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Review article
Signaling pathways governing stem-cell fate
Ulrika Blank,1 Göran Karlsson,1 and Stefan Karlsson1
1Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund
University Hospital, Lund, Sweden
Hematopoietic stem cells (HSCs) are historically the most thoroughly characterized type of adult stem cell, and the
hematopoietic system has served as a
principal model structure of stem-cell biology for several decades. However, paradoxically, although HSCs can be defined
by function and even purified to nearhomogeneity, the intricate molecular machinery and the signaling mechanisms
regulating fate events, such as selfrenewal and differentiation, have re-
mained elusive. Recently, several developmentally conserved signaling pathways
have emerged as important control devices of HSC fate, including Notch,
Wingless-type (Wnt), Sonic hedgehog
(Shh), and Smad pathways. HSCs reside
in a complex environment in the bone
marrow, providing a niche that optimally
balances signals that control selfrenewal and differentiation. These signaling circuits provide a valuable structure
for our understanding of how HSC regula-
tion occurs, concomitantly with providing information of how the bone marrow
microenvironment couples and integrates
extrinsic with intrinsic HSC fate determinants. It is the focus of this review to
highlight some of the most recent developments concerning signaling pathways
governing HSC fate. (Blood. 2008;111:
492-503)
© 2008 by The American Society of Hematology
Introduction
In tissues with a high cellular turnover, stem-cell populations
are essential for lifelong maintenance of organ function. At
present, somatic stem cells have been identified in several
self-renewing organs, including intestinal and epidermal epithelia as well as the blood cell system. Residing in the bone marrow
(BM) of adults, hematopoietic stem cells (HSCs) are ultimately
responsible for the continuous daily production of all mature
blood cell lineages.1 HSCs are functionally defined at the
single-cell level by their dual capacity for self-renewal and
multipotential differentiation. Self-renewal pertains to the process whereby at least one daughter cell of a dividing HSC retains
stem-cell fate and may be either symmetric or asymmetric in its
nature (Figure 1). A symmetric self-renewing division refers to
the process whereby both daughter cells retain stem cell
properties. Theoretically, this type of cell division expands the
stem-cell pool and is therefore thought to be important after
transplantation or after hematopoietic injury. In an asymmetric
self-renewing division, the 2 daughter cells adopt different
fates, resulting in one cell maintaining stem-cell properties.
On a population basis, this allows for steady-state maintenance of the HSC compartment. Self-renewal is critical
for sustaining the HSC compartment and thus is a prerequisite for
lifelong hematopoiesis.
Hematopoietic hierarchy
The hematopoietic system is hierarchically organized, with a
rare population of HSCs giving rise to progeny that progressively lose self-renewal potential and successively become more
Submitted July 20, 2007; accepted September 30, 2007. Prepublished online as
Blood First Edition paper, October 3, 2007; 10.1182/blood-2007-07-075168.
492
and more restricted in their differentiation capacity, finally
generating functionally mature cells (Figure 2). Phenotypic
characterization of HSCs has allowed for their prospective
isolation, and several groups have reported the successful
regeneration of all blood cell lineages after transplantation of a
single HSC into lethally irradiated mice.2,3 All functional HSCs
are associated with lack of expression of a range of cell surface
markers normally found on differentiating or mature blood cells
while displaying high levels of Sca1 and c-kit.4,5 This population
of cells is commonly referred to as the LSK compartment
(lineage⫺/Sca1⫹/c-kit⫹) and 100 LSK cells have been shown to
be sufficient for long-term multilineage repopulation of lethally
irradiated hosts.5 Based on their ability to self-renew, HSCs can
be divided into long-term and short-term reconstituting HSCs
(LT-HSCs and ST-HSCs, respectively). LT-HSCs have extensive
self-renewal capacity and can sustain lifelong hematopoiesis,
whereas ST-HSCs are restricted in self-renewal potential,
sustaining hematopoiesis only for a limited time in vivo.6-8 With
respect to self-renewal, the LSK compartment is heterogeneous,
containing a mixture of LT-HSCs, ST-HSCs, and multipotent
progenitor cells (MPPs). Further fractionation based on differential expression of CD34 and Flt3 has been shown to correlate
with distinct functional properties, with LT-HSCs being negative for both CD34 and Flt3.3,6,7 In addition to cell-surface
staining, exclusion of fluorescent dyes, such as Hoechst and
rhodamine, has been shown to correlate with HSC activity.9 The
side population defines a small and distinct subset of cells
with LT-HSC characteristics.10 More recently, Kiel et al identified the SLAM family of cell surface receptors that were
shown to be differentially expressed among stem and
progenitor populations.11
© 2008 by The American Society of Hematology
BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2
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BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2
SIGNALING PATHWAYS AND STEM CELL FATE
493
Symmetric
Blood vessel
HSC
HSC
HSC
HSC
Migration
Self-renewal
Apoptosis
Asymmetric
HSC
Differentiation
Figure 1. HSC fate decisions. During or after cell division, the 2 daughter cells of an HSC are faced with several options, or fate decisions. These include the possibility to
remain a HSC (the process of self-renewal), which can be either symmetric or asymmetric in its nature, to commit along the path of differentiation or to go through apoptosis. In
addition, the option to move to other anatomic sites in or outside the BM (migration) can be regarded as a fate decision, which is particularly important during specific stages of
ontogeny when HSCs seed the fetal liver or later the BM.
