SPECIAL ISSUE REVIEWS–A PEER REVIEWED FORUM

The Proliferating Field of Neural Crest StemCellsMariana Delfino-Machın, Thomas R. Chipperfield, Frederico S.L.M. Rodrigues, andRobert N. Kelsh1*

Neural crest stem cells were first isolated from early embryonic neural crest in the early 1990s, but in thepast 5 years, there has been a burst of discoveries of neural crest-derived stem cells from diverse locations.Here, we summarize these data, highlighting the characteristics of each stem cell type. These cells varywidely in the markers they express and the variety of cell types they appear to generate. They occupydiverse locations, but in some cases multiple stem cell types apparently occupy physically proximate niches.To date, few molecular similarities can be identified between these stem cells, although a systematiccomparison is required. We note other issues worthy of attention, including aspects of the in vivo behaviorof these stem cells, their niches, and their lineage relationships. Together, analysis of these issues willclarify this expanding, but still young, field and contribute to exploration of the important therapeuticpotential of these cells. Developmental Dynamics 236:3242–3254, 2007. © 2007 Wiley-Liss, Inc.

Key words: neural crest; stem cell; progenitor; eNCSC; SKP; EPI-NCSC; MSC; self-renewal; cell culture; Sox10; p75;melanocyte; embryo; mouse; chick

Accepted 2 August 2007

STEM CELLS AND THENEURAL CREST

The neural crest (NC) is a transientstructure, generated by delaminationof cells from the dorsal neural tube.Neural crest cells (NCCs) briefly restin a dorsal or dorsolateral position tothe neural tube before migratingalong defined pathways to populatevarious regions of the embryo (LeDouarin and Kalcheim, 1999). Re-markably, NCCs contribute to a di-verse array of distinct cell types, in-cluding multiple skeletal cells (e.g.,chondrocytes, osteoblasts, and odonto-blasts), pigment cells (e.g., melano-cytes, but also four or more othertypes in anamniotes), and peripheralneurons (e.g., sensory and sympa-

thetic) and glia (e.g., Schwann andsatellite cells).

NC development is widely consid-ered to involve a process of progres-sive fate restriction, whereby pluripo-tent cells tend to become increasinglylimited in their potencies over time(reviewed in Weston, 1991; Le Doua-rin and Dupin, 2003; Crane andTrainor, 2006). Single cell labellingstudies in vivo generally indicate a di-versity of cells even in the premigra-tory neural crest, with some beingmultipotent and others apparentlyfate-restricted (Bronner-Fraser et al.,1980; Bronner-Fraser and Fraser,1991; Raible and Eisen, 1994; Ser-bedzija et al., 1994; Dutton et al.,2001). Consistent with this finding,

molecular markers may show hetero-geneous patterns of expression inpremigratory neural crest cells (Ci-ment and Weston, 1982; Girdlestoneand Weston, 1985; Marusich et al.,1986). The differing potentialities ofneural crest cells have been testedmore extensively and rigorously invitro under culture conditions suitablefor differentiation of diverse cell types;these studies reinforce a picture of adiversity of cell types within the neu-ral crest, both in premigratory andpostmigratory locations (Sieber-Blumand Cohen, 1980; Baroffio et al., 1988;Sieber-Blum, 1989; Baroffio et al.,1991; Ito and Sieber-Blum, 1993; He-nion and Weston, 1997). Furthermore,the occasional identification in pri-

Centre for Regenerative Medicine and Department of Biology and Biochemistry, University of Bath, Bath, United KingdomGrant sponsors: BBSRC; MRC; Wellcome Trust; University of Bath.*Correspondence to: Robert N. Kelsh, Centre for Regenerative Medicine and Department of Biology and Biochemistry,University of Bath, Bath, BA2 7AY, UK. E-mail: bssrnk@bath.ac.uk

DOI 10.1002/dvdy.21314Published online 6 September 2007 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 236:3242–3254, 2007

© 2007 Wiley-Liss, Inc.

mary neural crest cell culture of singlecells apparently able to generate fiveor more distinct cell types has led tothe widespread assumption that fullymultipotent neural crest cells, able togenerate all neural crest derivatives,exist at least transiently (Baroffio etal., 1991). The huge diversity of neuralcrest-derived fates and the inevitablefocus on the best-studied derivativesmeans, however, that this attractiveassumption remains unproven.

Our understanding of mechanismsof neural crest fate choice has beenfocused by the concept of stem cells,and this has opened up a vision of atherapeutic approach to congenitaland degenerative diseases of the neu-ral crest (“neurocristopathies”; Bo-lande, 1974). However, the stem cellconcept is plagued by multiple, par-tially overlapping definitions. Re-cently, Smith proposed a unifyingstem cell biology vocabulary (Smith,2006). He defined a stem cell as “a cellthat can continuously produce unal-tered daughters and also has the abil-ity to produce daughter cells that havedifferent, more restricted properties.”Thus, a stem cell is a cell that under-goes self-renewal, but that can gener-ate differentiated progeny. In this ar-ticle, we will follow this simpledefinition of a stem cell. There is nowabundant evidence that undifferenti-ated neural crest-derived cells capableof self-renewal can be isolated frompostmigratory positions and, indeed,in some cases even from adult tissues.

A further criterion often used is thata stem cell must show multipotential-ity (e.g., Merkle and Alvarez-Buylla,2006), but given that progressive re-striction in developmental potential isa key feature of neural crest develop-ment, it seems logical to consider thepossibility that a neural crest-derivedstem cell might generate only one fate.Other recent reviews (e.g., Crane andTrainor, 2006) have used the termprogenitor for cells that are “more. . .limited in their capacity for self-renewal and differentiation poten-tial,” but as we shall see in many casesthe full potential of NC-derived stemcells is still to be explored and thecapacity for self-renewal is rarelytested extensively enough to definewhether it is “indefinite.” Instead,Smith distinguishes multipotent (canform all lineages constituting a tis-

sue), oligopotent (forms two or morelineages), and unipotent (one lineageonly) stem cells (Smith, 2006), and wewill do so here, although we note thatthis issue is confounded by our incom-plete knowledge of the conditions re-quired to specify each individual fate,so that definitive testing of full poten-tial has thus far not been achieved.We will use the term progenitor as ageneric term for precursor cells,whether stem cells or not, principallyin discussion of experimental data inwhich self-renewal capacity was notreported. A further issue regardingcell potency concerns the identifica-tion of derivative cell types, especiallyin vitro. In most cases, a small num-ber of markers, often only one, havebeen used to identify specific fates. Al-though understandable from a practi-cal perspective, it means that addedcaution is required when interpretingcell potencies.

