Physiol Rev 92: 75–99, 2012
doi:10.1152/physrev.00040.2010
CONTROLLING THE STEM CELL COMPARTMENT
AND REGENERATION IN VIVO: THE ROLE OF
PLURIPOTENCY PATHWAYS
Kirsty Greenow and Alan R. Clarke
School of Biosciences, Cardiff University, Cardiff, United Kingdom
L
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
INTRODUCTION
PLURIPOTENCY FACTORS
THE EPIGENETIC REGULATION...
IPS CELLS IN THERAPY
PLURIPOTENCY PATHWAYS
REGENERATIVE PATHWAYS
REGENERATION OR TRANSFORMATION?
SUMMARY
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I. INTRODUCTION
Embryonic stem (ES) cells and induced pluripotent stem
(iPS) cells have the capacity to self-renew, and the differentiation of these cells can yield over 200 diversely functional
cell types. The ability of pluripotent cells to differentiate
and produce a potential source of somatic cells makes these
cells an obvious choice for regenerative medicine. Unfortunately, before the potential of pluripotent cells can be harnessed for clinical application, it is essential that we understand the mechanisms involved in maintaining pluripotency
and inducing differentiation. The recent discovery of somatic cell reprogramming with key pluripotency factors has
provided essential information on the role of transcription
factors in pluripotency maintenance. These pluripotency
factors are thought to be capable of initiating most, if not
all, of the necessary developmental signaling pathways required for somatic cell reprogramming. The generation of
these reprogrammed iPS cells has opened up the prospect of
deriving patient-specific cells for regenerative medicine and
may resolve the ethical issues and the immune rejection
risks associated with the use of human ES cells.
While the generation of iPS cells and the identification of
key pluripotency factors has been a breakthrough in understanding pluripotency, somewhat less is known of the signaling networks surrounding these key transcription factors. Without a clearer understanding of the signaling network involved in pluripotency maintenance and also
differentiation, the use of iPS cells and ES cells in cellular
therapy will be limited, as will our capacity to modulate
existing stem cells in situ. It is therefore essential that further mechanistic and functional studies are carried out,
which will inevitably continue to identify factors that are
capable of defining stemness and mediating reprogramming
in culture. This also raises the much wider question of the
relevance of these mechanisms in adult stem cells and to
normal physiology, either in maintaining tissue homeostasis, or more critically in mediating the repair response to
disease and injury. Here, we will review the potential path-
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Greenow K, Clarke AR. Controlling the Stem Cell Compartment and Regeneration In Vivo: The Role of Pluripotency Pathways. Physiol Rev 92: 75–99, 2012;
doi:10.1152/physrev.00040.2010.—Since the realization that embryonic stem
cells are maintained in a pluripotent state through the interplay of a number of key signal
transduction pathways, it is becoming increasingly clear that stemness and pluripotency are defined by the complex molecular convergence of these pathways. Perhaps this has most
clearly been demonstrated by the capacity to induce pluripotency in differentiated cell types, so
termed iPS cells. We are therefore building an understanding of how cells may be maintained in a
pluripotent state, and how we may manipulate cells to drive them between committed and pluripotent compartments. However, it is less clear how cells normally pass in and out of the stem cell
compartment under normal and diseased physiological states in vivo, and indeed, how important
these pathways are in these settings. It is also clear that there is a potential “dark side” to
manipulating the stem cell compartment, as deregulation of somatic stem cells is being increasingly implicated in carcinogenesis and the generation of “cancer stem cells.” This review explores
these relationships, with a particular focus on the role played by key molecular regulators of
stemness in tissue repair, and the possibility that a better understanding of this control may open
the door to novel repair strategies in vivo. The successful development of such strategies has the
potential to replace or augment intervention-based strategies (cell replacement therapies), although it is clear they must be developed with a full understanding of how such approaches might
also influence tumorigenesis.
STEM CELL COMPARTMENT, REGENERATION, AND PLURIPOTENCY PATHWAYS
ways that may be downstream of pluripotency factors identified in ES cells and iPS cells (152, 255, 261) and also
examine their functional relevance in adult somatic stem
cells. Such an improved understanding will not only enhance our fundamental understanding of the regulation of
tissue maintenance, but may also open the possibility of
directly manipulating these pathways to mediate efficient
regeneration following disease or injury.
Pluripotent
Progenitor cells
Glossary
Adult stem cell
Embryonic
stem cells
Differentiation
Induced
pluripotent
stem cells
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Cancer cells that possess
characteristics associated with normal stem
cells, such as self-renewal, and also produce
differentiated progeny
that form the bulk of the
cancer.
Undifferentiated cells derived from the inner cell
mass of a developing
blastocyst that are capable of long-term selfrenewal and have the potential to differentiate
into all cell types.
The process of development by which a primitive, unspecialized cell
commits to becoming a
specific cell in the body.
A type of pluripotent stem
cell artificially derived
from a nonpluripotent
cell, such as an adult somatic cell, by inducing a
“forced” expression of
key pluripotency genes.
Quiescence
Regenerative
medicine
Reprogramming
Self-renewal
Stem cell niche
Stemness
Teratoma
The use of stem cells to replace or repair damaged
or diseased cells, tissues, and organs.
The process of creating an
induced pluripotent stem
cell through the introduction of a combination
of three to four genes,
into an adult cell.
The division of a cell that
repeatedly generates
at least one daughter cell with identical
properties to the parent cell, thereby allowing the population to
be replenished indefinitely.
The supporting microenvironment in which stem
cells are found.
An essential characteristic of a stem cell that
distinguishes it from a
normal cell.
A tumor composed of tissues from the three embryonic germ layers.
II. PLURIPOTENCY FACTORS
A. Embryonic Stem Cells
As described above, ES cells are pluripotent cells that are
capable of unlimited self-renewal and are able to differentiate into all three embryonic lineages. To maintain their
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Cancer stem
cell
An undifferentiated cell
found in the different tissues or organs of the developed, adult organism
that can renew itself
and differentiate to yield
all the specialized cell
types of that tissue or
organ.
Ability of a single stem cell
to differentiate into the
majority of the cells of
the body.
An early, partially differentiated, descendant of a
stem cell that is capable
of division and further
differentiation into the
various cell types of a
tissue, but is unable to
renew itself.
The state of a cell when it
is not dividing.
KIRSTY GREENOW AND ALAN R. CLARKE
ables them to maintain the ES cell transcriptome through a
tight regulatory network (FIG. 1).
Many other transcription factors have been identified to
closely cooperate with these three essential pluripotency factors including Sal14, Dax1, Essrb, Tcf4, Klf4, Tbx3, Tcl1,
Rif1, Nac1, and Zfp281 (40, 43, 102, 117, 155, 257). These
transcription factors also regulate each other, forming a complicated transcriptional regulatory network in ES cells (291).
Essrb, Rif1, Zic3, and Tcl1 are also primary targets of both
Oct4 and Nanog (148, 155). In addition, RNAi-mediated
knockdown has confirmed that eight genes, including Nanog,
Oct4, and Sox2, are essential for maintaining an undifferentiated state in ES cells (102). Expression profile analysis has also
identified developmental genes (Otx2, Pitx2, and Sox18) regulated by these eight factors, which induce ES cell differentiation when overexpressed (102).
B. Induced Pluripotent Stem Cells
From the above, it is clear that ES cell studies have implicated a number of factors in pluripotency, the requirement
FIGURE 1. Schematic of the core stem cell network in ES cells. ES cells require several key transcription
factors to maintain their ability to self-renew and remain undifferentaited. In addition to the key pluripotency
factors Oct4, Sox2, Nanog, and Klf4, several other transcription factors closely cooperate with these factors
to maintain pluripotency and self-renewal.
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ability to self-renew and remain undifferentiated, ES cells
require the cooperation of several regulatory factors, including a unique set of transcription factors (26, 117), Polycomb complexes (27, 36), microRNAs (164), and histone
modification enzymes (23). Although the precise role of
these mechanisms is still unclear, several studies have identified the core transcription factors that are necessary for
pluripotency in ES cells. Oct4 and Nanog are two homeodomain transcription factors and were the first to be identified as essential for early embryo development and pluripotency maintenance in ES cells (37, 168, 176). In addition,
Sox2, an HMG-box transcription factor, which heterodimerizes with Oct4, has also been shown to be central
to the transcriptional network regulating pluripotency in
mouse ES cells (26). All three of these transcription factors
act as either transcriptional activators or repressors and
share a substantial fraction of their target genes (26, 155).
The ability of these factors to activate or repress genes allows them to maintain pluripotency by promoting the expression of downstream self-renewal genes while simultaneously repressing the activity of differentiation-promoting
genes. These factors also function interdependently and
demonstrate an autoregulatory feedback loop which en-
STEM CELL COMPARTMENT, REGENERATION, AND PLURIPOTENCY PATHWAYS
In addition to the groups of pluripotency factors used to
reprogram iPS cells, the levels and ratios of the reprogramming factors have a critical role in the efficiency of reprogramming. For example, increasing Oct4 levels increases
reprogramming efficiency, whilst increasing Sox2, Klf4, or
c-Myc individually causes a significant decrease in reprogramming efficiency (47). The optimal ratio of the reprogramming factors will vary depending on the cell type used
and also the endogenous expression of any one of the re-
programming factors within the starting cell population. A
summary of some of the reprogramming scenarios that have
been used successfully in both mouse and human are given
in TABLE 1.
