Hematopoiesis

Michael A. Rieger1 and Timm Schroeder2

1
Georg-Speyer-Haus, Institute for Biomedical Research, Frankfurt (Main), Germany
2
Research Unit Stem Cell Dynamics, Helmholtz Zentrum Muenchen-German Research Center for Environmental
Health, Neuherberg, Germany
Correspondence: timm.schroeder@helmholtz-muenchen.de

SUMMARY

Enormous numbers of adult blood cells are constantly regenerated throughout life from he-
matopoietic stem cells through a series of progenitor stages. Accessibility, robust functional
assays, well-established prospective isolation, and successful clinical application made he-
matopoiesis the classical mammalian stem cell system. Most of the basic concepts of stem cell
biology have been defined in this system. At the same time, many long-standing disputes in
hematopoiesis research illustrate our still limited understanding. Here we discuss the embry-
onic development and lifelong maintenance of the hematopoietic system, its cellular compo-
nents, and some of the hypotheses about the molecular mechanisms involved in controlling
hematopoietic cell fates.

Outline

1 Introduction 4 Conclusion/outlook
2 Embryonic hematopoiesis References
3 Adult hematopoiesis

Editors: Patrick P.L. Tam, W. James Nelson, and Janet Rossant

Additional Perspectives on Mammalian Development available at www.cshperspectives.org
Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a008250
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1
M.A. Rieger and T. Schroeder

1 INTRODUCTION correlation of surface marker expression patterns with

functional tests examining self-renewal capacity, clonoge-
The blood system contains more than 10 different blood nicity, and lineage potential, leading to successful prospec-
cell types (lineages) with various functions: Leukocytes tive enrichment of distinct HSPC populations (Morrison
represent many specialized cell types involved in innate and Weissman 1994; Osawa et al. 1996; Kondo et al. 1997;
and acquired immunity. Erythrocytes provide O2 and Akashi et al. 2000; Kiel et al. 2005, Inlay et al. 2009). Last,
CO2 transport, whereas megakaryocytes generate platelets HSPCs grow into colonies from single cells under appro-
for blood clotting and wound healing. All blood cell types priate culture conditions, allowing assays at the clonal level.
arise from hematopoietic stem cells (HSCs) that reside Only functional tests in vivo can retrospectively deter-
mainly in the bone marrow (BM), a major site of adult mine the true identity of HSCs by demonstrating their
hematopoiesis. Blood is one of the most regenerative and unique abilities of long-term or even lifelong self-renewal
plastic tissues, and millions of “old” blood cells are replen- and multilineage differentiation. Robust HSPC transplants
ished with new ones each second during life. In emergency between congenic mouse strains, in which the donor cells
situations such as anemia or infections, blood cell counts differ from the recipient cells by only a single surface mark-
rapidly increase. The cell number then declines back to er, allow quantitative functional HSC readouts. Once in-
normal after recovery. The lifetimes of various mature jected intravenously, even single transplanted HSCs can
blood cell types range from hours to years. find their way to the appropriate location in the BM to
The hematopoietic system is a prime example of suc- initiate lifelong blood regeneration (Osawa et al. 1996).
cessful applied regenerative medicine. For more than 30 Serial transplantation shows that HSC self-renewal can
years, stem cell transplantation has become a routine treat- last longer than the normal lifetime of the organism in
ment for blood disorders and malignant diseases. After the the mouse model (Morrison and Weissman 1994). The
eradication of the patient’s own hematopoietic system, the HSC frequency within a mixture of undefined cells can
transplanted donor hematopoietic stem and progenitor be quantified by transplanting limiting dilutions of cells
cells (HSPCs) provide lifelong reconstitution of the blood (Szilvassy et al. 1990).
system of the patient. The experimental evidence that HSCs The murine hematopoietic system is by far the best
naturally migrate back and forth from the BM periodically, understood of all species. The generation of genetically
as well as the identification of agents that increase HSC modified mouse strains for gain- and loss-of-function
mobilization (e.g., granulocyte colony-stimulating factor studies and for lineage and cell tracing enables the study
[G-CSF]), have opened new avenues for stem cell trans- of hematopoiesis in vivo. Conditional and inducible sys-
plantation. However, although stem cell transplants work tems allow the timed and tissue-specific manipulation of
successfully in the clinic, further improvement of the meth- gene expression under homeostatic conditions. For this
od is needed to minimize engraftment failure and post- approach, mouse strains expressing Cre recombinase only
transplant infections. The ex vivo expansion of HSCs in specific hematopoietic cell types have proven extremely
would be beneficial for grafts with limiting numbers of valuable (Kuhn et al. 1995; de Boer et al. 2003). Murine
HSCs (umbilical cord blood) and for gene therapy ap- transplantation models for murine and human HSPCs
proaches for monogenetic inherited blood disorders. How- have been established for decades. The homing and engraft-
ever, despite decades of research, the robust expansion (or ment of murine cells transplanted into congenic mouse
even maintenance) of HSCs ex vivo is not yet routinely strains is very efficient, although not absolute. Sophisticat-
achieved. ed humanized mouse strains with an incompetent adaptive
HSCs are the most-studied adult stem cells. Five de- immune system have been developed to prevent xenograft
cades ago researchers passed the stage of providing purely rejection for reconstitution studies with human BM cells
descriptive data to start quantitative HSC research (Till and (McDermott et al. 2010). Existing species incompatibili-
McCulloch 1961; Becker et al. 1963; Siminovitch et al. ties in receptor–ligand or adhesion molecule binding can
1963). Several properties of hematopoietic cells are favor- be partly compensated for by intrafemoral cell injection
able for stem cell research. First, they are not tightly inter- (Notta et al. 2011) or by transgenic recipient mice express-
connected in a tissue. Therefore, cells can be physically ing human growth factors (Rongvaux et al. 2011). Less
separated without too much stress. The isolation from pe- commonly used but extremely valuable model organisms
ripheral blood is minimally invasive, and millions of cells for hematopoiesis research are Danio rerio (zebrafish) (Jing
can easily be harvested. Many blood cell types are naturally and Zon 2011), and increasingly also Drosophila mela-
capable of extravasating into tightly packed tissues and nogaster (fruit fly) (Martinez-Agosto et al. 2007). The ge-
sustaining enormous shear forces. Therefore, they tolerate netic manipulation of these organisms is faster and cheaper
isolation by flow cytometry well. This enabled the early than in the murine model, and genetically modified