In this review, we will summarizethe current knowledge of all stem celltypes, both embryonic and adult, thathave been shown to be neural crest-derived (Fig. 1; Table 1). Numerousinfluential studies of the propertiesand response to various growth fac-tors of neural crest cells have beenfundamental in revealing the biologyof the NC. However, often these stud-ies have not distinguished betweencells of different types. Hence, here,we are restricting our discussion tothose studies that examined specificpopulations of at least partially char-acterized cells that have been shownto self-renew. We will highlight ourunderstanding of their potencies, de-gree of self-renewal, and characteris-tic markers and niche. We will indi-cate the holes in our understandingand suggest future research direc-tions.

EARLY NEURAL CRESTSTEM CELLS

Stemple and Anderson first coined theterm neural crest stem cell (NCSC) forself-renewing, oligopotent cells iso-lated from rat neural tube explants byselection for low affinity nerve growthfactor (LNGFR or p75LNTR) expres-sion (Stemple and Anderson, 1992).These cells expressed nestin, lackedneural differentiation markers, andhave been shown to give rise to sen-

sory, sympathetic, and parasympa-thetic neurons; Schwann cells; andsatellite glia and myofibroblasts(Stemple and Anderson, 1992; Whiteand Anderson, 1999; Lee et al., 2004).These cells are now clearly one of mul-tiple types of neural crest-derivedstem cells (Table 1 summarizes all thecharacterized NC-derived stem cells),and we shall refer to them as earlyNCSCs (or eNCSCs; Lee, 2004). Cellswith similar properties to eNCSCshave been isolated from postmigra-tory phases of NC development, in-cluding from rat embryonic sciaticnerve and gut, again by expression ofp75. Because the peripheral nervoussystem (PNS) glia at these stages alsoshow p75 expression, the authors alsoselected against the peripheral myelinprotein, P0 (Morrison et al., 1999). Im-portantly, upon transplantation backinto chicken embryos, these postmi-gratory NCSCs give rise to cells ex-pressing markers characteristic ofsensory, sympathetic, and parasym-pathetic neurons, and Schwann cellsand satellite glia (Morrison et al.,1999; White and Anderson, 1999;Mosher et al., 2007). As all these post-migratory NC-derived cells show ex-pression of early NC markers, andalso migrate appropriately and con-tribute to similar structures as eNC-SCs, they are regarded here as persis-tent eNCSCs. However, quantitativeand qualitative changes in their fatepotential have been demonstrated(Kruger et al., 2002; Kubu et al., 2002;Mosher et al., 2007). For instance, sci-atic nerve-derived NCSCs morereadily form glia (Kubu et al., 2002).Furthermore, sciatic nerve and gutNCSCs transplanted into chick em-bryos displayed different migratorybehaviors and potencies, with sciaticnerve NCSCs unable to populate thegut or differentiate into enteric neu-rons, whereas gut NCSCs contributedfrequently to enteric neurons (Mosheret al., 2007). Although transplantedgut and sciatic nerve cells both con-tributed to chick dorsal root ganglia(DRGs), their adoption of sensory neu-ron fates both in vivo and in vitro isvery rare at best, even under cultureconditions optimized for sensory neu-ron survival or stimulation of sensoryneurogenesis (Mosher et al., 2007).The concept of progressive changes inNC-derived stem cell properties is fur-

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3244 DELFINO-MACHIN ET AL.

ther reinforced by analysis of gut-de-rived NCSCs from postnatal mouse(Kruger et al., 2002). NCSCs could beisolated from the plexus layers of thepostnatal gut, even as late as 110 dayspostparturition. These cells expressedp75 and could be strongly enriched byselection for this marker. Using condi-tions similar to those for the study ofeNCSCs, these cells were shown tobe self-renewing, although this abil-ity was reduced in cells from olderanimals. Furthermore, they weremultipotent, generating mixed neu-ronal/glial/myofibroblast clones, butthe spectrum of neuronal subtypeschanged with increasing age, becom-ing biased toward late-developingenteric neuron subtypes, as well asto glial cells. Importantly, the au-thors showed that the cells’ responseto neurogenic (bone morphogeneticprotein [BMP]) and gliogenic (Delta)factors declined and increased, re-spectively, compared with cells iso-lated from earlier stages.

Multipotent cells expressing theeNCSC marker p75 and responding toinstructive growth factors in a man-ner akin to eNCSCs have been iso-lated from embryonic rat DRG, buttheir self-renewal capacity was nottested (Hagedorn et al., 1999). Re-cently adult NC-derived stem cellshave been derived from rat DRG (Li etal., 2007). These cells can be grown asspheres in culture, and have beenmaintained for over 3 months by sub-cloning. These cells stably expressp75, nestin, and ErbB2 and ErbB4. Inculture, they can generate neurons,glia, and myofibroblasts. Reversetranscription-polymerase chain reac-tion (RT-PCR) analysis of various NC,DRG, and neural stem cell markergenes identified many expressed inthe spheres, but the authors did notdistinguish which were expressed spe-cifically in undifferentiated stem cells.Importantly, the authors provide evi-dence that these DRG-derived stemcells originate from satellite glial cells.

Analysis of the behavior of eNCSCsfrom gut has provided an interestinginsight into how eNCSC propertieschange with time, as well as the basisof Hirschsprung’s disease, a humanneurocristopathy associated with de-fective RET signalling (Carlomagno etal., 1996; Ivanchuk et al., 1996; Seri etal., 1997). RET and its coreceptor

GFRA1 mediate glial-derived neuro-trophic factor (GDNF) signalling, andthis finding is important for migrationof NCCs along the developing gut(Natarajan et al., 2002; Barlow et al.,2003; Yan et al., 2004). Iwashita andcolleagues identified expression of Retand Gfr1 in rat gut eNCSCs; interest-ingly, RET expression was not foundin other eNCSCs (Iwashita et al.,2003). Importantly, they showed thatmigration, but probably not prolifera-tion, survival, or differentiation, of guteNCSCs was stimulated in a cultureassay by GDNF and that Ret mutantmice show highly decreased numbersof eNCSCs in distal gut regions.