Although the mechanisms behind reprogramming somatic
cells back to a pluripotent state are largely unknown, recent
work has shown that the reprogramming process with defined factors is a gradual, time-dependent process, and studies by Brambrink et al. (28) and Stadfield et al. (227) have
identified the sequence of key events necessary for the reprogramming of mouse embryonic fibroblasts. The minimal
time for full reprogramming of mouse somatic cells has
been defined as 8 –12 days, and reprogramming appears to
follow a sequential path consisting of early and late events.
The reprogramming factors progressively reestablish the
core circuitry of pluripotency, and as reprogramming progresses, the properties of pluripotent stem cells begin to
appear.
III. THE EPIGENETIC REGULATION OF
PLURIPOTENCY
Gene regulation during pluripotency also requires epigenetic changes to chromatin, and it has been well established
that ES cells and differentiated cells have significantly different epigenetic signatures (22, 23). Pluripotent cells require a dynamic and transcriptionally permissive chromatin
structure whereby structural proteins are loosely associated, enabling rapid reorganization of the chromatin structure in response to differentiation signals (166). Lineagespecific genes are therefore poised for transcriptional activation but are held in check by repressive chromatin
FIGURE 2. Schematic of iPS cell derivation. Adult somatic cells can be reprogrammed to pluripotent iPS cells
by introducing a specific combination of key transcription factors such as the Yamanaka factors, Oct4, Sox2,
Klf4, and c-myc. iPS cells display similar characteristics to ES cells such as DNA methylation and expression
profiles and are also able to differentiate into advanced derivatives of all three primary germ layers.
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for which has subsequently been confirmed by reprogramming somatic cells with specific genes to generate iPS cells
(FIG. 2). For example, ectopic expression of Oct4, Sox2,
c-Myc, and Klf4 together was shown to be sufficient to
reprogram both mouse embryonic and adult fibroblasts to a
pluripotent state (160, 186, 237, 263). These reprogrammed cells were able to yield live born chimeras and
were also able to produce iPS cell progeny through germline
transmission. In addition, they displayed similar epigenetic
and transcriptional profiles compared with ES cells, such as
their status with respect to DNA methylation, histone H3
methylation, X chromosome activation, and global gene
expression profiles (160, 186, 263). These studies also revealed reactivation of the endogenous Oct4, Sox2, and
Nanog genes, all of which are markers for undifferentiated
ES cells. Oct4, Sox2, c-Myc, and Klf4 have also been shown
to successfully reprogram human dermal fibroblasts (191,
236), whereas others have shown that Oct4, Sox2, Nanog,
and Lin28 were sufficient to establish pluripotent cells from
human somatic cells (283). In all three of these studies, the
transcriptional and epigenetic profiles were similar to human ES cells and the iPS cells formed derivatives of all three
germ layers when differentiated in vitro and in teratoma
assays.
KIRSTY GREENOW AND ALAN R. CLARKE
Table 1. Examples of the various combinations of transcription factors and cell types that have been used in the production of iPS cells
Cell Type
Human
HNF
HDF
HFLS
BJ cells
HEFs
Keratinocytes
CD34⫹ blood cells
Adult fibroblasts
Foreskin fibroblasts
H1F cells
MSCs
Adult fibroblasts
HDFs
NHDF
BJ cells
MEFs and tail tip fibroblasts
MEFs
Tail tip fibroblasts and hepatocytes
Hepatocytes and gastric epithelial cells
MEFs and tail tip fibroblasts
MEFs
NSCs
NPCs
MEFs
MEFs and NSCs
MEFs
NSCs
Mouse
Factors
Oct4, Sox2, Klf4, c-Myc
Reference Nos.
119
237, 289
Oct4, Sox2, Nanog, LIN28
107, 269
1, 186
154
146, 283
Oct4, Sox2, Klf4, c-Myc, hTERT, SV40 large T
191
Oct4, Sox2, Klf4
Oct4, Sox2
Oct4, Sox2, Klf4, c-Myc
170
94
160, 165, 237
107, 186, 199, 263
228
8
165
93, 163, 263
119
61, 221
220
223
68
118
Oct4, Sox2, Klf4
Oct4, Klf4
Oct4, Sox2
Oct4
methylation until the appropriate signals are received (23).
The epigenetic status of iPS cells and their cells of origin are
also markedly different, suggesting that reprogramming involves dynamic chromatin changes, and it has been demonstrated that during reprogramming the epigenetic status
progresses from a somatic to a pluripotent level (120).
This specialized chromatin state is established partly by the
activity of ATP-dependent nucleosome remodeling complexes, such as Swi2/Snf2. A number of ATP-dependent
nucleosome remodeling complexes have been shown to be
important for ES cell self-renewal and differentiation, and
recent studies have demonstrated that treatment with various chromatin modifiers can improve the frequency of iPS
cell generation (93, 94). ES cells contain a family of functionally and structurally specialized chromatin remodeling
complexes that are critical for the self-renewal of ES cells
and maintain the stem cell state (90). Several BAF complexes, including a unique complex, esBAF, are expressed in
ES cells, and ES cells depleted of various known subunits of
BAF have been shown to have defects in gene regulation,
self-renewal, and pluripotency (72, 277). The catalytic ATPase SWI/SNF subunit Brg acts as the catalytic subunit of
esBAF and has been identified as an essential factor in nu-
clear reprogramming (87). esBAF plays a direct role in mediating the gene regulatory function of several core pluripotency factors. The Brg subunit selectively regulates the expression of several factors of the core network as well as
interacting directly with Oct4, Sox2, and Nanog (90). Brg
also interacts with several other proteins that have been
implicated in ES cell maintenance such as Rif1, Sall4,
Dppa2, and Dppa4, and it is thought that the surface of
esBAF may be tailored for interaction with factors specifically found in ES cells. esBAF also functions to repress pluripotency genes such as Oct4, Sox2, and Nanog; this inhibition is thought to refine their levels and maintain pluripotency. In addition to BAF, several other chromatin
modifiers have recently been identified as vital factors in
maintaining ES cell pluripotency, such as Chd1 (73), the
histone deacetylase NuRD (106, 292), and Tip60-p400
(65). Similarly to BAF, these factors interact and regulate
the core pluripotency transcription factors of ES cells.
As described above, pluripotency factors and epigenetic
regulators engage in cross-talk with one another to maintain pluripotency. Several pluripotency factors that are regulated by epigenetic regulators also regulate the genes encoding the epigenetic control factors; for example, Oct4,
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Species
STEM CELL COMPARTMENT, REGENERATION, AND PLURIPOTENCY PATHWAYS
Sox2, and Nanog coregulate certain genes encoding chromatin remodeling and histone-modifying complexes, such
as Ehmt1, SMARCAD1, MYST3, and SET in mouse and
human ES cells (26). Also, pluripotency factors interact
with histone-modifying enzymes and chromatin remodeling
complexes; for example, Nanog and Oct4 interact directly
and indirectly with the polycomb group (YY1, Rnf2, and
Rybp) and SWI/SNF chromatin remodeling (BAF155) factors and KAP1 (Tif1␤) (257). In addition, several other
genes regulated by pluripotency factors are subjected to
epigenetic regulation. For example, two downstream targets of Oct4, Jmjd1a and Jmjd2c, both of which are histone
demethylases, positively regulate several pluripotency-associated genes including Tcl1, Tcfcf2l1, Zfp57, and Nanog
(155, 156).
