mutations in candidate genes suspected to participate in IgE receptor signaling and using
these lines to generate mast cells for functional studies144,146.
Eosinophils.
Hamaguchi-Tsuru et al generated eosinophils (~50% enriched) by differentiating ES cells
on OP9 stroma with IL-5 and either IL-3 or granulocyte-macrophage colony stimulating
factor (GM-CSF)147. The ability to generate eosinophil-enriched populations and study
their development and function at defined stages has implications for human disorders
such as allergies and asthma. For example, this approach may be a useful basis for
screens to identify new anti-inflammatory agents that inhibit eosinophil development or
function.
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T and B Lymphocytes.
Nakano et al cultured ES cells on OP9 stroma with IL-7 to generate early B cells. Most
of these were IgM– and completed DJ gene rearrangement, but a small proportion
differentiated into IgM+ cells that expressed the complete µ chain mRNA49. B cell
maturation from ES cells was developed further by stimulating differentiation in the
presence of IL-7 and Flt3 ligand148,149. This provides an efficient method to produce
relatively large amounts of mature Ig-secreting B cells, which, in principle, could be
adapted to engineer and produce specific murine or human antibodies.
It has been particularly challenging to differentiate ES cells into T lymphocytes, probably
because their development requires complex thymic stromal interactions that are not
easily recapitulated in vitro. Zuniga-Pflucker’s group differentiated ES cells on OP9
stroma and then reseeded Flk1+CD45- cells into fetal thymic organ cultures (FTOCs).
Although the fraction of T cells was low, normal CD4 and CD8 thymic subsets were
generated150. Recognizing that Notch signaling is uniquely important for the production
of T cells, the same group engineered OP9 cells to express the delta-like 1 (DL-1) Notch
ligand151. Differentiation of ES cells on this modified stroma produced more extensive
and complete T cell maturation including γδ and αβ T cell receptor (TCR)-bearing cells,
as well as mature CD8+ cells that express the TCR and produce interferon (IFN)-γ in
response to stimulation. However, further maturation in FTOCs is required to render
these ES cell-derived late stage T cell progenitors competent to functionally reconstitute
immunodeficient NOD/SCID mice. This system represents an important technical
advance in the manipulation of ES cells and should be useful for examining the cell-
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autonomous and environmental factors that promote T lymphocyte differentiation and
maturation. The finding that ES cell-derived T lymphocyte progenitors can become self-
tolerant and functional after transplantation into mice has potential clinical utility if these
experimental methods can be adapted to humans.
Macrophages.
Macrophages can be produced from ES cells using either EB or OP9 culture systems. ES
cell-derived macrophages express Mac1 and lysozyme RNA, and stain with F4/80, a
specific macrophage antibody49,50. In addition, they exhibit numerous properties of
arterial lesion macrophages, and therefore, offer research opportunities in the field of
atherosclerosis152,153. In vitro differentiation of ES cells has been used to study the
effects of specific gene knockouts on macrophage function. For example, Guillemot et al
used this approach to demonstrate that GDID4, a guanine diphosphate dissociation
inhibitor that regulates the cycling of Rho family GTP binding proteins, is required for
normal superoxide production by macrophages154.
Dendritic Cells.
DCs are potent antigen presenting cells that can either promote immune response or self-
tolerance. ES cells offer a potential source of normal and genetically manipulated DCs
that could be used for a variety of clinical applications155-157. ES cell-derived DCs
expand in the presence of IL-3 and GM-CSF and are induced to mature by tumor necrosis
factor (TNF) α and IL-4 plus lipopolysaccharide or anti-CD40. Similar to their bone
marrow-derived counterparts, ES cell-derived DCs express characteristic markers,
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process foreign antigen and stimulate primary T-cell responses in the mixed leukocyte
reaction. In one interesting application, Senju et al randomly introduced a reporter gene
flanked by loxP recombination sequences into ES cells and identified a clone in which
reporter expression was particularly high in DC progeny generated by in vitro
differentiation155. Then, they introduced ovalbumin cDNA into this active locus by CRE-
mediated recombination to generate a DC line that efficiently presented antigen to prime
ovalbumin-specific cytotoxic T-lymphocytes in vivo. This method has potential clinical
applications if it can be widely applied to other antigens.
Natural Killer (NK) Lymphocytes.
NK cells, an important arm of the innate immune system, destroy cells that fail to present
the correct MHC class I receptor, including some virally infected and malignant clones.
Lian et al. induced ES cells to differentiate into functional NK cells that killed certain
tumor lines and MHC class I-deficient lymphoblasts158. These findings provide a
mechanism to further examine NK cell biology and possibly to manufacture these cells
for antiviral and anticancer therapies.
Osteoclasts.