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 Hematopoiesis

offspring can be generated within days and weeks. More- their nuclei and express fetal forms of hemoglobin. Other
over, their transparent embryos, which develop extremely primitive blood cells like macrophages and megakaryocytes
fast and ex utero, allow relatively easy live imaging. exist, but this field of hematopoiesis remains poorly under-
stood and many questions await investigation (Palis et al.
1999; Chen and Zon 2009). In addition, definitive ery-
2 EMBRYONIC HEMATOPOIESIS
throid/myeloid progenitors have been described before
The analysis of how blood cells are first produced during HSC formation (Bertrand et al. 2010b). These very recent
embryonic development is not only of great scientific in- discoveries of previously unrecognized embryonic cell
terest, but it can also offer important insights into the types and mechanisms of their generation (Chen et al.
cellular and molecular mechanisms that lead to the speci- 2011) well illustrate how little we still know.
fication of clinically important HSCs. This could lead to The first cells with functional properties of adult HSCs
their improved expansion or even de novo generation in (i.e., regeneration of the hematopoietic system upon trans-
vitro from other cells like embryonic stem cells or induced plantation into adult recipient mice; see below) are gener-
pluripotent stem cells by improved differentiation proto- ated in the intraembryonic AGM region (Muller et al. 1994;
cols or from any other cell type by direct reprogramming. Medvinsky and Dzierzak 1996; Ivanovs et al. 2011) and the
The earliest stages of embryonic development actually placenta (Gekas et al. 2005; Ottersbach and Dzierzak 2005).
happen in the absence of any blood cells. Only after the Interestingly, their location quickly changes throughout
embryo gets too big to be supplied with oxygen and other development. After being generated in the AGM, they
essential factors by diffusion are blood cells first generated soon migrate to the placenta and fetal liver, which are major
(Fig. 1). The sites of both de novo HSPC generation and sites of their expansion. Soon thereafter they migrate to the
maintenance/expansion are constantly changing in the spleen, and around birth to the BM. BM, and to a reduced
embryo (Fig. 1). Anatomically, the first hematopoietic cells extent the spleen, then remain the main sites of HSC-
are generated in the extraembryonic yolk sac before the first derived hematopoiesis after birth.
heartbeat and later in the allantois and placenta. Finally, The development of the blood system in the embryo is a
HSCs are then generated de novo in the intraembryonic very complex and dynamic process, and in contrast to most
aorta-gonad-mesonephros (AGM) region and the placen- other tissues, different cell types constantly appear in dif-
ta. Interestingly, although all adult and many of the later ferent intra- and extraembryonic tissues (Fig. 1). The tran-
embryonic blood cells are produced from HSCs (see be- sient generation of different embryonic cell types before
low), there is a transient population of so-called primitive establishment of HSCs, which then exclusively drive later
blood cells in the early embryo. These cells emerge in the hematopoiesis, complicates the analysis of how exactly this
extraembryonic yolk sac and placenta and possibly also in tissue is generated. This has led to many decades-old dis-
intraembryonic sites before the first HSCs. They do not putes about the cellular sources of their de novo generation,
propagate into the adult and are soon replaced by their differentiation hierarchy, and molecular control (Medvin-
definitive counterparts in the embryo. They have spe- sky et al. 2011). One of those disputes concerned the cel-
cific effector functions in the embryo and differ from their lular source of the first HSCs. Because of the facts that
adult counterparts. As an example, primitive erythrocytes, hematopoietic cells (both primitive and definitive) are al-
in contrast to adult “definitive” erythrocytes, mostly retain ways found initially in proximity to endothelial cells and

Yolk sac and placenta

Yolk
sac Liver and placenta

Allantois AGM Spleen

placenta placenta Bone marrow

Mouse 7.5 10.5 11.5 14.5 18.5 Birth

dpc dpc dpc dpc dpc

Man ~30 ~4 ~5 ~12 Birth

dpc wpc wpc wpc
Figure 1. Embryonic hematopoietic development. Anatomical sites and timing of hematopoietic cell generation,
maintenance, and expansion during embryonic development. Hematopoietic cells are generated de novo in both
extra- (yolk sac, allantois, placenta) and intraembryonic (AGM region) tissues. Cells are migrating to other sites for
expansion and ultimately long-term maintenance throughout adult life. dpc, days after conception; wpc, weeks after
conception.

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M.A. Rieger and T. Schroeder

that these cell types share many molecular markers, it had adult HSCs, they are actively cycling and regenerate hema-
been hypothesized for decades that the first hematopoietic topoiesis faster and more robustly upon transplantation.
cells are actually generated from endothelium (reviewed by The switch from this fetal to adult HSC behavior occurs in a
Dieterlen-Lievre et al. 2006; Medvinsky et al. 2011). These very short time window a few weeks after birth of a mouse
hemogenic endothelial cells would be integrated into vessel (Bowie et al. 2007). This sharp switch at a defined time is
walls but would be able, under the influence of unknown useful for the identification of the involved molecular
molecular signals, to generate hematopoietic cells. Howev- mechanisms (Kim et al. 2007; He et al. 2011), which could
er, proving the existence of hemogenic endothelial cells was be of great interest for the induction of a clinically preferred
surprisingly difficult. For decades, static analyses of cells fetal phenotype in HSCs.
either before or after the endothelial-to-hematopoietic
transition yielded data that could also be explained by the
alternative conclusion that hematopoietic cells were gener- 3 ADULT HEMATOPOIESIS
ated elsewhere and then migrated toward endothelial cells.
3.1 The Hematopoietic Differentiation Hierarchy
Final proof of direct generation of hematopoietic cells from
hemogenic endothelium came from continuous single-cell In adult mammals, HSCs are at the apex of a hierarchy of
observations of endothelial-to-hematopoietic transitions numerous progenitor cell stages with increasingly restricted
by time-lapse microscopy (Eilken et al. 2009). Subsequent lineage potentials that give rise to all blood cell lineages
studies could then also detect this direct transition in vivo (Fig. 2). Of all blood cells, only HSCs fulfill the criteria
in the zebrafish embryo (Bertrand et al. 2010a; Kissa and for somatic stem cells, namely, long-term (and possibly
Herbomel 2010). It is worth mentioning that these studies lifelong) self-renewal and differentiation potential. In the
do not rule out the possible existence of other sources of murine system they robustly self-renew ( produce more
hemogenic cell types in the embryo. Importantly, compa- HSCs) while also reconstituting all blood cell lineages for
rable timing and location of hemogenic activity in human .24 weeks in congenic transplant recipients (Benveniste
embryos makes it likely that the same cells, molecules, and et al. 2010), and are further able to repopulate secondary
mechanisms are also involved in human embryonic hema- recipients. These functional assays have also revealed multi-
topoiesis (Ivanovs et al. 2011). potent progenitor (MPP) populations with intermediate-
The proof of the existence of hemogenic endothelium or short-term repopulating capacity (formerly called IT-
now allows the improved analysis of the molecular mech- and ST-HSCs) with a finite self-renewal potential (Osawa
anisms involved in the generation of the first blood cells, et al. 1996; Benveniste et al. 2010). The stepwise identifica-
including the therapeutically important HSCs. This mo- tion of multiple surface markers in the last 25 years enabled
lecular mechanism is only partially understood. It involves the prospective isolation of defined stem and progenitor
complex signaling from different cell types surround- cell populations by flow cytometry-based cell sorting. Im-
ing developing hemogenic endothelium at different sites mature multipotent cells express CD117 (c-Kit) and stem
and times in the fast-developing and -changing embryo. cell antigen (Sca)-1 and are low in mature cell marker ex-
Mesodermal cells expressing Flk1 and ETV2 were recently pression (lineage markers) (Spangrude et al. 1988; Okada
identified as the common precursors for endothelial and et al. 1992; Uchida and Weissman 1992; Morrison and
hematopoietic cells (Lee et al. 2008; Kataoka et al. 2011a). Weissman 1994). About one in 30 cells within this so-called
The signals inducing hemogenic fate in these cells and their KSL (c-Kit+/Sca-1+/Lineage2) population (0.06% in BM)
progeny are the subject of ongoing research. Molecules is an HSC (0.002% in BM), and there is no HSC activity
like membrane-bound Notch ligands or soluble hedgehog, outside the KSL fraction. These findings laid the ground
bone morphogenetic protein, or vascular endothelial for the identification of additional surface markers for
growth factor, but also shear stress and nitric oxide signal- improved prospective HSC identification (Table 1). Cur-
ing, have been involved in the induction of the hemogenic rently, the highest purity of at least 50% HSCs in the sorted
program in endothelial cells—possibly even in mesodermal fraction is obtained with CD150+/CD482/CD34low KSL
precursors before they have reached endothelial identity cells (Kiel et al. 2005). Moreover, CD150 expression levels
(Medvinsky et al. 2011). Within the hemogenic endothelial might indicate distinct subpopulations of HSCs with pre-
cell, a number of transcription factors (TFs) seem to be disposed myeloid- or lymphoid-biased reconstitution pat-
crucial for the induction of the hemogenic program, with terns (see also Sec. 3.2.1) (Morita et al. 2010). CD49b
Runx1 and CBFb apparently being critical core factors expression could be attributed to intermediate-term re-
(Chen et al. 2011). populating MPPs, a population that shares many function-
Importantly, fetal HSCs differ from adult HSCs in their al properties of myeloid-biased HSCs (Benveniste et al.
regeneration behavior. In contrast to mostly quiescent 2010). In addition to surface markers, distinct functional