Evidence that NCSCs show self-re-newal was paramount to their originalclassification as stem cells (Stempleand Anderson, 1992). This study dem-onstrated that eNCSCs showed atleast a limited capacity for self-re-newal, because they generated mostlyoligopotent clones after two rounds ofsubcloning, estimated to correspond toperhaps 6–10 cell generations. Simi-larly, postmigratory, p75�P0� NCSCsdissociated from embryonic day (E)14.5 or E16.5 sciatic nerves and cul-tured for up to 11 days also give rise tooligopotent secondary subclones (Mor-rison et al., 1999). These authors alsopresented evidence indicating thatp75�P0� sciatic nerve cells incorpo-rate bromodeoxyuridine in vivo, andare therefore mitotic, consistent withpersisting NCSCs being self-renewing(Morrison et al., 1999). NCSCs iso-lated from other postmigratory posi-tions (gut, DRGs, and sympatheticganglia) have all been shown to gen-erate oligopotent daughter cells aftersubcloning (Bixby, 2002; Kruger,2002). Studies to clarify whether earlyNCSCs in mammalian and avian sys-tems might be capable of sustainedself-renewal remain to be performed(Stemple and Anderson, 1992; Bixbyet al., 2002; Kruger et al., 2002).

The abundance of NC-derived stemcells at different stages is a key ques-tion that remains largely unanswered.This question was addressed directlyin a study of gut eNCSCs, whichshowed that only 1–2% of E14.5 ratgut cells are strongly p75��4integrin�

(Iwashita et al., 2003). Importantly,when these cells were plated clonally,only some 60% formed colonies, and ofthese, only 80% generated mixed neu-

ron/glial/myofibroblast colonies. Thisfinding illustrates nicely one complex-ity of these stem cell studies, namelythat even fluorescence-activated cellsorting–purified cells remain a mix-ture and are simply enriched for stemcells. Studies examining early NCCprimary cultures have shown that themajority of NCCs that grow out from,for example, mouse E9 neural tubeexplants are Sox10�p75� (Hari et al.,2002), yet in vivo studies of lineagemarkers suggest that these cells are amixture of cell types, including, for ex-ample, melanocyte precursors (Wilsonet al., 2004).

Experiments in culture have iden-tified several key growth factors af-fecting the fates selected by eNCSCs(reviewed in Basch and Bronner-Fraser, 2006). Initially these studieshave focused on whether these fac-tors were instructive (driving fatespecification of stem cells) or selec-tive (allowing survival of cells speci-fied only to certain fates). In all casesidentified to date, they work in an in-structive mode, with Wnt/�-catenin,BMP2/4, Neuregulin1 and Notch/Delta,and transforming growth factor-beta(TGF�) signalling driving sensoryneuronal, sympathetic neuronal, glial,and myofibroblast fate, respectively.Importantly, these in vitro studies onisolated eNCSCs have generally beenextended convincingly to the in vivosituation, with evidence implicatingeach of these factors in these samefate choices in the endogenous NC(Shah et al., 1994, 1996; Lo et al.,1997; Shah and Anderson, 1997; Mor-rison et al., 2000; Wakamatsu et al.,2000; Hari et al., 2002; Lee et al.,2004). One fate conspicuously absentfrom current reports on eNCSCs is themelanocyte. Surprisingly, exposure ofeNCSCs to Wnt1 is not reported toinduce melanocytes in culture (Lee etal., 2004; L. Sommer, personal com-munication), despite the observationthat inactivation of �-catenin in NCCscauses almost total loss of melano-cytes, as well as sensory neurons, invivo (Hari et al., 2002). This finding ismore remarkable considering thatother oligopotent NC-derived stemcells clearly have melanocyte poten-tial (see below) and given that Wntsignalling is likely to directly regulatemelanocyte fate by activation of themaster switch gene Mitf (Dorsky et

NEURAL CREST STEM CELLS 3245

al., 2000; Takeda et al., 2000). Thesimplest explanation would seem to bethat it reflects an inadequacy of theculture conditions used. An alterna-tive, intriguing possibility is that me-lanocyte fate specification or commit-ment may be specific to the Wnt used,because Wnt3a, but not Wnt1, biasesNCCs to the melanocyte fate (Jin etal., 2001; Dunn et al., 2005). Further-more, where �-catenin signalling isstabilized in vivo in the NC, sensoryneurons are made and other celltypes, including melanocytes, are lost;apparently sensory neuron specifica-tion is driven to the exclusion of otherfates (Lee et al., 2004). This resultmight originate from altered NCC mi-gration affecting fate choice, but alsocan be readily reconciled with the�-catenin inactivation data if the sen-sory neuron fate decision precedes themelanocyte fate decision, or if melano-cyte specification requires purelytransient, rather than prolonged, sig-nalling. Hence, it will be interesting tosee whether eNCSCs and pigment cellprecursors segregate from a trulymultipotent neural crest stem cell.Such a scenario would be consistentwith the identification of segregatedmelanocyte progenitors in mouse dor-sal neural tube (Wilson et al., 2004)and early migrating NCCs from avianneural tube explants (Henion andWeston, 1997; Luo et al., 2003).

STEM CELLS IN AVIANNEURAL CREST

Extensive characterization of avianNC by primary culture of NCCs hasrevealed many types of embryonic NCprogenitors (Sieber-Blum and Cohen,1980; Baroffio et al., 1988, 1991;Sieber-Blum, 1989; Ito and Sieber-Blum, 1993; Henion and Weston,1997). These influential studies haveconvincingly demonstrated the diver-sity of potencies among even earlyNCCs and have led to a major model ofthe lineage relationships of progenitorsegregation in the NC (Baroffio et al.,1988, 1991; Dupin et al., 2007). How-ever, these studies did not address thecapacity for self-renewal among theseprogenitors. Hence, an important de-velopment of this work has been tobegin to evaluate the variety of stemcells among these progenitors by clas-sifying them according to their result-