The reprogramming of human somatic cells to a pluripotent
state has significant implications for the generation of patient-specific, genetically compatible cells for transplantation. TABLE 2 lists a number of disease models in which iPS
cells have already been used as a potential therapy. iPS cells
have been generated from the cells of various mouse tissues
including blood (85), liver (8), stomach (8), pancreas (227),
brain (61, 119, 221), and the intestine and adrenals (262) as
well as also human fibroblasts (158, 191, 236), keratinocytes (1), and blood progenitor cells (154). Recent studies
using iPS cells to alleviate the symptoms of Parkinson’s
disease and sickle cell anemia in mouse models have demonstrated the potential of these cells in cellular therapy (86,
264). Furthermore, Raya et al. (203) reprogrammed genetically corrected Fanconi anemia fibroblasts and obtained
corrected Fanconi anemia-specific iPS cells that can give rise
to phenotypically normal hematopoietic progenitors of the
myeloid and erythroid lineages. In addition to neural and
blood diseases, iPS cells have been differentiated into many
types of cardiovascular cells, including arterial, venous, and
lymphatic endothelial cells as well as cardiomyocytes (172,
285). Another potential therapeutic use of iPS cells is in the
treatment of infertile patients. Primordial germ cells (PGCs)
can be generated through in vitro differentiation of human
iPS and ES cells, and these in vitro differentiated PGCs show
gene expression similarities to in vivo PGCs (192). If these
V. PLURIPOTENCY PATHWAYS
Signal transduction pathways are critical in maintaining
pluripotency and controlling the lineage-specific differentiation of stem cells. Although ES cell research began more
than 20 years ago, the regulatory network of ES cell maintenance is still largely unknown. Several extrinsic growth
factors support the pluripotency of ES cells in vitro, with
mouse ES cells usually being cultured in the presence of
leukemia inhibitory factor (LIF) and bone morphogenetic
proteins (BMPs). LIF is thought to activate STAT3 signaling
pathways to maintain pluripotency, whilst BMPs act to inhibit differentiation by activating members of the Id gene
(inhibition of differentiation) family (177, 279). Importantly, recent work has also indicated that ES cells can be
cultured in the absence of LIF and BMP. These ES cells
represent a ground state, a basal proliferative state that is
free of epigenetic restriction and has minimal requirements
for extrinsic stimuli (280). In this state, the pluripotency
network, headed by Oct4/Sox2, Nanog, and KLF2/KLF4, is
intrinsically stable but extremely sensitive to destabilization
by exogenous signals, with ES cells requiring the presence of
MEK and GSK3 inhibitors to suppress innate differentiation stimuli and maintain the stability of the ground state
(84, 271).
The commitment of ES cells to a specific lineage is therefore
regulated by specific signaling pathways, such as the JAKSTAT pathway mentioned above. The Wnt/␤-catenin signaling pathway is also heavily implicated, for example,
through regulating cardiomyocyte differentiation of mouse
ES cells, while the induction of ERK1/2 signaling triggers
the differentiation of mouse ES cells towards the primitive
endoderm lineage (39, 170). A schematic showing the proposed stem cell states is given in FIGURE 3.
Table 2. Examples of the disease models in which iPS cells have been used as a potential therapy for treatment
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Disease
Cell of Origin
Sickle cell anemia
Parkinson’s Disease
Fanconi anemia
Spinal muscular atrophy
Duchenne muscular dystrophy
Tail-tip fibroblasts
Patient-derived fibroblasts
Patient-derived fibroblasts
Patient-derived fibroblasts
Mouse embryonic fibroblasts
Patient-derived fibroblasts
Factors
Oct4,
Oct4,
Oct4,
Oct4,
Oct4,
Oct4,
Sox2,
Sox2,
Sox2,
Sox2,
Sox2,
Sox2,
Klf4, c-Myc
Klf4, c-Myc
Klf4, c-Myc
Nanog, Lin28
Klf4
Klf4, c-Myc
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Reference Nos.
86
226
203
59
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IV. IPS CELLS IN THERAPY
PGCs can be further differentiated into sperm and oocytes,
it will be valuable for infertile patients. While these studies
demonstrate the potential therapeutic application of iPS
cells, our current limited understanding of the reprogramming process hinders the ultimate goal of using these cells in
cellular therapy.
KIRSTY GREENOW AND ALAN R. CLARKE
Signaling pathways therefore play a critical role in the induction of pluripotency during somatic cell reprogramming. However, unfortunately, rather little is known about
the signaling pathways regulated by the pluripotency factors Oct4, Sox2, Klf4, and c-Myc, although recent work in
mouse ES cells has identified key pathways that are regulated by these factors to maintain pluripotency (152). For
example, work in ES cells has identified that two key signaling pathways are integrated into the Oct4, Sox2, and
Nanog circuitries through Smad1 and STAT3 (40). Liu et
al. (152) have also demonstrated that these pluripotency
factors regulated a crucial developmental signaling network, which is comprised of 16 signaling pathways, including 9 with previously unknown roles in the maintenance of
ES cell pluripotency. In addition, further work by Huang et
al. (92) has identified a core signaling network regulated by
these factors to induce pluripotency in reprogrammed somatic cells. This study identified eight pathways as essential
pathways in iPS and ES cells, including transforming
growth factor (TGF)-␤, Hedgehog, Wnt, p53, and JAKSTAT, and it is thought that the key pluripotency factors
synergistically regulate these pathways in a balanced way to
induce pluripotency (92). In addition, several other developmental signaling pathways, including mitogen-activated
protein kinase (MAPK), vascular epidermal growth factor
(VEGF), Notch, ErbB, and mTOR were identified as pathways supporting the core network (92).
We therefore have an emerging picture of pluripotency regulating factors which impinge on a broad range of signaling
networks (FIG. 4). In the next section of this review, we will
examine some of the key pathways identified as being important for iPS cell induction. We will also investigate the
established role of these pathways in maintaining ES cell
pluripotency and their role in somatic stem cells. Although
the core pluripotency network is thought to differ in adult
stem cells, these key pathways have established roles in
maintaining the somatic stem cells and in regulating tissue
homeostasis; therefore, we will also examine the potential
of these signaling pathways in tissue regeneration.
A. TGF-␤
The TGF-␤ superfamily plays a major role in the biology of
mammalian development and regulates many cellular processes including cell fate, proliferation, senescence, apoptosis, and tissue repair. TGF-␤ and associated members of the
family are mainly pleiotropic growth factors and are comprised of two main groups, the bone morphogenetic proteins (BMPs) and the growth differentiation factors (GDFs),
which includes TGF-␤, activin, and nodal and can be further divided according to sequence homologies and functional similarities. The canonical signaling cascade of
TGF-␤ growth factors involves the ligands of the TGF-␤
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FIGURE 3. The various states of embryonic stem cells. Both human and mouse stem cells are derived from
the blastocyst but appear to have different characteristics and require different growth conditions (shown in
red). Human ES cells appear to correspond to mouse-derived epiblast stem cells (EpiSCs), and recent work by
Hanna et al. (84) has suggested that a more immature hES cell state (the ground state) exists that shares
defining features with mouse ES cells. Reprogramming to the ground state requires GSK3␤ and MAPK/ERK
inhibitors in addition to the key stem cell factors.
STEM CELL COMPARTMENT, REGENERATION, AND PLURIPOTENCY PATHWAYS
superfamily binding to cell surface receptors that activate
the Smad proteins in the cytoplasm, which leads to their
nuclear translocation and transcriptional activation of target genes. This pathway is also regulated by other key signaling pathways such as Wnt and Hedgehog, and also other
tyrosine kinase-linked growth receptor signaling pathways.
TGF-␤ signaling has been shown to be important in the
maintenance of self-renewal and pluripotency of both
mouse and human ES cells, although the role of these signaling molecules does appear to differ between the two
species (258). In mouse ES cells, BMP4 maintains self-renewal through inhibition of the MAPK/ERK pathway by
LIF and the expression of Id protein via the Smad pathway
(198, 279). Furthermore, BMP4 promotes mouse ES cell
proliferation via an increase in Wnt expression by Smad
activation (137). In contrast, in human ES cells, BMP4 promotes stem cell differentiation through the downregulation
of Nanog and Oct4, and activated Smad is only found at
low levels (274). Long-term maintenance of human ES cell
pluripotency therefore requires the downregulation of BMP
activity by Noggin and basic fibroblast growth factor
(bFGF) (6, 275). Other TGF-␤ family members are also
thought to play an important role in the maintenance of ES
82
cells. Phosphorylation and nuclear localization of Smad2
induced by TGF-␤, activin, or nodal signaling was observed
in undifferentiated human ES cells and was decreased upon
early differentiation (103). The inhibition of these pathways initiated differentiation and resulted in the decreased
expression of stem cell markers (103, 250). In addition,
Xiao et al. (272) have demonstrated that activin is able to
support long-term feeder-free culture and maintenance of
pluripotency in human ES cells by inducing the expression
of Oct-4 and Nanog, and suppressing BMP. Nodal expression also plays a role in the maintenance of human ES cell
pluripotency through the inhibition of neuroectodermal
differentiation, a default differentiation pathway of ES cells
(251). In addition, activin and nodal signaling has been
shown to promote mouse ES cell self-renewal in serum-free
conditions (183). It is therefore clear that TGF-␤ signaling
plays an important role in the maintenance of self-renewal
and pluripotency, although the exact mechanism of action
for this family of growth factors appears to differ between
family members and species.
BMPs are also potent inhibitors of neural differentiation in
vertebrate embryos (267), and it is thought that BMP regulation of ES cell pluripotency may be primarily through the
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FIGURE 4. Key developmental signaling pathways regulated by the core stem cell factors. These pathways
have been shown to have a role in both ES cells and iPS cells and also in tumorigenesis (⫹). These pathways
have also been shown to be essential in somatic stem cell maintenance and vertebrate limb regeneration.
Examples of the identified role of these various pathways in key cellular systems are shown in the table.