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 Hematopoiesis

CD150+ α β γ Human
CD48– LT-HSC
CD34lo CD90+
CD135– CD45RA–
CD49b– CD49f+

Self-renewal
Myeloid biased Lymphoid biased
CD201+ CD150 Rholo
CD110+ Hoechst
CD150+ MPP (IT)
CD117hi CD48–
Sca1hi CD34lo
Lin– CD135– CD34+
CD49b+
CD38–
CD117hi
CD150–
CD48– MPP (ST) Lin–
CD34hi CD90–
CD135– CD45RA–
CD49f–
CD150–
CD150– CD48+
CD48+ CD34hi
CD34hi
MPP LMPP CD135+ CD34+
CD135– CD110– CD38–
CD90–
CD45RA+
CD135+
CD7+/–
CD10+
ELP
CD127– CLP
CMP CD117hi CD127+ CMP
Sca1mid CD117mid CD123lo
Sca1mid CD45RA–
CD150+ CD135+
CD117+ CD10–
Sca1– MEP CD34+
CD16/32– CD150– MEP CD38+
CD117+
GMP CD123–
CD34lo CD90–
Lin– Sca1– MDP CD45RA–
CD16/32+ CD135– Lin–
CD34hi
CDP
CD115 + CD10–
Lin–
CD117 +
GMP
CD135 + +
Sca1 – Pro T Pro NK Pro B CD123
Megakaryocyte (Basophil) Monocyte CD45RA+
Dendritic cell CD135+
Erythrocyte (Neutrophil) CD10–

(Eosinophil) Macrophage Osteoclast

T cell NK cell B cell
Platelets Granulocytes

Figure 2. Adult hematopoietic differentiation hierarchy. Long-term self-renewing HSCs are at the apex of a hierarchy
of multiple progenitor cell stages giving rise to all blood cell lineages. Distinct HSPC stages have been described by
correlating surface marker expression and functional properties for prospective isolation, both in the murine and
human systems. Murine hematopoiesis is currently defined in more detail and therefore displayed here. Corre-
sponding human HSPC populations with their markers are indicated. HSCs differentiate into all blood cell lineages
via long described (bold arrows) and potentially also or alternatively more recently described differentiation routes
(thin arrows). It is important to point out that this model is only a simplified representation of current knowledge
and will continue to change. Individual murine HSCs show an intrinsic lineage-biased repopulating ability, gen-
erating either more myeloid or lymphoid cells or a balanced mixture. These HSC subgroups can be enriched by
detecting various levels of CD150 expression or Hoechst efflux. HSC, hematopoietic stem cell; MPP, multipotent
progenitor; LT-, long-term repopulating; IT-, intermediate-term repopulating; ST-, short-term repopulating; LMPP,
lymphoid-primed MPP; ELP, early lymphoid progenitor; CLP, common lymphoid progenitor; CMP, common
myeloid progenitor; GMP, granulocyte – macrophage progenitor; MEP, megakaryocyte–erythrocyte progenitor;
CDP, common dendritic progenitor; MDP, monocyte –dendritic cell progenitor; NK, natural killer cell.

properties of HSCs have been used for prospective iso- reconstitution behavior depends on the chosen transplan-
lation, like the increased efflux of the dye Hoechst 33342 tation model. This is particularly problematic for xenograft
(so-called side population, or SP) (Goodell et al. 1996) and settings with human HSCs repopulating murine recipients.
low staining for Rhodamine123 dye (Bertoncello et al. Although this is an extremely important methodology for
1985). A further discrimination of SPlower and SPhigher analyzing human HSCs, it is important to remember that
HSCs also dissected HSCs with myeloid- and lymphoid- HSC properties and behavior in such a different environ-
biased repopulation capacity, respectively (Challen et al. ment could differ massively from those under conditions in
2010). It is important to point out that although trans- the human niches (McDermott et al. 2010).
plantation experiments functionally identify HSC proper- HSCs differentiate into a cascade of progenitor cell
ties, these are stress conditions for the HSCs and for the stages with declining multilineage potential before uniline-
organism. They therefore may not reflect functional prop- age commitment occurs (Kondo et al. 1997; Akashi et al.
erties of homeostatic HSCs. Furthermore, functional HSC 2000; Adolfsson et al. 2005; Wilson et al. 2008; Rieger et al.

