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CONTROLLING THE STEM CELL COMPARTMENTAND REGENERATION IN VIVO: THE ROLE OFPLURIPOTENCY PATHWAYSKirsty Greenow and Alan R. Clarke

School of Biosciences, Cardiff University, Cardiff, United Kingdom

LGreenow K, Clarke AR. Controlling the Stem Cell Compartment and Regener-ation In Vivo: The Role of Pluripotency Pathways. Physiol Rev 92: 7599, 2012;doi:10.1152/physrev.00040.2010.Since the realization that embryonic stemcells 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 pluripo-tency are defined by the complex molecular convergence of these pathways. Perhaps this has mostclearly 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 apluripotent state, and how we may manipulate cells to drive them between committed and pluri-potent 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 importantthese pathways are in these settings. It is also clear that there is a potential dark side tomanipulating the stem cell compartment, as deregulation of somatic stem cells is being increas-ingly 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 ofstemness 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 thepotential to replace or augment intervention-based strategies (cell replacement therapies), al-

though it is clear they must be developed with a full understanding of how such approaches mightalso influence tumorigenesis.

I. INTRODUCTION 75

II. PLURIPOTENCY FACTORS 76III. THE EPIGENETIC REGULATION... 78

IV. IPS CELLS IN THERAPY 80

V. PLURIPOTENCY PATHWAYS 80

VI. REGENERATIVE PATHWAYS 89

VII. REGENERATION OR TRANSFORMATION? 90

VIII. SUMMARY 91

I. INTRODUCTION

Embryonic stem (ES) cells and induced pluripotent stem(iPS) cells have the capacity to self-renew, and the differen-

tiation of these cells can yield over 200 diversely functionalcell types. The ability of pluripotent cells to differentiateand produce a potential source of somatic cells makes thesecells an obvious choice for regenerative medicine. Unfortu-nately, before the potential of pluripotent cells can be har-nessed for clinical application, it is essential that we under-stand the mechanisms involved in maintaining pluripotencyand inducing differentiation. The recent discovery of so-matic cell reprogramming with key pluripotency factors hasprovided essential information on the role of transcriptionfactors in pluripotency maintenance. These pluripotency

factors are thought to be capable of initiating most, if notall, of the necessary developmental signaling pathways re-quired for somatic cell reprogramming. The generation ofthese reprogrammed iPS cells has opened up the prospect ofderiving patient-specific cells for regenerative medicine andmay resolve the ethical issues and the immune rejectionrisks associated with the use of human ES cells.

While the generation of iPS cells and the identification ofkey pluripotency factors has been a breakthrough in under-standing pluripotency, somewhat less is known of the sig-naling networks surrounding these key transcription fac-tors. Without a clearer understanding of the signaling net-work involved in pluripotency maintenance and also

differentiation, the use of iPS cells and ES cells in cellulartherapy will be limited, as will our capacity to modulateexisting stem cells in situ. It is therefore essential that fur-ther mechanistic and functional studies are carried out,which will inevitably continue to identify factors that arecapable of defining stemness and mediating reprogrammingin culture. This also raises the much wider question of therelevance of these mechanisms in adult stem cells and tonormal physiology, either in maintaining tissue homeosta-sis, or more critically in mediating the repair response todisease and injury. Here, we will review the potential path-

Physiol Rev 92: 7599, 2012doi:10.1152/physrev.00040.2010

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ways that may be downstream of pluripotency factors iden-tified in ES cells and iPS cells (152, 255, 261) and alsoexamine their functional relevance in adult somatic stemcells. Such an improved understanding will not only en-hance our fundamental understanding of the regulation oftissue maintenance, but may also open the possibility ofdirectly manipulating these pathways to mediate efficientregeneration following disease or injury.

Glossary

Adult stem cell An undifferentiated cellfound in the different tis-sues or organs of the de-veloped, adult organismthat can renew itselfand differentiate to yieldall the specialized celltypes of that tissue ororgan.

Cancer stemcell Cancer cells that possess

characteristics associ-ated with normal stemcells, such as self-re-newal, and also producedifferentiated progenythat form the bulk of thecancer.

Embryonicstem cells Undifferentiated cells de-

rived from the inner cellmass of a developingblastocyst that are ca-pable of long-term self-renewal and have the po-tential to differentiateinto all cell types.

Differentiation The process of develop-ment by which a primi-tive, unspecialized cellcommits to becoming a

specific cell in the body.Induced

pluripotentstem cells A type of pluripotent stem

cell artificially derivedfrom a nonpluripotentcell, such as an adult so-matic cell, by inducing aforced expression ofkey pluripotency genes.

Pluripotent Ability of a single stem cellto differentiate into themajority of the cells ofthe body.

Progenitor cells An early, partially differen-tiated, descendant of astem cell that is capableof division and further

differentiation into thevarious cell types of atissue, but is unable torenew itself.

Quiescence The state of a cell when itis not dividing.

Regenerativemedicine The use of stem cells to re-

place or repair damagedor diseased cells, tis-sues, and organs.

Reprogramming The process of creating aninduced pluripotent stemcell through the intro-duction of a combinationof three to four genes,into an adult cell.

Self-renewal The division of a cell thatrepeatedly generatesat least one daugh-ter cell with identicalproperties to the par-ent cell, thereby allow-

ing the population tobe replenished indefi-nitely.

Stem cell niche The supporting microenvi-ronment in which stemcells are found.

Stemness An essential characteris-tic of a stem cell thatdistinguishes it from anormal cell.

Teratoma A tumor composed of tis-

sues from the three em-bryonic germ layers.

II. PLURIPOTENCY FACTORS

A. Embryonic Stem Cells

As described above, ES cells are pluripotent cells that arecapable of unlimited self-renewal and are able to differen-tiate into all three embryonic lineages. To maintain their

STEM CELL COMPARTMENT, REGENERATION, AND PLURIPOTENCY PATHWAYS

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ability to self-renew and remain undifferentiated, ES cellsrequire the cooperation of several regulatory factors, in-cluding a unique set of transcription factors (26, 117), Poly-comb complexes (27, 36), microRNAs (164), and histonemodification enzymes (23). Although the precise role ofthese mechanisms is still unclear, several studies have iden-tified the core transcription factors that are necessary forpluripotency in ES cells. Oct4 and Nanog are two home-

odomain transcription factors and were the first to be iden-tified as essential for early embryo development and pluri-potency maintenance in ES cells (37, 168, 176). In addition,Sox2, an HMG-box transcription factor, which het-erodimerizes with Oct4, has also been shown to be centralto the transcriptional network regulating pluripotency inmouse ES cells (26). All three of these transcription factorsact as either transcriptional activators or repressors andshare a substantial fraction of their target genes (26, 155).The ability of these factors to activate or repress genes al-lows them to maintain pluripotency by promoting the ex-pression of downstream self-renewal genes while simulta-neously repressing the activity of differentiation-promoting

genes. These factors also function interdependently anddemonstrate an autoregulatory feedback loop which en-

ables them to maintain the ES cell transcriptome through atight regulatory network (FIG. 1).

Many other transcription factors have been identified toclosely cooperate with these three essential pluripotency fac-tors including Sal14, Dax1, Essrb, Tcf4, Klf4, Tbx3, Tcl1,Rif1, Nac1, and Zfp281 (40, 43, 102, 117, 155, 257). Thesetranscription factors also regulate each other, forming a com-

plicated transcriptional regulatory network in ES cells (291).Essrb, Rif1, Zic3, and Tcl1 are also primary targets of bothOct4 and Nanog (148, 155). In addition, RNAi-mediatedknockdown has confirmed that eight genes, including Nanog,Oct4, and Sox2, are essential for maintaining an undifferenti-ated state in ES cells (102). Expression profile analysis has alsoidentified developmental genes (Otx2, Pitx2, and Sox18) reg-ulated by these eight factors, which induce ES cell differentia-tion when overexpressed (102).

B. Induced Pluripotent Stem Cells

From the above, it is clear that ES cell studies have impli-cated 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.

