Download creating hematopoietic lineages from embryonic

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Review in translational hematology
Designer blood: creating hematopoietic lineages from embryonic stem cells
Abby L. Olsen, David L. Stachura, and Mitchell J. Weiss
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 used murine ES cells for
basic research to elucidate fundamental
properties of blood-cell development and
establish methods to derive specific ma-
ture 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 biologic
properties of ES cells, and generating ES
cell–derived hematopoietic stem cells that
are competent to repopulate adult hosts.
The ultimate medical goal is to create patientspecific 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.
(Blood. 2006;107:1265-1275)
© 2006 by The American Society of Hematology
Introduction
Embryonic stem (ES) cells, first isolated in 1981, are primary cell lines
originally derived from the inner cell mass (ICM) of preimplantation or
peri-implantation mouse blastocysts.1,2 ES cells are distinguished by
their capacity to self-renew in vitro and differentiate into tissues derived
from all 3 embryonic germ-cell layers. Following microinjection into
host blastocysts, donor ES cells contribute to all tissues of chimeric
mice, including the germ line.3 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 tissue 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. Studies of murine hematopoiesis provide a paradigm for using 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.
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 pathways.4,5
From the Cell and Molecular Biology Graduate Program and the Combined
Degree Graduate Program, The University of Pennsylvania School of
Medicine; and The Children’s Hospital of Philadelphia, Division of Hematology;
Philadelphia, PA.
Submitted September 9, 2005; accepted October 9, 2005. Prepublished online
as Blood First Edition Paper, October 27, 2005; DOI 10.1182/blood-2005-093621.
BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
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 complex.6 Recent studies
demonstrate that a combination of LIF and bone morphogenic protein 4
(BMP4) can bypass serum and feeder requirements to allow selfrenewal of murine ES cells in defined medium.7,8
Human ES cells can be maintained on stromal cells in the
presence of basic fibroblast growth factor (b-FGF).9 In contrast to
its effects on murine ES cells, LIF does not promote self-renewal of
human ES cells.10,11 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 understood.12-14 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 stroma,15,16 and numerous signaling pathways and
transcription factors that participate in ES-cell self-renewal have
been exploited for developing feeder-free culture systems.17-20 This
includes the use of b-FGF17,21 and Wnt signaling agonists.22,23 The
nuclear proteins Sox2, Oct3/4, and Nanog are critical for both
human and murine ES-cell maintenance, and manipulation of their
expression can bypass some cytokine and stromal requirements for
self-renewal.5,24-26 It appears that these 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 proteins.27
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
Supported by National Institutes of Health (NIH) grants R01 DK064037
(M.J.W.) and T32-HL007971-04 (D.L.S.).
Reprints: Mitchell J. Weiss, Division of Hematology, 3615 Civic Center Blvd,
Abramson Research Center, Philadelphia, PA 19104; e-mail: weissmi@email.
chop.edu.
© 2006 by The American Society of Hematology
1265
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1266
OLSEN et al
enormous amount of data on gene-expression profiling of human
and mouse ES cell lines have been generated.21,28-36 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.
Creating blood lineages from ES cells
Major technologic advances in deriving mature tissues from ES
cells were established through studies of the hematopoietic system
using murine cells (for practical reviews see Kitajima et al,37
Kennedy and Keller,38 and Fraser et al39). 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 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).40,41 BMP4 also promotes mesoderm formation from
human ES cells, although these experiments were performed with
serum present.42 Mesoderm formation is regulated by numerous positive
and negative factors.43,44 An ultimate goal is to recapitulate these
influences in defined culture systems for human and murine ES cells.