Efficient isolation of human HSCs has proven more difficult,
primarily as a consequence of lack of appropriate in vivo assays.
As of today, the rhodaminelow fraction of the Lin⫺CD34⫹CD38⫺
population of human cord blood (CB), containing putative HSCs
at a frequency of 1 in 30 cells, provides the most stringent
isolation of human HSCs.12 Populations containing putative
human HSCs can be functionally analyzed in nonobese diabetic/
severe combined immune deficient (NOD/SCID) mouse strains.13
SCID repopulating cells (SRCs) represent the most primitive
hematopoietic population assessable, but NOD/SCID assays are
hampered by the generally low engraftment of human cells and
the difficulty in keeping NOD/SCID mice for long-term analyses. This predicament can be overcome by serial NOD/SCID
transplantations, allowing for analysis of even more primitive
populations. However, such assays keep HSCs under constant
stress, which is why caution should be taken when interpreting
obtained data.
Regulation of hematopoietic stem cells, a
dynamic state of balance
To meet the need of a variety of situations, ranging from normal
homeostasis to acute blood loss or infection, hematopoiesis must be
rapidly yet meticulously controlled. Whether regarded as stochastic
mainly regulating probabilities or deterministic providing more
precise instructions, both intrinsic and extrinsic signals undoubtedly participate in the regulation of HSCs.1,14-16 Through reductionistic strategies, a wide variety of factors critical for HSC regulation
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BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2
BLANK et al
B cell
T cell
CLP
LSK compartment
LT-HSC
ST-HSC
NK cell
Granulocyte
MPP
GMP
Thy1.1 low
FLT3CD34Endoglin+
SP+
Rho low
CD150+
Thy1.1 low
FLT3CD34+
CD11b low
Macrophage
Thy1.1FLT3+
CD34+
CD11b low
CD4 low
CMP
Platelet
MEP
Erythrocyte
Figure 2. The hematopoietic hierarchy and phenotypic markers associated with HSCs. The LSK compartment contains LT-HSCs, ST-HSCs, and MPPs, each
subpopulation of which is associated with distinct phenotypic features as indicated. CLP indicates common lymphoid progenitor; CMP, common myeloid progenitor; GMP,
granulocyte/macrophage progenitor; and MEP, megakaryocyte/erythrocyte progenitor.
have been identified, including cytokines, growth factors, transcription factors, chromatin modifiers, and cell-cycle regulators. Delineating the signaling pathways that integrate these diverse components remains an enduring challenge of foremost importance,
which would have tremendous impact on regenerative medicine.
During the course of development, a handful of signaling pathways
shape the structures of the embryo and determine cell fate and
identity. In the adult organism, these signaling pathways are
reiterated, and it is becoming increasingly clear that most somatic
stem-cell compartments are under the influence of developmentally
conserved signals.
Regulation of HSCs by classic hematopoietic
cytokines
Numerous attempts have been made to use classic hematopoietic
cytokines for the purpose of expanding HSCs in vitro. Many of the
interleukins, particularly, interleukin (IL)-3, IL-6, and IL-11, as
well as Flt-3 ligand, thrombopoietin (TPO) and stem-cell factor
(SCF) have been investigated. In most cases, efforts to expand
HSCs have failed because of differentiation of HSCs and subsequent loss of reconstitution capacity. In one study, a modest
expansion of repopulating murine HSCs was observed using a
10-day culture protocol in the presence of IL-11, Flt-3 ligand, and
SCF.17 However, the same conditions failed to expand HSCs
derived from the fetal liver and later studies have found that the
Flt-3 receptor is not expressed on LT-HSCs.7,18 Furthermore, the
gp130 protein, which is a part of the receptor for IL-6 and IL-11
was suggested to play a role in the self-renewal of HSCs.19
However, elimination of the IL-11 receptor does not affect
hematopoiesis, making conclusions of the in vivo relevance
uncertain.20 Detailed studies of purified murine HSCs in recent
years have shown that the receptors for TPO and SCF, c-mpl and
c-kit, respectively, are both expressed on repopulating HSCs,6,7,21-24
and it has been shown that mice with genetic mutations in TPO or
c-mpl have a reduction in HSCs.21,22 Moreover, TPO has been
reported to support viability25 and suppressed apoptosis of HSCs.26
Thus, the main function of TPO may be to counteract apoptosis of
HSCs rather than promote their expansion.24
A further role for TPO in regulating HSC self-renewal was recently
suggested through the association with Lnk, an intracellular adaptor
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BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2
SIGNALING PATHWAYS AND STEM CELL FATE
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Figure 3. Notch and Wnt signaling pathways. (A) Notch pathway: When ligands of the Delta (Delta1-3) or Jagged (Jagged 1-2) families bind to the Notch receptor,
proteolytic events involving ␥-secretase lead to release and translocation of the intracellular domain of the receptor (NICD) to the nucleus. Subsequently, NICD will form
a complex with the transcription factor CSL and cofactors of the Mastermind-like (MAML) family to activate transcription of target genes.138 (B) Wnt pathway: Wnt
signaling is initiated when a ligand binds to the Frizzled and lipoprotein receptor-related protein (LRP) receptors at the cell surface. In the absence of Wnt ligands, the
downstream signal transducer ␤-catenin is trapped by adenomatous polyposis coli (APC) and Axin in a destruction complex, where it is phosphorylated by
casein-kinase 1␣ (CK1␣) and glycogen synthase kinase (GSK3␤). Phosphorylation ultimately leads to ubiquitination and degradation of ␤-catenin. On ligand binding,
Frizzled forms a complex with disheveled (Dsh), whereas LRP is phosphorylated, resulting in Axin relocation to the cell membrane. Subsequently, the destruction
complex is dispersed and ␤-catenin accumulates and translocates to the nucleus where it interacts with the T-cell factor/lymphoid enhancer factor (TCF) transcription
factors to regulate gene expression.139,140
molecule functioning as a broad inhibitor of several cytokine signaling
pathways, including TPO, SCF, erythropoietin, IL-3, and IL-7.24,27-30
Mice deficient in Lnk have been reported to exhibit increased HSC
numbers and their repopulation function was elevated, a phenotype
attributable to augmented proliferation and self-renewal.24,27,31 Interestingly, the increased self-renewal capacity of Lnk⫺/⫺ HSCs was dependent on TPO, and Lnk-negative HSCs were shown to be hypersensitive
to stimulation by TPO.24,27 Thus, TPO and Lnk serve opposing roles in
the regulation of HSC self-renewal and Lnk acts as a powerful inhibitor
of TPO signaling in HSCs. To conclude, of the classic hematopoietic
cytokines, the most important positive regulators of HSCs are
TPO and SCF.
