Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin.
ABSTRACT Given their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell-like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and display extensive self-renewal capacity in sphere cultures. To determine the origin of these cells, we genetically mapped the fate of neural crest cells in face and trunk skin of mouse. In whisker follicles of the face, many mesenchymal structures are neural crest derived and appear to contain cells with sphere-forming potential. In the trunk skin, however, sphere-forming neural crest-derived cells are restricted to the glial and melanocyte lineages. Thus, self-renewing cells in the adult skin can be obtained from several neural crest derivatives, and these are of distinct nature in face and trunk skin. These findings are relevant for the design of therapeutic strategies because the potential of stem and progenitor cells in vivo likely depends on their nature and origin.
- [Show abstract] [Hide abstract]
ABSTRACT: Neural crest cells (NCCs) are unique to vertebrates and emerge from the border of the neural plate and subsequently migrate extensively throughout the embryo after which they differentiate into many types of cells. This multipotency is the main reason why NCCs are regarded as a versatile tool for stem cell biology and have been gathering attention for their potential use in stem cell based therapy. Multiple sets of networks comprised of signaling molecules and transcription factors regulate every developmental phase of NCCs, including maintenance of their multipotency. Pluripotent stem cell lines, such as embryonic stem cells and induced pluripotent stem (iPS) cells, facilitate the induction of NCCs in combination with sophisticated culture systems used for neural stem cells, although at present, clinical experiments for NCC-based cell therapy need to be improved. Unexpectedly, the multipotency of NCCs is maintained after they reach the target tissues as tissue neural crest stem cells (NCSCs) that may contribute to the establishment of NCC-derived multipotential stem cells. In addition, under specific culture conditions, fate-restricted unipotent descendants of NCCs, such as melanoblasts, show multipotency to differentiate into melanocytes, neurons, and glia cells. These properties contribute to the additional versatility of NCCs for therapeutic application and to better understand NCC development. Birth Defects Research (Part C), 2014. © 2014 Wiley Periodicals, Inc.Birth Defects Research Part C Embryo Today Reviews 09/2014; · 4.44 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Resident neural precursor cells (NPCs) have been reported for a number of adult tissues. Understanding their physiological function or, alternatively, their activation after tissue damage or in vitro manipulation remains an unsolved issue. Here, we investigated the source of human dermal NPCs in adult tissue. By following an unbiased, comprehensive approach employing cell-surface marker screening, cell separation, transcriptomic characterization, and in vivo fate analyses, we found that p75NTR+ precursors of human foreskin can be ascribed to the Schwann (CD56+) and perivascular (CD56-) cell lineages. Moreover, neural differentiation potential was restricted to the p75NTR+CD56+ Schwann cells and mediated by SOX2 expression levels. Double-positive NPCs were similarly obtained from human cardiospheres, indicating that this phenomenon might be widespread.Stem Cell Reports. 11/2014; 3(5):1-15.
- [Show abstract] [Hide abstract]
ABSTRACT: The neural crest is the name given to the strip of cells at the junction between neural and epidermal ectoderm in neurula-stage vertebrate embryos, which is later brought to the dorsal neural tube as the neural folds elevate. The neural crest is a heterogeneous and multipotent progenitor cell population whose cells undergo EMT then extensively and accurately migrate throughout the embryo. Neural crest cells contribute to nearly every organ system in the body, with derivatives of neuronal, glial, neuroendocrine, pigment, and also mesodermal lineages. This breadth of developmental capacity has led to the neural crest being termed the fourth germ layer. The neural crest has occupied a prominent place in developmental biology, due to its exaggerated migratory morphogenesis and its remarkably wide developmental potential. As such, neural crest cells have become an attractive model for developmental biologists for studying these processes. Problems in neural crest development cause a number of human syndromes and birth defects known collectively as neurocristopathies; these include Treacher Collins syndrome, Hirschsprung disease, and 22q11.2 deletion syndromes. Tumors in the neural crest lineage are also of clinical importance, including the aggressive melanoma and neuroblastoma types. These clinical aspects have drawn attention to the selection or creation of neural crest progenitor cells, particularly of human origin, for studying pathologies of the neural crest at the cellular level, and also for possible cell therapeutics. The versatility of the neural crest lends itself to interlinked research, spanning basic developmental biology, birth defect research, oncology, and stem/progenitor cell biology and therapy. Birth Defects Research (Part C), 2014. © 2014 Wiley Periodicals, Inc.Birth Defects Research Part C Embryo Today Reviews 09/2014; · 4.44 Impact Factor
T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $8.00
The Journal of Cell Biology, Vol. 175, No. 6, December 18, 2006 1005–1015
Embryonic, fetal, and adult tissues are used as sources to inves-
tigate the developmental and therapeutic potential of stem cells.