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M.A. Rieger and T. Schroeder

Table 1. Prospective HSC isolation

Transplanted HSCs
HSPC population cells Assay/model (%) References
Murine
Thy-1low/Sca-1+/Lin2 30 Competitive 1.5 –3 Spangrude et al. 1988
KSL Limiting Competitive 3 –7 Okada et al. 1992; Bryder et al.
dilutions 2006
Thy-1low KSL 5– 10 Competitive 10 –20 Morrison and Weissman 1994
CD342 KSL 1 Competitive 21 Osawa et al. 1996
SP Rholow/CD45mid/Lin2 1 Sublethal W-41 42/33 Uchida et al. 2003; Dykstra et al.
2006
SP CD201+ 10 Competitive .10 Balazs et al. 2006
CD150+/CD482/CD412 KSL 1 Competitive 47 Kiel et al. 2005
CD150+/CD482/CD49blow KSL 1 Sublethal W-41 29 Benveniste et al. 2010
competitive
CD150+/CD482/CD201+/CD45+ 1 Sublethal W-41 43 Kent et al. 2009

Human
CD34+/CD382/Lin2 Limiting NOD/SCID ,1 Bhatia et al. 1997
dilutions
CD34+/CD382/CD90+/CD45RA2/Lin2 Limiting Sublethal NSG 5 Majeti et al. 2007; Notta et al.
dilutions 2011
Rholow/CD49f+/CD34+/CD382/CD90+/ 1 Sublethal NSG 15 Notta et al. 2011
CD45RA2/Lin2
HSPC, hematopoietic stem or progenitor cell; KSL, CD117+, Sca-1+, and Lin2 cells; SP, side population; Rho, Rhodamine123; W-41, mouse line with homo-
zygous Kit W-41J/Kit W-41J mutation; NOD/SCID, nonobese diabetic/severe combined immune deficiency mouse line; NSG, NOD/SCID/Il2 g-chain knockout
mouse line; competitive, transplantation in lethally irradiated recipients with “competitor-recipient” HSPCs.

2009b). Current models of the hematopoietic hierarchy According to current data, the human hematopoietic
describe a successive loss of individual lineage potentials differentiation hierarchy closely resembles the murine one
(Adolfsson et al. 2005; Arinobu et al. 2007; Mansson et al. (Majeti et al. 2007; Doulatov et al. 2010). However, mostly
2007; Pronk et al. 2007). MPPs first lose their erythroid/ as a result of more difficult experimentation, and despite
megakaryocytic potential and develop into lymphoid- their clinical importance, purification methods for human
primed MPPs (LMPPs) and early lymphoid progenitors HSPCs are far less advanced than for murine HSPCs (Table
(ELPs) (Adolfsson et al. 2005). Then they lose their myeloid 1). Human HSCs express the surface molecule CD34, in
potential and become common lymphoid progenitors contrast to their murine counterparts (Osawa et al. 1996;
(CLPs) (Kondo et al. 1997; Inlay et al. 2009; Schlenner Dick 2008). However, although many reports describe
et al. 2010), and finally differentiate into lymphoid cells CD34+/CD382/Lineage2 cells as human HSCs, it is very
(Fig. 2). However, the successive restriction to solely lym- important to point out that this population comprises
phoid fate should not imply a picture of default lymphoid ,1% functional HSCs (as determined by xenograft trans-
cell fate. The opposite might be the case, namely, that active plantation assays). Only recently have improved methods
repression of the myeloid cell fate is required to induce for the prospective enrichment of human HSCs been
lymphoid development. Interestingly, B- and T-cell pro- described that now allow enrichment to purities of ~15%
genitors retain, at least in vitro, some myeloid potential (Notta et al. 2011) (Table 1). Prospective isolation of
(Kawamoto et al. 2010). In contrast, lineage-tracing exper- human hematopoietic progenitors has also recently been
iments revealed only a minor contribution of myeloid cells improved, allowing the functional and molecular charac-
from interleukin-7 (IL-7) receptor–positive CLPs in mu- terization of these important cell types with much im-
rine homeostatic hematopoiesis (Schlenner et al. 2010). It proved resolution (Manz et al. 2002; Majeti et al. 2007;
is important to point out that this model of the hemato- Doulatov et al. 2010). However, the percentage of pro-
poietic differentiation hierarchy clearly only reflects current spectively isolated human HSCs may be underestimated
knowledge and will continue to change over time. In par- by xenotransplantations because the murine environment
ticular, the early progenitor stages are still ill defined, and is less permissive to human HSCs, and advanced human-
numerous novel maturation stages and differentiation ized mouse models have improved engraftment efficiency
pathways will certainly be described in coming years. (Rongvaux et al. 2011).

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 Hematopoiesis

3.2 Molecular Control of Hematopoietic stages in sufficient numbers and purity, (2) continuous
Fate Choices quantitative measurements of functional molecular activi-
ty during differentiation, and (3) the observation of mul-
To continuously regenerate the hematopoietic system, the tiple factors in complex genetic programs. Many of these
right number of specific cell types must permanently be requirements have been achieved individually, but their
generated at the right time and place. To achieve this, cor- integrated analysis is still limited by current technologies.
rect fate decisions constantly have to be chosen in HSPCs: Hematopoiesis is usually analyzed in heterogeneous
quiescence versus proliferation, self-renewal versus differ- populations and at individual time points of the process
entiation, lineage choice, survival versus death, and sessility of interest. However, these snapshot analyses only allow
versus migration (Fig. 3A). Exact timing and sequential limited conclusions about the involved sequence of indi-
order of all choices in each cell underpin normal hemato- vidual molecular and cellular events (Fig. 3B). Experimen-
poiesis, both in steady-state and injury situations. Disrup- tal data can then usually be explained by a multitude of
tions of normal cell fate decisions underlie hematological hypotheses, leading to ambiguous conclusions. Only when
disorders. A comprehensive analysis of their molecular the behavior and fate of each individual cell and its progeny
control and the exact interplay of these fate decisions are are continuously known can a comprehensive understand-
therefore crucial for a full understanding of normal and ing of developmental steps and their molecular control be
malignant hematopoiesis and the development of novel drawn (Fig. 3B). Ideally, therefore, cellular and molecular
therapies. HSPC behavior is continuously quantified at the single-cell
The molecular circuitries underlying HSPC fate choice level and throughout the process of interest (Schroeder
must enable cells to respond to changing external stimuli 2008). One approach to fulfill these requirements is long-
but also allow specific cellular states to be stable indepen- term video microscopy of HSPCs and computer-aided
dent of their environment. As an example, plasticity must single-cell tracking (Eilken et al. 2009; Rieger et al. 2009a).
enable the choice of HSPCs between multiple lineages but However, despite its potential, this field is still in its infancy,
ensure stability of lineage commitment after the choice has and because of the required high level of interdisciplinary
been made. The expression, activity, or changing function technical know-how, its use is still limited to few expert
of fate-control molecules therefore must be well timed, cell laboratories (Schroeder 2011).
type specific, and controllable by other factors. For a com-
prehensive understanding, these parameters have to be
3.2.1 A Stem Cell’s Decision: Self-Renewal
quantified in a cell-stage-specific manner, at the single-
versus Differentiation
cell level, and ideally over time. Experimental analysis of
molecular cell-fate control requires demanding technical The HSC’s choice between self-renewal and differentiation
prerequisites: (1) the prospective isolation of distinct cell must be tightly regulated to enable both the generation of

A Lineage choice/ B Start

Cell cycle/
differentiation
proliferation

Polarization/
migration Self-renewal
…many other
Apoptosis possibilities
ECM

Endpoint

Niche cells

Figure 3. Continuous single-cell observations are required for a comprehensive understanding of dynamic cellular
behavior. (A) Individual cell-fate decisions are the reason for the behavior of the complete hematopoietic system in
health and disease, and must therefore be quantified. They are influenced by factors like cytokines, extracellular
matrix (ECM), or membrane-bound signaling molecules in many different niches. (B) Only continuous single-cell
observation allows unambiguous conclusions about cell-fate choices in complex cell systems. Discontinuous input/
output analyses—even when done at the single-cell level—cannot distinguish between different combinations of
cell-fate decisions leading to the same output of a cellular system, and therefore lead to ambiguous conclusions.