Notch pathway: at the interface of osteoblasts
and HSCs
In humans, Notch was discovered 15 years ago as an oncogene
that, after chromosomal translocation, gave rise to T-cell
leukemia.32 Since then, several loss- and gain-of-function
studies have demonstrated a critical role for Notch in lineage
specification of lymphopoiesis, where active Notch signaling
(Figure 3A) drives differentiation toward T cells.33 It was soon
suggested that Notch could have a role in HSC biology as
primitive hematopoietic cells as well as cells in the putative
HSC microenvironment expressed both Notch and Notch-ligand
mRNA.34,35 Indeed, through various approaches, several groups
have shown that members of the Notch ligand families, Delta
and Jagged, can expand both murine and human hematopoietic
progenitors in vitro as measured by reconstitution assays.35-41
Importantly, enforced activation of the Notch signaling pathway
or the downstream target Hes-1, have been demonstrated to
increase the self-renewal capacity of long-term in vivo repopulating HSCs42,43 or even to immortalize primitive hematopoietic
progenitor cells.44 When Calvi et al established osteoblasts as
putative members of the HSC niche, they demonstrated how
constitutively active parathyroid hormone receptor expanded
these cells and stimulated their expression of Jagged-1.45 The
activated osteoblasts were able to increase self-renewal of
primitive hematopoietic cells as measured by LTC-ICs and
competitive repopulation assays.45 Moreover, LSK cells from
these transgenic mice had increased levels of Notch intracellular
domain (NICD), and the elevated LTC-IC numbers could be
normalized with a ␥-secretase inhibitor.45 Supporting these data
were the observation by Duncan et al that a Notch reporter was
active in c-kit⫹ hematopoietic cells in the trabecular bone and
isolated LSK and side population cells.46
Together these findings indicate not only that osteoblasts are
part of the so-called HSC niche but also that Jagged/Notch
signaling activated by these cells might be important in the
extracellular regulation of HSC self-renewal.45 However, HSCs
deficient in Notch-1 could reconstitute Jagged-1 deficient hosts in a
normal fashion, suggesting that Notch signaling through mechanisms including these molecules is dispensable for in vivo HSC
function.47 It is probable that the lack of phenotype observed in this
setting is the result of compensatory mechanisms mediated by
other Notch receptors and ligands, which is why approaches to
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496
BLANK et al
broadly eliminate Notch signaling would be informative. Regardless of what the case may be, manipulation of the Notch signaling
pathway holds great promise for ex vivo expansion of HSCs in
future clinical protocols.
HSC self-renewal under control by the Wnt
signaling pathway
A role for Wnt signaling in the regulation of murine HSCs was
suggested a decade ago when expression of different Wnts was
discovered at sites of fetal hematopoiesis and Wnt5a was shown to
stimulate proliferation and self-renewal of fetal HSCs/progenitors
in vitro.48 Adult HSCs also appear to respond to Wnt treatment
because exposure to purified Wnt3a was recently shown to increase
self-renewal of murine HSCs in vitro, as determined by in vivo
reconstituting assays.49 Moreover, Van den Berg et al demonstrated
that the positive effects on HSCs were not exclusive to the murine
setting because human Lin⫺CD34⫹ cells exposed to WNTs
also expanded in vitro as measured by immunophenotype and
colony assays.50
Not only is there evidence supporting the hypothesis that Wnts
could be used as tools for expanding HSCs in culture, but WNT
proteins have also been reported to be expressed in both human
fetal BM stroma and adult BM, where WNT5A has been shown to
be expressed in the primitive Lin⫺CD34⫹ population.50 This would
suggest that Wnts are physiologically important and act on HSCs
through both paracrine and autocrine mechanisms. In support of the
latter statement was the finding that exposure of HSCs in vitro to a
soluble Wnt binding antagonist reduced their proliferation capacity.51 However, the intracellular mechanisms through which Wnts
exercise their effect on HSCs are somewhat inconclusive. Canonical Wnt signaling (Figure 3B) occurs through mechanisms involving the stabilization and nuclear translocation of ␤-catenin, followed by transcriptional regulation of target genes by a ␤-catenin/
T-cell factor complex. Reya et al detected active canonical Wnt
signaling, as measured by a T-cell factor reporter construct, in a
large proportion of the HSC enriched LSK compartment in adult
BM.51 Intriguingly, retroviral–mediated enforced expression of
␤-catenin resulted in a 102 to 103 fold increase in HSCs during
long-term cultures of KTLS cells as defined by both phenotype and
function.51 In accordance with the observed phenotype resulting
from forced expression of ␤-catenin was an up-regulation in
expression of the HSC self-renewal stimulating genes Notch1 and
HoxB4.51 However, when ␤-catenin was conditionally expressed in
vivo under the control of the ROSA26 locus, no increase in HSC
self-renewal was observed.52 Adding to the contradiction, deletion
of the ␤-catenin gene in HSCs failed to result in an in vivo
phenotype.53
The reasons for these discrepancies are unclear but may reflect a
stronger role for Wnt signaling in in vitro expansion of HSCs as
opposed to its function in vivo. The more complex and enduring in
vivo setting might allow for redundant mechanisms to develop.