Because of their accessibility and the possibility that the pa-
tient could act as a stem cell donor, adult stem cells from the
skin have received particular attention (Slack, 2001). Apart
from multipotent epithelial stem cells that form hair follicles,
sebaceous glands, and epidermis (Taylor et al., 2000; Oshima
et al., 2001; Blanpain et al., 2004; Claudinot et al., 2005) and
so-called melanocyte stem cells that generate pigmented cells
(Nishimura et al., 2002), a multipotent cell dubbed skin-
derived precursor cell (SKP) has been isolated from both the
murine and human skin (Toma et al., 2001). SKPs have the po-
tential to produce in vitro cell types normally not found in the
skin, such as neuronal cells. Subsequently, several laboratories
reported the existence of self-renewing cells present in the
skin of mice, pigs, and humans and able to differentiate in vitro
into cells expressing neuronal, glial, osteoblast, chondrocyte,
smooth muscle, melanocyte, and adipocyte lineage markers
(Belicchi et al., 2004; Dyce et al., 2004; Joannides et al., 2004;
Sieber-Blum et al., 2004; Amoh et al., 2005; Shih et al., 2005;
Toma et al., 2005).
The formation of cells normally not present in skin might
be due to transdifferentiation, which describes the conversion of
a cell type of a specifi c tissue lineage into a cell type of another
lineage (Wagers and Weissman, 2004). Alternatively, cells from
a given lineage might dedifferentiate into a more naive state that
allows the cell to redifferentiate along new lineages. Finally,
multipotent cells with stem cell features might persist until
adulthood, able to generate a broad variety of cells, depending
on their environment. To distinguish among these possibilities,
the origin and nature of the cell in question has to be determined
and its developmental potential has to be analyzed at the single
cell level (Wagers and Weissman, 2004).
Neural crest–derived cells with stem cell features can
be traced back to multiple lineages in the adult skin
Christine E. Wong,1 Christian Paratore,1 María T. Dours-Zimmermann,2 Ariane Rochat,3 Thomas Pietri,4 Ueli Suter,1
Dieter R. Zimmermann,2 Sylvie Dufour,4 Jean Paul Thiery,4 Dies Meijer,5 Friedrich Beermann,6 Yann Barrandon,3
and Lukas Sommer1
1Department of Biology, Institute of Cell Biology, Swiss Federal Institute of Technology (ETH) Zurich, CH-8093 Zurich, Switzerland
2Molecular Biology Laboratory, Department of Pathology, University Hospital of Zurich, CH-8091 Zurich, Switzerland
3Laboratory of Stem Cell Dynamics, School of Life Sciences, Ecole Polytechnique Féderale de Lausanne and Lausanne University Hospital, CH-1015 Lausanne, Switzerland
4UMR144, Centre National de la Recherche Scientifi que, Institut Curie, 75248 Paris, Cedex 05, France
5Department of Cell Biology and Genetics, Erasmus University Medical Center, 3000DR Rotterdam, Netherlands
6Swiss Institute for Experimental Cancer Research, 1066 Epalinges, Switzerland
tent stem cell–like cells from the adult trunk skin of mice
and humans that express the neural crest stem cell mark-
ers p75 and Sox10 and display extensive self-renewal
capacity in sphere cultures. To determine the origin of
these cells, we genetically mapped the fate of neural crest
cells in face and trunk skin of mouse. In whisker follicles of
the face, many mesenchymal structures are neural crest
iven their accessibility, multipotent skin-derived
cells might be useful for future cell replacement
therapies. We describe the isolation of multipo-
derived and appear to contain cells with sphere-forming
potential. In the trunk skin, however, sphere-forming neu-
ral crest–derived cells are restricted to the glial and mela-
nocyte lineages. Thus, self-renewing cells in the adult skin
can be obtained from several neural crest derivatives, and
these are of distinct nature in face and trunk skin. These
fi ndings are relevant for the design of therapeutic strate-
gies because the potential of stem and progenitor cells
in vivo likely depends on their nature and origin.
C.E. Wong and C. Paratore contributed equally to this paper.
Correspondence to Lukas Sommer: email@example.com
T. Pietri’s present address is Institute of Neuroscience, University of Oregon,
Eugene, OR 97403.