Osteoclasts, which are derived from HSCs, are involved in bone resorption and
remodeling. Therefore, understanding the biology of this lineage has important
implications for various bone disorders. Methods to derive osteoclasts from ES cells in
vitro have been developed and refined, mainly by Hayashi’s group159-163. Cytokines that
promote osteoclast differentiation and maturation from ES cells include M-CSF and
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Receptor Activator of Nuclear factor Kappa B Ligand (RANKL). Osteoclast production
is also augmented by ascorbic acid, dexamethasone and 1α, 25-dihydroxyvitamin D3.
Analysis of gene targeted ES cells utilizing these culture methods identified several
signaling pathways that participate in osteoclastogenesis. For example, ES cells lacking
transcription factor NFATc1 are deficient in RANKL-induced osteoclastogenesis,
suggesting that NFATc1 is downstream of RANKL signaling164. In a different study,
Tsuneto et al showed that enforced expression of the myeloid transcription factor PU.1
stimulates osteoclast production in ES cells lacking SCL (Tal-1), a nuclear protein
essential for early stages of hematopoiesis165. This indicates that PU.1 is not only
important for osteoclast development, but it may also bypass some early hematopoietic
requirements for SCL.
Recent progress with human ES cells
In 1998, Thomson et al derived human ES cells from embryos4. More recently, human
and murine ES cells were developed by somatic cell nuclear transfer (SCNT), a method
in which the nucleus of an adult somatic cell (commonly from the skin for humans) is
injected into a normal enucleated oocyte to generate a blastocyst that can be used to
derive ES cells5,166. This technology opens up new opportunities to generate patient-
specific ES cell lines.
Blood from hES cells.
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Olsen et al. Producing blood lineages from ES cells 21
Methods previously established for examining hematopoiesis in murine ES cells are
applicable to human studies167,168. Human ES cells differentiate to hematopoietic
lineages via EBs or culture on stromal cell lines169. It appears that human ES cell
differentiation cultures accurately mirror early human embryonic hematopoiesis, similar
to what is observed in murine systems. For example, distinct sequential waves of
primitive and definitive erythropoiesis occur in human EBs. Moreover, cells within
human EBs give rise to colonies that contain mesoderm, hematopoietic and endothelial
cells arranged into structures reminiscent of yolk sac blood islands. The nature of the
progenitor which gives rise to these colonies remains to be determined170. It is possible
that hemangioblasts originate from within these colonies. Along these lines, Wang et al
showed that CD45–, PECAM-1+, Flk-1+, VE-cadherin+ (CD45negPFV) cells within human
EBs give rise to hematopoietic and endothelial lineages171. This hints at the existence of
human hemangioblasts. However, in clonality studies, the viability and capacity for
hemato-endothelial differentiation of CD45negPFV cells were low. Hence, it will be
important to further fractionate these cells and optimize conditions for their culture and
differentiation.
While extensive studies of murine ES cells provide a broad basis for analyzing
hematopoiesis in human lines, it is important to point out that there are significant
interspecies differences that could influence culture protocols13,172. For example,
differences in self-renewal pathways are discussed earlier in the section on ES cell
culture. There are also differences in colony morphology, cloning efficiency, surface
markers and patterns of gene expression. In contrast to murine ES cells, serum free
methods to generate mesoderm and hematopoietic lineages from human ES cells are not
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Olsen et al. Producing blood lineages from ES cells 22
yet reported. It is likely that protocols to specifically optimize hematopoiesis from
human ES cells will be further developed and refined over the next few years.
Generating human HSCs.
As in murine systems, generating a human ES cell-derived HSC capable of long-term
multi-lineage engraftment is a vexing problem. It has been possible to produce human
ES cell-derived progenitors with some features of adult-type HSCs including CD34
expression, multi-lineage hematopoietic potential in clonal assays, the ability to efflux
dyes such as Hoechst 3334 and Rhodamine 123, and high aldehyde dehydrogenase
(ALDH) activity167. However, these cells exhibit limited repopulating activity after
transplantation into immunodeficient mice. Wang et al demonstrated that a population of
human ES cell-derived progenitors incapable of repopulating the marrow after
intravenous injection exhibits limited regional repopulation after intrafemoral
injections173. At minimum, this suggests defects in migratory capacity, homing or niche
interactions, similar to what is observed in pre-HSCs of the early mouse embryo174.
Consistent with this interpretation, multipotential progenitors derived from human ES
cells exhibit a distinct gene expression signature that resembles primitive embryonic
progenitors relative to adult HSCs found in bone marrow or cord blood173,175.