KIRSTY GREENOW AND ALAN R. CLARKE

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for which has subsequently been confirmed by reprogram-ming 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 toreprogram both mouse embryonic and adult fibroblasts to apluripotent state (160, 186, 237, 263). These repro-grammed cells were able to yield live born chimeras andwere also able to produce iPS cell progeny through germline

transmission. In addition, they displayed similar epigeneticand transcriptional profiles compared with ES cells, such astheir status with respect to DNA methylation, histone H3methylation, X chromosome activation, and global geneexpression profiles (160, 186, 263). These studies also re-vealed reactivation of the endogenous Oct4, Sox2, andNanog genes, all of which are markers for undifferentiatedES cells. Oct4, Sox2, c-Myc, and Klf4 have also been shownto successfully reprogram human dermal fibroblasts (191,236), whereas others have shown that Oct4, Sox2, Nanog,and Lin28 were sufficient to establish pluripotent cells fromhuman somatic cells (283). In all three of these studies, thetranscriptional and epigenetic profiles were similar to hu-

man ES cells and the iPS cells formed derivatives of all threegerm layers when differentiated in vitro and in teratomaassays.

In addition to the groups of pluripotency factors used toreprogram iPS cells, the levels and ratios of the reprogram-ming factors have a critical role in the efficiency of repro-gramming. For example, increasing Oct4 levels increasesreprogramming efficiency, whilst increasing Sox2, Klf4, orc-Myc individually causes a significant decrease in repro-gramming efficiency (47). The optimal ratio of the repro-gramming factors will vary depending on the cell type usedand also the endogenous expression of any one of the re-

programming factors within the starting cell population. Asummary of some of the reprogramming scenarios that havebeen used successfully in both mouse and human are givenin TABLE 1.

Although the mechanisms behind reprogramming somaticcells back to a pluripotent state are largely unknown, recentwork has shown that the reprogramming process with de-

fined factors is a gradual, time-dependent process, and stud-ies by Brambrink et al. (28) and Stadfield et al. (227) haveidentified the sequence of key events necessary for the re-programming of mouse embryonic fibroblasts. The minimaltime for full reprogramming of mouse somatic cells hasbeen defined as 812 days, and reprogramming appears tofollow a sequential path consisting of early and late events.The reprogramming factors progressively reestablish thecore circuitry of pluripotency, and as reprogramming pro-gresses, the properties of pluripotent stem cells begin toappear.

III. THE EPIGENETIC REGULATION OFPLURIPOTENCY

Gene regulation during pluripotency also requires epige-netic changes to chromatin, and it has been well establishedthat ES cells and differentiated cells have significantly dif-ferent epigenetic signatures (22, 23). Pluripotent cells re-quire a dynamic and transcriptionally permissive chromatinstructure whereby structural proteins are loosely associ-ated, enabling rapid reorganization of the chromatin struc-ture in response to differentiation signals (166). Lineage-specific genes are therefore poised for transcriptional acti-vation 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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methylation until the appropriate signals are received (23).The epigenetic status of iPS cells and their cells of origin arealso markedly different, suggesting that reprogramming in-volves dynamic chromatin changes, and it has been demon-strated that during reprogramming the epigenetic statusprogresses from a somatic to a pluripotent level (120).

This specialized chromatin state is established partly by theactivity of ATP-dependent nucleosome remodeling com-plexes, such as Swi2/Snf2. A number of ATP-dependentnucleosome remodeling complexes have been shown to beimportant for ES cell self-renewal and differentiation, andrecent studies have demonstrated that treatment with vari-

ous chromatin modifiers can improve the frequency of iPScell generation (93, 94). ES cells contain a family of func-tionally and structurally specialized chromatin remodelingcomplexes that are critical for the self-renewal of ES cellsand maintain the stem cell state (90). Several BAF com-plexes, including a unique complex, esBAF, are expressed inES cells, and ES cells depleted of various known subunits ofBAF have been shown to have defects in gene regulation,self-renewal, and pluripotency (72, 277). The catalytic AT-Pase SWI/SNF subunit Brg acts as the catalytic subunit ofesBAF and has been identified as an essential factor in nu-

clear reprogramming (87). esBAF plays a direct role in me-diating the gene regulatory function of several core pluripo-tency factors. The Brg subunit selectively regulates the ex-pression of several factors of the core network as well asinteracting directly with Oct4, Sox2, and Nanog (90). Brgalso interacts with several other proteins that have beenimplicated in ES cell maintenance such as Rif1, Sall4,Dppa2, and Dppa4, and it is thought that the surface ofesBAF may be tailored for interaction with factors specifi-cally found in ES cells. esBAF also functions to repress plu-ripotency genes such as Oct4, Sox2, and Nanog; this inhi-bition is thought to refine their levels and maintain pluripo-tency. In addition to BAF, several other chromatin

modifiers have recently been identified as vital factors inmaintaining ES cell pluripotency, such as Chd1 (73), thehistone deacetylase NuRD (106, 292), and Tip60-p400(65). Similarly to BAF, these factors interact and regulatethe core pluripotency transcription factors of ES cells.

As described above, pluripotency factors and epigeneticregulators engage in cross-talk with one another to main-tain pluripotency. Several pluripotency factors that are reg-ulated by epigenetic regulators also regulate the genes en-coding the epigenetic control factors; for example, Oct4,

Table 1. Examples of the various combinations of transcription factors and cell types that have been used in the production of iPS cells

Species Cell Type Factors Reference Nos.

Human HNF Oct4, Sox2, Klf4, c-Myc 119

HDF 237, 289

HFLS

BJ cells

HEFs 107, 269Keratinocytes 1, 186

CD34 blood cells 154

Adult fibroblasts Oct4, Sox2, Nanog, LIN28 146, 283

Foreskin fibroblasts

H1F cells Oct4, Sox2, Klf4, c-Myc, hTERT, SV40 large T 191

MSCs

Adult fibroblasts

HDFs

NHDF Oct4, Sox2, Klf4 170

BJ cells Oct4, Sox2 94

Mouse MEFs and tail tip fibroblasts Oct4, Sox2, Klf4, c-Myc 160, 165, 237

MEFs 107, 186, 199, 263

Tail tip fibroblasts and hepatocytes 228

Hepatocytes and gastric epithelial cells 8

MEFs and tail tip fibroblasts Oct4, Sox2, Klf4 165

MEFs 93, 163, 263

NSCs Oct4, Klf4 119

NPCs 61, 221

MEFs 220

MEFs and NSCs 223

MEFs Oct4, Sox2 68

NSCs Oct4 118




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Sox2, and Nanog coregulate certain genes encoding chro-matin remodeling and histone-modifying complexes, suchas Ehmt1, SMARCAD1, MYST3, and SET in mouse andhuman ES cells (26). Also, pluripotency factors interactwith histone-modifying enzymes and chromatin remodelingcomplexes; for example, Nanog and Oct4 interact directlyand indirectly with the polycomb group (YY1, Rnf2, andRybp) and SWI/SNF chromatin remodeling (BAF155) fac-

tors and KAP1 (Tif1

) (257). In addition, several othergenes regulated by pluripotency factors are subjected toepigenetic regulation. For example, two downstream tar-gets of Oct4, Jmjd1a and Jmjd2c, both of which are histonedemethylases, positively regulate several pluripotency-asso-ciated genes including Tcl1, Tcfcf2l1, Zfp57, and Nanog(155, 156).

IV. IPS CELLS IN THERAPY

The reprogramming of human somatic cells to a pluripotentstate has significant implications for the generation of pa-tient-specific, genetically compatible cells for transplanta-tion. TABLE 2 lists a number of disease models in which iPScells have already been used as a potential therapy. iPS cellshave been generated from the cells of various mouse tissuesincluding blood (85), liver (8), stomach (8), pancreas (227),brain (61, 119, 221), and the intestine and adrenals (262) aswell as also human fibroblasts (158, 191, 236), keratino-cytes (1), and blood progenitor cells (154). Recent studiesusing iPS cells to alleviate the symptoms of Parkinsonsdisease and sickle cell anemia in mouse models have dem-onstrated the potential of these cells in cellular therapy (86,264). Furthermore, Raya et al. (203) reprogrammed genet-ically corrected Fanconi anemia fibroblasts and obtained

corrected Fanconi anemia-specific iPS cells that can give riseto phenotypically normal hematopoietic progenitors of themyeloid and erythroid lineages. In addition to neural andblood diseases, iPS cells have been differentiated into manytypes of cardiovascular cells, including arterial, venous, andlymphatic endothelial cells as well as cardiomyocytes (172,285). Another potential therapeutic use of iPS cells is in thetreatment of infertile patients. Primordial germ cells (PGCs)can be generated through in vitro differentiation of humaniPS and ES cells, and these in vitro differentiated PGCs showgene expression similarities to in vivo PGCs (192). If these

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 iPScells, our current limited understanding of the reprogram-ming process hinders the ultimate goal of using these cells in

cellular therapy.