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M.A. Rieger and T. Schroeder

differentiated cells and the accurate maintenance of the renewal and differentiation controlled by the environ-
right HSC number. The BM provides the environment ment, but intrinsic regulators of HSC functionality exist
for sustained HSC function, and HSCs rapidly lose their (Table 2).
self-renewal capacity once isolated from their in vivo Remarkably, ectopic expression of HoxB4 and in par-
niches. Extrinsic signals from membrane-bound, soluble, ticular of Hox fusion proteins to NUP98 (NUP98–HoxB4
or extracellular matrix-associated ligands from the niche and NUP98–HoxA10 homeodomain) lead to a massive
are necessary for appropriate HSC behavior. Two major net expansion of HSCs in vitro, while still maintaining their
HSC niches are currently proposed to exist in BM: the normal multilineage differentiation in vivo (Antonchuk
endosteal osteoblastic niche (Calvi et al. 2003; Zhang et al. 2002). However, despite years of research, the exact
et al. 2003; Lo Celso et al. 2009; Xie et al. 2009) and the
perivascular endothelial niche (Kiel et al. 2005; Ding et al.
2012). It is unclear if both niches, although spatially sepa- Table 2. Examples of HSC intrinsic molecules implicated in control
of self-renewal
rated, fulfill a similar function; if both niches provide dis-
tinct properties, for example, for dormant versus cycling Molecule Effect on self-renewal (SR) References
HSCs; or if the niche comprises both osteoblasts and en- Transcription factors
dothelial cells working synergistically on HSC function. HoxB4 Expression enhances SR Antonchuk et al. 2002
Also, BM comprises a heterogeneous mixture of various Gfi1 Loss reduces SR upon Hock et al. 2004; Zeng
HSC exhaustion et al. 2004
cell types including blood cells, mesenchymal cells, osteo- Evi1 Loss reduces SR Kataoka et al. 2011b
blasts, osteoclasts, endothelial cells, reticular cells, fat cells, c-Myc Loss leads to HSC Laurenti et al. 2008
and many other less-defined types, which will also influ- accumulation with
ence hematopoietic fates. A multitude of signaling path- differentiation defect
ways have been shown to be activated in HSCs by the niche Signaling modulators
(e.g., the cytokine receptors c-Kit and Mpl, Wnt, Notch, Pten Loss reduces SR upon Yilmaz et al. 2006
Sonic hedgehog, and integrin signaling). However, their HSC exhaustion
precise involvement in HSC maintenance remains sur- Lnk Loss increases SR Ema et al. 2005; Seita
prisingly controversial. For most of them, contradictory et al. 2007
conclusions have been drawn in separate studies. Our in- STAT5A/B Loss reduces SR Wang et al. 2009
complete understanding of the molecular control of HSC Epigenetic modifiers
self-renewal is well illustrated by the fact that therapeuti- Dnmt1 Loss reduces SR by reduced Broske et al. 2009
cally relevant robust maintenance or even expansion of DNA methylation
these clinically important cells ex vivo remains elusive Dnmt3a/b Loss alters SR and/or Tadokoro et al. 2007;
without genetic manipulation. Nevertheless, several recent differentiation Challen et al. 2011
Bmi-1 Loss reduces SR, expression Lessard and Sauvageau
studies with murine and human HSCs describe very prom-
(PRC1) increases SR 2003; Park et al. 2003;
ising approaches for the ex vivo expansion of HSCs without Iwama et al. 2004
genetic manipulation (Zhang et al. 2008; Boitano et al. Suz12 Loss increases SR Majewski et al. 2010
2010). A comprehensive discussion of all potential molec- (PRC2)
ular HSC self-renewal modulators goes beyond the scope of Ezh2 Expression enhances SR, Herrera-Merchan et al.
this chapter, and can be found elsewhere (Ehninger and (PRC2) loss reduces SR (mainly in 2012
Trumpp 2011; Mercier et al. 2011). fetal liver)
At the single-cell level, even HSCs with retrospective- Cell cycle regulators
ly proven functionality (.1% long-term contribution p57KIP2 Loss reduces SR upon Matsumoto et al. 2011;
to mature myeloid and lymphoid cells in peripheral HSC exhaustion Zou et al. 2011
blood) are heterogeneous in their reconstitution efficacy p18Ink4c Loss increases SR Yuan et al. 2004
and their lineage contribution pattern. They range from microRNAs
1% to almost 100% total contribution, with varying ratios miR125a Loss reduces SR and Guo et al. 2010
of myeloid and lymphoid lineages (Muller-Sieburg et al. increases apoptosis
2002, 2004; Sieburg et al. 2006; Dykstra et al. 2007; Kent miR125b Expression enhances SR O’Connell et al. 2010;
by blocking apoptosis Ooi et al. 2010
et al. 2009). Importantly, these patterns are conserved
through serial transplantations, indicating the existence RNA-binding proteins
of stable inheritable stem cell intrinsic programs (Muller- Msi2 Loss reduces SR Hope et al. 2010; Ito et al.
Sieburg et al. 2002, 2004; Sieburg et al. 2006; Dykstra 2010; Kharas et al. 2010
et al. 2007; Kent et al. 2009). Therefore, not only are self- PRC, polycomb repressive complex.