KIRSTY GREENOW AND ALAN R. CLARKE
TGF-␤ signaling also participates in the cell fate decisions of
ES cells, and these factors can direct the fate of ES cells
towards multiple cell lineages of the three embryonic layers,
including neural, hematopoietic, cardiomyogenic, and hepatic (121). Specifically hematopoietic cell differentiation is
dependent on BMP4 in mouse ES cells (190). BMP4 regulates mesodermal cell commitment to the hematopoietic
lineage and specifies blood lineages at the later stages of
differentiation (139, 190, 210). Furthermore, a recent study
by Lengerke et al. (138) demonstrated that the role of
BMP4 in hematopoietic differentiation is conserved in human iPS cells, and stimulation of BMP4 directs iPS cell
differentiation toward blood lineages.
TGF-␤ signaling also has a role during endothelial cell proliferation and vascular development in mouse ES cells (124,
180, 235, 259). BMP4 and activin are thought to induce
mesoderm differentiation into the cardiac lineage and induce human ES cells to differentiate into cardiomyocytes
(132). In addition, BMP2-induced mesodermal and cardiac
specification results in full cardiogenic differentiation, leading to an enrichment of cardiomyocytes within embryoid
bodies (20). This ability of the TGF-␤ family members to
commit mouse ES cells toward a mesodermal fate is due to
Smad-mediated regulation of the Oct4 promoter, further
implicating a role for Smad signaling in the regulation of the
core self-renewal network in ES cells (284). This role for
TGF-␤ family members in cardiomyocyte differentiation is
of potential interest for the treatment of cardiac disease,
particularly as Narazaki et al. (172) have successfully differentiated mouse iPS cells into cardiovascular progenitor
cells, which subsequently generated arterial, venous, and
lymphatic endothelial cells as well as self-beating cardiomyocytes. It may therefore be possible to potentially manipulate the TGF-␤ pathway to increase the efficiency of cardiovascular cell differentiation from patient-specific iPS cells.
In addition, the TGF-␤ pathway could potentially be used
in the clinical treatment of other diseases by exploiting its
ability to direct the cell fate of ES cells and potentially iPS
cells. For example, recent progress in obtaining endoderm
cells by treating differentiating ES cells with activin and
BMP4 (78, 127) could potentially lead the way for the development of iPS cell use to obtain endoderm derivative
cells such as pancreatic and hepatic cells for use in the
treatment of type I diabetes and liver diseases, respectively.
B. TGF-␤ and In Vivo Regeneration
In addition to its role in ES cells, the TGF-␤ signaling family
also has a functional role in the maintenance of somatic
stem cells and also tissue regeneration (258). This role of the
TGF-␤ pathway is of particular significance as the potential
use of iPS cells in regenerative therapy requires a clearer
understanding of the regulatory signaling networks maintaining somatic stem cells and driving tissue regeneration.
BMP signaling plays a vital role in the regeneration of the
intestinal epithelium. The BMP pathway maintains intestinal stem cell quiescence and regulates terminal differentiation by negatively regulating the Wnt signaling pathway
through PTEN activation. The conditional deletion of
Bmpr1a results in the expansion of the stem and progenitor
cell populations leading to hyperproliferative crypts, and
the inhibition of BMP signaling on the villus through overexpression of Noggin results in ectopic crypt formation
(88). BMP antagonism of the Wnt pathway also maintains
the hair follicle stem cells (74, 122, 144), the mesenchymal
stem cells (104, 153, 174), and neural stem cells (64). In all
these systems, BMP signaling molecules antagonize Wnt
signaling to maintain quiescent stem cells.
In addition to its role in somatic stem cell maintenance,
BMP signaling also has a functional role during limb regeneration. BMP is required for Xenopus tail and limb regrowth (17, 80) and also mammalian digit tip regeneration
(83). During Xenopus tail regeneration, BMP signaling is
required to induce cellular proliferation in the notochord
and the spinal cord (17). BMP also has the ability to rescue
regeneration of the entire tail when overexpressed, suggesting that BMP regulation of these structures may be the
driving force behind tail tissue regeneration (17). BMP is
also essential for bone matrix deposition by regulating the
proliferation and/or differentiation of scleroblasts during
the outgrowth phase of zebrafish fin regeneration (200). In
addition to appendage regeneration, BMP signaling has
also been shown to be crucial in the regulation of early
events during liver regeneration in zebrafish (108).
C. WNT
Similar to TGF-␤ signaling, the Wnt pathway has an important role in tissue development and also regulates cellular
mechanisms such as adhesion, morphology, proliferation,
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inhibition of differentiation rather than as a direct stimulation of self-renewal (279). The knockdown of Smad1 and
Smad4 expression in ES cells did not impair the self-renew
ability of these cells but led to a change in the expression
pattern of germ layer markers during differentiation (66).
Furthermore, Smad4⫺/⫺ ES cells show no defect in their
self-renewal ability (225). It has been suggested that BMP/
Smad signaling interacts with the core self-renewal network
in ES cells and modulates the balance between self-renewal
and differentiation by regulating development-associated
target genes. Consistent with this hypothesis, several Smad
target genes overlap with genes bound by the key pluripotency factors, for example, Smad4-regulated genes have a
substantial overlap with those of Sox2, NR0B1/Dax1, and
Klf4 (66). In addition, a recent study has demonstrated that
several Smad targets were mapped to Nanog, Oct4, and
TCF3-bound genes (43).
STEM CELL COMPARTMENT, REGENERATION, AND PLURIPOTENCY PATHWAYS
migration, and structural remodeling (21, 204). The Wnt
pathway consists of over 30 extracellular ligands that bind
to the Frizzled (FZD) and low-density lipoprotein receptorrelated protein (LRP) receptors at the cell surface (24). The
Wnt ligands are able to activate the canonical Wnt pathway
or the noncanonical pathway (265). The activation of Wnt
signaling in the canonical pathway results in the downregulation of ␤-catenin degradation and subsequent nuclear
translocation, which enables gene activation by the TCF/
LEF family of transcription factors. The noncanonical pathway is independent of ␤-catenin and involves the activation
of several other signaling pathways such as the JNK pathway.
In addition to maintaining pluripotency, Wnt signaling regulates differentiation in ES cells. Wnt signaling has been
found to specifically inhibit neural differentiation (12, 82),
and several other studies have implicated that Wnt signaling
may be sufficient to inhibit differentiation altogether (214).
ES cells with a mutated form of Apc were found to exhibit
differentiation defects both in vitro and in teratomas, and
this inhibitory effect was shown to be dose-dependent with
the severity of the Apc mutation (115). ES cells with highly
elevated ␤-catenin levels also have a compromised ability to
differentiate (54), although even mutant ES cells with the
highest levels of ␤-catenin activity were capable of some
differentiation, which is consistent with previous reports
that the activation of Wnt signaling alone is not sufficient to
support long-term ES cell self-renewal (182, 280).
The downstream mediators of ␤-catenin activation are the
TCF/LEF family of transcription factors. In ES cells, Tcf3 is
the most abundantly expressed member of this transcription factor family, and Tcf3-null ES cells have an increased
resistance to differentiation (195). Tcf3-null ES cells
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Recent studies have also established a role for Wnt signaling
in the generation of iPS cells. Soluble Wnt3a promotes the
derivation of iPS cells in the absence of c-Myc (163). Also,
the simultaneous inhibition of MAPK pathway and glycogen synthase kinase-3 (GSK3) promotes iPS cell formation
from brain-derived NSCs, using only Oct4 and Klf4 (223).
These pathways may facilitate iPS cell generation by activating endogenous reprogramming factors or pluripotencyassociated genes, and further investigations are required to
understand how Wnt may regulate somatic cell reprogramming.
D. Wnt and In Vivo Regeneration
The role of Wnt in ES cells and iPS cell reprogramming
suggests that manipulation of this pathway could have the
potential to be beneficial for regenerative therapies. Wnt
signaling has a vital role in regulating cell growth and differentiation and is essential in the maintenance and selfrenewal of various somatic stem cells and is therefore vital
to the regeneration of these tissues. For example, the constant proliferative state of the stem cells in the intestinal
crypt is under the control of the Wnt pathway, and loss of
Wnt signaling results in loss of the stem cell compartment
(125, 128). Knockout mice deficient in Tcf4 lack crypt
structures, and the entire intestinal epithelium is composed
of nondividing villus cells (125). Similar to the situation in
ES cell maintenance, Oct4 plays an important role in intestinal crypt maintenance as ectopic, intestinal expression of
Oct4 results in an increase in ␤-catenin expression, dysplasia, and an expansion of crypt progenitor cells (91). This
phenotype is similar to that seen following conditional loss
of Apc (213). Critically, Wnt signaling has also been shown
to play a direct role in the regenerative response after injury.
Thus treatment with the positive Wnt modulator R-Spondin1 was able to restore the damaged mucosa in both experimental colitis and radiation-induced oral mucositis
(287, 288). This role in the regenerative response has recently shown to be Wnt dependent, being mediated downstream of c-Myc by focal adhesion kinase (11).