V. PLURIPOTENCY PATHWAYS

Signal transduction pathways are critical in maintaining

pluripotency and controlling the lineage-specific differenti-ation of stem cells. Although ES cell research began morethan 20 years ago, the regulatory network of ES cell main-

tenance is still largely unknown. Several extrinsic growthfactors support the pluripotency of ES cells in vitro, withmouse ES cells usually being cultured in the presence of

leukemia inhibitory factor (LIF) and bone morphogeneticproteins (BMPs). LIF is thought to activate STAT3 signalingpathways to maintain pluripotency, whilst BMPs act to in-

hibit differentiation by activating members of the Id gene(inhibition of differentiation) family (177, 279). Impor-tantly, recent work has also indicated that ES cells can be

cultured in the absence of LIF and BMP. These ES cellsrepresent a ground state, a basal proliferative state that isfree of epigenetic restriction and has minimal requirements

for extrinsic stimuli (280). In this state, the pluripotencynetwork, headed by Oct4/Sox2, Nanog, and KLF2/KLF4, isintrinsically stable but extremely sensitive to destabilization

by exogenous signals, with ES cells requiring the presence ofMEK and GSK3 inhibitors to suppress innate differentia-tion stimuli and maintain the stability of the ground state

(84, 271).

The commitment of ES cells to a specific lineage is thereforeregulated by specific signaling pathways, such as the JAK-

STAT pathway mentioned above. The Wnt/-catenin sig-naling pathway is also heavily implicated, for example,through regulating cardiomyocyte differentiation of mouseES cells, while the induction of ERK1/2 signaling triggers

the differentiation of mouse ES cells towards the primitiveendoderm lineage (39, 170). A schematic showing the pro-posed 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

Disease Cell of Origin Factors Reference Nos.

Sickle cell anemia Tail-tip fibroblasts Oct4, Sox2, Klf4, c-Myc 86

Parkinsons Disease Patient-derived fibroblasts Oct4, Sox2, Klf4, c-Myc 226

Fanconi anemia Patient-derived fibroblasts Oct4, Sox2, Klf4, c-Myc 203

Spinal muscular atrophy Patient-derived fibroblasts Oct4, Sox2, Nanog, Lin28 59

Duchenne muscular dystrophy Mouse embryonic fibroblasts Oct4, Sox2, Klf4 113

Patient-derived fibroblasts Oct4, Sox2, Klf4, c-Myc




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Signaling pathways therefore play a critical role in the in-duction of pluripotency during somatic cell reprogram-ming. However, unfortunately, rather little is known aboutthe signaling pathways regulated by the pluripotency fac-tors Oct4, Sox2, Klf4, and c-Myc, although recent work inmouse ES cells has identified key pathways that are regu-lated by these factors to maintain pluripotency (152). Forexample, work in ES cells has identified that two key sig-naling pathways are integrated into the Oct4, Sox2, andNanog circuitries through Smad1 and STAT3 (40). Liu etal. (152) have also demonstrated that these pluripotencyfactors regulated a crucial developmental signaling net-work, which is comprised of 16 signaling pathways, includ-ing 9 with previously unknown roles in the maintenance ofES cell pluripotency. In addition, further work by Huang etal. (92) has identified a core signaling network regulated by

these factors to induce pluripotency in reprogrammed so-matic cells. This study identified eight pathways as essentialpathways in iPS and ES cells, including transforminggrowth factor (TGF)-, Hedgehog, Wnt, p53, and JAK-STAT, and it is thought that the key pluripotency factorssynergistically regulate these pathways in a balanced way toinduce pluripotency (92). In addition, several other devel-opmental signaling pathways, including mitogen-activatedprotein kinase (MAPK), vascular epidermal growth factor(VEGF), Notch, ErbB, and mTOR were identified as path-ways supporting the core network (92).

We therefore have an emerging picture of pluripotency reg-ulating factors which impinge on a broad range of signalingnetworks (FIG. 4). In the next section of this review, we willexamine some of the key pathways identified as being im-portant for iPS cell induction. We will also investigate theestablished role of these pathways in maintaining ES cellpluripotency and their role in somatic stem cells. Althoughthe core pluripotency network is thought to differ in adultstem cells, these key pathways have established roles inmaintaining the somatic stem cells and in regulating tissuehomeostasis; therefore, we will also examine the potentialof these signaling pathways in tissue regeneration.

A. TGF-

The TGF- superfamily plays a major role in the biology ofmammalian development and regulates many cellular pro-cesses including cell fate, proliferation, senescence, apopto-sis, and tissue repair. TGF- and associated members of thefamily are mainly pleiotropic growth factors and are com-prised of two main groups, the bone morphogenetic pro-teins (BMPs) and the growth differentiation factors (GDFs),which includes TGF-, activin, and nodal and can be fur-ther divided according to sequence homologies and func-tional similarities. The canonical signaling cascade ofTGF- growth factors involves the ligands of the TGF-

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 inred). 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.


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superfamily binding to cell surface receptors that activatethe Smad proteins in the cytoplasm, which leads to theirnuclear translocation and transcriptional activation of tar-get genes. This pathway is also regulated by other key sig-naling pathways such as Wnt and Hedgehog, and also othertyrosine kinase-linked growth receptor signaling pathways.

TGF- signaling has been shown to be important in themaintenance of self-renewal and pluripotency of bothmouse and human ES cells, although the role of these sig-naling molecules does appear to differ between the twospecies (258). In mouse ES cells, BMP4 maintains self-re-newal 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 cellproliferation via an increase in Wnt expression by Smadactivation (137). In contrast, in human ES cells, BMP4 pro-motes stem cell differentiation through the downregulationof Nanog and Oct4, and activated Smad is only found atlow levels (274). Long-term maintenance of human ES cellpluripotency therefore requires the downregulation of BMPactivity by Noggin and basic fibroblast growth factor(bFGF) (6, 275). Other TGF- family members are alsothought to play an important role in the maintenance of ES

cells. Phosphorylation and nuclear localization of Smad2induced by TGF-, activin, or nodal signaling was observedin undifferentiated human ES cells and was decreased uponearly differentiation (103). The inhibition of these path-ways initiated differentiation and resulted in the decreasedexpression of stem cell markers (103, 250). In addition,Xiao et al. (272) have demonstrated that activin is able tosupport long-term feeder-free culture and maintenance ofpluripotency in human ES cells by inducing the expressionof Oct-4 and Nanog, and suppressing BMP. Nodal expres-sion also plays a role in the maintenance of human ES cellpluripotency through the inhibition of neuroectodermaldifferentiation, a default differentiation pathway of ES cells

(251). In addition, activin and nodal signaling has beenshown to promote mouse ES cell self-renewal in serum-freeconditions (183). It is therefore clear that TGF- signalingplays an important role in the maintenance of self-renewaland pluripotency, although the exact mechanism of actionfor this family of growth factors appears to differ betweenfamily members and species.

BMPs are also potent inhibitors of neural differentiation invertebrate embryos (267), and it is thought that BMP reg-ulation of ES cell pluripotency may be primarily through the

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 pathwayshave 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.




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inhibition of differentiation rather than as a direct stimula-tion of self-renewal (279). The knockdown of Smad1 andSmad4 expression in ES cells did not impair the self-renewability of these cells but led to a change in the expressionpattern of germ layer markers during differentiation (66).Furthermore, Smad4/ ES cells show no defect in theirself-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-renewaland differentiation by regulating development-associatedtarget genes. Consistent with this hypothesis, several Smadtarget genes overlap with genes bound by the key pluripo-tency factors, for example, Smad4-regulated genes have asubstantial overlap with those of Sox2, NR0B1/Dax1, andKlf4 (66). In addition, a recent study has demonstrated thatseveral Smad targets were mapped to Nanog, Oct4, andTCF3-bound genes (43).