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 Hematopoiesis

molecular mechanism and the relevant target genes induc- for this endeavor. Most of these studies have elucidated far
ing HSC expansion remain poorly defined. more binding sites and DNA motifs than originally pre-
Although all the discussed and numerous additional dicted, often at sites far from expected promoter regions.
molecules could be identified to be involved in regulating These potential cis- or trans-acting sites would have been
HSC self-renewal, their exact interplay remains to be un- undetected by conventional methods. However, the techni-
raveled. More sensitive large-scale methods will likely be a cal requirement for high cell numbers interferes with the
key to drawing a more complete picture of the stem cell self- necessity to analyze distinct primary cell populations at
renewal network and to identifying a potential core mech- many different time points during differentiation. These
anism of self-renewal control. It is interesting that deletion usually are very infrequent, and enrichments still yield in-
of many factors that are essential for fetal HSC generation sufficient numbers and purity. Technical improvement is
and self-renewal (e.g., Runx1, Tal1, Notch1 and -2, RBP-J, eagerly awaited to allow these comprehensive analyses
b-catenin, and HoxB4) only have minimal consequences from small cell numbers and ideally from single cells (Islam
upon deletion in adult HSCs, whereas overexpression often et al. 2011).
results in enhanced HSC maintenance, self-renewal, and
leukemia (Mikkola et al. 2003; Ichikawa et al. 2004). 3.2.2.1 Stability of Lineage Commitment. In con-
trast to other tissues, plasticity between lineages, with fre-
quent physiological “transdifferentiation” of cells of one
3.2.2 Hematopoietic Lineage Choice and Stability
lineage into another, has not been observed in the hema-
Differentiation of multipotent cells into different lineages topoietic system. Although maintenance of lineage choice
must be well controlled to enable the timely production of is critical for normal hematopoiesis, its targeted manipu-
the right number and type of mature cells. Despite inten- lation could also be used to induce dedifferentiation or
sive research over decades, we are only just beginning to differentiation in another lineage for therapeutic purposes.
understand how cells manage to establish and maintain a The expulsion of the nucleus during late stages of ery-
lineage-committed stage at the molecular level. The molec- throid maturation is—although implemented for other
ular mechanisms of lineage stability are better defined than reasons—probably the most drastic way of preventing the
those of lineage choice. Here we discuss some exemplary activation of genetic programs of another lineage. In most
mechanisms of intrinsic and extrinsic control of lineage other cell types, lineage commitment is not entirely an
choice. irreversible state. In contrast, it must be actively maintained
The differentiation of a multipotent cell to a specific by sustained commitment factor expression or network
lineage involves a global change of gene expression. Lineage stability in committed cells and their progeny. Positive au-
choice and commitment are accompanied by the induction toregulation of a lineage-specific factor while inhibiting
and maintenance of lineage-affiliated genetic programs. opposing factors leads to stable situations with one factor
These include not only the expression of lineage-specific expressed and the other repressed (Kerenyi and Orkin
genes but also the repression of those specific for other 2010). One excellent example is the switch from high levels
lineages. Stable gene expression requires the presence and of GATA2 to high levels of GATA1, which precedes eryth-
activity of a set of distinct TFs, which are integrated in ropoiesis from HSCs and is called the “GATA switch”
complex networks with other TFs, modulating cofactors, (Fig. 4A). GATA2 is mainly expressed in early progenitors
chromatin modifiers, microRNAs, and other regulatory and induces GATA1 expression, which in turn activates its
RNAs (Davidson 2010). own expression and represses GATA2. The switch is medi-
Most current knowledge about TF function in hemato- ated by the displacement of GATA2 from its own upstream
poiesis has been gained in static gene-by-gene analyses. Ge- enhancer by increasing levels of the interacting TF pair
netically modified mouse models dissect TF function in GATA1 and Friend of GATA1 (FOG1) (Grass et al. 2003).
distinct cell stages during hematopoiesis. These analyses Moreover, the displacement of GATA2 by GATA1–FOG1 at
unravel central players of genetic networks but ignore less the c-Kit locus results in rearranged chromatin looping and
prominent components that are vital for the orchestration down-regulated c-Kit expression, demonstrating the ability
of the whole program and the dynamic regulation of these of TFs to directly alter long-range chromatin interactions
networks. In the future we will also need to systemically (Jing et al. 2008). These switches can be implemented rap-
understand the dynamic changes of expression or activity idly, taking only one specific S phase for their implemen-
of the whole ensemble of lineage regulators. Deep parallel tation, thus driving cells to the next stage of maturation in
sequencing methods allowing quantitative whole-genome very short time (Pop et al. 2010).
information about TF binding to DNA, chromatin status, Lineage commitment in B-cell development is orches-
and resulting transcriptional activity will be of great use trated in a regulatory network of key TFs with feed-forward

Cite this article as Cold Spring Harb Perspect Biol 2012;4:a008250 9

M.A. Rieger and T. Schroeder

Extrinsic signal
A C
Notch
GATA2 GATA1

TCF1 Non-T-cell genes

FOG1 Erythroid genes

Bcl11b GATA3
B
PU.1 IKAROS IL-7Rα
T-cell genes

D
E2A EBF1
PU.1 GATA1

B-cell
genes PAX5 Non-B-cell genes Myeloid genes Erythroid genes

Figure 4. Network motifs for induction and maintenance of lineage commitment. Simplified examples of molecular
mechanisms and networks of stable commitment induction and propagation in erythroid (A), B-lymphoid (B), T-
lymphoid (C), and myeloid (D) cells. Direct or indirect activation (arrows) and repression (barred lines) of
individual factor expression are indicated. Transcription factors are depicted in white, surface receptors in gray.
Dashed lines represent indirect interactions.

regulatory cascades (Fig. 4B). PU.1 and E2A are critical Rolink et al. 1999; Mikkola et al. 2002; Delogu et al. 2006;
for lymphoid cell-fate determination and induce specific Cobaleda et al. 2007). Pax5 is directly involved in epigenetic
B-lineage commitment factors like early B-cell factor 1 alterations of B-lineage-specific genes (Schebesta et al.
(EBF1) and Pax5. EBF1 is a primary determinant of B- 2007; Gao et al. 2009; McManus et al. 2011). Interestingly,
cell fate, and its expression is controlled by high expression ectopic expression of EBF1 in Pax5-deficient hematopoi-
of IKAROS and E2A in conjunction with PU.1 and by etic progenitors restricts their alternate lineage potential
extrinsic signals of the IL-7 receptor. EBF1-deficient cells in vivo (Nutt et al. 1999; Rolink et al. 1999; Mikkola et al.
arrest in early B-cell development (before the immunoglob- 2002; Delogu et al. 2006; Pongubala et al. 2008). This re-
ulin heavy-chain rearrangement) with a failure to initiate pression of other lineage-specific molecular programs by
the early B-lineage program of gene expression. E2A might Pax5 and partners is an excellent example of how a lineage
promote the generation of LMPPs by antagonizing mega- choice can be kept stable. This mechanism also requires
karyocyte/erythroid-lineage programs and priming the sustained expression and activity of these factors and might
transcription of the B-cell-specific factors EBF1 and Pax5 therefore be less stable against perturbations than other
(Dias et al. 2008). B-cell differentiation in the absence of mechanisms, for example, involving chromatin modifica-
E2A can be rescued with EBF1 and Pax5 (Seet et al. 2004; tions. Subtle changes and misregulation of individual TFs
Kwon et al. 2008). Progenitors with genetically ablated E2A will directly impact normal differentiation and can lead to
or EBF1 retain their multilineage potential (with the excep- leukemia (Rosenbauer et al. 2006).
tion of the erythroid lineage) (Dias et al. 2008; Semerad et al. Epigenetic activation or silencing of lineage-restricting
2009). EBF1 can antagonize the expression of myeloid de- programs by chromatin and DNA modifications may ulti-
termining factors such as PU.1, C/EBPa, and Id2 and in- mately determine lineage commitment. As one example,
duce synergistically with E2A the expression of Pax5 as the the c-fms locus (coding for the macrophage CSF [M-CSF]
main determinant factor of B-cell commitment. The stable receptor) becomes epigenetically silenced during B-cell
B-cell fate is locked in by a feedback loop of Pax5 and differentiation in a gradual process, inhibiting remaining
IKAROS to sustain EBF1 expression. All factors function myeloid potential (Tagoh et al. 2004). However, sustained
in a concerted manner in pro-B cells to ensure the repression Pax5 expression is still required for maintaining c-fms locus
of myeloid genes and thus ensure stability of B-cell commit- silencing, because mature B cells rapidly up-regulate M-
ment (Pridans et al. 2008; Decker et al. 2009). Pax5 deletion CSF receptor expression upon Pax5 deletion. These find-
leads not only to a B-cell differentiation block at the pre-/ ings well illustrate that TFs function by altering chromatin
pro-B-cell stage, but it also enables B cells to dedifferentiate states and directly recruiting epigenetic modifying com-
into an LMPP-like stage and to allow entry into normal plexes (McManus et al. 2011).
alternative differentiation paths into various myeloid and In contrast to the intrinsically stabilized B-cell commit-
lymphoid lineages in vivo and in vitro (Nutt et al. 1999; ment network, T-lineage fate choice remains provisional for