The requirement for Wnt signaling is, however, not constrained to the gastrointestinal tract, with other epithelial
organs such as the lung (286) showing Wnt-dependent control of the balance between progenitor expansion and epi-
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The Wnt signaling pathway is coupled directly with the core
transcriptional network of pluripotency. Wnt signaling is
activated in ES cells and is downregulated during differentiation (214). Sato et al. (214) demonstrated that the activation of the canonical Wnt pathway is sufficient to maintain self-renewal of both human and mouse embryonic stem
cells and is independent from the LIF/STAT pathway. During this study, they demonstrated that the activation of the
Wnt signaling pathway maintained pluripotency and also
sustained the expression of pluripotency factors including
Oct4, Nanog, and Rex1. Further studies also demonstrated
that the conditioned medium from mouse fibroblasts expressing Wnt maintained the pluripotency of mouse ES cells
(182). In addition, a study overexpressing an activated mutant of ␤-catenin confirmed the role of Wnt in maintaining
the expression levels of Oct4, Nanog, and Rex1 and the
long-term proliferation of ES cells (238). This study also
demonstrated that the ␤-catenin upregulation of Nanog
was in an Oct4-dependent manner and that ␤-catenin physically associates with Oct4 (238).
showed upregulation of various Oct4- and Nanog-regulated genes and was also shown to cooccupy the promoters
of several Oct4 and Nanog targets (43). TCF3 is thought to
function to oppose the effects of the core pluripotency machinery and limit the expression of key regulators. Activation of Wnt converts TCF3 into an activator, elevating the
expression of these same targets and suppressing differentiation (43). TCF3 may also suppress the expression of Oct4
(240) and Nanog (195), although its ability to activate these
targets is unclear.
KIRSTY GREENOW AND ALAN R. CLARKE
thelial differentiation, implying a broad role for the Wnt
pathway in normal development and regeneration. A further example of such a role is seen in normal hair follicle
development, with deletion of ␤-catenin in mice impairing
hair follicle morphogenesis and resulting in the loss of the
follicle stem cell niche (7, 96). Stabilization of ␤-catenin also
drives hair follicle stem cells to proliferate (157), and overexpression of Wnt ligand in the epidermis is able to induce
hair follicle regeneration after wounding (100).
Several studies have also demonstrated the essential role of
Wnt signaling in the regeneration of mammalian muscle
and bone (219, 290). For example, the Wnt pathway plays
a vital role in bone homeostasis and has a role in the normal
embryonic development of the vertebrate limb (95). Wnt
signaling is also activated during fracture repair and enhances bone formation (222). Furthermore, gain-of-function mutations in the Wnt coreceptor LRP5 in humans gives
rise to high bone mass (13). A recent study has also demonstrated that modulation of Wnt levels through Dkk1 inhibition was sufficient to repair the bone lesions in multiple
myeloma and rheumatoid arthritis (287). Wnt signaling has
also been shown to be key to the regeneration of limbs in
lower vertebrates. These have the capacity to regenerate
complex structures consisting of multiple cell types either
through the reprogramming of differentiated cells or
through the activation of resident stem cells. Recent work in
amphibian and fish appendages has demonstrated a role for
Wnt in whole appendage regeneration, and the inhibition of
Wnt/␤-catenin signaling during the early stages of limb regeneration in both the Xenopus and axolotl completely inhibits this regeneration (112, 282). Furthermore, the expression of ␤-catenin in later-stage Xenopus limbs, where
the regenerative response is reduced and in nonregenerative
developing chick limbs, is sufficient to rescue the regenera-
Work in zebrafish has also demonstrated a functional role
for Wnt/␤-catenin in heart regeneration, which has been
confirmed by studies in mice. In vivo studies using ␤-catenin
conditional heart specific null mice has demonstrated a role
for the canonical Wnt signaling pathway in the differentiation of cardiomyocytes (3, 131). The Wnt11 ligand is required for cardiac differentiation and heart formation in
both chick and Xenopus embryos (60, 189). This function
of Wnt11 in heart development has also been shown to be
conserved in higher vertebrates as exogenous Wnt11 promotes the differentiation of murine ES cells into cardiomyocytes (245).
From the studies discussed above, it is clear that Wnt/␤catenin signaling plays a central role in many regenerative
processes, suggesting that Wnt/␤-catenin signaling may be a
conserved feature of regeneration. The manipulation of this
pathway should therefore be key in both manipulating somatic stem cells in situ and also in the development of iPS
cells for regenerative therapy.
E. Hedgehog
In vertebrates the Hedgehog (Hh) gene family encodes three
highly conserved, homologous signaling proteins: Sonic
(Shh), Indian (Ihh), and Desert (Dhh). These signaling molecules bind to the membrane receptor Patched (Ptc), which
in the absence of Hh inhibits the activity of another transmembrane protein, Smoothened (Smo) (99). The downstream effectors of this pathway are the Gli family of zincfinger transcription factors, and the alteration of the balance between activator and repressor forms of these
proteins results in regulation of Hh target genes.
Hh signaling is vital for many developmental processes, and
Shh has been shown to be involved in the determination of
cell fate and embryonic patterning during early vertebrate
development.
F. Hedgehog and In Vivo Regeneration
Hh signaling is important for the maintenance of stem or
progenitor cells in many adult tissues, including the epithe-
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Wnt signaling has also been suggested to play a role in
mammalian liver regeneration as patients with mutations in
the APC gene have an increased risk of developing hepatoblastoma, indicating that Wnt affects the hepatic progenitor
cells (75, 130, 181). Work in zebrafish has also demonstrated that an increase in Wnt activity significantly accelerated liver regeneration after a partial hepatectomy (77).
Furthermore, liver regeneration is significantly enhanced in
ApcMin/⫹ mice, and a conditional knockout model of
␤-catenin in the mouse liver has demonstrated decreased
hepatocyte proliferation and suboptimal regeneration
(241). In addition, Wnt signaling is upregulated in adult
hepatic progenitor cells during regeneration (101, 278).
Wnt signaling can also promote the renewal of the hematopoietic stem cells, and studies have shown that the
overexpression of activated ␤-catenin results in stem cell
survival and proliferation (205). In addition, during the
regenerative process following injury, hematopoietic cells
reactivate growth signals and show an increased activation in Wnt signaling (46).
tive process and induce regeneration (112). Similar to Xenopus limb regeneration, studies in zebrafish have demonstrated that Wnt/␤-catenin signaling has a role in all three
phases of fin regeneration and is sufficient to enhance regenerative outgrowth; also, ␤-catenin-independent Wnt signaling functions as an antagonist of fin regeneration, allowing a negative feedback loop to control fin regrowth (232).
Similarly, a crucial role for Wnt has been demonstrated in
Xenopus tail regeneration with the molecular mechanism
being similar to that of the zebrafish fin (149, 234). The only
mammalian organ capable of complete regeneration, deer
antlers, also requires ␤-catenin expression to maintain the
survival of antler progenitor cells (169).
STEM CELL COMPARTMENT, REGENERATION, AND PLURIPOTENCY PATHWAYS
Hh signaling also functions to maintain adult hematopoietic stem cells (HSCs) in both Drosophila and vertebrates by
regulating the HSC cell cycle and by balancing hematopoietic homeostasis and regeneration in vivo. Loss of Hh signaling in Drosophila results in the depletion of blood progenitor cells in the lymph gland due to premature differentiation (162) and the inhibition of hematopoiesis and
vasculogenesis in early postimplantation mouse embryos
(58). In adult mice, Ihh is expressed in differentiated epithelial cells of the small intestine, and conditional of Ihh from
these cells results in a wound healing response, thought to
be mediated through mesenchymal signaling (252). Ihh is
also expressed in bone marrow stroma, while Shh expression is found in lymph node and spleen stroma. Ihh overexpression in stromal cells promotes hematopoietic regeneration after bone marrow transplantation (123), and Hh signal activation induces cycling and expansion of primitive
hematopoietic cells (247).
The Hh pathway has also been extensively analyzed in neural stem and progenitor cells and has several roles in the
specification, proliferation, and differentiation of neural
precursors during embryogenesis. Hh signaling is also required for the maintenance of quiescent neural stem cells in
the adult brain (2, 14, 188). Shh has been shown to be
crucial in regulating stem cell niches and neural stem cell
proliferation in the postnatal telecephalon (244), the adult
hippocampus (133), and the SVZ (2) and plays a critical
role in the survival and patterning of neural progenitor cells
in the ventral spinal cord (151).
Hh signaling also has a role in vertebrate limb development
and is vital to both bone formation and bone remodeling,
which is primarily mediated through the regulation of both
86
chondrocyte and osteoblast differentiation (50). This function of the Hh pathway, in addition to its role in wound
healing, suggests a potential role for the Hh pathway in
limb regeneration. Consistent with this, work in urodeles
has demonstrated an increase in Shh expression after limb
amputation (98, 246) and also in the regenerating limb buds
of anurans (62). Functional evidence also suggests that Shh
is important for anterior-posterior axis patterning in the
regenerating limb (208, 209). Furthermore, activating Shh
in Xenopus froglets improves limb regeneration and results
in the formation of multiple cartilaginous structures (276).