ing cell fates and testing their self-renewal capacity by in vitro serialsubcloning (Trentin et al., 2004).These authors found that NCCs fromthe head and trunk contained differ-ent kinds of progenitors, and theseprogenitors also differ in their abilityto self-maintain and to respond to en-dothelin 3 (ET3), previously shown tobe a proliferation and survival-induc-ing factor for avian NCCs (Lahav etal., 1996, 1998). For instance, cranialprimary colonies of bipotent cells(glia–melanocyte or GM) could bepropagated by subcloning twice, butthis was increased to four subcloningrounds in the presence of ET3. Glia–fibroblast (GF) progenitors were ableto self-renew for up to three subclon-ings with or without ET3. In contrast,trunk primary colonies were only suc-cessfully subcloned when ET3 wasadded; one oligopotent (GMF) and twobipotent progenitors (GM and GF) dis-played capacity for self-renewal. Fur-ther evaluation of the effect of cultureconditions on self-renewal in this sys-tem will be critical. Likewise, investi-gation of the self-renewal capacity ofsuch cells in vivo will provide an in-teresting comparison. Finally, thissystem presents a unique opportunityto test whether EDNRB signallingmight function in melanocyte fatespecification from multipotent precur-sors, because, in contrast to mam-mals, melanocyte fate specification inquail is accompanied by a switch fromEDNRB to EDNRB2 expression(Lecoin et al., 1998). Studies of mouseEdnrb mutant NCCs provide qualita-tive evidence that, in mammals, Ed-nrb signalling has no role in melano-cyte fate specification from NCCs, butfunctions in melanocyte differentia-tion (Lee et al., 2003; Hou et al., 2004),as well as affecting proliferation ofsome sort of melanocyte progenitor(Reid et al., 1996). The equivalentavian NCC studies are consistent withthis finding, but do not rule out ED-NRB signalling having an instructiverole in melanocyte fate specification.

MELANOCYTE STEM CELLS

In mammals, hair pigmentation is de-rived from melanocytes localized inthe hair follicle (Fig. 1B), which se-crete pigmented organelles, melano-somes. The cyclical turnover of hair

during the moult cycle is accompaniedby a similar turnover of melanocytes;hence, a stem cell source of melano-cyte renewal was anticipated. Candi-date melanocyte stem cells (MSCs)were first identified in the lower per-manent portion or bulge of the hairfollicle as Dct-LacZ� cells resistant totreatment with ACK2, a monoclonalc-Kit blocking antibody (Nishimura etal., 2002). In the resting phase (telo-gen), MSCs are quiescent, but theirdivision is activated during thegrowth phase of the hair follicle (ana-gen) to supply proliferating and differ-entiating progeny to pigment the ma-turing hair. These cells colonized thebulge of early hair follicles by postna-tal day 0.5, were shown to be slowcycling, and lacked melanin granules(Nishimura et al., 2002). Unlike otherneural crest-derived stem cells, thepotency of MSCs has not been exam-ined; although they generate melano-cytes in vivo, it remains to be seenwhether they could generate other celltypes in culture. Evidence supportingthe self-renewal of MSCs in the hairfollicle throughout the lifetime of themouse has been presented (Nishimuraet al., 2002), but this finding needs tobe tested directly in culture with pu-rified MSCs.

The ability to identify MSCs in theirniche has allowed some characteriza-tion of their expression profile in vivo(Nishimura et al., 2002; Lang et al.,2005; Osawa et al., 2005). Using bothimmunofluorescent staining of sec-tioned mouse skin and single-cell ex-pression profiling of microdissected,genetically labeled MSCs, a molecularsignature (Dct�, Pax3�,Tyr�, Si�,Tyrp1�, Kitlow/�, Mitf�, Sox10�,Lef1�, and Mki67�) has been pro-posed as distinguishing MSCs frommelanoblasts or melanocytes, whichwere generally positive for all thesemarkers (Osawa et al., 2005). It is per-haps surprising that MSCs expressthe melanocyte marker Dct, while notexpressing key activators of Dct ex-pression, Mitf and Sox10 (Ludwig etal., 2004). This finding suggests thatDct expression can be induced inde-pendently of Mitf and Sox10 by meansof another pathway. It has been pro-posed that MSCs are derived fromSox10�, Mitf�, Dct� embryonic mela-noblasts, and hence, once activated,the Dct chromatin may adopt a config-

3246 DELFINO-MACHIN ET AL.

uration in which expression becomesSox10- and Mitf-independent (Osawaet al., 2005). Alternatively, Dct ex-pression may be activated by another

combination of transcription factors inthese cells. A study by Lang et al.(2005) indicated that Pax3 is likely tobe a key component of this; they iden-

tified a set of Pax3�, Dct� neuralcrest-derived cells in the bulge asMSCs and suggested that these cellsmight themselves be derived fromPax3�, Dct� cells in response to Wntsignalling. They suggest that Pax3 isa key component of a molecular ge-netic mechanism maintaining MSCsin an undifferentiated state (Lang etal., 2005). Pax3 is known to act syner-gistically with Sox10 to activate ex-pression of Mitf (Bondurand et al.,2000; Potterf et al., 2000), thus pro-moting melanocyte specification. Butin addition, at least in cultured celllines, Pax3 can also bind to the Dctpromoter in a manner preventingMitf-dependent activation; thus, Pax3may repress Dct transcription andthus melanocyte differentiation (Langet al., 2005). Hence, Pax3 is proposedto initiate a differentiation cascade inmelanocyte precursors, while inhibit-ing actual differentiation by repress-ing transcription of key differentiationgene(s). It is unlikely that repressionof Dct is sufficient to inhibit melano-cyte differentiation, so that studiesaddressing whether Pax3 binding af-fects transcription of other Mitf targetgenes are now required. The authorsalso show that Wnt signalling in thehair follicle is required for Dct expres-sion in vivo in the MSCs, consistentwith their repression model. During

Fig. 1. Origins of neural crest (NC) -derived stem cells. A: Schematic representation of a murine embryo and the locations from which NC-derivedstem cells have been isolated. B: Identified stem cells in the hair follicle and dermis. The hair follicle contains a diverse population of neural crest stemcells (NCSCs) including melanocyte stem cells (MSCs, blue), SKin-derived Precursors (SKPs, purple), epidermal neural crest stem cells (EPI-NCSCs,green), and non–NC-derived stem cells such as keratinocyte stem cells (not shown for clarity).

Fig. 2. Markers of melanocyte stem cells (MSCs), epidermal neural crest stem cells (EPI-NCSCs),and SKin-derived Precursors (SKPs). A Venn diagram highlighting the examined and sharedmarkers of the three neural crest stem cell (NCSC) populations identified in the hair follicle. Markersdetected are shown in green, markers in plain text have not been detected. References: (1)Nishimura et al., 2002; (2) Osawa et al., 2005; (3) Lang et al., 2005; (4) Toma et al., 2001; (5)Fernandes et al., 2004; (6) Sieber-Blum et al., 2001; (7) Hu et al., 2006; and (8) Wong et al., 2006.

