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Signaling Pathways Governing Stem-Cell Fate: Review Article
Signaling Pathways Governing Stem-Cell Fate: Review Article
Hematopoietic stem cells (HSCs) are his- mained elusive. Recently, several develop- tion occurs, concomitantly with provid-
torically the most thoroughly character- mentally conserved signaling pathways ing information of how the bone marrow
ized type of adult stem cell, and the have emerged as important control de- microenvironment couples and integrates
hematopoietic system has served as a vices of HSC fate, including Notch, extrinsic with intrinsic HSC fate determi-
principal model structure of stem-cell bi- Wingless-type (Wnt), Sonic hedgehog nants. It is the focus of this review to
Introduction
In tissues with a high cellular turnover, stem-cell populations and more restricted in their differentiation capacity, finally
are essential for lifelong maintenance of organ function. At generating functionally mature cells (Figure 2). Phenotypic
present, somatic stem cells have been identified in several characterization of HSCs has allowed for their prospective
self-renewing organs, including intestinal and epidermal epithe- isolation, and several groups have reported the successful
lia as well as the blood cell system. Residing in the bone marrow regeneration of all blood cell lineages after transplantation of a
(BM) of adults, hematopoietic stem cells (HSCs) are ultimately single HSC into lethally irradiated mice.2,3 All functional HSCs
responsible for the continuous daily production of all mature are associated with lack of expression of a range of cell surface
blood cell lineages.1 HSCs are functionally defined at the markers normally found on differentiating or mature blood cells
single-cell level by their dual capacity for self-renewal and while displaying high levels of Sca1 and c-kit.4,5 This population
multipotential differentiation. Self-renewal pertains to the pro- of cells is commonly referred to as the LSK compartment
cess whereby at least one daughter cell of a dividing HSC retains (lineage⫺/Sca1⫹/c-kit⫹) and 100 LSK cells have been shown to
stem-cell fate and may be either symmetric or asymmetric in its be sufficient for long-term multilineage repopulation of lethally
nature (Figure 1). A symmetric self-renewing division refers to irradiated hosts.5 Based on their ability to self-renew, HSCs can
the process whereby both daughter cells retain stem cell be divided into long-term and short-term reconstituting HSCs
properties. Theoretically, this type of cell division expands the (LT-HSCs and ST-HSCs, respectively). LT-HSCs have extensive
stem-cell pool and is therefore thought to be important after self-renewal capacity and can sustain lifelong hematopoiesis,
transplantation or after hematopoietic injury. In an asymmetric whereas ST-HSCs are restricted in self-renewal potential,
self-renewing division, the 2 daughter cells adopt different sustaining hematopoiesis only for a limited time in vivo.6-8 With
fates, resulting in one cell maintaining stem-cell properties. respect to self-renewal, the LSK compartment is heterogeneous,
On a population basis, this allows for steady-state main- containing a mixture of LT-HSCs, ST-HSCs, and multipotent
tenance of the HSC compartment. Self-renewal is critical progenitor cells (MPPs). Further fractionation based on differen-
for sustaining the HSC compartment and thus is a prerequisite for tial expression of CD34 and Flt3 has been shown to correlate
lifelong hematopoiesis. with distinct functional properties, with LT-HSCs being nega-
tive 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
Hematopoietic hierarchy side population defines a small and distinct subset of cells
with LT-HSC characteristics.10 More recently, Kiel et al identi-
The hematopoietic system is hierarchically organized, with a fied the SLAM family of cell surface receptors that were
rare population of HSCs giving rise to progeny that progres- shown to be differentially expressed among stem and
sively lose self-renewal potential and successively become more progenitor populations.11
Submitted July 20, 2007; accepted September 30, 2007. Prepublished online as © 2008 by The American Society of Hematology
Blood First Edition paper, October 3, 2007; 10.1182/blood-2007-07-075168.
Blood vessel
Symmetric
HSC
HSC HSC
HSC
Migration
Asymmetric
Apoptosis 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, stress, which is why caution should be taken when interpreting
primarily as a consequence of lack of appropriate in vivo assays. obtained data.