Shh is also required for dorsoventral patterning of the regenerating spinal cord and surrounding mesodermal tissues
during Xenopus tail regeneration (216). While this function
is similar to that seen in the regenerating limb, it is clear that
the Shh signaling pathway has distinct roles in different
regenerating organs. Zebrafish fin regeneration also requires Shh during the outgrowth phase, and studies have
implicated a role for this pathway in the proliferation
and/or differentiation of scleroblasts (200). Shh has also
been shown to be important in axolotl tail regeneration,
where it is required for dorsoventral patterning of the regenerating spinal cord and for the regeneration of surrounding mesodermal tissues (216).
From the studies described above, it is clear that the Hedgehog pathway is a fundamental regulator of somatic stem
cells that maintain tissue integrity in mature organisms. The
importance of hedgehog signaling in regeneration has been
emphasized by recent work developing hedgehog-related
therapies for restoring tissue function where the normal
capacity to regenerate has been compromised (256).
G. JAK-STAT
The JAK-STAT pathway plays an essential role in mediating the biological response to growth factors and cytokines,
and characterization of IFN signaling resulted in the initial
identification of this pathway. Currently, four Janus kinase
(JAK) family members have been identified, and seven signal transducers and activators of transcription (STATs) are
known. The binding of ligands to the dimeric cytokine receptor results in the activation of two receptor-associated
JAKs and subsequent phosphorylation of the ligand-bound
receptor. The phosphorylation of the receptor tyrosine residues promotes the recruitment of a STAT family member,
which becomes phosphorylated and forms a homo- or heterodimer, which translocates into the nucleus and activates
target genes.
The JAK-STAT pathway plays a vital role in maintaining
the pluripotent state of mouse ES cells. As described above,
in vitro culture of mouse ES cells requires LIF to maintain
pluripotency. The JAK-STAT pathway is a key downstream
mediator of LIF, and the activation of the STAT3 pathway
by LIF induces transcription of self-renewal genes (34, 177).
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lia of many internal organs and the brain (16, 254). In the
small intestine, Hh signaling is important for the correct
overall crypt-villi structure of the epithelium, although it is
not directly involved in the fate of the epithelial cells (159).
Inhibition of Hh signaling in the intestinal epithelium results in reduced villus formation and mislocalization of subepithelial stromal cells. In addition, loss of Hh signaling has
a secondary effect by enhancing proliferation through Tcf4
and ␤-catenin gene activity and the formation of ectopic
precrypt structures on villus tips (159). In addition to tissue
maintenance through Hh-mediated stem or progenitor cell
self-renewal, Hh signaling also plays a critical role in the
regenerative responses to tissue injury, for example, in the
regeneration of the pulmonary epithelium (260), prostate
epithelium (111), and exocrine pancreas (67). In addition, a
recent study has demonstrated a role for Hh signaling during gastric regeneration, where this pathway is essential for
the differentiation of gastric progenitor cells (110). Recent
work has also suggested a role for the Hh pathway in hepatic remodeling as liver injury increases the activity of Hh
signaling in progenitor cells (69, 187).
KIRSTY GREENOW AND ALAN R. CLARKE
H. JAK-STAT and In Vivo Regeneration
In addition to maintaining pluripotency, the JAK-STAT
pathway stimulates cell proliferation, differentiation, cell
migration, and apoptosis and is vital in regulating various
developmental processes (97).
Recent work has demonstrated a key role for JAK-STAT
signaling in the homeostasis of the Drosophila midgut. The
JAK-STAT pathway regulates ISC self-renewal and differentiation, and compromised JAK-STAT signaling causes
ISC quiescence and loss, whereas signaling overactivation
produces extra ISC-like and progenitor cells (150, 239). In
addition to midgut ISCs, Stat92e is also specifically activated in the Drosophila hindgut stem cell population (239),
indicating a similar role of JAK/STAT signaling in hindgut
ISCs. Several recent studies have also suggested a role for
Drosophila JAK-STAT signaling in mediating injury- or
infection-induced ISC proliferation and regeneration (5, 31,
49, 105, 179). Enterocytes that are subjected to stress produce cytokines that activate the JAK-STAT pathway in the
intestinal stem cells, which results in proliferation and regeneration (105). Furthermore, we have shown that conditional deletion of Stat3 in the mouse intestinal epithelium
leads to loss of the stem cell compartment (unpublished
data), while others have shown that in a mouse model of
DSS-induced colitis, colon epithelial damage triggers an immune response and the expressed cytokines activate
STAT3, which subsequently promotes cell proliferation
and regeneration. In addition, inhibition of the STAT inhibitor SOCS3 in the mouse gut increased the proliferative
response to DSS (207).
The canonical JAK-STAT pathway also regulates germline
stem cell self-renewal, maintenance, and differentiation in
the Drosophila (29, 116, 248). The JAK-STAT pathway
maintains germline stem cell self-renewal by activating the
expression of self-renewal genes, and conditional loss of
JAK-STAT signaling results in germline stem cells differentiating into spermatogonia without self-renewal (29). Furthermore, the conditional restoration of JAK-STAT signaling in differentiated spermatogonia that are undergoing
transit-amplifying divisions are able to dedifferentiate to
become functional germline stem cells that are able to repopulate the niche (29).
STAT3 influences cell survival, metabolism, growth, differentiation, and migration in multiple organs, and recent
work has indicated a vital role for STAT3 in liver regeneration (145). In the normal liver, the interleukin (IL)-6/
STAT3 pathway is thought to play a central role in regeneration as disruption of this pathway in IL-6-deficient mice
results in impaired liver regeneration after 70% partial hepatectomy (PH) (48). STAT3 signaling also has a vital role in
wound healing, and in vivo studies have revealed that Stat3
expression in keratinocytes contributes to the regeneration
of the epidermis and hair cycle process (212). This potential
role of the STAT3 pathway in wound healing has also been
observed in the injured spinal cord. After injury, STAT3
activation (phosphorylation and nuclear localization) was
observed in reactive astrocytes surrounding the lesion, implicating a role for this pathway in tissue repair and functional recovery (185).
I. Notch
The Notch pathway is an evolutionary conserved pathway
that regulates cell fate determination during development.
This pathway is essential in the control of embryonic organogenesis and is also vital for postnatal tissue repair (10).
The Notch pathway consists of a single transmembrane
receptor, which undergoes proteolytic cleavage upon binding by members of the Jagged and Delta ligand families.
Cleavage involves the activity of the ␥-secretase protease
complex and releases a Notch intracellular domain (NICD)
that translocates to the nucleus. In the nucleus, NICD heterodimerizes with CSL transcription factors and activates
Notch target genes (30).
Notch signaling is required for the differentiation of ESCs;
however, the precise function of endogenous Notch signaling in mammalian ESCs is still unclear, although several
components of the Notch signaling pathway are highly expressed in both mESCs and hESCs (206, 215). Notch signaling is thought to be relatively inactive in undifferentiated
hESCs, and not necessary for their proliferation, although
many hESC-derived differentiated cells require Notch
(179). Neural specification of ESCs is influenced by Notch
signaling, and Notch1 activation promotes neural lineages
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STAT3 is the primary STAT protein activated in mouse ES
cells, and constitutively active STAT3 removes the need for
LIF to promote self-renewal (177, 178). STAT3 has multiple roles in the regulation of ES cell pluripotency including
gene activation, cell cycle regulation, and the inhibition of
differentiation pathways. STAT3 has also been reported to
function through the regulation of c-Myc (35), Klf4 (147),
and Klf5 (25), although these target genes have not been
shown to be completely sufficient to replace the effect of
LIF. This ability of LIF-mediated activation of STAT3 to
support the long-term self-renewal of mouse ES cells in vitro
has been supported in vivo by the requirement for this pathway in embryonic diapause (175). In addition to STAT3
homodimers, STAT1 is able to heterodimerize with STAT3
in mouse ES cells, although it is thought that it is unlikely to
be required for self-renewal as Stat1⫺/⫺ cells remain LIFdependent for undifferentiated growth (56). In human ES
cells, STAT3 does not appear to be sufficient to maintain
pluripotency (214), indicating once again that the signaling
pathways responsible for maintaining pluripotency may be
species-specific. Also, recent work by Griffiths et al. (79) has
identified a STAT3-independent JAK pathway that is able
to directly regulate pluripotency genes and maintain selfrenewal through chromatin phosphorylation.
STEM CELL COMPARTMENT, REGENERATION, AND PLURIPOTENCY PATHWAYS
and suppresses ESC differentiation into mesodermal cell
fates (173, 218). Conversely, the inhibition of Notch reduces neural lineage differentiation and increases nonneural ESC fate. For example, ESC differentiation into cardiomyocytes required Notch1 inhibition (173).