Abbreviations used in this paper: BMP, bone morphogenic protein; CNS, central
nervous system; FN, fi bronectin; GFAP, glial fi brillary acidic protein; GM,
growth medium; NCSC, neural crest stem cell; NRG, neuregulin; SKP, skin-
derived precursor cell; SMA, smooth muscle actin.
The online version of this article contains supplemental material.
on May 31, 2012
Published December 11, 2006
Supplemental Material can be found at:
JCB • VOLUME 175 • NUMBER 6 • 2006 1006
The developmental origin and exact localization of skin
cells giving rise to neural and nonneural progeny is unclear in
many of the reported cases. Multipotent skin-derived cells have
been enriched by means of markers found on hematopoietic
stem cells (Belicchi et al., 2004) or have been isolated from
transgenic animals expressing GFP from promoter elements of
Nestin (Amoh et al., 2005), a gene also expressed in neural pro-
genitor cells. One source that has been associated with sphere-
forming SKPs is the dermal papilla from whisker follicles
(Fernandes et al., 2004). Whisker follicles are large hair follicles
of the face that serve as sensory organs for a wide range of
mammals, excluding humans. Genetic in vivo cell fate mapping
revealed that the dermal papilla of these follicles is of neural
crest origin (Fernandes et al., 2004). Similarly, culturing ex-
plants of bulge and dermal sheath of whisker follicles allowed
the identifi cation of neural crest–derived multipotent cells in the
upper part of the whisker follicle (Sieber-Blum and Grim, 2004;
Sieber-Blum et al., 2004).
A neural crest origin might explain the multipotency of at
least some stem and progenitor cells in the skin. Indeed, the
neural crest contributes during vertebrate development to a va-
riety of tissues, including the peripheral nervous system and
nonneural cell types such as melanocytes in the skin (Le Douarin
and Dupin, 2003). Clonal analysis revealed that multipotent,
self-renewing neural crest stem cells (NCSCs) cannot only be
isolated from migratory neural crest but also from different tis-
sues at later stages and even from the adult organism (Stemple
and Anderson, 1992; Bixby et al., 2002; Kruger et al., 2002).
Thus, it is conceivable that apart from the whisker follicle, other
neural crest–derived compartments in the skin might contain
multipotent neural crest–derived cells.
p75/Sox10-positive neural crest–derived
cells with stem cell properties
can be isolated from the adult murine
and human skin
Floating sphere cultures have previously been used to identify
self-renewing cells in both murine and human skin (Toma et al.,
2001, 2005; Belicchi et al., 2004; Fernandes et al., 2004; Joannides
et al., 2004). To further characterize sphere-forming cells de-
rived from the trunk skin of adult mice, dorsal and ventral
skin biopsies comprising both dermis and epidermis were dis-
sociated and cultured, and formation of spheres was observed
within 4–7 d of culture. These spheres could be passaged for
several months without overt morphological changes (Fig. 1 A),
pointing to the self-renewing capacity of cells present in the
spheres. Intriguingly, unlike SKPs enriched by marker selection
(Belicchi et al., 2004) or cultured in slightly different conditions
than used here (Fernandes et al., 2004), 100% of all primary,
secondary, and later passage spheres generated from mouse
trunk skin (n > 50 spheres) contained cells expressing the low-
affi nity neurotrophin receptor p75 and the transcription factor
Sox10, both markers for NCSCs (Fig. 1, C and E; Stemple and
Anderson, 1992; Paratore et al., 2001). In spheres passaged >20
times, 67.0 ± 10.5% of all cells expressed p75, 76.6 ± 4.5% of
all cells expressed Sox10, and 58.6 ± 10.5% of all cells were
double positive for p75 and Sox10. 15.0 ± 6.2% of all cells
were negative for these markers, pointing to a cellular heteroge-
neity within skin-derived spheres, as also observed in sphere
cultures from other tissues (Reynolds and Rietze, 2005). Thus,
skin-derived cells expressing NCSC markers can be propagated
in culture for prolonged time periods.