TGF- signaling also participates in the cell fate decisions ofES cells, and these factors can direct the fate of ES cellstowards multiple cell lineages of the three embryonic layers,including neural, hematopoietic, cardiomyogenic, and he-patic (121). Specifically hematopoietic cell differentiation isdependent on BMP4 in mouse ES cells (190). BMP4 regu-lates mesodermal cell commitment to the hematopoieticlineage and specifies blood lineages at the later stages ofdifferentiation (139, 190, 210). Furthermore, a recent studyby Lengerke et al. (138) demonstrated that the role ofBMP4 in hematopoietic differentiation is conserved in hu-man iPS cells, and stimulation of BMP4 directs iPS celldifferentiation toward blood lineages.

TGF- signaling also has a role during endothelial cell pro-

liferation and vascular development in mouse ES cells (124,180, 235, 259). BMP4 and activin are thought to inducemesoderm differentiation into the cardiac lineage and in-duce human ES cells to differentiate into cardiomyocytes(132). In addition, BMP2-induced mesodermal and cardiacspecification results in full cardiogenic differentiation, lead-ing to an enrichment of cardiomyocytes within embryoidbodies (20). This ability of the TGF- family members tocommit mouse ES cells toward a mesodermal fate is due toSmad-mediated regulation of the Oct4 promoter, furtherimplicating a role for Smad signaling in the regulation of thecore self-renewal network in ES cells (284). This role for

TGF- family members in cardiomyocyte differentiation isof potential interest for the treatment of cardiac disease,particularly as Narazaki et al. (172) have successfully dif-ferentiated mouse iPS cells into cardiovascular progenitorcells, which subsequently generated arterial, venous, andlymphatic endothelial cells as well as self-beating cardiomy-ocytes. It may therefore be possible to potentially manipu-late the TGF- pathway to increase the efficiency of cardio-vascular cell differentiation from patient-specific iPS cells.In addition, the TGF- pathway could potentially be usedin the clinical treatment of other diseases by exploiting its

ability to direct the cell fate of ES cells and potentially iPScells. For example, recent progress in obtaining endodermcells by treating differentiating ES cells with activin andBMP4 (78, 127) could potentially lead the way for the de-velopment of iPS cell use to obtain endoderm derivativecells such as pancreatic and hepatic cells for use in thetreatment 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 familyalso has a functional role in the maintenance of somaticstem cells and also tissue regeneration (258). This role of theTGF- pathway is of particular significance as the potentialuse of iPS cells in regenerative therapy requires a clearerunderstanding of the regulatory signaling networks main-taining somatic stem cells and driving tissue regeneration.

BMP signaling plays a vital role in the regeneration of theintestinal epithelium. The BMP pathway maintains intesti-

nal stem cell quiescence and regulates terminal differentia-tion by negatively regulating the Wnt signaling pathwaythrough PTEN activation. The conditional deletion ofBmpr1a results in the expansion of the stem and progenitorcell populations leading to hyperproliferative crypts, andthe inhibition of BMP signaling on the villus through over-expression of Noggin results in ectopic crypt formation(88). BMP antagonism of the Wnt pathway also maintainsthe hair follicle stem cells (74, 122, 144), the mesenchymalstem cells (104, 153, 174), and neural stem cells (64). In allthese systems, BMP signaling molecules antagonize Wntsignaling to maintain quiescent stem cells.

In addition to its role in somatic stem cell maintenance,BMP signaling also has a functional role during limb regen-eration. BMP is required for Xenopus tail and limb re-growth (17, 80) and also mammalian digit tip regeneration(83). During Xenopus tail regeneration, BMP signaling isrequired to induce cellular proliferation in the notochordand the spinal cord (17). BMP also has the ability to rescueregeneration of the entire tail when overexpressed, suggest-ing that BMP regulation of these structures may be thedriving force behind tail tissue regeneration (17). BMP isalso essential for bone matrix deposition by regulating theproliferation and/or differentiation of scleroblasts during

the outgrowth phase of zebrafish fin regeneration (200). Inaddition to appendage regeneration, BMP signaling hasalso been shown to be crucial in the regulation of earlyevents during liver regeneration in zebrafish (108).

C. WNT

Similar to TGF- signaling, the Wnt pathway has an impor-tant role in tissue development and also regulates cellularmechanisms such as adhesion, morphology, proliferation,


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migration, and structural remodeling (21, 204). The Wntpathway consists of over 30 extracellular ligands that bindto the Frizzled (FZD) and low-density lipoprotein receptor-related protein (LRP) receptors at the cell surface (24). TheWnt ligands are able to activate the canonical Wnt pathwayor the noncanonical pathway (265). The activation of Wntsignaling in the canonical pathway results in the downregu-lation of -catenin degradation and subsequent nuclear

translocation, which enables gene activation by the TCF/LEF family of transcription factors. The noncanonical path-way is independent of-catenin and involves the activationof several other signaling pathways such as the JNK path-way.

The Wnt signaling pathway is coupled directly with the coretranscriptional network of pluripotency. Wnt signaling isactivated in ES cells and is downregulated during differen-tiation (214). Sato et al. (214) demonstrated that the acti-vation of the canonical Wnt pathway is sufficient to main-tain self-renewal of both human and mouse embryonic stemcells and is independent from the LIF/STAT pathway. Dur-ing this study, they demonstrated that the activation of theWnt signaling pathway maintained pluripotency and alsosustained the expression of pluripotency factors includingOct4, Nanog, and Rex1. Further studies also demonstratedthat the conditioned medium from mouse fibroblasts ex-pressing Wnt maintained the pluripotency of mouse ES cells(182). In addition, a study overexpressing an activated mu-tant of-catenin confirmed the role of Wnt in maintainingthe expression levels of Oct4, Nanog, and Rex1 and thelong-term proliferation of ES cells (238). This study alsodemonstrated that the -catenin upregulation of Nanogwas in an Oct4-dependent manner and that -catenin phys-

ically associates with Oct4 (238).

In addition to maintaining pluripotency, Wnt signaling reg-ulates differentiation in ES cells. Wnt signaling has beenfound to specifically inhibit neural differentiation (12, 82),and several other studies have implicated that Wnt signalingmay be sufficient to inhibit differentiation altogether (214).ES cells with a mutated form of Apc were found to exhibitdifferentiation defects both in vitro and in teratomas, andthis inhibitory effect was shown to be dose-dependent withthe severity of the Apc mutation (115). ES cells with highlyelevated -catenin levels also have a compromised ability to

differentiate (54), although even mutant ES cells with thehighest levels of -catenin activity were capable of somedifferentiation, which is consistent with previous reportsthat the activation of Wnt signaling alone is not sufficient tosupport long-term ES cell self-renewal (182, 280).

The downstream mediators of-catenin activation are theTCF/LEF family of transcription factors. In ES cells, Tcf3 isthe most abundantly expressed member of this transcrip-tion factor family, and Tcf3-null ES cells have an increasedresistance to differentiation (195). Tcf3-null ES cells

showed upregulation of various Oct4- and Nanog-regu-lated genes and was also shown to cooccupy the promotersof several Oct4 and Nanog targets (43). TCF3 is thought tofunction to oppose the effects of the core pluripotency ma-chinery and limit the expression of key regulators. Activa-tion of Wnt converts TCF3 into an activator, elevating theexpression of these same targets and suppressing differen-tiation (43). TCF3 may also suppress the expression of Oct4

(240) and Nanog (195), although its ability to activate thesetargets is unclear.

Recent studies have also established a role for Wnt signalingin the generation of iPS cells. Soluble Wnt3a promotes thederivation of iPS cells in the absence of c-Myc (163). Also,the simultaneous inhibition of MAPK pathway and glyco-gen synthase kinase-3 (GSK3) promotes iPS cell formationfrom brain-derived NSCs, using only Oct4 and Klf4 (223).These pathways may facilitate iPS cell generation by acti-vating endogenous reprogramming factors or pluripotency-associated genes, and further investigations are required tounderstand how Wnt may regulate somatic cell reprogram-

ming.

D. Wnt and In Vivo Regeneration

The role of Wnt in ES cells and iPS cell reprogrammingsuggests that manipulation of this pathway could have thepotential to be beneficial for regenerative therapies. Wntsignaling has a vital role in regulating cell growth and dif-ferentiation and is essential in the maintenance and self-renewal of various somatic stem cells and is therefore vitalto the regeneration of these tissues. For example, the con-stant proliferative state of the stem cells in the intestinalcrypt is under the control of the Wnt pathway, and loss ofWnt signaling results in loss of the stem cell compartment(125, 128). Knockout mice deficient in Tcf4 lack cryptstructures, and the entire intestinal epithelium is composedof nondividing villus cells (125). Similar to the situation inES cell maintenance, Oct4 plays an important role in intes-tinal crypt maintenance as ectopic, intestinal expression ofOct4 results in an increase in -catenin expression, dyspla-sia, and an expansion of crypt progenitor cells (91). Thisphenotype is similar to that seen following conditional lossof Apc (213). Critically, Wnt signaling has also been shownto play a direct role in the regenerative response after injury.