J. Notch and In Vivo Regeneration
In the small intestine, the Notch pathway plays a central
function in the cell fate decisions of intestinal stem cells, and
similar to the Wnt pathway, the Notch pathway is essential
to maintain the crypt compartment in its undifferentiated,
proliferative state. Inhibition of various components of the
Notch pathway in the intestine results in the rapid and
complete conversion of all epithelial cells into secretory
goblet cells (167, 270), an effect which is also seen in intestinal adenomas in ApcMin/⫹ mice (253). Consistent with
this, the constitutive activation of the Notch pathway in
the intestinal epithelium results in the depletion of goblet
cells and a reduction in enteroendocrine and Paneth cell
differentiation (70, 229). In addition to the loss of absorptive lineage cells, the in vivo phenotype of Notch inhibition resulted in the loss of the entire epithelial layer,
suggesting that Notch not only appears to control the
absorptive versus secretory cell fate decisions but is able
to promote the proliferation of crypt progenitor cells in
the transit-amplifying region by regulating the expansion
of intestinal stem cells. In addition, the ability of Notch
activation to suppress goblet cell differentiation and promote proliferation is vital to the regenerative response of
the intestinal epithelial cells. Thus the Notch pathway is
activated in the murine intestinal epithelium during ulcerative colitis and contributes to the regeneration of the
damaged epithelia by regulating differentiation, proliferation, and also the antimicrobial response of the epithelial
cells (184). This function of the Notch pathway was also
seen in the human intestine, both under normal conditions and when tissue damage has occurred (184).
The ability of Notch to expand the stem cell population in
response to injury has also been shown in several other
tissues including the brain (242), and various studies have
confirmed that the capacity of Notch to enhance tissue regeneration is due to its ability to activate progenitor cell
populations, in addition to regulating the differentiation
status of cells and enhancing proliferation. Therefore, the
activation of Notch signaling may provide a promising tar-
88
Notch signaling is also vital for the functional recovery of
vertebrate appendages. The inhibition of Notch prevents
anuran tail regeneration, while the overexpression of NICD
stimulates the regeneration of a tail that contains notochord
and spinal cord but little or no muscle (18). Notch also has
a role in mammalian skeletal muscle regeneration, and
Notch signaling is induced in activated muscle progenitor
cells, satellite cells, 2 days after injury both in culture and in
vivo (45). Expression of a constitutively active Notch receptor results in the inhibition of differentiation markers and
increased proliferation of progenitor cells in culture (45).
Importantly, the inhibition of Notch signaling in vivo impairs muscle regeneration (44). The induction of Notch signaling in activated satellite cells appears to be triggered by
the upregulation of the Delta ligand in satellite cells themselves and in muscle fibres adjacent to the damage (44). In
addition, the upregulation of Delta and the activation of
Notch are diminished in old mice, whose satellite cells proliferate less in response to injury, resulting in reduced efficacy of regeneration. Forced activation of Notch signaling
at the site of injury in old mice is sufficient to improve
muscle regeneration, rendering it similar to that of young
mice (44).
K. FGF/ERK MAPK Pathway
The MAPKs are a family of serine/threonine kinases that
have a functional role in connecting the activation of cellsurface receptors to various cellular functions such as proliferation, differentiation, migration, and apoptosis (38,
194). MAPKs are a part of a three-tiered kinase module that
consists of a MAPK, an upstream MEK, and a MEKK that
is coupled to cell surface receptors via adaptor proteins. The
ERK MAPKs can be activated by a wide variety of extracellular ligands, including growth factors, serum, cytokines,
and stress signals. One important upstream activator of the
ERK MAPK pathway is the fibroblast growth factor (FGF)
family of signaling molecules. FGFs are a large family of
signaling molecules with various functions in development
and adult physiology, and the ERK pathway mediates the
effects of this growth factor.
In human ES cells, it has been well established that exogenous FGF-2 is required to sustain self-renewal, and it is
widely used to culture human ES cells and iPS cells (6, 52,
273). Furthermore, in human ES cells, elevated levels of
exogenous FGF-2 alone are sufficient to maintain undifferentiated hESC in the absence of fibroblast feeders or fibroblast-conditioned media (141, 275). Inhibition of FGF signaling in hESC results in rapid differentiation (57), an effect
that is similarly seen in mouse ES cells (268). In human ES
cells, exogenous FGF-2 activates the ERK MAPK pathway,
which is thought to be necessary for the maintenance of
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In addition to a role in embryonic stem cells, Notch signaling is required for the maintenance and repair of various
organ systems. The Notch pathway is required for selfrenewal and cell-fate commitment of tissue stem cells and
has been shown to play a vital role in the regenerative response to injury in several adult tissues including the kidney, liver, bone marrow, skin, muscle, intestine, heart, trachea, and pancreas.
get for regenerative therapy in adult tissue in response to
injury.
KIRSTY GREENOW AND ALAN R. CLARKE
pluripotency, although the mechanism of action is still unclear (57, 143).
L. FGF/ERK and In Vivo Regeneration
As described above, the ERK MAPK family is activated by
several growth factors, one of which is FGF. The FGF pathway plays a central role in development and tissue regeneration, and FGF signaling has been implicated in the regulation of cell proliferation and specification in the majority of
regenerating systems. Components of the FGF pathway are
expressed in various somatic stem cell compartments, and
FGFs appear to be universal regulators of regeneration.
In the mouse small intestine, FGFs have been shown to have
an active role in murine colon development and have been
shown to promote the proliferation and survival of intestinal cells (211). The FGF receptor FGFR3 has been shown to
be strictly expressed in the intestinal progenitor cell compartment, where it has a role in regulating the expansion of
intestinal crypt cells (9). Also, in irradiated mice, treatment
with FGF7 increases crypt survival (114), and FGF1 and
FGF2 have a protective effect after intestinal injury (51, 71).
In humans, various FGF receptors have been shown to be
expressed in the gastrointestinal tract, and the altered expression of FGFR3-IIIb in intestinal epithelial cells is associated with differentiation (109).
In addition to tissue maintenance, FGF signaling is important in limb regeneration after injury. FGFs have several
essential roles during epimorphic regeneration of anuran
VI. REGENERATIVE PATHWAYS
The signaling networks that play a vital role in stem cell
maintenance of ES cells and somatic stem cells are essential
for progenitor cell formation and for the differentiation of
cells leading to tissue homeostasis. By looking at the pathways that regulate pluripotency, self-renewal, and stem cellmediated regenerative processes, it should enable us to
progress the dual tracks of in situ stem cell manipulation
and the development of iPS cells for regenerative therapy. A
key goal of these studies must be the identification of small
molecules that can be used to precisely manipulate these
regenerative processes.
Although we have briefly discussed some of the potential
pathways involved in pluripotency maintenance and also
their potential role in tissue regeneration, it is important to
remember that these pathways do not function in isolation
from each other or from other signaling pathways (e.g., see
FIG. 4). For example, the effects of Wnt signaling on stem
cells are modulated through association with Notch, Sonic
Hedgehog, and TGF-␤ (41). In addition, several other key
pathways regularly appear to be essential in the various
regenerative processes discussed above that have not been
discussed in this review. For example, cell cycle control
pathways have a vital role in tissue regeneration, and several studies have demonstrated that checkpoint proteins
such as p21 and Rb also appear to be important in tissue
regeneration in mice (19, 89, 217, 230, 266).
In addition, it is important to note that stem cells also depend on the extrinsic signaling pathways of the stem cell
niche. This stem cell microenvironment plays a vital role in
deciding the fate of these cells. The niche provides supportive growth factors and cytokines for the stem cell and con-
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In contrast to most cell lines and human ES cells, undifferentiated mouse ES cells do not require the ERK pathway for
proliferation and self-renewal (33), and remarkably,
ERK1/2 signaling actually inhibits the self-renewal of these
ES cells and triggers differentiation towards the primitive
endoderm lineage (39). Furthermore, reducing ERK activity
has been shown to enhance the efficiency of ES cell derivation by promoting the retention of Oct4-positive epiblast
during the outgrowth phase (32). Also, the differentiation
of established ES cells and their requirement for LIF can be
diminished by the addition of synthetic inhibitors of ERK
signaling (34). The mechanism behind the ability of the
ERK pathway to promote differentiation is unclear, although recent studies have suggested that ERK1/2 activation may act as a stimulus for ground-state ES cells, which
are intrinsically self-maintaining, to exit the self-renewal
program and enter a state that is responsive to inductive
stimuli (129). As mentioned already, the direct consequence
of this is that blocking the ERK inductive differentiation
pathway can maintain the ground state of the epiblast
(280). As perhaps a direct consequence of the above, this
inhibition of Erk1/2 MAPK pathway has been shown to
significantly improve the generation efficiency of iPS cells
(92, 223).
tails, urodele limbs, and fish fins and regulate the various
stages including wound epidermis formation, blastema formation, and proliferation and also positional memory
(231). The FGF pathway is vital during Xenopus limb regeneration, and the treatment of a nonregenerative stage
Xenopus limb stump after amputation with FGF8 results in
partial regeneration (281). Furthermore, treatment with
FGF10 results in significant regeneration of the limb due to
the activation of expression of several genes that are expressed in regenerating limbs such as Shh and Fgf10 (281).