Similarly, spheres readily formed from dissociated surgi-
cal samples of adult human thigh and face skin (Fig. 1 B). These
spheres could be expanded by passaging, such that after 3 mo
>109 cells had been generated from a 16-cm2 skin sample used
as starting material. Similar to mouse cultures, all spheres
contained p75/Sox10-positive cells, which accounted for >60%
of all cells (Fig. 1, D and F). However, other markers for pre-
migratory or migratory NCSCs, such as Sox9 and HNK-1, were
As p75 and Sox10 are markers for NCSCs (Stemple and
Anderson, 1992; Paratore et al., 2001), we next examined
whether the mouse trunk skin–derived spheres originate from
the neural crest. The fate of neural crest cells was mapped
in vivo by mating ROSA26 Cre reporter (R26R) mice, which
express β-galactosidase upon Cre-mediated recombination,
with mice expressing Cre recombinase under the control of the
Wnt1 promoter (Jiang et al., 2000; Lee et al., 2004). In Wnt1-
Cre/R26R double-transgenic mice, virtually all NCSCs express
β-galactosidase (Brault et al., 2001; Lee et al., 2004). Importantly,
despite the transient expression of Cre recombinase, the progeny
Figure 1. Skin-derived spheres contain cells that express the NCSC markers
p75 and Sox10. Spheres were generated from murine adult trunk skin
(A) and human adult thigh skin (B). Spheres passaged >22 times were al-
lowed to spread on FN-coated plates (C–F) and fi xed after 8 h. Immuno-
cytochemical analysis revealed that both murine and human skin–derived
spheres contain numerous cells expressing p75 (visualized by Alexa 488
fl uorescence, green) and Sox10 (revealed by Cy3 fl uorescence, red; E and F).
The arrow shows a double-positive cell, and the arrowhead shows a nega-
tive cell. (C and D) Corresponding DAPI staining.
on May 31, 2012
Published December 11, 2006
MULTIPOTENT NEURAL CREST–DERIVED CELLS IN THE ADULT SKIN • WONG ET AL.1007
of neural crest cells continue to express β-galactosidase because
of the genomic recombination event. Anti–β-galactosidase
antibody staining revealed that all primary and late passage
spheres generated from the back skin of adult Wnt1-Cre/R26R
double-transgenic mice were composed of neural crest–derived
cells (Fig. 2 and not depicted). In particular, 100% of all p75-
positive cells coexpressed β-galactosidase, as revealed by a
typical punctuated staining pattern (Lutolf et al., 2002).
Because 87.3 ± 6.0% of all p75-positive cells also expressed
Sox10 (three independent experiments with spheres obtained
after 20–35 passages), the data demonstrate that sphere-forming,
p75/Sox10-expressing cells from the adult mouse skin are
neural crest derivatives.
To test the developmental potential of sphere cells derived
from murine and human skin, spheres containing p75/Sox10-
positive neural crest cells were allowed to differentiate at high
cellular density. The formation of glia expressing glial fi brillary
acidic protein (GFAP), βIII tubulin (TuJ1)–positive neuronal
cells, and smooth muscle actin (SMA)–expressing nonneural
cells was readily detectable in both mouse and human cell
cultures (Fig. 3, A–C and G–I), although the number of neuronal
cells generated was highly variable and low in comparison to
that of glia and smooth muscle cells. Upon addition of ascorbic
acid and bone morphogenic protein (BMP) 2, the generation of
chondrocytes was observed (Fig. 3, D and J), whereas treatment
with stem cell factor and endothelin-3 resulted in formation of a
few melanocytes (Fig. 3, E and K). Finally, occasional adipo-
cytes were detected (Fig. 3, F and L). However, we never ob-
served the generation of keratinocytes as assessed by staining
with a pan-keratin antibody (unpublished data), demonstrating
that neural crest–derived sphere-forming cells are distinct from
epithelial stem cells of the skin.
The aforementioned data are consistent with the idea that
skin-derived spheres contain multipotent cells capable of gener-
ating neural and nonneural cell types. In analogy to NCSCs iso-
lated from other stages and locations, it is likely that this broad
potential is inherent to the p75/Sox10-expressing neural crest–
derived cells found in the spheres. To address this hypothesis,
we plated cells from mouse trunk skin–derived spheres at clonal
density and prospectively identifi ed and mapped single undif-
ferentiated, unpigmented p75-positive clone founder cells
(Stemple and Anderson, 1992; Hagedorn et al., 1999; Lee et al.,
2004; Kleber et al., 2005). The clone founder cells were then
incubated in culture conditions permissive for neurogenesis,
gliogenesis, and nonneural cell formation (Stemple and Anderson,
1992). 57.9% of all p75-positive founder cells were at least
tripotent, giving rise to clones consisting of neural and nonneu-
ral cell types (Fig. 4 A). Virtually no p75-positive cell was re-
stricted to a single cell lineage. Thus, p75/Sox10-positive neural
crest–derived cells prepared from the adult trunk skin are multi-
potent and can be expanded in culture. Upon isolation, these
cells therefore exhibit properties of NCSCs.