Alternatively, the HSC expansion observed when ␤-catenin was
retrovirally overexpressed might be affected by the use of Bcl-2
transgenic HSCs in that particular study.51 Thus, even though Wnts
may possibly be useful for expanding HSCs in vitro, the unresolved
mechanisms behind the effect and the question pertaining to the
role of canonical Wnt signaling remains to be determined.
BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2
Integration of Wnt and Notch signals in
HSC regulation
Recently, an intriguing report presented evidence that Notch and
Wnt pathways act in synergy to maintain the HSC pool as both
signaling circuits were shown to be active simultaneously in a large
fraction of cells in the trabecular bone.46 Even though Notch- and
Wnt signaling both are implied to stimulate self-renewal of HSCs,
Duncan et al propose that they do so through different mechanisms.46 As Wnt signaling seems to induce proliferation and
support viability of LSK cells, inhibition of the Notch pathway had
no effect on these functions.46 Instead, Notch signaling was
demonstrated to be imperative in maintaining HSCs in an undifferentiated state, even if Wnt3a was present in the culture.46 These
findings suggest that Wnt and Notch signaling together could play a
role in self-renewal of HSCs. Moreover, observations that Wnt3a
regulate expression of established Notch target genes46 and that
inhibition of the Wnt signaling component GSK-3 affect HSC fate
options through mechanisms involving regulation of both Wnt and
Notch target genes54 suggest that the 2 pathways belong to a
network of regulatory circuits controlling the HSC pool.
Smad signaling pathway
The Smad signaling pathway embodies an evolutionary ancient
signaling circuitry, which functions to transduce signals downstream of the transforming growth factor-␤ (TGF-␤) family of
ligands (Figure 4). Apart from TGF-␤, activins and bone morphogenetic proteins (BMPs) also belong to this superfamily, which
regulate a bewildering array of fundamental biologic processes
during development and postnatally. Because of the highly redundant nature of the Smad pathway and early embryonic lethality of
most knockout mouse models, the precise role of the Smad
circuitry in hematopoiesis has remained nebulous. However, recently several reports have shed new light suggesting that this
pathway plays a pivotal role in the regulation of HSC fate
decisions.
TGF-␤, key negative modulator of HSCs in vitro
TGF-␤ is generally catalogued as one of the most potent inhibitors
of HSC growth in vitro, and ample evidence from a variety of
culture systems exist to support this fact.55-57 A hallmark feature of
HSCs is their relative quiescence, and given the strong growth
inhibitory properties of TGF-␤, it has naturally been hypothesized
to be a cardinal regulator of HSC quiescence in vivo. In keeping
with this, neutralization of TGF-␤ in vitro was shown to release
early hematopoietic progenitor cells from quiescence.58-60 Several
molecular mechanisms have been proposed to account for TGF-␤–
mediated growth inhibition, including alterations in cytokine
receptor expression and up-regulation of cyclin-dependent kinase
inhibitors, such as p21 and p57.59,61-69
TGF-␤ can affect most cell types throughout the hematopoietic
hierarchy and depending on the context and differentiation stage of
the target cell different biologic responses are ultimately elicited.70-72 In vivo, TGF-␤ plays a principal role as regulator of
immune cell homeostasis and function, as unambiguously evidenced by the development of a lethal inflammatory disorder of
both TGF-␤1- ligand and receptor knockout mice.73-75 Furthermore, Tgf-␤1 null mice exhibited enhanced myelopoiesis, suggesting that TGF-␤ acts as a negative regulator of myelopoiesis in
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BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2
Figure 4. Smad signaling pathway. Smad pathway:
TGF-␤ family members bind and signal through 2 types
of serine/threonine kinase receptors, type I and type II,
both of which are necessary for signal transduction. On
ligand binding and receptor activation, the Smad proteins are activated through phosphorylation by type I
receptors.141,142 Three groups of Smads have been
identified: receptor-activated Smads (R-Smads), commonpartner Smads (Co-Smads), and inhibitory Smads
(I-Smads). In general, TGF-␤ and activin signal via
R-Smad2 and 3, whereas BMP signals are transduced
through R-Smad1, 5, and 8. Phosphorylated R-Smads
subsequently associate with the Co-Smad4, creating a
complex that translocates to the nucleus where target
gene transcription is modified. In contrast to R- and
Co-Smads, the I-Smads, Smad6 and Smad7, function
in a negative feedback loop to prevent activation of
R-Smads.143-145 Divergence and convergence of the
Smad signaling circuitry are depicted. Commonly used
alternative names include: ALK2/Activin type I receptor,
ALK3/BMP type IA receptor, ALK4/Activin type IB receptor, ALK5/TGF-␤ type I receptor, and ALK6/BMP type IB
receptor. The shaded portion indicates the nucleus.