In contrast to murine ES cells, ectopic expression of HoxB4 does not promote the
expansion of bone fide HSCs from human ES cells173. This important finding could be
due to technical issues or could reflect intrinsic differences in the biologies of human and
mouse HSC development. In case of the latter, it is possible that manipulated expression
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Olsen et al. Producing blood lineages from ES cells 23
of different proteins in human ES cell cultures will stimulate HSC production. Candidate
molecules include Hox paralogues and numerous other nuclear regulators176-178. In
considering this experimental approach for human HSC production, it will be important
to utilize strategies that avoid stable genetic alterations by transgenes or viral vectors,
which pose a risk for malignant transformation. One innovative approach fused the cell
penetrating peptide, TAT, to HoxB4 protein179. This fusion protein was taken up into
murine bone marrow-derived HSCs and stimulated their expansion ex vivo. This
method, which allows cells to be manipulated by transcription factors without altering the
genome, could be applied to ES cells.
The future
The ability to create hematopoietic cells from human ES cells opens up profound
therapeutic possibilities (Figure 1). Currently, bone marrow transplantation (BMT) can
cure many hematopoietic diseases, but HSC availability is often limiting and many
patients lack matched tissue donors. Human ES cells offer a potentially renewable source
of tissue-compatible HSCs for BMT. In addition, compared to somatic HSCs used
currently for BMT, human ES cells are potentially more amenable to genetic
manipulation for rescuing specific defects or for altering antigenicity to augment
transplantation. Eventually, it should be possible to use SCNT to generate individualized
ES cell lines from patients with various inherited or acquired disorders, treat the defect by
genetic manipulation using homologous recombination, lentiviral or siRNA approaches,
and then generate transplantable HSCs by in vitro differentiation. However, numerous
unsolved questions related to the biology of ES cells derived from adult somatic tissues
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Olsen et al. Producing blood lineages from ES cells 24
must be further investigated. These include the nature of epigenetic influences such as
X-chromosome inactivation and genomic imprinting, telomere status and genomic
stability5.
It is also possible that replacement therapies using mature hematopoietic lineages derived
from human ES cells could be of clinical utility. For example, in vitro manipulated
human DCs are being explored as a mechanism to tolerize the immune system against
autoimmune disease or to stimulate anti-tumor immune responses. However, the number
of DCs that can be isolated from patients using current technologies is limiting180. Using
human ES cells as a source for generating DCs could circumvent this problem.
Additionally, ES cells appear to be a particularly robust source for expanding erythroid
progenitors compared to fetal liver and adult tissues130. By merging several established
technologies, it may be possible to produce large quantities of mature red blood cells
from human ES cells181. In principle, this represents a limitless source for erythrocyte
transfusions and for laboratory reagents used in blood bank testing. This may be
especially useful for rare red blood cell types with limited donor availability. In addition,
it may be possible through genetic manipulation to design improved universal donor
erythrocytes with a limited antigenic repertoire. Similarly, functional granulocytes and
megakaryocytes or their committed progenitors could be used to treat neutropenia and
thrombocytopenia, respectively. One potentially interesting application for the latter
would be to manipulate hemostasis by engineering hES cells to produce modified
platelets that deliver ectopically expressed pro or anticoagulant factors directly to the site
of blood clots182,183.
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Olsen et al. Producing blood lineages from ES cells 25
Several additional problems must be further addressed to facilitate the transit of ES cells
from bench to bedside. Like all organs, hematopoietic tissues develop in the context of a
complex and poorly understood three-dimensional microenvironment comprised of
paracrine factors, stromal contacts and physical forces that vary during development.
Defining these microenvironments and recapitulating them in vitro may provide a key to
more efficiently recapitulating hematopoiesis, particularly HSC generation. Along the
same lines, continued refinement of serum free and stroma free culture systems will
minimize pathogen contamination and provide tighter control of cellular differentiation.
It is also critical to develop new approaches for testing ES cell-based therapies. For
example, transplantation into NOD/SCIDS immunodeficient mice may not be the most
sensitive or effective method to evaluate ES-cell derived human hematopoietic cells. To
gain more information on the safety and utility of ES cell-based therapies, it is important
to use model systems that more closely approximate humans. Along these lines, several
groups are examining hematopoietic development from non-human primate ES cells184-
188. In general, these lines exhibit hematopoiesis similar to what is observed from human
ES cells. While these studies are relatively preliminary, continued investigation of non-
human primate ES cells will provide improved preclinical models for ES cell-derived
hematopoietic transplantation therapies.
In addition to therapeutic applications, new technologies for making blood from human
ES cells will shed light on the biology of hematopoietic ontogeny, which is difficult to
study in the human embryo for ethical and practical reasons. Due to inherent
developmental differences, not all conclusions drawn from murine ES cells will be
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Olsen et al. Producing blood lineages from ES cells 26
applicable to the human embryo. Comparative studies to examine the onset of
hematopoiesis in human and murine ES cell differentiation cultures should elucidate
fundamental similarities and differences between the two species.