Several instructive growth factors, including Wnt, BMP,
neuregulin (NRG), and TGFβ, have been shown to promote
specifi c fate decisions in NCSCs at the expense of other possi-
ble fates. Although Wnt responsiveness is lost at later develop-
mental stages (Kleber et al., 2005), postmigratory NCSCs
isolated from various structures maintain their responsiveness
to BMP2, NRG1, and TGFβ, although the biological activity of
these factors changes with time and location (Bixby et al., 2002;
Figure 2. Skin-derived spheres are of neural crest origin. Neural crest–
derived cells were identifi ed by lineage tracing in spheres generated from
Wnt1-Cre/R26R adult mouse skin samples. Primary spheres (not depicted)
and spheres at passage 20 were analyzed immunocytochemically for p75
(A, green) and β-galactosidase (B, red) expression. Overlay of A and B
reveals that spheres contain cells coexpressing p75 and β-galactosidase (D).
(C) Corresponding DAPI staining. Bar, 20 μm.
Figure 3. Cells from skin-derived spheres dif-
ferentiate into neural crest cell lineages and
adipocytes. Late-passage spheres obtained
from murine and human skin were allowed to
spread at high densities on different substrates
and were incubated in media permissive for
cell differentiation. Marker expression indi-
cated the generation of GFAP-positive glia
(A and G), TuJ1-positive neuronal cells (B and H),
and SMA-positive smooth muscle cells (C and I).
Some plates (A, B, G, and H) were counter-
stained with DAPI. Cells with features of chon-
drocytes, melanocytes, and adipocytes were
identifi ed by Alcian blue staining (D and J),
DOPA reaction (E and K), or with oil red O
(F and L), respectively. Bar, 20 μm.
on May 31, 2012
Published December 11, 2006
JCB • VOLUME 175 • NUMBER 6 • 2006 1008
Kruger et al., 2002). Similarly, single prospectively identifi ed
p75-positive neural crest cells isolated from the adult back skin
were sensitive to BMP2, NRG1, and TGFβ (Fig. 4, B–D). All
three instructive growth factors suppressed multipotency with-
out affecting survival of founder cells and promoted the genera-
tion of clones containing nonneural cells that were mostly SMA
positive. However, we were unable to identify growth factors
inducing exclusively neuro- or gliogenesis in skin-derived neu-
ral crest cells, whereas NCSCs isolated from other sources give
rise to neurons and glia, respectively, in response to BMP2 and
NRG1 (Sommer, 2001; Le Douarin and Dupin, 2003). Hence,
adult skin–derived neural crest cells, although displaying NCSC
features, are intrinsically different from other types of NCSCs
and show altered factor responsiveness.
Multiple sources of sphere-forming neural
crest–derived cells in the whisker follicle
Apart from back skin–derived p75/Sox10-positive multipotent
cells (Figs. 1 and 2), the neural crest origin of sphere-forming
cells in the adult skin has been demonstrated for whisker fol-
licle–derived SKPs, which, however, are negative for the NCSC
markers p75 and Sox10 (Fernandes et al., 2004). This could
either refl ect differential regulation of NCSC markers in the
same cell type because of varying culture conditions or indi-
cate sphere-forming capacity of skin cells from different neural
crest derivatives. To address this issue, we fi rst mapped neu-
ral crest derivatives in the adult skin and investigated which of
these neural crest derivatives express the NCSC marker Sox10
in vivo. We initially focused on the whisker follicle because this
structure has been identifi ed before as a source of multipotent
neural crest–derived cells (Fernandes et al., 2004; Sieber-Blum
et al., 2004). In the head, the neural crest contributes to many
mesenchymal structures (Santagati and Rijli, 2003). Thus,
many mesenchymal structures in whisker follicles isolated
from Wnt1-Cre/R26R double-transgenic mice expressed β-
galactosidase (Fig. 5 A). In particular, the capsula, the ringwulst,
the dermal sheath, and, as previously published (Fernandes
et al., 2004; Sieber-Blum et al., 2004), the dermal papilla turned
out to be neural crest derived. The neural crest origin of all
these structures was confi rmed by fate mapping experiments
performed in human tissue plasminogen activator (Ht-PA)-Cre/
R26R mice, in which Cre recombinase is expressed in neural
crest cells independently from Wnt1 promoter activity (Pietri
et al., 2003; Fig. 5 B). As revealed by X-gal staining of whis-
ker follicles isolated from Sox10lacZ mice (that express β-galac-
tosidase from the Sox10 locus; Britsch et al., 2001), capsula,
ringwulst, and dermal papilla did not express Sox10 in vivo,
whereas the dermal sheath, glial cells in nerve endings, and
melanocytes were Sox10 positive (Fig. 5 C). Thus, the whisker
follicle comprises various Sox10-positive and -negative tissues
of neural crest origin.