Thus treatment with the positive Wnt modulator R-Spon-din1 was able to restore the damaged mucosa in both ex-perimental colitis and radiation-induced oral mucositis(287, 288). This role in the regenerative response has re-cently shown to be Wnt dependent, being mediated down-stream of c-Myc by focal adhesion kinase (11).

The requirement for Wnt signaling is, however, not con-strained to the gastrointestinal tract, with other epithelialorgans such as the lung (286) showing Wnt-dependent con-trol of the balance between progenitor expansion and epi-


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thelial differentiation, implying a broad role for the Wntpathway in normal development and regeneration. A fur-ther example of such a role is seen in normal hair follicledevelopment, with deletion of-catenin in mice impairinghair follicle morphogenesis and resulting in the loss of thefollicle stem cell niche (7, 96). Stabilization of-catenin alsodrives hair follicle stem cells to proliferate (157), and over-expression of Wnt ligand in the epidermis is able to induce

hair follicle regeneration after wounding (100).

Wnt signaling has also been suggested to play a role inmammalian liver regeneration as patients with mutations inthe APC gene have an increased risk of developing hepato-blastoma, indicating that Wnt affects the hepatic progenitorcells (75, 130, 181). Work in zebrafish has also demon-strated that an increase in Wnt activity significantly accel-erated liver regeneration after a partial hepatectomy (77).Furthermore, liver regeneration is significantly enhanced inApcMin/ 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 hema-

topoietic 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 activa-

tion in Wnt signaling (46).

Several studies have also demonstrated the essential role ofWnt signaling in the regeneration of mammalian muscle

and bone (219, 290). For example, the Wnt pathway playsa vital role in bone homeostasis and has a role in the normalembryonic development of the vertebrate limb (95). Wntsignaling is also activated during fracture repair and en-hances bone formation (222). Furthermore, gain-of-func-tion mutations in the Wnt coreceptor LRP5 in humans givesrise to high bone mass (13). A recent study has also demon-strated that modulation of Wnt levels through Dkk1 inhi-bition was sufficient to repair the bone lesions in multiplemyeloma and rheumatoid arthritis (287). Wnt signaling hasalso been shown to be key to the regeneration of limbs inlower vertebrates. These have the capacity to regenerate

complex structures consisting of multiple cell types eitherthrough the reprogramming of differentiated cells orthrough the activation of resident stem cells. Recent work inamphibian and fish appendages has demonstrated a role forWnt in whole appendage regeneration, and the inhibition ofWnt/-catenin signaling during the early stages of limb re-generation in both the Xenopus and axolotl completely in-hibits this regeneration (112, 282). Furthermore, the ex-pression of-catenin in later-stage Xenopus limbs, wherethe regenerative response is reduced and in nonregenerativedeveloping chick limbs, is sufficient to rescue the regenera-

tive process and induce regeneration (112). Similar to Xe-nopus limb regeneration, studies in zebrafish have demon-strated that Wnt/-catenin signaling has a role in all threephases of fin regeneration and is sufficient to enhance re-generative outgrowth; also,-catenin-independent Wnt sig-naling functions as an antagonist of fin regeneration, allow-ing 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 mechanismbeing similar to that of the zebrafish fin (149, 234). The onlymammalian organ capable of complete regeneration, deerantlers, also requires -catenin expression to maintain thesurvival of antler progenitor cells (169).

Work in zebrafish has also demonstrated a functional rolefor Wnt/-catenin in heart regeneration, which has beenconfirmed by studies in mice. In vivo studies using-cateninconditional heart specific null mice has demonstrated a rolefor the canonical Wnt signaling pathway in the differentia-tion of cardiomyocytes (3, 131). The Wnt11 ligand is re-quired for cardiac differentiation and heart formation in

both chick and Xenopus embryos (60, 189). This functionof Wnt11 in heart development has also been shown to beconserved in higher vertebrates as exogenous Wnt11 pro-motes the differentiation of murine ES cells into cardiomy-ocytes (245).

From the studies discussed above, it is clear that Wnt/-catenin signaling plays a central role in many regenerativeprocesses, suggesting that Wnt/-catenin signaling may be aconserved feature of regeneration. The manipulation of thispathway should therefore be key in both manipulating so-matic stem cells in situ and also in the development of iPScells for regenerative therapy.

E. Hedgehog

In vertebrates the Hedgehog (Hh) gene family encodes threehighly conserved, homologous signaling proteins: Sonic(Shh), Indian (Ihh), and Desert (Dhh). These signaling mol-ecules bind to the membrane receptor Patched (Ptc), whichin the absence of Hh inhibits the activity of another trans-membrane protein, Smoothened (Smo) (99). The down-stream effectors of this pathway are the Gli family of zinc-finger transcription factors, and the alteration of the bal-ance between activator and repressor forms of these

proteins results in regulation of Hh target genes.

Hh signaling is vital for many developmental processes, andShh has been shown to be involved in the determination ofcell fate and embryonic patterning during early vertebratedevelopment.

F. Hedgehog and In Vivo Regeneration

Hh signaling is important for the maintenance of stem orprogenitor cells in many adult tissues, including the epithe-


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lia of many internal organs and the brain (16, 254). In thesmall intestine, Hh signaling is important for the correctoverall crypt-villi structure of the epithelium, although it isnot directly involved in the fate of the epithelial cells (159).Inhibition of Hh signaling in the intestinal epithelium re-sults in reduced villus formation and mislocalization of sub-epithelial stromal cells. In addition, loss of Hh signaling hasa secondary effect by enhancing proliferation through Tcf4

and -catenin gene activity and the formation of ectopicprecrypt structures on villus tips (159). In addition to tissuemaintenance through Hh-mediated stem or progenitor cellself-renewal, Hh signaling also plays a critical role in theregenerative responses to tissue injury, for example, in theregeneration of the pulmonary epithelium (260), prostateepithelium (111), and exocrine pancreas (67). In addition, arecent study has demonstrated a role for Hh signaling dur-ing gastric regeneration, where this pathway is essential forthe differentiation of gastric progenitor cells (110). Recentwork has also suggested a role for the Hh pathway in he-patic remodeling as liver injury increases the activity of Hhsignaling in progenitor cells (69, 187).

Hh signaling also functions to maintain adult hematopoi-etic stem cells (HSCs) in bothDrosophilaand vertebrates byregulating the HSC cell cycle and by balancing hematopoi-etic homeostasis and regeneration in vivo. Loss of Hh sig-naling in Drosophila results in the depletion of blood pro-genitor cells in the lymph gland due to premature differen-tiation (162) and the inhibition of hematopoiesis andvasculogenesis in early postimplantation mouse embryos(58). In adult mice, Ihh is expressed in differentiated epithe-lial cells of the small intestine, and conditional of Ihh fromthese cells results in a wound healing response, thought to

be mediated through mesenchymal signaling (252). Ihh isalso expressed in bone marrow stroma, while Shh expres-sion is found in lymph node and spleen stroma. Ihh overex-pression in stromal cells promotes hematopoietic regenera-tion after bone marrow transplantation (123), and Hh sig-nal activation induces cycling and expansion of primitivehematopoietic cells (247).

The Hh pathway has also been extensively analyzed in neu-ral stem and progenitor cells and has several roles in thespecification, proliferation, and differentiation of neuralprecursors during embryogenesis. Hh signaling is also re-

quired for the maintenance of quiescent neural stem cells inthe adult brain (2, 14, 188). Shh has been shown to becrucial in regulating stem cell niches and neural stem cellproliferation in the postnatal telecephalon (244), the adulthippocampus (133), and the SVZ (2) and plays a criticalrole in the survival and patterning of neural progenitor cellsin the ventral spinal cord (151).