Two different experimental systems are used for the majority of
experiments to generate blood precursors from ES cells: formation
of embryoid bodies (EBs)45,46 and induction of hematopoietic
differentiation on stromal cells.47 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
al46 noted that EBs in liquid culture form cystic structures that
contain blood islands analogous to those found in the embryonic
yolk sac. Subsequent studies established that erythroid and myeloid
lineages develop within EBs.48,49 The emergence of hematopoietic
progenitors in this culture system can be tracked by the appearance
of lineage-specific markers through gene expression analysis and
immunohistochemistry.45,50-52 In addition, disruption of EBs by
trypsin or collagenase produces single-cell suspensions from which
hematopoietic progenitors can be analyzed 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
factors.38,45 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 calvariae of newborn op/op
mice, which lack functional macrophage colony-stimulating factor
(M-CSF).47,53 OP9 cells enhance hematopoietic development by
providing a supportive microenvironment for differentiation. The
absence of M-CSF inhibits the survival 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
BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
on OP9 stroma without exogenous growth factors, although
cytokine supplementation influences lineage output. The EB and
OP9 systems exhibit similar kinetics and efficiencies for many
aspects of hematopoietic development, although each approach
may offer unique advantages.54
Hematopoietic progenitor and stem cells develop optimally in
vivo through cellular interactions and paracrine effects provided by
stromal niches.55,56 Recapitulating these microenvironments in
vitro could enhance cell production. Toward this goal, 3-dimensional culture systems incorporating extracellular matrix proteins
have been used to provide a scaffold for EB development.57-59 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 hematopoiesis.60 Incorporation of recombinant
mKirre into artificial matrix systems might, 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 tissues.61-64 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).65,66 Around
midgestation, the major site of HSC activity and production of
mature blood cells shifts to the fetal liver where definitive
erythrocytes and other adult-type lineages are produced.67
Numerous studies in avian and murine species suggest that the earliest
embryonic hematopoietic progenitors and endothelial cells derive
from a common progenitor termed the hemangioblast.68-70
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 embryos.45 Moreover, Keller and colleagues
(Kennedy et al71 and Choi et al72) discovered a transient progenitor
in early EBs, termed blast-colony–forming cell (BL-CFC), which
gives rise to primitive erythrocytes, definitive lineages, and endothelial cells. Nishikawa et al73 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 VEcadherin. They also identified single progenitor cells capable of
hematopoietic and endothelial differentiation. 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, VEGF. Gene-targeting studies have identified numerous
transcription factors and signaling molecules important for the
generation or maintenance of murine ES cell–derived
hemangioblasts.74-79
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BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
Recently, Huber et al80 identified hemangioblasts from the
primitive streak of the early mouse embryo. 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 fewer 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
biologic 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, they repopulate animals only weakly and inconsistently in
most reported studies.81,82 Burt et al83 recently reported more robust
in vivo reconstitution from purified EB-derived CD45⫹ c-Kit⫹ progenitors. 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.
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 niches.84,85 Accordingly, “preHSCs” are found in the yolk sac and the para-aortic splanchnopleura region of early embryos.86-90 Presumably, during fetal
maturation, pre-HSCs are somehow made competent to repopulate
Figure 1. In vitro hematopoiesis from 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 indicates hematopoietic
stem cell.
PRODUCING BLOOD LINEAGES FROM ES CELLS
1267
adult hosts, either through 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
manipulation.91,92 The homeobox (Hox) family of transcription
factors participates in normal hematopoiesis and leukemogenesis.93-98 Work by Humphries’ group (Antonchuk et al,99,100 Sauvageau et al,101 Buske et al,102 and Helgason et al103) showed that
ectopic expression of HoxB4 can enhance the self-renewal of
hematopoietic progenitors and HSCs from mice and ES cells.
Daley and colleagues (Kyba et al104) 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 hosts. 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 warranting
further examination.104,105 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, structurefunction studies identified a HoxB4 mutant with more potent
regenerating activity.106 In addition, it is important to define the
HoxB4 transcriptional program associated with pre-HSC maturation.107 It is possible that this line of research will identify important
cell signaling interactions that can be modulated by drugs.
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BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
OLSEN et al
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 embryogenesis.108 In support of this, multilineage progenitors and HSCs
develop autonomously in ex vivo organ cultures of the para-aortic
splanchnopleura and AGM regions.87,89,90,109 Additionally recent
work by several groups suggests that HSC activity is induced and
supported by stromal lines from various embryonic sources.110-112 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 effects.113 Ultimately, the goal is to
reconstruct these inductive events using recombinant molecules.
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
various hematopoietic cell types 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. This application has been used extensively.