P indicates phosphorylation.
SIGNALING PATHWAYS AND STEM CELL FATE
Ligand
BMP
TGF-beta
Activin/Nodal
497
Receptor
ActR-IIA
ActR-IIB
BMPR-II
ActR-IIA
ActR-IIB
TGF-b RII
ALK4
ALK7
ALK1
ALK5
ALK2
ALK3
ALK6
I-Smads
Smad2
R-Smads
Smad3
Smad5
Smad7
Smad4
R-Smads
Smad8
Co-Smad
Cytoplasm
Nucleus
P
DNA-binding partner
R-Smad
Smad4
P
R-Smad
vivo.76 However, despite the pronounced role of TGF-␤ in vitro,
mice deficient of the TGF-␤ type I receptor displayed normal HSC
self-renewal and regenerative capacity in vivo, even under extreme
hematopoietic stress.77,78 The apparent discrepancy between in
vitro and in vivo findings may reflect mechanisms of redundancy
between other type I receptors or alternatively other ligands such as
activin, which signals through the same R-Smad pathway.
Smad1
Smad6
Smad4
Target gene
formation was assessed, indicative of increased self-renewal.
However, conditional deletion of Smad5 in adult mice does not
affect hematopoiesis, and HSCs lacking Smad5 have a normal
differentiation and regenerative potential in vivo.88 It is possible
that BMP–activated Smad signaling plays a more pronounced role
early in hematopoietic development, as opposed to adult hematopoiesis. Alternatively, redundant mechanisms within the Smad pathway may explain the lack of phenotype in the adult setting.
BMP promotes maintenance of HSCs in culture
BMPs have been implicated as key regulators of hematopoietic development in a variety of species, from zebrafish to mouse.79-83 In the context
of adult hematopoiesis, BMP-4 was shown to promote maintenance of
HSCs in culture, whereas lower concentrations of BMP-4 induced
proliferation and differentiation of human hematopoietic progenitors.84
Furthermore, Shh induced proliferation of primitive human hematopoietic progenitors in vitro, apparently through a BMP-4–dependent
mechanism.85 However, although BMP-4 has been shown to maintain
human NOD/SCID repopulating cells in culture, it does not seem to
cause an expansion, suggesting that Shh may act through additional
mechanisms. In the murine system, BMP-4 does not appear to affect
proliferation of purified HSCs in vitro, although it is currently unclear
whether BMP-4 can extend the maintenance of murine HSCs in
culture.86 Furthermore, BMPs function to regulate the HSC niche, thus
indirectly controlling HSC numbers as discussed in “HSC niche.”
A role for Smad5, traditionally characterized as a mediator of
BMP signals, in self-renewal was implicated through a series of in
vitro studies. Interestingly, the number of colonies, derived from
yolk sacs or ES cell differentiated embryoid bodies deficient of
Smad5, were found to be increased.87 Smad5 deficient colonies also
had an enhanced regenerative potential when secondary colony
Smad signaling: toward a deeper understanding
To block the entire Smad signaling pathway, thus sidestepping
redundant mechanisms, the inhibitory Smad7 was overexpressed in
murine HSCs using a retroviral gene transfer approach.89 Forced
expression of Smad7 significantly increased the self-renewal
capacity of HSCs in vivo, indicating that the Smad pathway
negatively regulates self-renewal in vivo. In a similar approach
using human SCID repopulating cells (SRCs), overexpression of
Smad7 resulted in a shift from lymphoid dominant engraftment
toward increased myeloid contribution.90 Thus, in the xenograft
model system, forced expression of Smad7 modulates cell fate
decisions of primitive multipotent human SRCs.
It is beyond doubt that the Smad signaling pathway lies at the
very core of mediating signals from the TGF-␤ family of ligands.
However, a considerably more diversified picture is now emerging,
which indicates that the Smad circuitry is more varied than
previously thought.91 Using a conditional knockout mouse model,
disruption of the entire Smad pathway at the level of Smad4 was
recently investigated. Intriguingly, Smad4 deficient HSCs displayed a significantly reduced repopulative capacity of primary and
secondary recipients, indicating that Smad4 is critical for HSC
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BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2
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self-renewal in vivo.92 Because overexpression of Smad7 versus
deletion of Smad4 would be anticipated to yield similar hematopoietic phenotypes, it is conceivable that Smad4 functions as a
positive regulator of self-renewal independently of its role as a
central mediator of the canonical Smad pathway. The precise
molecular mechanism for this is currently unknown, but it is
possible that Smad4 participates in other signaling cascades such as
Wnt or Notch.93-95 Moreover, He et al proposed an elegant model
for how TGF-␤ mediates erythroid differentiation concomitantly
with balancing growth inhibition in human hematopoietic stem/
progenitor cells.96 According to this model, transcriptional intermediary factor1␥ (TIF1␥) selectively binds Smad2 and Smad3 in
competition with Smad4. In response to TGF-␤, the TIF1␥/
Smad2/3 complex was shown to stimulate erythroid differentiation
of human hematopoietic progenitors, whereas Smad2/3 in association with Smad4 inhibited proliferation of the same cells. Thus, the
relative abundance of Smad4 and TIF1␥ is likely to be important
for determining the effect of TGF-␤ stimulation. Interestingly, the
zebrafish homolog of TIF1␥, encoded by moonshine, has been
shown to be essential for blood formation with mutants displaying
severe red cell aplasia, indicating that the function of TIF1␥ may be
preserved across species.97
However, the mechanism through which TIF1␥ cooperates
with Smads is not entirely clear. Evidence from Xenopus has
suggested that TIF1␥, known as Ectodermin, acts as a ubiquitin
ligase for Smad4, thus functioning as a direct inhibitor of Smad4
downstream of TGF-␤ and BMP signaling.98 This dichotomy
may reflect differences in cellular context or alternatively
species or temporal differences. Another interesting observation
is the association between Smad4 and Hox proteins. Homeobox
(hox) genes encode transcription factors that function as paramount regulators of hematopoiesis and are frequently dysregulated in human leukemia, particularly acute myeloid leukemia.