Hh signaling also has a role in vertebrate limb developmentand is vital to both bone formation and bone remodeling,which is primarily mediated through the regulation of both

chondrocyte and osteoblast differentiation (50). This func-tion of the Hh pathway, in addition to its role in woundhealing, suggests a potential role for the Hh pathway inlimb regeneration. Consistent with this, work in urodeleshas demonstrated an increase in Shh expression after limbamputation (98, 246) and also in the regenerating limb budsof anurans (62). Functional evidence also suggests that Shhis important for anterior-posterior axis patterning in the

regenerating limb (208, 209). Furthermore, activating Shhin Xenopus froglets improves limb regeneration and resultsin the formation of multiple cartilaginous structures (276).Shh is also required for dorsoventral patterning of the re-generating spinal cord and surrounding mesodermal tissuesduringXenopus tail regeneration (216). While this functionis similar to that seen in the regenerating limb, it is clear thatthe Shh signaling pathway has distinct roles in differentregenerating organs. Zebrafish fin regeneration also re-quires Shh during the outgrowth phase, and studies haveimplicated a role for this pathway in the proliferationand/or differentiation of scleroblasts (200). Shh has alsobeen shown to be important in axolotl tail regeneration,

where it is required for dorsoventral patterning of the re-generating spinal cord and for the regeneration of sur-rounding mesodermal tissues (216).

From the studies described above, it is clear that the Hedge-hog pathway is a fundamental regulator of somatic stemcells that maintain tissue integrity in mature organisms. Theimportance of hedgehog signaling in regeneration has beenemphasized by recent work developing hedgehog-relatedtherapies for restoring tissue function where the normalcapacity to regenerate has been compromised (256).

G. JAK-STAT

The JAK-STAT pathway plays an essential role in mediat-ing the biological response to growth factors and cytokines,and characterization of IFN signaling resulted in the initialidentification of this pathway. Currently, four Janus kinase(JAK) family members have been identified, and seven sig-nal transducers and activators of transcription (STATs) areknown. The binding of ligands to the dimeric cytokine re-ceptor results in the activation of two receptor-associated

JAKs and subsequent phosphorylation of the ligand-boundreceptor. The phosphorylation of the receptor tyrosine res-

idues promotes the recruitment of a STAT family member,which becomes phosphorylated and forms a homo- or het-erodimer, which translocates into the nucleus and activatestarget genes.

The JAK-STAT pathway plays a vital role in maintainingthe pluripotent state of mouse ES cells. As described above,in vitro culture of mouse ES cells requires LIF to maintainpluripotency. The JAK-STAT pathway is a key downstreammediator of LIF, and the activation of the STAT3 pathwayby LIF induces transcription of self-renewal genes (34, 177).


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STAT3 is the primary STAT protein activated in mouse EScells, and constitutively active STAT3 removes the need forLIF to promote self-renewal (177, 178). STAT3 has multi-ple roles in the regulation of ES cell pluripotency includinggene activation, cell cycle regulation, and the inhibition ofdifferentiation pathways. STAT3 has also been reported tofunction 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 ofLIF. This ability of LIF-mediated activation of STAT3 tosupport the long-term self-renewal of mouse ES cells in vitrohas been supported in vivo by the requirement for this path-way in embryonic diapause (175). In addition to STAT3homodimers, STAT1 is able to heterodimerize with STAT3in mouse ES cells, although it is thought that it is unlikely tobe required for self-renewal as Stat1/ cells remain LIF-dependent for undifferentiated growth (56). In human EScells, STAT3 does not appear to be sufficient to maintainpluripotency (214), indicating once again that the signalingpathways responsible for maintaining pluripotency may bespecies-specific. Also, recent work by Griffiths et al. (79) has

identified a STAT3-independent JAK pathway that is ableto directly regulate pluripotency genes and maintain self-renewal through chromatin phosphorylation.

H. JAK-STAT and In Vivo Regeneration

In addition to maintaining pluripotency, the JAK-STATpathway stimulates cell proliferation, differentiation, cellmigration, and apoptosis and is vital in regulating variousdevelopmental processes (97).

Recent work has demonstrated a key role for JAK-STATsignaling in the homeostasis of the Drosophila midgut. The

JAK-STAT pathway regulates ISC self-renewal and differ-entiation, and compromised JAK-STAT signaling causesISC quiescence and loss, whereas signaling overactivationproduces extra ISC-like and progenitor cells (150, 239). Inaddition to midgut ISCs, Stat92e is also specifically acti-vated in the Drosophila hindgut stem cell population (239),indicating a similar role of JAK/STAT signaling in hindgutISCs. Several recent studies have also suggested a role forDrosophila JAK-STAT signaling in mediating injury- orinfection-induced ISC proliferation and regeneration (5, 31,49, 105, 179). Enterocytes that are subjected to stress pro-duce cytokines that activate the JAK-STAT pathway in the

intestinal stem cells, which results in proliferation and re-generation (105). Furthermore, we have shown that condi-tional deletion of Stat3 in the mouse intestinal epitheliumleads to loss of the stem cell compartment (unpublisheddata), while others have shown that in a mouse model ofDSS-induced colitis, colon epithelial damage triggers an im-mune response and the expressed cytokines activateSTAT3, which subsequently promotes cell proliferationand regeneration. In addition, inhibition of the STAT inhib-itor SOCS3 in the mouse gut increased the proliferativeresponse to DSS (207).

The canonical JAK-STAT pathway also regulates germlinestem cell self-renewal, maintenance, and differentiation inthe Drosophila (29, 116, 248). The JAK-STAT pathwaymaintains germline stem cell self-renewal by activating theexpression of self-renewal genes, and conditional loss of

JAK-STAT signaling results in germline stem cells differen-tiating into spermatogonia without self-renewal (29). Fur-thermore, the conditional restoration of JAK-STAT signal-

ing in differentiated spermatogonia that are undergoingtransit-amplifying divisions are able to dedifferentiate tobecome functional germline stem cells that are able to re-populate the niche (29).

STAT3 influences cell survival, metabolism, growth, differ-entiation, and migration in multiple organs, and recentwork has indicated a vital role for STAT3 in liver regener-ation (145). In the normal liver, the interleukin (IL)-6/STAT3 pathway is thought to play a central role in regen-eration as disruption of this pathway in IL-6-deficient miceresults in impaired liver regeneration after 70% partial hep-atectomy (PH) (48). STAT3 signaling also has a vital role in

wound healing, and in vivo studies have revealed that Stat3expression in keratinocytes contributes to the regenerationof the epidermis and hair cycle process (212). This potentialrole of the STAT3 pathway in wound healing has also beenobserved in the injured spinal cord. After injury, STAT3activation (phosphorylation and nuclear localization) wasobserved in reactive astrocytes surrounding the lesion, im-plicating a role for this pathway in tissue repair and func-tional recovery (185).

I. Notch

The Notch pathway is an evolutionary conserved pathwaythat regulates cell fate determination during development.This pathway is essential in the control of embryonic or-ganogenesis and is also vital for postnatal tissue repair (10).The Notch pathway consists of a single transmembranereceptor, which undergoes proteolytic cleavage upon bind-ing by members of the Jagged and Delta ligand families.Cleavage involves the activity of the -secretase proteasecomplex and releases a Notch intracellular domain (NICD)that translocates to the nucleus. In the nucleus, NICD het-erodimerizes with CSL transcription factors and activatesNotch target genes (30).

Notch signaling is required for the differentiation of ESCs;however, the precise function of endogenous Notch signal-ing in mammalian ESCs is still unclear, although severalcomponents of the Notch signaling pathway are highly ex-pressed in both mESCs and hESCs (206, 215). Notch sig-naling is thought to be relatively inactive in undifferentiatedhESCs, and not necessary for their proliferation, althoughmany hESC-derived differentiated cells require Notch(179). Neural specification of ESCs is influenced by Notchsignaling, and Notch1 activation promotes neural lineages


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and suppresses ESC differentiation into mesodermal cellfates (173, 218). Conversely, the inhibition of Notch re-duces neural lineage differentiation and increases nonneu-ral ESC fate. For example, ESC differentiation into cardio-myocytes required Notch1 inhibition (173).

J. Notch and In Vivo Regeneration

In addition to a role in embryonic stem cells, Notch signal-ing is required for the maintenance and repair of variousorgan systems. The Notch pathway is required for self-renewal and cell-fate commitment of tissue stem cells andhas been shown to play a vital role in the regenerative re-sponse to injury in several adult tissues including the kid-ney, liver, bone marrow, skin, muscle, intestine, heart, tra-chea, and pancreas.