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 Regulation of hematopoietic stem cells, a
human HSCs can be functionally analyzed in nonobese diabetic/ dynamic state of balance
severe combined immune deficient (NOD/SCID) mouse strains.13
SCID repopulating cells (SRCs) represent the most primitive To meet the need of a variety of situations, ranging from normal
hematopoietic population assessable, but NOD/SCID assays are homeostasis to acute blood loss or infection, hematopoiesis must be
hampered by the generally low engraftment of human cells and rapidly yet meticulously controlled. Whether regarded as stochastic
the difficulty in keeping NOD/SCID mice for long-term analy- mainly regulating probabilities or deterministic providing more
ses. This predicament can be overcome by serial NOD/SCID precise instructions, both intrinsic and extrinsic signals undoubt-
transplantations, allowing for analysis of even more primitive edly participate in the regulation of HSCs.1,14-16 Through reduction-
populations. However, such assays keep HSCs under constant istic strategies, a wide variety of factors critical for HSC regulation
494 BLANK et al BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2
B cell
T cell
CLP
GMP
Thy1.1 low Thy1.1 low Thy1.1- Macrophage
FLT3- FLT3- FLT3+
CD34- CD34+ CD34+
Endoglin+ CD11b low CD11b low
SP+ CD4 low
Rho low CMP
CD150+
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, transcrip- HSCs have failed because of differentiation of HSCs and subse-
tion factors, chromatin modifiers, and cell-cycle regulators. Delin- quent loss of reconstitution capacity. In one study, a modest
eating the signaling pathways that integrate these diverse compo- expansion of repopulating murine HSCs was observed using a
nents remains an enduring challenge of foremost importance, 10-day culture protocol in the presence of IL-11, Flt-3 ligand, and
which would have tremendous impact on regenerative medicine. SCF.17 However, the same conditions failed to expand HSCs
During the course of development, a handful of signaling pathways derived from the fetal liver and later studies have found that the
shape the structures of the embryo and determine cell fate and Flt-3 receptor is not expressed on LT-HSCs.7,18 Furthermore, the
identity. In the adult organism, these signaling pathways are gp130 protein, which is a part of the receptor for IL-6 and IL-11
reiterated, and it is becoming increasingly clear that most somatic was suggested to play a role in the self-renewal of HSCs.19
stem-cell compartments are under the influence of developmentally However, elimination of the IL-11 receptor does not affect
conserved signals. 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
Regulation of HSCs by classic hematopoietic c-kit, respectively, are both expressed on repopulating HSCs,6,7,21-24
cytokines 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
Numerous attempts have been made to use classic hematopoietic reported to support viability25 and suppressed apoptosis of HSCs.26
cytokines for the purpose of expanding HSCs in vitro. Many of the Thus, the main function of TPO may be to counteract apoptosis of
interleukins, particularly, interleukin (IL)-3, IL-6, and IL-11, as HSCs rather than promote their expansion.24
well as Flt-3 ligand, thrombopoietin (TPO) and stem-cell factor A further role for TPO in regulating HSC self-renewal was recently
(SCF) have been investigated. In most cases, efforts to expand suggested through the association with Lnk, an intracellular adaptor
BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2 SIGNALING PATHWAYS AND STEM CELL FATE 495
molecule functioning as a broad inhibitor of several cytokine signaling progenitors in vitro as measured by reconstitution assays.35-41
pathways, including TPO, SCF, erythropoietin, IL-3, and IL-7.24,27-30 Importantly, enforced activation of the Notch signaling pathway
Mice deficient in Lnk have been reported to exhibit increased HSC or the downstream target Hes-1, have been demonstrated to
numbers and their repopulation function was elevated, a phenotype increase the self-renewal capacity of long-term in vivo repopu-
attributable to augmented proliferation and self-renewal.24,27,31 Interest- lating HSCs42,43 or even to immortalize primitive hematopoietic
ingly, the increased self-renewal capacity of Lnk⫺/⫺ HSCs was depen- progenitor cells.44 When Calvi et al established osteoblasts as
dent on TPO, and Lnk-negative HSCs were shown to be hypersensitive putative members of the HSC niche, they demonstrated how
to stimulation by TPO.24,27 Thus, TPO and Lnk serve opposing roles in constitutively active parathyroid hormone receptor expanded
the regulation of HSC self-renewal and Lnk acts as a powerful inhibitor these cells and stimulated their expression of Jagged-1.45 The
of TPO signaling in HSCs. To conclude, of the classic hematopoietic activated osteoblasts were able to increase self-renewal of
cytokines, the most important positive regulators of HSCs are
primitive hematopoietic cells as measured by LTC-ICs and
TPO and SCF.