Enforced expression of HoxA9 immortalizes and blocks myeloid differentiation, eventually causing acute myeloid leukemia.99 Recently, Wang et al described a mechanism whereby
TGF-␤/BMP inhibited the bone marrow transformation capacity
of HoxA9 and HoxA9-Nup98 fusion protein through a Smad4dependent mechanism. Accordingly, Smad4 was shown to
interact directly with HoxA9 and Nup98-HoxA9 fusion protein,
thus precluding their DNA binding capacity and subsequent
transcriptional activity.100 Whether Smad4 mutations facilitate
leukemogenesis through increased Hox activity remains an
unanswered question.
Angiopoietin-like proteins
Building on the fact that the fetal liver (FL) is a powerful site of
HSC expansion during development, Lodish et al embarked on an
approach to identify “stromal” cells and their expression pattern of
molecules responsible for stimulation of HSC self-renewal in the
murine FL. Ter119⫹CD3⫹ cells were recognized as a population of
cells derived from the FL that could support expansion of HSCs in
vitro. Insulin-like growth factor 2 (IGF-2) was identified on the
basis of being expressed in CD3⫹ FL cells and was shown to
modestly expand HSCs in vitro. More interestingly, they found that
several angiopoietin-like (Angptl) proteins were expressed in these
support cells for HSC growth. When highly enriched HSCs were
cultured under serum free conditions in the presence of TPO, SCF,
IGF-2, and Angptl protein 2 or 3, a robust increase in repopulating
HSCs was observed after 10 days of in vitro culture.101 More
specifically, a 24- to 30-fold increase in HSC number was
observed. Angptl proteins 5 and 7 were also reported to expand
murine HSCs, whereas Angptl protein 4 failed to show a similar
effect. The molecular mechanisms and the signaling events downstream of Angptl proteins remain unidentified and their receptors
are currently unknown, which is why it is not clear how the Angptl
proteins expand HSCs. However, Angptl protein 2 has been shown
to bind to a highly enriched population of HSCs. Therefore, the
expansion effect attributable to these proteins is likely to be a result
of a direct action on HSCs. It will be exciting to see whether the
Angptl proteins can also be used for robust expansion of human
HSCs, particularly HSCs derived from cord blood.
These findings are important especially in light of the convenience of
adding these factors to cultures of HSCs in vitro, and they can therefore
be used in future attempts to expand HSCs for the clinic and possibly
also to increase the efficiency of gene delivery to HSCs.
HSC niche
On the role of osteoblasts
The spatial organization of hematopoietic cells within the BM,
with primitive cells distributed closer to the bone surface and
more differentiated cells positioned closer to the BM longitudinal axis of the femur, has long been recognized.102,103 As early as
1978, Schofield proposed the niche hypothesis of a physiologically limited microenvironment supporting stem cells in vivo.104
Because of the anatomic complexity of mammalian stem cell
niches, the precise location as well as the cellular and molecular
basis for these structures has only recently begun to be
uncovered. Several lines of evidence now converge to suggest
that the osteoblasts, cells of mesenchymal origin positioned at
the endosteal surface of bone, are essential components of the
HSC niche.105 Using a transgenic mouse model expressing a
constitutively active Parathyroid hormone receptor under an
osteoblast specific promoter (␣1(I) collagen), Calvi et al demonstrated that an increase in trabecular osteoblasts was accompanied by an increase in HSC numbers.45 Importantly, Calvi et al45
propose a model in which increased parathyroid hormone
signaling in osteoblasts results in elevated levels of the Notch1
ligand, Jagged1, ultimately generating increased activation of
Notch1 signaling in HSCs. An independent study investigated
the role of the BMP signaling pathway to regulate HSC
numbers, by conditional inactivation of the BMP type IA
receptor Alk3.106 In the absence of Alk3 the number of spindle
shaped osteoblastic cells expressing N-cadherin were increased.
Interestingly, this correlated with an increase in the number of
HSCs, which were found attached to N-cadherin⫹ osteoblasts.