NEURAL CREST STEM CELLS 3247

Wnt signalling, �-catenin acts to dis-place Pax3 from the Dct promoter and,therefore, promotes Mitf-dependentactivation of Dct expression (Lang etal., 2005). The authors suggest thatthese mechanisms enable MSCs toprepare for rapid differentiation whenrequired during the hair cycle. Thisexplanation is a fascinating idea, sup-ported by analogy with developmentof embryonic NCSCs, in which sympa-thetic neuron specification (activationof MASH-1 and Phox2b expression) isinitiated, but differentiation is tran-siently inhibited, by Sox10 (Kim et al.,2003). However, the recent demon-stration of other neural crest-derivedstem cells with much broader poten-tial, including melanocytes, in theskin and hair follicles (see below;Sieber-Blum et al., 2004), suggests thepossibility that MSCs might in fact begenerated by differentiation of otherneural crest stem cells in the hair fol-licle. In this context, it would be ofgreat interest to know whether otherNC-derived stem cells in the hair fol-licle (see below) express Pax3 and howthese relate, if at all, to the MSCs. Inaddition, it now becomes particularlyimportant to test definitively the stemcell status of isolated Pax3� MSCs toeliminate the alternative idea thatthese cells are melanocyte progenitorsin a very early stage of differentiation,generated from oligopotent stem cellsnearby in the bulge.

EPIDERMAL NCSCs

MSCs are found in the skin and areexpected to be unipotent, althoughthis finding has yet to be explored.However, in recent years, severalother types of stem cells, of unexpect-edly broad potential, have also beenidentified in the skin. These cells havebeen labeled epidermal neural creststem cells (EPI-NCSCs, formerly epi-dermal NCSCs or even eNCSCs) andSKin-derived Precursors (SKPs; Tomaet al., 2001; Sieber-Blum et al., 2004;Wong et al., 2006).

EPI-NCSCs were identified by not-ing large numbers of neural crest-de-rived cells in the facial and back skinof adult mouse and were isolated bymicrodissection from the bulge area ofwhisker follicles (Sieber-Blum et al.,2004). These cells expressed Sox10and nestin and were serially cultured.

Interestingly, EPI-NCSCs showedvery broad potential, generating cellsexpressing markers appropriate forneurons, glia, myofibroblasts, chon-drocytes, and melanocytes. Thus, theyhave currently the best claim to betruly multipotent neural crest stemcells (see Table 1). Like eNCSCs, EPI-NCSCs respond to neuregulin-1 bygenerating Schwann cells. However,in contrast to eNCSCs, in response toBMP-2 they form chondrocytes.

The transcriptional profile of EPI-NCSCs in culture has been character-ized by long serial analysis of geneexpression (Long SAGE) and has beendirectly compared with that of eNC-SCs and a mixture of differentiatedneural crest progeny (Hu et al., 2006).As noted by the authors, this studyhas the limitation that samples weretaken from cells of 48-hr primary cul-tures (EPI-NCSC and eNCSC) and7-day differentiated primary cultures(neural crest progeny). Nevertheless,the authors identify a small suite of 19genes shared by EPI-NCSCs and em-bryonic NCSCs, but absent from epi-dermal stem cells (Fig. 2). This findingis important at least in demonstratingthat the NC-derived stem cells are dis-tinct from epidermal stem cells (whichgenerate keratinocytes), despite shar-ing a niche in the bulge. Furthermore,it provides the basis for future com-parison with other NC-derived stemcell types.

SKPs

Highly oligopotent SKPs were isolatedand characterized from postnatal andadult mouse dermis (Toma et al.,2001). Subsequently, the same groupshowed they originate from dermalpapillae of whisker follicles and arederived from neural crest; similarcells derived from dorsal skin are also,at least in part, likely to be neuralcrest-derived (Fernandes et al., 2004).Of interest, from a therapeutic pointof view, SKPs have also been isolatedfrom both human scalp and foreskin(Toma et al., 2001, 2005). These cellshave been grown as floating spherecultures for over 50 generations, butafter dissociation and plating on adhe-sive substrates will generate mixedfate clones containing undefined neu-rons, glia, adipocytes, and rarelymyofibroblasts, but apparently not

melanocytes (but see below) or chon-drocytes (Toma et al., 2001; Fer-nandes et al., 2004). In spheres, SKPsexpress nestin, fibronectin, Sca1,Dermo-1, SHOX2, slug, snail, twist,Pax3, and Sox9, but not Sox10, p75,and PSA–nerve cell adhesion mole-cule (NCAM; Toma et al., 2001; Fer-nandes et al., 2004; Fig. 2). Theauthors distinguish SKPs from mes-enchymal stem cells on the basis ofmarker expression, behavior in float-ing culture and morphology, and fromeNCSCs by lack of p75 (very low levelin human SKPs; Toma et al., 2001,2005; Fernandes et al., 2004) andNCAM expression. They can be distin-guished from EPI-NCSCs by their ex-pression of SHOX2 and Dermo-1 (Fer-nandes et al., 2004; Sieber-Blum etal., 2004). However, their relationshipto other stem cells, including mesen-chymal stem cells, is unknown (Fer-nandes et al., 2007). SKPs have beentested for their in vivo potential aftertransplantation into chick embryo(Fernandes et al., 2004). Thus, whenyellow fluorescent protein-labeledtransgenic SKP spheres were placedin the neural crest migration paths,some cells dispersed along neuralcrest cell migration routes. Subse-quently, they contribute at least toDRG glia and probably also to periph-eral nerves. Of interest, they do mi-grate into the skin, a behavior associ-ated with specified melanoblasts(Erickson and Goins, 1995), yet de-spite the lack of melanocytes in SKPcultures, the fate of these cells was nottested (Fernandes et al., 2004).

A further type of NC-derived pre-cursor capable of forming floatingspheres was identified from mousewhisker skin and from both mouseand human adult trunk skin, andthese have also been called SKPs(Wong et al., 2006). However, in clearcontrast to the SKPs identified byToma and colleagues, cells of theseskin-derived spheres expressed theNCSC markers Sox10 and p75 (Tomaet al., 2001; Wong et al., 2006). Self-renewal was demonstrated by mainte-nance of Sox10/p75 expression in mostcells over several months of passages.In common with Sox10-expressingEPI-NCSCs, Sox10�p75� SKPs dis-played broad potency, forming glia,neurons, myofibroblasts, and rare adi-pocytes in culture; chondrocytes and a

3248 DELFINO-MACHIN ET AL.

few melanocytes were formed withmodified culture conditions. Althoughsharing Sox10 and p75 expressionwith eNCSCs, when p75� SKP cellswere isolated from spheres and ex-posed to growth factors, includingBMP2 and NRG1, they displayed verydifferent growth factor responsive-ness, differentiating into myofibro-blasts. This shift in response is similarto, but rather more dramatic than,that noted before in eNCSCs (see, forexample, Kruger et al., 2002). The invivo role of these precursors has yet tobe identified.