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
Notch pathway: at the interface of osteoblasts normalized with a ␥-secretase inhibitor.45 Supporting these data
and HSCs were the observation by Duncan et al that a Notch reporter was
active in c-kit⫹ hematopoietic cells in the trabecular bone and
In humans, Notch was discovered 15 years ago as an oncogene
isolated LSK and side population cells.46
that, after chromosomal translocation, gave rise to T-cell
leukemia.32 Since then, several loss- and gain-of-function Together these findings indicate not only that osteoblasts are
studies have demonstrated a critical role for Notch in lineage part of the so-called HSC niche but also that Jagged/Notch
specification of lymphopoiesis, where active Notch signaling signaling activated by these cells might be important in the
(Figure 3A) drives differentiation toward T cells.33 It was soon extracellular regulation of HSC self-renewal.45 However, HSCs
suggested that Notch could have a role in HSC biology as deficient in Notch-1 could reconstitute Jagged-1 deficient hosts in a
primitive hematopoietic cells as well as cells in the putative normal fashion, suggesting that Notch signaling through mecha-
HSC microenvironment expressed both Notch and Notch-ligand nisms including these molecules is dispensable for in vivo HSC
mRNA.34,35 Indeed, through various approaches, several groups function.47 It is probable that the lack of phenotype observed in this
have shown that members of the Notch ligand families, Delta setting is the result of compensatory mechanisms mediated by
and Jagged, can expand both murine and human hematopoietic other Notch receptors and ligands, which is why approaches to
496 BLANK et al BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2
Smad4 Co-Smad
Nucleus Cytoplasm
DNA-binding partner
P
R-Smad
Smad4
P
R-Smad
Smad4
Target gene
vivo.76 However, despite the pronounced role of TGF- in vitro, formation was assessed, indicative of increased self-renewal.
mice deficient of the TGF- type I receptor displayed normal HSC However, conditional deletion of Smad5 in adult mice does not
self-renewal and regenerative capacity in vivo, even under extreme affect hematopoiesis, and HSCs lacking Smad5 have a normal
hematopoietic stress.77,78 The apparent discrepancy between in differentiation and regenerative potential in vivo.88 It is possible
vitro and in vivo findings may reflect mechanisms of redundancy that BMP–activated Smad signaling plays a more pronounced role
between other type I receptors or alternatively other ligands such as early in hematopoietic development, as opposed to adult hematopoi-
activin, which signals through the same R-Smad pathway. esis. Alternatively, redundant mechanisms within the Smad path-
way may explain the lack of phenotype in the adult setting.
BMP promotes maintenance of HSCs in culture
Smad signaling: toward a deeper understanding
BMPs have been implicated as key regulators of hematopoietic develop-
ment in a variety of species, from zebrafish to mouse.79-83 In the context To block the entire Smad signaling pathway, thus sidestepping
of adult hematopoiesis, BMP-4 was shown to promote maintenance of redundant mechanisms, the inhibitory Smad7 was overexpressed in
HSCs in culture, whereas lower concentrations of BMP-4 induced murine HSCs using a retroviral gene transfer approach.89 Forced
proliferation and differentiation of human hematopoietic progenitors.84 expression of Smad7 significantly increased the self-renewal
Furthermore, Shh induced proliferation of primitive human hematopoi- capacity of HSCs in vivo, indicating that the Smad pathway
etic progenitors in vitro, apparently through a BMP-4–dependent negatively regulates self-renewal in vivo. In a similar approach
mechanism.85 However, although BMP-4 has been shown to maintain using human SCID repopulating cells (SRCs), overexpression of
human NOD/SCID repopulating cells in culture, it does not seem to Smad7 resulted in a shift from lymphoid dominant engraftment
cause an expansion, suggesting that Shh may act through additional toward increased myeloid contribution.90 Thus, in the xenograft
mechanisms. In the murine system, BMP-4 does not appear to affect model system, forced expression of Smad7 modulates cell fate
proliferation of purified HSCs in vitro, although it is currently unclear decisions of primitive multipotent human SRCs.
whether BMP-4 can extend the maintenance of murine HSCs in It is beyond doubt that the Smad signaling pathway lies at the
culture.86 Furthermore, BMPs function to regulate the HSC niche, thus very core of mediating signals from the TGF- family of ligands.