In the small intestine, the Notch pathway plays a centralfunction in the cell fate decisions of intestinal stem cells, andsimilar to the Wnt pathway, the Notch pathway is essential

to maintain the crypt compartment in its undifferentiated,proliferative state. Inhibition of various components of theNotch pathway in the intestine results in the rapid andcomplete conversion of all epithelial cells into secretorygoblet cells (167, 270), an effect which is also seen in intes-tinal adenomas in ApcMin/ mice (253). Consistent withthis, 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 absorp-

tive lineage cells, the in vivo phenotype of Notch inhibi-

tion 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 pro-

mote proliferation is vital to the regenerative response of

the intestinal epithelial cells. Thus the Notch pathway is

activated in the murine intestinal epithelium during ulcer-

ative colitis and contributes to the regeneration of the

damaged epithelia by regulating differentiation, prolifera-

tion, 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 condi-tions and when tissue damage has occurred (184).

The ability of Notch to expand the stem cell population inresponse to injury has also been shown in several othertissues including the brain (242), and various studies haveconfirmed that the capacity of Notch to enhance tissue re-generation is due to its ability to activate progenitor cellpopulations, in addition to regulating the differentiationstatus of cells and enhancing proliferation. Therefore, theactivation of Notch signaling may provide a promising tar-

get for regenerative therapy in adult tissue in response toinjury.

Notch signaling is also vital for the functional recovery ofvertebrate appendages. The inhibition of Notch preventsanuran tail regeneration, while the overexpression of NICDstimulates the regeneration of a tail that contains notochordand spinal cord but little or no muscle (18). Notch also has

a role in mammalian skeletal muscle regeneration, andNotch signaling is induced in activated muscle progenitorcells, satellite cells, 2 days after injury both in culture and invivo (45). Expression of a constitutively active Notch recep-tor results in the inhibition of differentiation markers andincreased proliferation of progenitor cells in culture (45).Importantly, the inhibition of Notch signaling in vivo im-pairs muscle regeneration (44). The induction of Notch sig-naling in activated satellite cells appears to be triggered bythe upregulation of the Delta ligand in satellite cells them-selves and in muscle fibres adjacent to the damage (44). Inaddition, the upregulation of Delta and the activation ofNotch are diminished in old mice, whose satellite cells pro-

liferate less in response to injury, resulting in reduced effi-cacy of regeneration. Forced activation of Notch signalingat the site of injury in old mice is sufficient to improvemuscle regeneration, rendering it similar to that of youngmice (44).

K. FGF/ERK MAPK Pathway

The MAPKs are a family of serine/threonine kinases thathave a functional role in connecting the activation of cell-surface receptors to various cellular functions such as pro-liferation, differentiation, migration, and apoptosis (38,194). MAPKs are a part of a three-tiered kinase module thatconsists of a MAPK, an upstream MEK, and a MEKK thatis coupled to cell surface receptors via adaptor proteins. TheERK MAPKs can be activated by a wide variety of extracel-lular ligands, including growth factors, serum, cytokines,and stress signals. One important upstream activator of theERK MAPK pathway is the fibroblast growth factor (FGF)family of signaling molecules. FGFs are a large family ofsignaling molecules with various functions in developmentand adult physiology, and the ERK pathway mediates theeffects of this growth factor.

In human ES cells, it has been well established that exoge-nous FGF-2 is required to sustain self-renewal, and it iswidely used to culture human ES cells and iPS cells (6, 52,273). Furthermore, in human ES cells, elevated levels ofexogenous FGF-2 alone are sufficient to maintain undiffer-entiated hESC in the absence of fibroblast feeders or fibro-blast-conditioned media (141, 275). Inhibition of FGF sig-naling in hESC results in rapid differentiation (57), an effectthat is similarly seen in mouse ES cells (268). In human EScells, exogenous FGF-2 activates the ERK MAPK pathway,which is thought to be necessary for the maintenance of


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pluripotency, although the mechanism of action is still un-clear (57, 143).

In contrast to most cell lines and human ES cells, undiffer-entiated mouse ES cells do not require the ERK pathway forproliferation and self-renewal (33), and remarkably,ERK1/2 signaling actually inhibits the self-renewal of theseES cells and triggers differentiation towards the primitive

endoderm lineage (39). Furthermore, reducing ERK activityhas been shown to enhance the efficiency of ES cell deriva-tion by promoting the retention of Oct4-positive epiblastduring the outgrowth phase (32). Also, the differentiationof established ES cells and their requirement for LIF can bediminished by the addition of synthetic inhibitors of ERKsignaling (34). The mechanism behind the ability of theERK pathway to promote differentiation is unclear, al-though recent studies have suggested that ERK1/2 activa-tion may act as a stimulus for ground-state ES cells, whichare intrinsically self-maintaining, to exit the self-renewalprogram and enter a state that is responsive to inductivestimuli (129). As mentioned already, the direct consequence

of this is that blocking the ERK inductive differentiationpathway can maintain the ground state of the epiblast(280). As perhaps a direct consequence of the above, thisinhibition of Erk1/2 MAPK pathway has been shown tosignificantly improve the generation efficiency of iPS cells(92, 223).

L. FGF/ERK and In Vivo Regeneration

As described above, the ERK MAPK family is activated byseveral growth factors, one of which is FGF. The FGF path-way plays a central role in development and tissue regener-ation, and FGF signaling has been implicated in the regula-tion of cell proliferation and specification in the majority ofregenerating systems. Components of the FGF pathway areexpressed in various somatic stem cell compartments, andFGFs appear to be universal regulators of regeneration.

In the mouse small intestine, FGFs have been shown to havean active role in murine colon development and have beenshown to promote the proliferation and survival of intesti-nal cells (211). The FGF receptor FGFR3 has been shown tobe strictly expressed in the intestinal progenitor cell com-partment, where it has a role in regulating the expansion of

intestinal crypt cells (9). Also, in irradiated mice, treatmentwith FGF7 increases crypt survival (114), and FGF1 andFGF2 have a protective effect after intestinal injury (51, 71).In humans, various FGF receptors have been shown to beexpressed in the gastrointestinal tract, and the altered ex-pression of FGFR3-IIIb in intestinal epithelial cells is asso-ciated with differentiation (109).

In addition to tissue maintenance, FGF signaling is impor-tant in limb regeneration after injury. FGFs have severalessential roles during epimorphic regeneration of anuran

tails, urodele limbs, and fish fins and regulate the variousstages including wound epidermis formation, blastema for-mation, and proliferation and also positional memory(231). The FGF pathway is vital during Xenopus limb re-generation, and the treatment of a nonregenerative stageXenopus limb stump after amputation with FGF8 results inpartial regeneration (281). Furthermore, treatment withFGF10 results in significant regeneration of the limb due to

the activation of expression of several genes that are ex-pressed in regenerating limbs such as Shh and Fgf10 (281).Also, the treatment of the amputation surface of a nonre-generative chick limb bud with FGF2 or FGF4 induces aregenerative response (126, 243). FGF signaling is also ac-tive and important in all three stages of zebrafish fin regen-eration, similar to the Wnt pathway (231), and also for thelater phases of heart regeneration (140). FGF signaling isalso implicated in the regulation of cell proliferation anddifferentiation in regenerating skeletal muscle and mamma-lian liver (63, 233), although clarification of its in vivo roleis still required.

VI. REGENERATIVE PATHWAYS

The signaling networks that play a vital role in stem cellmaintenance of ES cells and somatic stem cells are essentialfor progenitor cell formation and for the differentiation ofcells leading to tissue homeostasis. By looking at the path-ways that regulate pluripotency, self-renewal, and stem cell-mediated regenerative processes, it should enable us toprogress the dual tracks of in situ stem cell manipulationand the development of iPS cells for regenerative therapy. Akey goal of these studies must be the identification of smallmolecules that can be used to precisely manipulate theseregenerative processes.

Although we have briefly discussed some of the potentialpathways involved in pluripotency maintenance and alsotheir potential role in tissue regeneration, it is important toremember that these pathways do not function in isolationfrom each other or from other signaling pathways (e.g., seeFIG. 4). For example, the effects of Wnt signaling on stemcells are modulated through association with Notch, SonicHedgehog, and TGF- (41). In addition, several other keypathways regularly appear to be essential in the variousregenerative processes discussed above that have not been

discussed in this review. For example, cell cycle controlpathways have a vital role in tissue regeneration, and sev-eral studies have demonstrated that checkpoint proteinssuch as p21 and Rb also appear to be important in tissueregeneration in mice (19, 89, 217, 230, 266).