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Olsen et al. Producing blood lineages from ES cells 27
Acknowledgements
We thank Gerd Blobel, Gordon Keller, Marcela Maus and Toru Nakano for comments
and suggestions on the manuscript.
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Olsen et al. Producing blood lineages from ES cells 28
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Table 1: Generation of Specific Hematopoietic Lineages from Murine ES cells.Hematopoietic
LineageMethod
ofCulture
Cytokines References Other
Megakaryocytes and platelets
OP9, EBs Major: TPO
Other: IL-6, IL-11
39,135-139 Megarkaryocytes show agonist dependent fibrinogen binding.Platelets exhibit agonist dependent fibrinogen binding, aggregation and spreading.
Erythroid OP9, EBs Major: EPO, KL Other: Insulin, IGF-1
39,47,49,116,130
Dexamethasone used for erythroid progenitor expansion..
Primitive progenitors (EryP) are KL-independent. Definitive progenitors (EryD) require EPO/KL.
Iron saturated transferrin added to improve hemoglobin synthesis during terminal maturation.
Mast cells EBs IL-3, KL 145,146 EB-derived mast cells proliferate extensively in culture and reconstitute mast cell-deficient KitW/KitW-v mice after adoptive transfer.
Macrophages OP9, EBs IL-1, IL-3, M-CSF, GM-CSF 49,152-154 Macrophage overgrowth tends to inhibit the production of other lineages in ES cell differentiation cultures
T lymphocytes OP9OP9-DL1
Flt-3 ligand, IL-7 150,151 Full T lymphocyte development requires maturation in thymic stroma (FTOC or RTOCs).
T lymphocyte production is augmented by cultivation on OP9 cells ectopically expressing the notch ligand, DL-1.
B lymphocytes OP9 Flt-3 ligand, IL-7 49,148,149 Stimulation with LPS augments production of IgM+
secreting B-lymphocytes.
Eosinophils OP9 IL-5, IL-3, GM-CSF, eotaxin 147 Express characteristic granules and cell surface markers, produce oxidative response to Leishmania.
Neutrophils EBs, OP9 Progenitor expansion:OSM, bFGF, IL-6, IL-11, LIF, KL
Neutrophil differentiation:G-CSF, GM-CSF, IL-6
142,143 Functional neutrophils produced. This includes characteristic markers and morphology, superoxide production and chemotactic responses.
Dendritic Cells (DC)
EBs, OP9 Progenitor expansion:GM-CSF, IL-3 Differentiation/maturation:IL-4, TNFα, LPS, anti-CD40
155,157,189 DC production facilitated by plating EBs on tissue culture dishes.Exhibit characteristic surface phenotype, process protein antigens and stimulate T lymphocytes in the mixed lymphocye reaction (MLR).
Natural Killer (NK) cells
OP9 Flt-3 ligand, IL-15, IL-6, IL-7, KL
149,158 Express characteristic NK proteins, kill selected tumor lines and MHC I-deficient lymphoblasts.
Osteoclasts OP9 ST2
M-CSF, RANKL 159-163 Recognized by TRAP expression and bone resorbtion activity. Production augmented by vitamins C and D and dexamethasone.
Abbreviations: TPO, thrombopoietin; EPO, erythropoietin; OP9, M-CSF–/– stromal line; EB, embryoid bodies; IL-1, interleukin 1; IL-3, interleukin 3; IL-5, interleukin 5; IL-6, interleukin 6; IL-7, interleukin 7; IL-11, interleukin 11; G-CSF, granulocyte colony stimulating factor; M-CSF, macrophage colony stimulating factor; GM-CSF granulocyte/macrophage colony stimulating factor; IGF-1, insulin-like growth factor 1; KL, c-kit ligand; OSM, oncostatin M; bFGF, basic fibroblast growth factor; LIF, leukemia inhibitory factor; FTOC, fetal thymic organ cultures; RTOC, reaggregate thymic organ cultures; OP9-DL-1, Notch ligand Delta-like-1 expressing OP9 cells; VEGF, vascular endothelial growth factor; LPS, lipopolysaccharide; TNFα, tumor necrosis factor α; ST2, bone marrow-derived stromal line; TRAP, tartrate-resistant acid phosphatase.
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Olsen et al. Producing blood lineages from ES cells 42
Figure legend
Figure 1. In vitro hematopoiesis from embryonic stem (ES) cells. The solid arrows
indicate established methods to produce specific hematopoietic lineages. The dashed
arrows indicate potential future therapeutic applications to be derived from human ES
cells. HSC, hematopoitic stem cell.
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