Moreover, depletion of osteoblasts in vivo was associated with
decreased BM cellularity and extramedullary hematopoiesis,
further emphasizing the importance of osteoblasts for HSC
function.107 In addition, Arai et al identified Tie2/Angiopoietin1
signaling to be critical for the maintenance of HSCs in a
quiescent state in the BM niche.108 Tie2 is a receptor tyrosine
kinase expressed on endothelial cells and HSCs, whereas
Angiopoietin1 (Ang1) is expressed by osteoblasts. Tie2⫹ HSCs
adhered to Ang1 expressing osteoblasts, supposedly through a
mechanism involving N-cadherin.
Even though osteoblasts clearly constitute part of the HSC
niche, they are probably not the only players. Apart from soluble
factors, matrix components and other cellular constituents are
likely to also have important functions. One example is
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BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2
Figure 5. The HSC niche. HSCs are positioned in
close proximity to osteoblasts at the endosteal surface
of bone. Other factors contributing to the in vivo
microenvironment of HSCs include extracellular matrix
(ECM), soluble compounds, as well as other cellular
components.
SIGNALING PATHWAYS AND STEM CELL FATE
499
Osteoblasts
Endosteal bone surface
ECM
Ca 2+
Ca 2+
Ca 2+
HSC
BM cell
Soluble factors
osteopontin, a matrix glycoprotein synthesized by osteoblasts, which
has been associated with negative regulation of the HSC compartment.109,110 Furthermore, HSCs deficient in the calcium-sensing receptor
were profoundly defective in localizing anatomically to the endosteal
niche, a behavior that correlated with diminished adhesion to the
extracellular matrix protein collagen I.111 These data indicate that the
local calcium gradient is involved in retaining HSCs in close physical
proximity to the endosteal surface of bone. Furthermore, it was recently
shown that a defective niche could result in HSC disorders further
emphasizing the regulatory function of the HSC niche in vivo.112,113
Hypoxia and reactive oxygen species (ROS)
Another peculiar property of the BM microenvironment is its
hypoxic nature, a fact recently highlighted by Parmar et al showing
that HSCs are distributed predominantly at the lowest end of an
oxygen gradient within the BM.114 This observation bears importance for several findings, which indicate that the level of oxidative
stress influences HSC function. For example, mice deficient of the
cell-cycle regulator Atm developed early onset BM failure, a
phenotype accompanied by elevated levels of ROS in HSCs.115
Interestingly, ROS appear to activate the p38/MAPK pathway
causing quiescent HSCs to cycle more frequently and eventually
become exhausted.116 Moreover, members of the FoxO subfamily
of forkhead transcription factors have been shown to protect HSCs
from oxidative stress by up-regulating genes involved in their
detoxification. Triple knockout mice of FoxO1, FoxO3, and FoxO4
exhibited defective long-term repopulating activity that correlated
with increased cycling and apoptosis of HSCs as well as increased
levels of ROS.117 Similarly, the HSC compartment of FoxO3a null
mice suffers from augmented levels of ROS.146 As previously
reported for mice deficient of Atm, the HSC defect resulting from
loss of FoxOs could be rescued by administration of the antioxidant
N-acetyl-L-cysteine. In light of these observations, it is conceivable that the hypoxic environment in which the HSCs reside may
serve to protect them from oxygen radicals ultimately keeping
them quiescent.
It’s not all in the niche
Despite the plethora of evidence pointing at the importance of the BM
milieu, it would be premature to assume that the microenvironment is
the sole regulator of HSC fate options. For instance, during ontogeny
before the establishment of the BM niche, HSCs undergo extensive
self-renewal in the fetal liver. Indeed, HSCs derived from fetal liver have
been shown to encompass a more efficient regenerative potential
compared with their adult counterparts when transplanted into irradiated
recipients.118 Recent evidence uncovered by Bowie et al suggest the
existence of an intrinsically regulated developmental switch, which
abruptly changes the self-renewing properties of HSCs at the age of 3 to
4 weeks in the mouse.119 At this time the HSC compartment rapidly
changes from a fully cycling population to a largely quiescent one.119
This change appears to be independent of the niche as it occurs well after
the migration of HSCs to the BM and because it is not affected by
transplantation into adult recipients.120 Interestingly, juvenile HSCs
exhibit a greater sensitivity to SCF; thus, signaling events downstream
of the c-kit receptor may be developmentally and intrinsically regulated,
ultimately determining symmetric versus asymmetric self-renewal.121
To summarize, HSC behavior is regulated by joint efforts from intrinsic
factors and environmental cues, both entities that are integrated in a
more comprehensive and holistic view by the concept of the stem-cell
niche (Figure 5).
Stem-cell expansion for cell and gene therapy
The process of finding donors that have compatible histocompatibility antigens for patients who need blood and marrow transplantation is often a challenge of significant magnitude. As of today,
umbilical CB is being used increasingly as a source of HSCs because of
the common availability of CB cells and the diversity of histocompatibility gene haplotypes that are available in banked CB samples.122 The
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BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2
BLANK et al
number of HSCs in each CB sample is limited, and it would therefore
greatly increase the applicability of this stem-cell source if the CB stem
cells could be expanded ex vivo before transplantation. Stem-cell
expansion can also theoretically be accomplished in vivo after engraftment of the transplanted cells, but this approach may involve greater
risks compared with ex vivo expansion unless the growth stimuli used in
vivo can be carefully controlled.123,124 Moreover, because culture
conditions can be precisely defined and transient modulation of regulatory pathways more easily used in vitro, stem-cell expansion ex vivo
presents an attractive approach. However, even this tactic has proven a
great challenge because a successful stem-cell expansion involves
symmetric self-renewal divisions of HSCs, where both daughter cells
retain HSC properties.123 More commonly, HSCs grown in vitro
undergo asymmetric divisions characterized by the production of one
HSC and a more differentiated progenitor, or alternatively, a symmetric
division where both progeny cells have lost their HSC potential.