The apparent niches of Sox10�p75�

SKPs were identified using geneticlineage labelling techniques. Thisfinding suggested that, in the whiskerfollicles, many structures containedNC-derived cells and could formspheres in culture. The source of SKPsin the trunk was more restricted, butincluded cells that had expressedDesert Hedgehog and that were likelyto be glial-derived and also cells thathad expressed Dct and were, thus,likely to be of the melanocyte lineage;both cell types were found in the folli-cle bulge. There is strong evidencethat the Sox10�, p75� SKP niche in-cludes dermal papilla, but the isola-tion of very similar cells from non-hairy skin (e.g., foreskin) suggeststhat another niche, presumably alsodermal, must be suitable (Fernandeset al., 2004; Toma et al., 2005).

Together, these reports identifycells with similar potential from mul-tiple locations in the skin (Toma et al.,2001, 2005; Fernandes et al., 2004).However, further work is required toclarify the relationships, if any, be-tween the trunk- and cranial skin-de-rived precursors identified by thesetwo groups and to reconcile the differ-ences in markers they express.

Both EPI-NCSCs and SKPs arehighly oligopotent cells and arereadily accessible. Consequently, at-tempts to test the therapeutic utilityof these highly potent cells are becom-ing an important research focus.Schwann cells derived from Sox10�

p75� SKPs can both integrate intoand generate myelin sheathing inboth the PNS and central nervous sys-tem (CNS) of shiverer mice (McKenzieet al., 2006), although less success hasyet been obtained with SKP-derivedneuronal cells (Fernandes et al.,

2006). Likewise, initial attempts toget Sox10�p75� SKPs to generatecells of the CNS failed even after le-sioning, suggesting that these oligopo-tent cells may not be readily transdif-ferentiated into CNS cell types (Wonget al., 2006).

CARDIAC NCSCs

Oligopotent cells from mouse cardiacNC generate myofibroblast, neurons,Schwann cells, melanocytes, and chon-drocytes in culture (Youn et al., 2003).The authors comment that self-renewalwas shown by serial cloning, but they donot give details of, for example, for howmany passages they could be main-tained. In the same study, other progen-itors of more restricted potential (givingmyofibroblast and rarely chondrocytesand Schwann cells, but not pigmentcells or neurons) were observed as wellas committed myofibroblast progeni-tors. The authors do not comment onwhether they tested these latter twoprogenitors for self-renewal (Youn etal., 2003).

A neural crest-derived stem cellpopulation has also been identifiedwithin the adult mice cardiac sidepopulation (SP; Tomita et al., 2005).SP cells are dormant, multipotentcells that are found in a variety oftissues and can be identified by theirdifferential ability to efflux theHoechst 33342 dye. In mice, a subsetof cardiac SP cells were able to gener-ate spheres (“cardiospheres”) in vitro(nestin�, Musashi-1�) and to differen-tiate into neurons, glia, melanocytes,chondrocytes, and myofibroblasts af-ter dissociation and in the absence ofendothelial growth factor and fibro-blast growth factor-2. When trans-planted into chick neural tube, cardio-sphere cells behaved as neural crestand migrated into the outflow tract,DRGs and spinal nerve and followedthe lateral migration pathway. To-gether, this evidence suggests that apopulation of neural crest-derivedstem cells remains in the heart andhas capacity to differentiate (Tomitaet al., 2005).

OTHER NC-DERIVED STEMCELLS

A further NC-derived and self-renew-ing SP stem cell has been identified

from mouse corneal precursors (Yo-shida et al., 2005, 2006). These cellsform spheres in culture; expressmarkers including nestin, Notch1,Musashi1, Twist, Slug, Snail, andSox9; and can be clonally expanded forover 18 passages (Yoshida, 2006).They generate keratocytes (cornealstroma cells), adipocytes, myofibro-blasts, neurons, and glia in culture.

Finally, a population of highly pro-liferative oligopotent stem cells hasbeen reported from human exfoliatedteeth (Miura et al., 2003). Known asSHED, these cells express two earlymesenchymal stem cell markers:STRO-1 and CD146. They have beenshown to generate odontoblasts, neu-ronal, glial, and adipocyte fates, andthe authors speculate that they mayhave an NC origin. This speculationremains untested, but is consistentwith the demonstration in mouse thatNCCs contribute extensively to mostcell types in teeth, including the den-tal pulp, although a cranial mesodermcontribution is likely too (Chai et al.,2000). These and other dental pulpstem cells can generate odontoblastsand dentine and have promise fortooth replacement therapies (Sloanand Smith, 2007). Lineage tracing ofall these cells is important for a fullunderstanding of NC-derived stemcells.

ARE THERE COMMONMOLECULAR FEATURES TONC-DERIVED STEM CELLS?

To date, universal molecular defini-tions of stem cells have been elusive. Asummary of the combination of stemcell and neural crest markers used inthe search for self-renewing, oligopo-tent neural crest cells is presented inTable 1. It would be expected thatgenes required for the two key pro-cesses of self-renewal and multipoten-tiality might be broadly seen amongNC-derived stem cells. Currently,most NC-derived stem cells have beenexamined in a slightly ad hoc mannerfor marker genes and this obscuresany generalities that may exist. Fewstudies have explicitly attempted toprofile these stem cells and only twohave done so in an unbiased way(Molofsky et al., 2003; Hu et al., 2006),but in addition, a wide range of candi-date markers have also been exam-

NEURAL CREST STEM CELLS 3249

ined for MSCs (Osawa et al., 2005).Markers identified by these studiesare summarized in Figure 2, but re-veal few commonalities. Sox10, p75,and nestin are widespread and mayhave a relatively general role as mark-ers of NC-derived stem cells. Sox10has been proposed to be necessary formaintenance of multipotentiality inNCCs (Paratore et al., 2002; Kim etal., 2003) and, thus, might be expectedto be a common feature of all multipo-tent NC stem cells. The apparent dis-crepancy of Sox10� SKPs (Toma et al.,2001; Fernandes et al., 2004) is, there-fore, enigmatic, especially becausesimilar cells expressing Sox10 havealso been well-characterized (Wong etal., 2006). MSCs have been character-ized as Sox10� (Osawa et al., 2005),which would be consistent with theassumption that these cells are unipo-tent, but again highlights the signifi-cance of a direct test of MSC potentialand self-renewal. Interestingly, nestinand p75 are both markers of neuralstem cells (Lendahl et al., 1990; An-dressen et al., 2001).