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 Hematopoiesis

an extended period of differentiation with high dependency Instruction Selection

on extrinsic stimulation from the thymic environment (Fig. “Black”
Multipotent signal
4C). T-cell fate initiation and determination requires per-
sistent extrinsic Notch receptor signaling, which is stimu-
Committed
lated by Delta-like 1 and 4 expressing cortical endothelial
cells in the thymus (Radtke et al. 1999). Notch signaling
turns LMPPs into early T-cell progenitors by inducing a
Mature
pro-T-cell developmental program with direct and indirect
activation of GATA3, Tcf7, and other T-cell-restricting
genes, which, once initiated, positively autoregulate their Figure 5. Possible influence of extracellular signals on production of
lineage-committed cells. Extrinsic signals by, for example, hemato-
own expression. The activation of a T-lineage program by poietic cytokines may instruct the lineage choice of multipotent cells.
Notch signaling is, however, not sufficient for T-cell fate Alternatively, extrinsic signals could merely enable survival, prolifer-
determination alone. Additionally, Notch suppresses B- ation, and maturation signals of cells that have already committed to
cell-lineage-determining EBF1 and E2A expression and di- one lineage independently of the extrinsic signal. Importantly, static
minishes remaining myeloid potential by down-regulation analyses at the start and end of an experiment would lead to identical
data in both situations, and continuous single-cell observations are
of PU.1 and C/EBPa (Ordentlich et al. 1998; Smith et al.
required for definitive conclusions about cytokine function.
2005; Laiosa et al. 2006b). Id2 expression is blocked by
Notch signaling to circumvent NK cell development (Ikawa
et al. 2001). Conversely, the TF leukemia/lymphoma-relat- IL-2 receptor converted CLPs from the lymphoid to the
ed factor (LRF) inhibits Notch signaling in the BM, thus myeloid lineage, probably through ectopic up-regulation
preventing T-cell development. Consequently, the condi- of granulocyte –macrophage CSF (GM-CSF) receptor up-
tional deletion of LRF in HSCs results in the generation of on IL-2 stimulation (Kondo et al. 2000; Iwasaki-Arai et al.
T-lineage progeny in the BM (Maeda et al. 2007). 2003). However, the effects of ectopically expressed mole-
cules may not be representing physiologically relevant
3.2.2.2 Extrinsic Regulation of Lineage Choice— programs of lineage choice. Only recently has lineage
Instructive versus Selective Function of Cytokines. instruction of unmanipulated primary hematopoietic
The mechanisms discussed above can explain how a lineage progenitors by cytokines to which they physiologically re-
choice, once it is made and implemented, can be main- spond been proven (Rieger et al. 2009a). Culture of gran-
tained. Before this state, molecular changes in uncommit- ulocyte –macrophage progenitors (GMPs) in only G-CSF
ted cells must lead to the actual lineage choice and the or M-CSF will eventually lead to production of only gran-
induction of these maintenance programs. One way of in- ulocytic or monocytic cells, respectively. In the selective
ducing lineage choice is by signals from outside the cells, model, GMPs would produce progeny of both lineages
for example, by hematopoietic cytokines. Cytokines are even if only one cytokine was present. The absence of one
very important regulators of hematopoiesis. They affect lineage-specific cytokine would then lead to the death of
multiple cell fates of every hematopoietic cell type, includ- cells that have committed to this “wrong” lineage, leading
ing survival, proliferation, maturation, and functional ac- to a monolineage output (Fig. 5). However, the continuous
tivation (Rieger and Schroeder 2009). In addition to these observation of individual GMPs and all of their progeny
functions, the instruction of hematopoietic lineage choice throughout the differentiation process demonstrated the
by cytokines has been postulated for decades (Metcalf 1998, lack of cell death events during the cytokine-dependent
2010). However, it proved to be surprisingly difficult to monolineage differentiation of GMPs (Fig. 5). A purely
show beyond doubt that cytokines do not merely allow selective effect could therefore be excluded, demonstrating
the survival and proliferation of precursors that have al- GMP lineage instruction by these cytokines (Rieger et al.
ready intrinsically—and independently of the presence of a 2009a). However, the involved molecular mechanisms are
cytokine—committed to one lineage ( permissive/selective poorly understood. A signal strength-dependent model of
model) (Fig. 5) (Enver et al. 1998). The pleiotropic, redun- dendritic cell development has recently been proposed,
dant, and cell-type-dependent effects of cytokines are one in which dendritic cell development signals are integrated
reason for the difficulty in precisely defining their effects on from cytokine receptor expression, downstream signal
cell fates (Rieger and Schroeder 2009). Loss-of-function transduction, and the presence of the cytokines M-CSF,
studies have thus provided limited insight. Most studies Flt3 ligand, and GM-CSF in specific niches (Schmid et al.
suggesting lineage instruction by cytokines have therefore 2010). This kind of model could have broader relevance in
relied on ectopic molecular overexpression and/or trans- understanding the role of cytokines in instructing lineage
formed cell lines. As an example, the ectopic expression of development.