Positive and negative regulators ultimately balance the transition from quiescence to proliferation of HSCs. Thus, the strategies
for stem-cell expansion should involve activation of regulators that
encourage HSC self-renewal and/or inhibition of pathways that
mediate quiescence, differentiation, or apoptosis of HSCs. Because
of the recent advances in understanding the mechanisms that
control HSC self-renewal, novel methods to expand stem cells can
now be developed.70,123,124 Some approaches require viral vectormediated gene transfer to HSCs for efficient expansion. Ideally, the
vectors should be nonintegrating and mediate transient gene
expression for safety reasons.125-127 However, the safest approaches
would involve soluble factors, for example, cytokines, developmental cues or factors like the Angptl proteins.
When the transcription factor HoxB4 was expressed in HSCs ex
vivo, a more than 40-fold net expansion of murine HSCs was generated
over a period of 10 to 14 days.128 Furthermore, modified HoxB4
proteins have been used successfully to stimulate proliferation of human
and murine HSCs ex vivo.129,130 The soluble form of the HoxB4 protein
could actively be taken up by HSCs and could be used without concerns
for insertional mutagenesis or the potential adverse effects that continuous production of HoxB4 in HSCs might generate in vivo. However, the
HSC expansion achieved by the soluble HoxB4 was quite modest
compared with overexpression of HoxB4 within HSCs. Developmental
cues that activate Notch and Wnt signaling in HSCs may also be useful
to increase HSC self-renewal ex vivo. As mentioned previously, Wnt3A
has been shown to expand murine repopulating HSCs and Wnt5A
injection into NOD/SCID mice repopulated with human hematopoietic
cells increased the reconstitution and number of primitive hematopoietic
cells. Similarly, a soluble form of the Notch ligand, Jagged 1, was
reported to stimulate growth of human SRCs and may therefore be used
to expand stem cells ex vivo.35 However, the Angptl proteins are by far
the most promising soluble factors identified to date for expansion of
murine HSCs.101 If the angiopoietin-like proteins would prove to expand
human HSCs as efficiently as in the murine system, they may become a
promising tool for future cell and gene therapy approaches. Furthermore, other positive regulatory factors, in combination with Angptl
proteins, may be able to expand human HSCs by a factor of 10 during a
relatively short and safe culture period, suitable for GMP conditions that
will be required for the clinic. Worthy of mention are also fibroblast
growth factors, which have been shown to sustain primitive murine
hematopoietic cells in culture and to maintain their primitive phenotype.131,132 Interestingly, fibroblast growth factor-1 in conjunction with
SCF, TPO, and IGF-2 resulted in a 20-fold increase of LT-HSCs during a
10-day culture period.133
It should be emphasized that most of the knowledge about stem- cell
expansion has come from studies in mice. There is a difference between
mouse and human HSCs with regards to cytokine receptors, telomere
biology, and proliferative capacity, and there will therefore be both
differences and similarities in the approaches used to expand mouse and
human HSCs. Recently, the protein nephroblastoma overexpressed
(NOV/CCN3) has been shown to expand primitive human HSCs, and
this protein should therefore be carefully evaluated for use in clinical
stem-cell expansion protocols.134 As more detailed knowledge is unearthed concerning the regulatory pathways that govern HSC selfrenewal, it may even be possible to modulate these pathways with small
molecule drugs as has been shown in the modulation of Wnt signaling
and the expression of p21 and HoxB4.54,135,136 As an example, chemicals
that enhance prostaglandin E2 synthesis were recently reported to
increase HSC numbers in zebrafish and mice.137 Undoubtedly, however,
a major challenge still ahead will be the integration of major signaling
pathways into networks of control mechanisms ultimately coupling
extrinsic with intrinsic regulatory fate determinants. Such knowledge
would open up new avenues for maintaining and expanding HSCs in
vitro.
Acknowledgments
This work was supported by the European Commission (INHERINET,
Gene Therapy of Hematopoietic Stem Cells for Inherited Diseases, and
CONSERT, Concerted Safety and Efficiency Evaluation of Retroviral
Transgenesis for Gene Therapy of Inherited Diseases); Swedish Gene
Therapy Program; Swedish Medical Research Council; Swedish Children Cancer Foundation; Clinical Research Award from Lund University Hospital; Joint Program on Stem Cell Research; Juvenile Diabetes
Research Foundation; and Swedish Medical Research Council (S.K.),
the Wenner-Gren Foundations (U.B.), and Kungliga Fysiografiska
Sällskapet (G.K. and U.B.). The Lund Stem Cell Center is supported by
a Center of Excellence grant in life sciences from the Swedish
Foundation for Strategic Research.
Authorship
Contribution: All authors contributed to the writing of this
manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Stefan Karlsson, Molecular Medicine and
Gene Therapy, Lund University Hospital, BMC A12, 221 84 Lund,
Sweden; e-mail: [email protected].
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2008 111: 492-503
doi:10.1182/blood-2007-07-075168 originally published
online October 3, 2007
Signaling pathways governing stem-cell fate
Ulrika Blank, Göran Karlsson and Stefan Karlsson
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