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
In vitro production of specific hematopoietic
lineages from ES cells
Early studies showed that mixtures of different hematopoieticcell types develop in ES-cell differentiation cultures.45-48,50,53,114
When 7- to 8-day-old EBs are disaggregated and plated into
methylcellulose cultures with erythropoietin (Epo) and Kit ligand
(KL), the majority of resultant colonies are erythroid.45 We used
this strategy to show that ES cells with disrupted Gata1, which
encodes an essential hematopoietic transcription factor, produce
Table 1. Generation of specific hematopoietic lineages from murine ES cells
Hematopoietic lineage
Method of
culture
Megakaryocytes and platelets OP9, EBs
Cytokines
Major: Tpo
References
38, 115-119
Other: IL-6, IL-11
Other
Megakaryocytes show agonist-dependent fibrinogen
binding. Platelets exhibit agonist-dependent fibrinogen
binding, aggregation, and spreading.
Erythroid
OP9, EBs
Major: Epo, KL
38, 45, 47, 114, 120 EryPs are KL independent. EryDs require Epo/KL. Ironsaturated transferrin added to improve hemoglobin
Other: Insulin, IGF-1, IL-3
synthesis during terminal maturation. Dexamethasone
can be used for erythroid progenitor expansion.
Mast cells
EBs
IL-3, KL
121, 122
EB-derived mast cells proliferate extensively in culture
and reconstitute mast cell-deficient Kit W/Kit W-v mice
after adoptive transfer.
Macrophages
OP9, EBs
IL-1, IL-3, M-CSF, GM-CSF
T lymphocytes
OP9, OP9-DL1 Flt-3 ligand, IL-7
47, 123-125
Macrophage overgrowth tends to inhibit the production
126, 127
Full T-lymphocyte development requires maturation in
of other lineages in ES-cell differentiation cultures.
thymic stroma (FTOCs 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
47, 128, 129
Stimulation with LPS augments production of lgM⫹-
Eosinophils
OP9
IL-5, IL-3, GM-CSF, eotaxin
130
Express characteristic granules and cell-surface
Neutrophils
EBs, OP9
Progenitor expansion: OSM, b-FGF, IL-6, 131, 132
secreting B lymphocytes.
markers, produce oxidative response to Leishmania.
IL-11, LIF, KL
Functional neutrophils produced. This includes
characteristic markers and morphology, superoxide
Neutrophil differentiation: G-CSF,
production, and chemotactic responses.
GM-CSF, IL-6
DCs
EBs, OP9
Progenitor expansion: GM-CSF, IL-3
133-135
DC production facilitated by plating EBs on tissue culture
dishes. Exhibit characteristic surface phenotype,
Differentiation/maturation: IL-4, TNF-␣,
process protein antigens, and stimulate T lymphocytes
LPS, anti-CD40
in the MLR.
NK cells
OP9
Flt-3 ligand, IL-15, IL-6, IL-7, KL
129, 136
Express characteristic NK proteins; kill selected tumor
Osteoclasts
OP9, ST2
M-CSF, RANKL
137-141
Recognized by TRAP expression and bone resorbtion
lines and MHC 1-deficient lymphoblasts.
activity. Production augmented by vitamins C and D
and by dexamethasone.
M-CSF⫺/⫺
stromal line; EB, embryoid bodies; Tpo, thrombopoietin; IL-6, interleukin 6; Epo, erythropoietin; KL, c-kit
DCs indicates dendritic cells; NK, natural killer; OP9,
ligand; IGF-1, insulin-like growth factor 1; EryP, primitive erythroid progenitor; EryD, definitive erythroid progenitor; M-CSF, macrophage colony-stimulating factor; GM-CSF
granulocyte/macrophage colony-stimulating factor; FTOCs, fetal thymic organ cultures; RTOCs, reaggregate thymic organ cultures; DL-1, Delta-like-1; LPS, lipopolysaccharide; OSM, oncostatin M; b-FGF, basic fibroblast growth factor; LIF, leukemia inhibitory factor; G-CSF, granulocyte colony-stimulating factor; TNF-␣, tumor necrosis factor ␣;
MLR, mixed lymphocyte reaction; ST2, bone marrow-derived stromal line; and TRAP, tartrate-resistant acid phosphatase.