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M.A. Rieger and T. Schroeder

Despite this accumulating evidence for lineage instruc- possibility is the instruction of lineage choice by cell-ex-
tion by cell-extrinsic signals in some of the more restricted trinsic signals. In this case, TFs are most likely the final
progenitors, their impact on earlier branching points of the executors, implementing the lineage decision, but are not
hematopoietic hierarchy still remains poorly understood. involved in the actual decision making. Another intriguing
Cytokines contribute in a major way to the survival and possible mechanism is the stochastic output of fluctuating
proliferation of different hematopoietic cell types, and not cell-intrinsic networks of TFs. In this case, TFs would not
all lineage-affiliated cytokines have a lineage-instructive only execute a previously made decision, they would also be
ability. Even if specific lineages can be instructed by cyto- the relevant components of the molecular machinery gen-
kines, this does not preclude the existence of additional erating the lineage choice. This hypothesis stems from the
cell-intrinsic lineage choice mechanisms that could lead initially surprising findings that multiple “lineage-specific”
to lineage choice in the absence of cell-extrinsic instructive TFs are coexpressed in multipotent cells before lineage
signals. The capacity of individual extrinsic signals to in- commitment (“lineage priming”; see, e.g., Miyamoto
struct lineage choice, as well as its integration with other et al. 2002). These factors are expressed only exclusively
cytokine signals and with other intracellular molecular in different mature lineages, and they can drive cells into
states like TF expression, must be further analyzed for this lineage upon overexpression. Initially, therefore, it was
each individual cytokine, in specific cell types, under chem- assumed that multipotent cells have the potential to differ-
ically defined conditions, and ideally continuously at the entiate into multiple lineages because they lack the expres-
single-cell level. sion of these lineage-specific TFs. However, with the
availability of improved prospective purification of multi-
3.2.2.3 Intrinsic Regulation of Lineage Choice by potent cells and more sensitive molecular methods for the
TFs—Decision Makers or Only Executors? It is clear detection of TF expression, it then became clear that the
that individual TFs can instruct lineage choice and are opposite could be true. Multipotency might in fact be char-
even able to reprogram committed cells, leading to cross- acterized not by the absence of lineage-specific properties
ing of lineage borders. For example, ectopic expression but by the presence of properties of multiple lineages, in
of GATA1 in multipotent cells or cells committed to other particular the coexpression of multiple “lineage-specific”
than the megakaryocyte–erythrocyte lineages (e.g., CLPs) TFs. The additional finding that these TFs often inhibit
enforces the development into erythroid and megakar- each other by binding mutually or to cofactors of opposing
yocytic cells (Heyworth et al. 2002). C/EBPa and PU.1 TFs led to the idea that these factors may neutralize each
are essential for the generation of granulocyte –macro- other in uncommitted cells, thus retaining multipotency.
phage progenitors. C/EBPa expression in committed lym- Random fluctuations in their expression could then lead to
phoid cells (B and T cells) instructs the development of one TF gaining dominance; positive autoregulation and
macrophages (Xie et al. 2004; Laiosa et al. 2006a,b). Fur- repression of other TFs would lead to a lineage-committed
thermore, committed T cells transdifferentiate into mye- state in which only one lineage-specific TF is expressed
loid dendritic cells upon ectopic PU.1 expression (Laiosa (Enver et al. 1998; Cantor and Orkin 2001; Graf and Enver
et al. 2006b). TFs also actively repress lineage programs. To 2009). The production of all lineages would be ensured by
this end, the myeloid potential as well as PU.1- or C/EBPa- the overall wiring of the TF network leading to stable sto-
dependent myeloid reprogramming of thymic precursors chastic lineage output. Although not predictable for an
can be blocked by active Notch signaling (Franco et al. individual cell, different cells within a population would
2006; Laiosa et al. 2006b; Rothenberg 2007), suggesting fluctuate into decisions for different lineages with specific
an instructive extracellular non-cytokine-mediated induc- probabilities. Adaptation of the blood system to stress or
tion of T-cell identity. Findings like these clearly demon- injury would then be regulated by the selection of existing
strate the ability of many lineage-specific TFs to drive cells lineage-committed progenitors for survival and prolifera-
into one lineage. Upon their up-regulation or activation, tion (Suda et al. 1984).
they will start the transcription of myriads of direct and Studies on transcriptional pathways that control binary
indirect target genes and chromatin modifications, which lineage choices in hematopoiesis revealed some similarities
lead to the changing phenotypes of lineage-committing in the gene regulatory network circuits of different lineages.
and maturing cells. Pairs of TFs that mutually antagonize each other’s activity
However, the ability of TFs to drive a cell into one and expression are often involved. One example is the an-
lineage does not yet explain how the initial decision for tagonistic interplay between the lineage-determining fac-
this lineage was made—it only explains how this decision tors PU.1 and GATA1 as a molecular mechanism of lineage
is then executed. What led to the up-regulation or activa- choice between myeloid and megakaryocytic –erythroid
tion of, for example, the executing TF remains unclear. One fate, respectively (Fig. 4D). PU.1 and GATA1 physically

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 Hematopoiesis

bind each other and cross-antagonize their activity (Zhang stem cell research have been defined in the hematopoietic
et al. 1999, 2000; Stopka et al. 2005; Liew et al. 2006). PU.1 system, and many novel technical approaches are first de-
directly inhibits GATA1 DNA-binding capacity, while veloped or applied in hematopoiesis research. Because of
GATA1 inhibits the transactivation potential of PU.1. the routine use of hematopoietic cells for clinical therapy,
Both PU.1 and GATA1 are autoregulatory for their own the chances for a quick transfer of novel basic insights to
expression (Nishimura et al. 2000; Okuno et al. 2005; Laio- patient benefit in the clinic are higher than for most other
sa et al. 2006a), thereby providing stability to their levels, tissues. Nevertheless, despite decades of successful research,
once expressed. The genetic ablation of these factors un- many questions posed very long ago are still awaiting an
derpins their central role in implementing lineage choice. answer, and long-standing disputes illustrate the need for
Loss of GATA1 demonstrates its absolute necessity for ever improving technological approaches. These continue
megakaryocyte/erythrocyte development, whereas PU.1 to be exciting times in hematopoiesis research.
deficiency leads to a lack of granulocytes, macrophages,
and B cells. Furthermore, both factors have instructive lin-
eage commitment ability by implementing lineage-affiliat- ACKNOWLEDGMENTS
ed gene programs (Iwasaki et al. 2003, 2006). However, M.A.R. is thankful for the support of the LOEWE Center
recent studies suggest a more complicated dynamic imple- for Cell and Gene Therapy Frankfurt (HMWK III L 4- 518/
mentation. In fish hematopoiesis, the interplay of PU.1 and 17.004 [2010]) and institutional funds of the Georg-
GATA1 differs in various cell stages during hematopoiesis Speyer-Haus. The Georg-Speyer-Haus is funded jointly
and is influenced by other factors, such as the transcription by the German Federal Ministry of Health (BMG) and
intermediate factor 1g (tif1g), a RING domain E3 ubiqui- the Ministry of Higher Education, Research and the Arts
tin ligase (Monteiro et al. 2011). Moreover, PU.1 showed of the state of Hessen (HMWK).
positive autoregulation in all analyzed cell stages, but
GATA1 only in some of them. C/EBPa and FOG1 have
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