indirectly controlling HSC numbers as discussed in “HSC niche.” However, a considerably more diversified picture is now emerging,
A role for Smad5, traditionally characterized as a mediator of which indicates that the Smad circuitry is more varied than
BMP signals, in self-renewal was implicated through a series of in previously thought.91 Using a conditional knockout mouse model,
vitro studies. Interestingly, the number of colonies, derived from disruption of the entire Smad pathway at the level of Smad4 was
yolk sacs or ES cell differentiated embryoid bodies deficient of recently investigated. Intriguingly, Smad4 deficient HSCs dis-
Smad5, were found to be increased.87 Smad5 deficient colonies also played a significantly reduced repopulative capacity of primary and
had an enhanced regenerative potential when secondary colony secondary recipients, indicating that Smad4 is critical for HSC
498 BLANK et al BLOOD, 15 JANUARY 2008 䡠 VOLUME 111, NUMBER 2
self-renewal in vivo.92 Because overexpression of Smad7 versus specifically, a 24- to 30-fold increase in HSC number was
deletion of Smad4 would be anticipated to yield similar hematopoi- observed. Angptl proteins 5 and 7 were also reported to expand
etic phenotypes, it is conceivable that Smad4 functions as a murine HSCs, whereas Angptl protein 4 failed to show a similar
positive regulator of self-renewal independently of its role as a effect. The molecular mechanisms and the signaling events down-
central mediator of the canonical Smad pathway. The precise stream of Angptl proteins remain unidentified and their receptors
molecular mechanism for this is currently unknown, but it is are currently unknown, which is why it is not clear how the Angptl
possible that Smad4 participates in other signaling cascades such as proteins expand HSCs. However, Angptl protein 2 has been shown
Wnt or Notch.93-95 Moreover, He et al proposed an elegant model to bind to a highly enriched population of HSCs. Therefore, the
for how TGF- mediates erythroid differentiation concomitantly expansion effect attributable to these proteins is likely to be a result
with balancing growth inhibition in human hematopoietic stem/ of a direct action on HSCs. It will be exciting to see whether the
progenitor cells.96 According to this model, transcriptional interme- Angptl proteins can also be used for robust expansion of human
diary factor1␥ (TIF1␥) selectively binds Smad2 and Smad3 in HSCs, particularly HSCs derived from cord blood.
competition with Smad4. In response to TGF-, the TIF1␥/ These findings are important especially in light of the convenience of
Smad2/3 complex was shown to stimulate erythroid differentiation adding these factors to cultures of HSCs in vitro, and they can therefore
of human hematopoietic progenitors, whereas Smad2/3 in associa- be used in future attempts to expand HSCs for the clinic and possibly
ECM
Ca 2+
Ca 2+
HSC
Soluble factors
number of HSCs in each CB sample is limited, and it would therefore relatively short and safe culture period, suitable for GMP conditions that
greatly increase the applicability of this stem-cell source if the CB stem will be required for the clinic. Worthy of mention are also fibroblast
cells could be expanded ex vivo before transplantation. Stem-cell growth factors, which have been shown to sustain primitive murine
expansion can also theoretically be accomplished in vivo after engraft- hematopoietic cells in culture and to maintain their primitive pheno-
ment of the transplanted cells, but this approach may involve greater type.131,132 Interestingly, fibroblast growth factor-1 in conjunction with
risks compared with ex vivo expansion unless the growth stimuli used in SCF, TPO, and IGF-2 resulted in a 20-fold increase of LT-HSCs during a
vivo can be carefully controlled.123,124 Moreover, because culture 10-day culture period.133
conditions can be precisely defined and transient modulation of regula- It should be emphasized that most of the knowledge about stem- cell
tory pathways more easily used in vitro, stem-cell expansion ex vivo expansion has come from studies in mice. There is a difference between
presents an attractive approach. However, even this tactic has proven a mouse and human HSCs with regards to cytokine receptors, telomere
great challenge because a successful stem-cell expansion involves biology, and proliferative capacity, and there will therefore be both
symmetric self-renewal divisions of HSCs, where both daughter cells differences and similarities in the approaches used to expand mouse and
retain HSC properties.123 More commonly, HSCs grown in vitro human HSCs. Recently, the protein nephroblastoma overexpressed
undergo asymmetric divisions characterized by the production of one (NOV/CCN3) has been shown to expand primitive human HSCs, and
HSC and a more differentiated progenitor, or alternatively, a symmetric this protein should therefore be carefully evaluated for use in clinical
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