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BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
normal-appearing proerythroblasts that subsequently undergo maturation arrest and apoptosis.142,143 In 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 maturation.144
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 development.145-153 Zheng et al154 generated ES cells in
which the endogenous Gata1 gene was ablated and a tetracyclineregulated Gata1 cDNA expression cassette was inserted, permitting conditional rescue of the gene defect. 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 (Carotta et al120), 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 months. This approach relies on prior
findings that dexamethasone promotes erythroid progenitor selfrenewal.155-157 When dexamethasone is removed and the cytokine
mixture is adjusted, terminal maturation ensues. Carotta et al120
used this approach to demonstrate an unrecognized function for the
cytokine receptor Flk-1 in erythroid development. This role was
not appreciated from in vivo studies, as Flk1⫺/⫺ embryos die early
with complex, non–cell-autonomous defects in hematopoiesis.158
PRODUCING BLOOD LINEAGES FROM ES CELLS
1269
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 vitro.45,48,121,161 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 cross-linking 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 studies.122,161
Eosinophils
Hamaguchi-Tsuru et al130 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).
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.
T and B lymphocytes
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 agonists.115-119 Eto et
al118 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 signaling. Fujimoto et al115 reported similar methods to derive functional megakaryocytes and platelets from ES
cells. 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 maturation.159 A defined
role at this stage of hematopoietic development is relevant to recent
findings that mutations in the GATA1 gene contribute to Down
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).160
Granulocytes
Lieber et al131,132 generated greater than 75% pure neutrophil
cultures from ES cells. 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.
Nakano et al47 cultured ES cells on OP9 stroma with IL-7 to
generate early B cells. Most of these were IgM⫺ and completed
diversity-joining (DJ) gene rearrangement, but a small proportion
differentiated into IgM⫹ cells that expressed the complete ␮ chain
mRNA.47 B-cell maturation from ES cells was developed further
by stimulating differentiation in the presence of IL-7 and Flt3
ligand.128,129 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 (de Pooter et al126) 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
generated.126 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 ligand.127 Differentiation of ES cells on this modified stroma produced more extensive
and complete T-cell maturation including ␥␦ and ␣␤ T-cell receptor
(TCR)–bearing cells, as well as mature CD8⫹ cells that express the
TCR and produce interferon ␥ (IFN-␥) 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 nonobese diabetic–severe combined immunodeficiency (NOD/SCID) mice. This system represents an important
technical advance in the manipulation of ES cells and should be
useful for examining the cell-autonomous and environmental
factors that promote T-lymphocyte differentiation and maturation.
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OLSEN et al
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 macrophage-specific
antibody.47,48 In addition, they exhibit numerous properties of
arterial lesion macrophages and, therefore, offer research opportunities in the field of atherosclerosis.123,124 In vitro differentiation of
ES cells has been used to study the effects of specific gene
knockouts on macrophage function. For example, Guillemot et
al125 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 macrophages.
Dendritic cells
Dendritic cells (DCs) are potent antigen-presenting cells that can
promote either 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 applications.133-135 ES
cell–derived DCs expand in the presence of IL-3 and GM-CSF and
are induced to mature by tumor necrosis factor ␣ (TNF-␣) and IL-4
plus lipopolysaccharide or anti-CD40. Similar to their bone
marrow–derived counterparts, ES cell–derived DCs express characteristic markers, process foreign antigens, and stimulate primary
T-cell responses in the mixed leukocyte reaction. In one interesting
application, Senju et al133 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 differentiation. Then,
they introduced ovalbumin cDNA into this active locus by Cremediated recombination to generate a DC line that efficiently
presented antigens to prime ovalbumin-specific cytotoxic T lymphocytes in vivo. This method has potential clinical applications if it
can be widely applied to other antigens.
NK lymphocytes
Natural killer (NK) cells, an important arm of the innate immune
system, destroy cells that fail to present the correct major histocompatibility complex (MHC) class I receptor, including some virally
infected and malignant clones. Lian et al136 induced ES cells to
differentiate into functional NK cells that killed certain tumor lines
and MHC class I–deficient lymphoblasts. 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 group (Yamane et al,137,138 Tsuneto et al,139
Okuyama et al,140 and Hemmi et al141). Cytokines that promote
osteoclast differentiation and maturation from ES cells include M-CSF
and receptor activator of nuclear factor ␬B ligand (RANKL). Osteoclast
production is also augmented by ascorbic acid, dexamethasone, and 1␣,
BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
25-dihydroxyvitamin D3. Analysis of gene-targeted ES cells using 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 signaling.162 In a different
study, Tsuneto et al163 showed that enforced expression of the myeloid
transcription factor PU.1 stimulates osteoclast production in ES cells
lacking stem-cell leukemia (SCL/Tal-1), a nuclear protein essential for
early stages of hematopoiesis. 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, Thompson et al9 derived human ES cells from embryos.