An important finding concerns therole for the polycomb family transcrip-tional repressor Bmi-1 in self-renewalof stem cells in the central and periph-eral nervous systems (Molofsky et al.,2003). Bmi-1 appears to act in post-natal stem cells by repressing thecyclin-dependent kinase inhibitors,p16Ink4a and p19Arf, thereby pro-moting self-renewal, but does not af-fect the proliferation of restrictedprogenitors (Molofsky et al., 2003).Whether Bmi-1 or related transcrip-tional repressors are a common fea-ture of all NC-derived stem cells re-mains to be examined.

As we have seen, many of the NC-derived stem cells reviewed here re-spond in a similar way to instructivesignals such as BMPs. An elegantstudy identified a crucial role for com-bined Wnt and BMP signals in main-tenance of eNCSCs (Kleber et al.,2005). Previous work showed thateach signal alone drives sensory neu-ron and autonomic neuron specifica-tion and differentiation respectively(Shah and Anderson, 1997; Lee et al.,2004), but a combination of Wnt1 andBMP2 represses specification of eitherneuronal fate (Kleber et al., 2005). In-deed, cells fail to become glia or myo-fibroblasts either, and instead main-

tain p75 and Sox10 expression andmultipotency. Intriguingly, althoughmultipotentiality was clearly main-tained, sensitivity to Wnt was specifi-cally lost, a finding paralleled by eNC-SCs prospectively isolated from twopostmigratory locations: sciatic nerveand DRGs. Such a shift in the intrin-sic properties of eNCSCs during devel-opment has also been reported in thecontext of responsiveness to BMPsand neuregulin (Bixby et al., 2002;Kruger et al., 2002). Thus, it seemsthat eNCSCs alter their phenotypeduring embryonic and postnatal de-velopment (Bixby et al., 2002; Krugeret al., 2002), a process that may befurther exaggerated in SKPs (Wong etal., 2006). This change may, at least inpart, explain why markers common toall neural crest-derived stem cells ap-pear so elusive.

Finally, a study of cardiac NCSCssuggests that TrkC function is re-quired for stem cell maintenance, per-haps by driving continuing prolif-eration, because premature fatespecification happens in mutants(Youn et al., 2003). It is currently un-clear how widespread is TrkC expres-sion in NC-derived stem cells.

PLASTICITY OF STEMCELL FATE?

An ongoing issue in the study of anystem cell is the extent to which cellsare reprogrammed or modified by cul-ture conditions. This is important fortwo distinct reasons. On the one hand,from the perspective of understandingnormal stem cell biology, avoidance ofculture conditions causing such arte-factual responses is required. On theother hand, from a therapeutic per-spective, controlled reprogramming ofstem cells in culture may have greatpotential in the treatment of disease.Work on avian NC derivatives dra-matically reinforces the necessity totake the possibility of reprogrammingseriously, because remarkably mela-nocytes have been generated from cul-tured sciatic nerve Schwann cells andSchwann cells can be formed by cul-turing of epidermal melanocytes(Dupin et al., 2000; Nataf and LeDouarin, 2000). Furthermore, this re-programming takes place by means ofdedifferentiation to form a precursorexpressing NC markers and showing

self-renewal and oligopotency, that is,a NC-like stem cell (Dupin et al., 2000,2003; Real et al., 2005, 2006).Whether NC-derived stem cells canalso be reprogrammed remains to beseen. Although important, determina-tion of whether artefactual modifica-tion of stem cell properties has oc-curred is difficult, especially because acomparison of the in vivo and in vitroderivatives of an oligopotent stem cellneed not be identical. Consequently,this issue has been rarely exploredand requires more attention in the fu-ture. It is perhaps best accomplishedby comparison of properties of stemcells freshly ex vivo with those thathave been maintained in culture forsome time.

There is clearly a close embryologi-cal relationship between the neuralcrest and the neural tube, and thisfinding may be reflected in the appar-ent ability of NC-derived stem cells togenerate neural stem cell derivatives(Sieber-Blum et al., 2006). Whetherthis represents true stem cell plastic-ity or simply the latent potential ofstem cells reflecting their shared em-bryological origin is unclear, but itcertainly encourages investigation ofthe therapeutic potential of adultstem cells.

PERSPECTIVES

There has been considerable effort inrecent years to identify NC-derivedstem cells from multiple sources, bothin the embryo and in the adult. A co-ordinated effort to test definitively therelationship between these differentstem cells is now paramount. There isat least some evidence that these di-verse stem cell types are derived fromother stem cells and, thus, that neuralcrest stem cells are somehow modifiedin time. This concept appears to chal-lenge the stem cell definition we areusing, because they are not “continu-ously producing unaltered daughters”(Smith, 2006). However, it is impor-tant to remember that stem cell be-havior in the changing in vivo envi-ronment might be expected to be lessconstant than that under standard-ized culture conditions; hence, long-term changes in stem cell propertiesin vivo are not to be considered incon-sistent with our stem cell definition. Asimilar view is increasingly well-sup-

3250 DELFINO-MACHIN ET AL.

ported in the field of neural stem cells(Merkle and Alvarez-Buylla, 2006).

Neural stem cells have turned out tobe surprisingly morphologically dis-tinctive (i.e., differentiated; Merkleand Alvarez-Buylla, 2006), and it willbe interesting to see to what extentthis is true for neural crest-derivedstem cells at different stages. Indeed,the parallel may be even stronger, be-cause neural stem cells in the fetusand adult appear to be specific glialcell types. Of interest, many markersof early neural crest stem cells arealso late markers of glia, most notablySox10, which is functionally associ-ated with stem cell maintenance andglial cell differentiation (Kelsh, 2006).Furthermore, some SKPs, at least inthe trunk, may actually have the char-acteristics of glial cells (Wong et al.,2006), and adult DRG-derived stemcells most likely derive from satelliteglial cells (Li et al., 2007). Thus, it willbe interesting to see if NC-derivedstem cells in at least the peripheralnervous system adopt “glial” morphol-ogies.

What is the in vivo role of the NC-derived stem cells? In most cases, thisfeature has hardly been addressed,but it is important in terms of under-standing to what extent stem cell po-tency is tightly tuned to the in vivofunction. For example, it is not clearhow neuronal potential is necessaryfor hair follicle cells, and yet SKPsshow this in vitro.