Also, the treatment of the amputation surface of a nonregenerative chick limb bud with FGF2 or FGF4 induces a
regenerative response (126, 243). FGF signaling is also active and important in all three stages of zebrafish fin regeneration, similar to the Wnt pathway (231), and also for the
later phases of heart regeneration (140). FGF signaling is
also implicated in the regulation of cell proliferation and
differentiation in regenerating skeletal muscle and mammalian liver (63, 233), although clarification of its in vivo role
is still required.
STEM CELL COMPARTMENT, REGENERATION, AND PLURIPOTENCY PATHWAYS
tributes to the maintenance of the undifferentiated stem cell
state. For example, regulation of the intestinal stem cell
involves constant cross-talk between the epithelial cells and
the underlying mesenchymal cells in the intestinal stem cell
niche. This cross-talk is mediated by some of the key pathways mentioned above, in addition to pathways such as
the phosphatidylinositol 3-kinase pathway. This additional
level of control emphasizes yet further the complex goal of
precisely manipulating the endogenous stem cell population.
VII. REGENERATION OR
TRANSFORMATION?
Several studies have made the experimental observation
that, in some circumstances, it is possible to enrich for cancer forming cells within a bulk population of tumor cells,
which has led to the concept that cancers may be sustained
through a small population of “cancer stem cells.” This
model suggests that tumors are generated and maintained
by a subset of undifferentiated cells, the cancer stem cells,
which are similar to adult tissue-specific stem cells and are
able to self-renew and differentiate into the bulk tumor
population (see FIG. 5). Although the origin and indeed the
existence of the cancer stem cell is still under debate (53, 76,
81, 202), it is thought that the cancer stem cell could possibly arise from adult stem or progenitor cells that, due to
their higher capacity to proliferate, are more prone to accumulate mutations during their lifespan. These observations
suggest that deregulation of the pathways such as the ones
discussed above, that control “stemness” may potentially
promote cancer, either by increasing the number of potential initiators of neoplasia, or by expanding an established
cancer stem cell.
The cancer stem cell hypothesis has gained strength through
the apparent identification of initiating cell populations in
several tumor types, including blood (134), breast (4), pan-
FIGURE 5. The cancer stem cell model. The cancer stem may occur as a result of acquired mutations in a
somatic stem cell. The cancer stem cells retain the ability to self-renew and through multiple asymmetric cell
divisions are able to produce daughter cells that may form the bulk tumor population.
90
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The discussion above has focused on the positive roles of six
key pathways in pluripotency and regeneration. However,
these self-renewal pathways are also key pathways that often become deregulated in tumorigenesis. For example, activating mutations in the Wnt/␤-catenin pathway have been
shown to initiate tumorigenesis in a variety of human epithelial carcinomas, possibly by expanding the stem cell population as has been suggested in the small intestine (204).
Similarly, activation of Wnt signaling has been shown in
non-small-cell lung cancer (249), and bronchioalveolar
stem cells have been shown to multiply in response to Wnt
activation (286). Therefore, although the manipulation of
these pathways may be vital to the development of iPS cell
technology and regenerative therapy, there is clear potential
for unwanted side effects.
KIRSTY GREENOW AND ALAN R. CLARKE
creas (135, 142), as well as colon and brain (224). This field
does, however, remain contentious, with some arguing that
the observation of such initiating cells occurs as an artifact
of the experimental protocols used. Despite this uncertainty, it remains clear that early progenitor or stem cells in
a range of tumor types can undergo oncogenic transformation, and it therefore seems highly likely that the modulation of the pluripotency pathways of these cell populations
will directly alter tumor predisposition.
Table 3. Examples of the mutations that occur in the key
self-renewal pathways and their incidence in various cancers
Pathway
TGF-␤
Wnt
Hedgehog
JAK-STAT
Notch
MAPK-ERK
Cancer/Tumor Type
2SMAD4 in 53% of pancreatic carcinomas
1BMP2 in 98% of lung carcinomas
1␤-Catenin in 48% of small intestinal
carcinomas
1␤-Catenin in 64% of gastric polyps
2APC in 90% of colon cancers
2APC in 76% of gastric adenomas
2Ptch in 67% of basal cell carcinomas
1Smo in 10% of basal cell carcinomas
2Ptch in 10-20% of sporadic
medulloblastomas
1STAT3 in 50% lung
95% head and neck
1Notch in 55-60% of T-cell acute
lymphoblastic leukemia
1RAS in 45% of colon and 90% of
pancreatic cancers
1RAF in 60% of melanoma
[Adapted from Dreesen and Brivanlou (55).]
The examples cited above are but a few of the many examples implicating aberrant control of stem cell signaling pathways in the initiation and progression of cancer, either
through the expansion of normal or preneoplastic stem
cells, or by the expansion of proposed cancer stem cell populations. The cancer stem cell hypothesis remains controversial, and while the discussion of both sides of the argument is beyond the scope of this review, the potential existence of a tumor initiating cell population must remain a
key consideration for any therapies based on either modifying stem cell signaling pathways and the stem cell niche
directly in vivo or the introduction of iPS cells.
VIII. SUMMARY
Manipulation of the stem cell compartment remains an attractive target for the development of novel regenerative
therapies, either by directly manipulating existing stem cells
in vivo, or by supplying exogenously developed iPS cells.
This problem might usefully be broken down into two fundamental questions: 1) Have we identified the key molecules and pathways that regulate stemness? 2) Can the manipulation of these pathways lead to controlled tissue regeneration? We have already made substantial progress
towards addressing the first question, with a good understanding of the factors that control pluripotency, together
with an understanding of the pathways that they compose.
The main difficulty in addressing the second question is
simply the ubiquitous nature of the programming machinery; as we have tried to summarize in this review, it spans
and interacts with many, if not all, of the key transcriptional
pathways that regulate cellular life. For example, many laboratories have demonstrated a role for the Wnt pathway in
both pluripotency and repair, but unfortunately, the Wnt
pathway is so broad in its downstream influence that we
will need very complete and precise knowledge of the
checks and balances that influence Wnt-mediated regeneration before we can think of using this pathway therapeutically. This problem is further compounded by the extra
level of control exerted epigenetically, and also by the clear
influence of the niche within which the stem cells sit.
Hence, the real difficulty may be in understanding and integrating these pathways in a single tissue type or setting.
For example, complete deregulation of the Wnt pathway
fails to lead to a simple enhancement of a stem cell or
regenerative phenotype, but rather to a more sinister preneoplastic phenotype. More specifically, overexpression of
the Wnt-related pluripotency gene Oct4 also leads to a preneoplastic phenotype rather than an enhanced regenerative
phenotype. The conclusion that can be perhaps drawn is
that simple deregulation of the stem cell program will default to an enhanced predisposition to neoplasia precisely
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Several of the pathways discussed in this review have already been shown to be deregulated in cancer stem cells,
and thus manipulating these pathways may be a doubleedged sword. Some examples of these involvements are
given in TABLE 3. For example, the Hedgehog pathway has
been shown to be active in the cancer stem cells of several
myeloma (193) and brain tumors (15, 42, 197, 201). Direct
modulation of tumor outcome has now been shown for the
Wnt pathway, which has been suggested to sustain stemness
of CSCs within cutaneous squamous cell carcinomas, such
that deletion of ␤-catenin in established tumors resulted in
rapid loss of the CSC compartment and tumor regression
(161). Conversely, increased BMP4 signaling has been
shown to block glioblastoma formation by inhibiting cell
proliferation and promoting CSC differentiation (196).
Critically, in this example, the in vivo delivery of BMP4 was
able to reduce tumor growth and the associated mortality in
mice inoculated with human glioblastoma cells (196). In a
similar way, epigenetic silencing of the BMP type IB receptor is thought to contribute to the pathogenesis of glioblas-
toma by blocking BMP-mediated astroglial differentiation
in a subpopulation of brain cancer stem cells (136).
STEM CELL COMPARTMENT, REGENERATION, AND PLURIPOTENCY PATHWAYS
because we have failed to understand the complete network
that regulates entry and exit into the stem cell compartment.
12. Aubert J, Dunstan H, Chambers I, Smith A. Functional gene screening in embryonic
stem cells implicates Wnt antagonism in neural differentiation. Nat Biotechnol 20:
1240 –1245, 2002.
Despite these clear difficulties, we are successfully beginning to understand these networks more fully, and we do
now have clear examples of pathway-driven regeneration in
mammalian tissues. Indeed, we absolutely know from other
organisms that regeneration driven by these pathways is
possible, and the challenge now must be to develop a systems understanding of the multiple processes governing regeneration so that we can ultimately utilize them therapeutically.
13. Balemans W, Devogelaer J, Cleiren E, Piters E, Caussin E, Van Hul W. Novel LRP5
missense mutation in a patient with a high bone mass phenotype results in decreased
DKK1-mediated inhibition of Wnt signaling. J Bone Miner Res 22: 708 –716, 2007.
ACKNOWLEDGMENTS
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DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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