More recently, murine ES cells were developed by somatic-cell
nuclear transfer (SCNT), a method in which the nucleus of an
adult somatic cell is injected into a normal enucleated oocyte to
generate cloned blastocysts.164 SCNT was also reportedly used
to generate human ES cell and this approach should eventually
open up new opportunities to generate generic and patientspecific lines.165,166
Blood from human ES cells
Methods previously established for examining hematopoiesis in
murine ES cells are applicable to human studies.167,168 Human
ES cells differentiate to hematopoietic lineages via EBs or
culture on stromal-cell lines.169 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 mesodermal, hematopoietic, and endothelial cells arranged into structures reminiscent of yolk sac blood islands. The nature of the
progenitor that gives rise to these colonies remains to be
determined.170 It is possible that hemangioblasts originate from
within these colonies. Along these lines, Wang et al171 showed
that CD45⫺, PECAM-1⫹, Flk-1⫹, VE-cadherin⫹ cells within
human EBs give rise to hematopoietic and endothelial lineages.
This hints at the existence of human hemangioblasts. However,
in clonality studies, the viability and capacity for hematoendothelial differentiation of these 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
protocols.12,172 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 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 HSCs 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
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BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
hematopoietic potential in clonal assays, the ability to efflux dyes
such as Hoechst 3334 and Rhodamine 123, and high aldehyde
dehydrogenase (ALDH) activity.167 However, these cells exhibit
limited repopulating activity after transplantation into immunodeficient mice. Wang et al173 demonstrated that a population of human
ES cell–derived progenitors incapable of repopulating the marrow
after intravenous injection exhibits limited regional repopulation
after intrafemoral injections. At minimum, this suggests defects in
migratory capacity, homing, or niche interactions, similar to what is
observed in pre-HSCs of the early mouse embryo.174 Consistent
with this interpretation, multipotential progenitors derived from
human ES cells exhibit a distinct gene expression signature that
resembles primitive embryonic progenitors.173,175
In contrast to murine ES cells, ectopic expression of HoxB4
does not promote the expansion of bona fide HSCs from human ES
cells.173 This 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 of different proteins in human ES-cell
cultures will stimulate HSC production. Candidate molecules
include Hox paralogues and numerous other nuclear regulators.176-178 In considering this experimental approach for human
HSC production, it will be important to use 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 protein.179 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 with 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 must be
further investigated. These include the nature of epigenetic influences such as X-chromosome inactivation and genomic imprinting,
telomere status, and genomic stability.
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 against autoimmune disease or to stimulate
antitumor immune responses. However, the number of DCs that can be
isolated from patients using current technologies is limiting.180 Using
human ES cells as a source for generating DCs could circumvent this
problem. Additionally, ES cells appear to be a particularly robust source
PRODUCING BLOOD LINEAGES FROM ES CELLS
1271
for expanding erythroid progenitors compared with fetal liver and adult
tissues.120 By merging several established technologies, it may be
possible to produce large quantities of mature red blood cells from
human ES cells.181 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 human ES cells to produce modified platelets that deliver
ectopically expressed procoagulant or anticoagulant factors directly to
the site of blood clots.182,183
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 3-dimensional microenvironment composed
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. In addition, 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/SCID
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. Accordingly, several groups are examining hematopoietic development from nonhuman primate ES
cells.184-188 In general, these lines exhibit hematopoiesis similar
to what is observed from human ES cells. While these studies
are relatively preliminary, continued investigation of nonhuman
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 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
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 these species.
Note added in proof: The generation of human ES cells by
somatic-cell nuclear transfer described in references 165 and 166
has recently come under question, and it seems likely that one or
both papers will be retracted. Nonetheless, we remain hopeful that
this technology is feasible and can eventually be utilized to develop
patient-specific ES cells for therapeutic purposes.
Acknowledgments
We thank Gerd Blobel, Gordon Keller, Marcela Maus, and Toru
Nakano for comments and suggestions on the manuscript.
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1272
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BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
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2006 107: 1265-1275
doi:10.1182/blood-2005-09-3621 originally published online
October 27, 2005
Designer blood: creating hematopoietic lineages from embryonic stem
cells
Abby L. Olsen, David L. Stachura and Mitchell J. Weiss
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