The importance of the stem cellniche in providing a cellular environ-ment maintaining stem cells is becom-ing increasingly clear. For the NC-de-rived stem cells discussed here, theprecise location of their niche is poorlydefined, let alone the details of thesignals provided by the niche that pro-mote stem cell self-renewal and pre-vent differentiation. The hair folliclebulge is apparently the niche forMSCs (Nishimura et al., 2002) alongwith EPI-NCSCs (Sieber-Blum et al.,2004), SKPs (Wong et al., 2006), aswell as the keratinocyte stem cells(Cotsarelis et al., 1990). There is,thus, an excellent opportunity to dis-sect the specialized cellular microen-vironment and its influences on thesediverse stem cell types (Nishikawaand Osawa, 2007). Stem cell popula-tions tend to become weakened withphysiological aging, and in the case of

MSCs, this weakening may result, atleast in part, from ectopic differentia-tion within the niche and result inhair graying (Nishimura et al., 2005).Identification of the changes in theniche that occur with ageing will,thus, help elucidate some of the keyfactors conferring stem cell character-istics. Conversely, it will be exciting tosee what factors activate stem cellproliferation and differentiation and,in the follicle bulge, how is this con-trolled with respect to the hair cycle.

The current list of growth factorsand their receptors that instruct NC-derived stem cells to adopt specificfates is certainly inadequate to ex-plain the full set of NC fate specifica-tion events. Furthermore, the re-sponse to the same signal orcombinations of signals varies withdifferent NC-derived stem cells. Weare mostly ignorant of the receptorsmediating growth factor signalling ina NC context. It is likely that thesesignals act combinatorially to drivespecification to individual fates. Like-wise, the cellular context of these sig-nals is also crucial for the effects theyhave (Paratore et al., 2001; Kruger etal., 2002; Lewis et al., 2004). A con-certed effort to analyse, for each classof stem cell, the response to all theknown instructive factors is required.Certainly, there are other factors inthe NC literature, including growthdifferentiation factor-7 and brain-de-rived neurotrophic factor (Sieber-Blum, 1991; Lo et al., 2005), thatshould be tested on defined NC-de-rived stem cells, but this problem de-serves a comprehensive and methodi-cal analysis of all candidates. Thedevelopment of NCSC-like immortal-ised cell lines, MONC-1 and especiallythe more stable JoMa1, expressingp75 and Sox10 and with very broadpotency, including autonomic neu-rons, glia, smooth muscle, melano-cytes and chondrocytes, promise to beinvaluable tools in this regard (Raoand Anderson, 1997; Maurer et al.,2007).

The lineage relationship of the var-ious NC-derived stem cells is a crucialissue that needs to be resolved.Whereas their origins at some stagefrom early NC is generally not indoubt, the idea that some may share acloser relationship is certainly plausi-ble. At the level of cell potential, a

nested hierarchy with EPI-NCSCsand SKPs showing broadest potency,eNCSCs a more restricted one, andMSCs an untested, but assumedunipotency, would be consistent withthis idea. The issue is particularly poi-gnant in the case of the three stemcells described from the hair folliclebulge. Are there really three distinctstem cell types in such close proximity(four, if the non-NC derived keratino-cyte stem cell is included)? We notethat both SKPs and MSCs have beenidentified as being labeled by lineagetracing using the Dct promoter andboth reside in the bulge (Nishimura etal., 2002; Osawa et al., 2005; Wong etal., 2006). Furthermore, SKPs can, atleast under some culture conditions,generate melanocytes (Wong et al.,2006). Indeed, they have been pro-posed to be identical (Wong et al.,2006), but discrepancies in the de-scribed marker characteristics (espe-cially Sox10) of these two cell typesneed to be resolved. It is highly plau-sible that they are lineally related. Di-rect tests of the potency, self-renewal,and molecular characteristics of SKPsand MSCs during late phases of thehair cycle when the stem cell-contain-ing bulge region and the melanocyte-containing hair papilla are physicallymost discrete should address this is-sue. Currently, understanding of thein vivo lineage relationships betweendifferent types of NCCs is limited, buta detailed model based on extensivecharacterization of pluripotentiality ofcultured NCCs has been proposed bythe Le Douarin lab (Le Douarin andDupin, 2003; Le Douarin et al., 2004;Dupin et al., 2007). As noted above,several of the intermediates they pro-posed show at least some capacity forself-renewal (Trentin et al., 2004); asin vivo lineage relationships and pro-genitor types become clearer, it will beimportant to establish their self-re-newal capacity.

A related issue is that of whetherthere is a truly multipotent NC-de-rived stem cell, as has been proposed(Dupin et al., 2007). Potentiality ofNC-derived stem cells has rarely beenevaluated systematically. eNCSCs doproduce multiple neuronal cell types,but not, apparently, chondrocytes ormelanocytes, so the relationship be-tween these cells, if any, needs to beevaluated; EPI-NCSCs and SKPs

NEURAL CREST STEM CELLS 3251

might be multipotent cells, but thisfinding remains to be tested defini-tively. A further question relates tohow potential changes with develop-ment; it is perhaps natural to tend toassume that potential decreases withdevelopmental age, but the initialstudies on eNCSCs argue more for ashift in responsiveness (White et al.,2001; Bixby et al., 2002; Mosher et al.,2007), while EPI-NCSCs appear tohave a broader potential than eNC-SCs. Again, understanding the lin-eage relationship between these stemcell types will help make sense of thevaried stem cell potencies.

Part of the problem is that we re-main ignorant about how many stemcells of any specific type are present atany stage in vivo. For example, theavian studies show that early NCCsconsist of a mixture of stem cells ofdifferent potencies and other progeni-tors. Conceivably, therefore, cells akinto EPI-NCSCs are mixed in with morerestricted eNCSCs in the embryonicNC. Differences in their relative abun-dance and in experimental techniquewill then affect our ability to culturethem from different source material.Stem cell numbers are likely to bevery variable for each stem cell type;for example, MSCs are described asbeing at very low abundance in thehair follicle (Nishimura et al., 2002;Mak et al., 2006), whereas SKPs inthe whisker follicles are more abun-dant (Fernandes et al., 2007). Identi-fication of both definitive markers andthe endogenous niche for each stemcell will be necessary to assess thisquestion, one which has great signifi-cance for the therapeutic use of thesestem cells. Therapeutic applications ofthese stem cells are only just beingexplored and consideration of all ofthe above issues are likely to influencethe success of these endeavors in thecoming years.

ACKNOWLEDGMENTSWe thank Sarah Colanesi, MariosStavridis, and two anonymous review-ers for comments on a draft of thismanuscript.

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