In addition, it is important to note that stem cells also de-pend on the extrinsic signaling pathways of the stem cellniche. This stem cell microenvironment plays a vital role indeciding the fate of these cells. The niche provides support-ive growth factors and cytokines for the stem cell and con-


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tributes to the maintenance of the undifferentiated stem cellstate. For example, regulation of the intestinal stem cellinvolves constant cross-talk between the epithelial cells andthe underlying mesenchymal cells in the intestinal stem cellniche. This cross-talk is mediated by some of the key path-ways mentioned above, in addition to pathways such asthe phosphatidylinositol 3-kinase pathway. This additionallevel of control emphasizes yet further the complex goal of

precisely manipulating the endogenous stem cell popula-tion.

VII. REGENERATION ORTRANSFORMATION?

The discussion above has focused on the positive roles of sixkey pathways in pluripotency and regeneration. However,these self-renewal pathways are also key pathways that of-ten become deregulated in tumorigenesis. For example, ac-tivating mutations in the Wnt/-catenin pathway have beenshown to initiate tumorigenesis in a variety of human epi-thelial carcinomas, possibly by expanding the stem cell pop-ulation as has been suggested in the small intestine (204).Similarly, activation of Wnt signaling has been shown innon-small-cell lung cancer (249), and bronchioalveolarstem cells have been shown to multiply in response to Wntactivation (286). Therefore, although the manipulation ofthese pathways may be vital to the development of iPS cell

technology and regenerative therapy, there is clear potentialfor unwanted side effects.

Several studies have made the experimental observationthat, in some circumstances, it is possible to enrich for can-cer forming cells within a bulk population of tumor cells,which has led to the concept that cancers may be sustainedthrough a small population of cancer stem cells. This

model suggests that tumors are generated and maintainedby a subset of undifferentiated cells, the cancer stem cells,which are similar to adult tissue-specific stem cells and areable to self-renew and differentiate into the bulk tumorpopulation (see FIG. 5). Although the origin and indeed theexistence of the cancer stem cell is still under debate (53, 76,81, 202), it is thought that the cancer stem cell could pos-sibly arise from adult stem or progenitor cells that, due totheir higher capacity to proliferate, are more prone to accu-mulate mutations during their lifespan. These observationssuggest that deregulation of the pathways such as the onesdiscussed above, that control stemness may potentiallypromote cancer, either by increasing the number of poten-tial initiators of neoplasia, or by expanding an establishedcancer stem cell.

The cancer stem cell hypothesis has gained strength throughthe apparent identification of initiating cell populations inseveral 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.




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creas (135, 142), as well as colon and brain (224). This fielddoes, however, remain contentious, with some arguing thatthe observation of such initiating cells occurs as an artifactof the experimental protocols used. Despite this uncer-tainty, it remains clear that early progenitor or stem cells ina range of tumor types can undergo oncogenic transforma-tion, and it therefore seems highly likely that the modula-tion of the pluripotency pathways of these cell populations

will directly alter tumor predisposition.

Several of the pathways discussed in this review have al-ready been shown to be deregulated in cancer stem cells,and thus manipulating these pathways may be a double-edged sword. Some examples of these involvements aregiven in TABLE 3. For example, the Hedgehog pathway hasbeen shown to be active in the cancer stem cells of severalmyeloma (193) and brain tumors (15, 42, 197, 201). Directmodulation of tumor outcome has now been shown for theWnt pathway, which has been suggested to sustain stemnessof CSCs within cutaneous squamous cell carcinomas, suchthat deletion of-catenin in established tumors resulted inrapid loss of the CSC compartment and tumor regression(161). Conversely, increased BMP4 signaling has beenshown to block glioblastoma formation by inhibiting cellproliferation and promoting CSC differentiation (196).Critically, in this example, the in vivo delivery of BMP4 wasable to reduce tumor growth and the associated mortality inmice inoculated with human glioblastoma cells (196). In asimilar way, epigenetic silencing of the BMP type IB recep-tor is thought to contribute to the pathogenesis of glioblas-

toma by blocking BMP-mediated astroglial differentiationin a subpopulation of brain cancer stem cells (136).

The examples cited above are but a few of the many exam-ples implicating aberrant control of stem cell signaling path-ways in the initiation and progression of cancer, eitherthrough the expansion of normal or preneoplastic stemcells, or by the expansion of proposed cancer stem cell pop-

ulations. The cancer stem cell hypothesis remains contro-versial, and while the discussion of both sides of the argu-ment is beyond the scope of this review, the potential exis-tence of a tumor initiating cell population must remain akey consideration for any therapies based on either modi-fying stem cell signaling pathways and the stem cell nichedirectly in vivo or the introduction of iPS cells.

VIII. SUMMARY

Manipulation of the stem cell compartment remains an at-tractive target for the development of novel regenerative

therapies, either by directly manipulating existing stem cellsin vivo, or by supplying exogenously developed iPS cells.This problem might usefully be broken down into two fun-damental questions: 1) Have we identified the key mole-cules and pathways that regulate stemness? 2) Can the ma-nipulation of these pathways lead to controlled tissue re-generation? We have already made substantial progresstowards addressing the first question, with a good under-standing of the factors that control pluripotency, togetherwith an understanding of the pathways that they compose.The main difficulty in addressing the second question issimply the ubiquitous nature of the programming machin-ery; as we have tried to summarize in this review, it spansand interacts with many, if not all, of the key transcriptionalpathways that regulate cellular life. For example, many lab-oratories have demonstrated a role for the Wnt pathway inboth pluripotency and repair, but unfortunately, the Wntpathway is so broad in its downstream influence that wewill need very complete and precise knowledge of thechecks and balances that influence Wnt-mediated regener-ation before we can think of using this pathway therapeu-tically. This problem is further compounded by the extralevel of control exerted epigenetically, and also by the clearinfluence of the niche within which the stem cells sit.

Hence, the real difficulty may be in understanding and in-tegrating these pathways in a single tissue type or setting.For example, complete deregulation of the Wnt pathwayfails to lead to a simple enhancement of a stem cell orregenerative phenotype, but rather to a more sinister pre-neoplastic phenotype. More specifically, overexpression ofthe Wnt-related pluripotency gene Oct4 also leads to a pre-neoplastic phenotype rather than an enhanced regenerativephenotype. The conclusion that can be perhaps drawn isthat simple deregulation of the stem cell program will de-fault to an enhanced predisposition to neoplasia precisely

Table 3. Examples of the mutations that occur in the keyself-renewal pathways and their incidence in various cancers

Pathway Cancer/Tumor Type

TGF- 2SMAD4 in 53% of pancreatic carcinomas

1BMP2 in 98% of lung carcinomas

Wnt 1-Catenin in 48% of small intestinalcarcinomas

1-Catenin in 64% of gastric polyps

2APC in 90% of colon cancers

2APC in 76% of gastric adenomas

Hedgehog 2Ptch in 67% of basal cell carcinomas

1Smo in 10% of basal cell carcinomas

2Ptch in 10-20% of sporadicmedulloblastomas

JAK-STAT 1STAT3 in 50% lung

95% head and neck

Notch 1Notch in 55-60% of T-cell acutelymphoblastic leukemia

MAPK-ERK 1RAS in 45% of colon and 90% ofpancreatic cancers

1RAF in 60% of melanoma

[Adapted from Dreesen and Brivan-lou (55).]




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because we have failed to understand the complete networkthat regulates entry and exit into the stem cell compartment.

Despite these clear difficulties, we are successfully begin-ning to understand these networks more fully, and we donow have clear examples of pathway-driven regeneration inmammalian tissues. Indeed, we absolutely know from otherorganisms that regeneration driven by these pathways is

possible, and the challenge now must be to develop a sys-tems understanding of the multiple processes governing re-generation so that we can ultimately utilize them therapeu-tically.

ACKNOWLEDGMENTS

Address for reprint requests and other correspondence:A. R. Clarke, School of Biosciences, Cardiff University,Cardiff CF10 3AX, UK (e-mail: [email protected]).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declaredby the authors.

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