To investigate which of these neural crest derivatives
contain cells with sphere-forming potential, dermal papilla, cap-
sula, the upper part of the dermal sheath (without the bulge), and
the lower part of the dermal sheath were isolated from whiskers
of adult Wnt1-Cre/R26R double-transgenic mice by microdis-
section, dissociated, and cultured in the same conditions as used
before for trunk skin–derived multipotent neural crest cells. In
addition, rat whiskers were used to dissect the ringwulst, which
in mice was too small to be isolated without contamination from
other tissues. Strikingly, all these whisker follicle structures ap-
pear to harbor cells with the capacity to generate spheres (Fig.
5 D). X-gal staining of Wnt1-Cre/R26R mouse cell cultures
confi rmed that the spheres were neural crest derived. Therefore,
Figure 4. Clonal analysis of multipotent neural crest–derived cells from
adult skin. Spheres derived from mouse skin were dissociated after pas-
sage 17, and single cells were plated at clonal density with or without the
addition of instructive growth factors. Cells were prospectively identifi ed
with p75 staining and mapped on the plate. A clone was counted posi-
tive for a given cell type when it contained at least one cell expressing
the appropriate marker (TuJ1, neuronal cells; NG2, glia; SMA, smooth
muscle). N, neuronal cells; G, glia; S, smooth muscle; O, other; death,
lost clones. (A) Culture without the addition of instructive growth factors
was permissive for the generation of heterogeneous clones. Multipotent
cells generated three or four different fates. Clones containing exclusively
smooth muscle, neuronal, or glia cells were not identifi ed. (B) Cultures
containing BMP2 generated S-only clones, S/O, and N/S/O clones.
Clone types containing glial cells were signifi cantly decreased. (C) Cul-
tures containing NRG1 also generated S/O and S-only clones. Clone
types containing neuronal cells were decreased. (D) TGFβ addition in-
creased the numbers of S/O clones and S-only clones. Each bar repre-
sents the mean ± SD of three independent experiments, counting at least
50 clones per experiment.
on May 31, 2012
Published December 11, 2006
MULTIPOTENT NEURAL CREST–DERIVED CELLS IN THE ADULT SKIN • WONG ET AL.1009
neural crest cells with sphere-forming potential are not confi ned
to a particular niche in the whisker follicle.
Glial cells as well as the melanocyte lineage
are associated with sphere-forming
p75/Sox10-positive cells in the adult
Unlike in the head, the mesenchyme in the trunk is not de-
rived from the neural crest (Santagati and Rijli, 2003), and
β-galactosidase expression in back skin of Wnt1-Cre/R26R mice
was thus restricted to a few locations (Fig. 6, A and C). The same
structures were also labeled in the back skin of Ht-PA-Cre/R26R
mice (Fig. 6, B and D). In particular, both in the anagen and telo-
gen stage, X-gal staining was found in the permanent part of the
pelage follicle, including the bulge region below the sebaceous
gland (Fig. 6, A and B; and Fig. S1, available at http://www.jcb.
org/cgi/content/full/jcb.200606062/DC1). This area comprises
the location of melanocyte stem cells (Nishimura et al., 2002)
and glial cells in nerve endings (Botchkarev et al., 1997). In addi-
tion, pigmented melanocytes in the bulb region (the lower part
of the hair follicles; Fig. 6, C and D) and nerves expressed
β-galactosidase. In contrast, other hair follicle structures such
as the dermal papilla, dermal sheath, and the outer and inner
root sheaths were X-gal negative and, in the trunk skin, do not
originate from the neural crest (Fig. 6, A–D).
To determine the potential origin of Sox10-positive
sphere-forming cells in the skin (Fig. 1), we assessed Sox10 ex-
pression by virtue of β-galactosidase activity in the back skin of
Sox10lacZ mice in vivo. Interestingly, Sox10-expressing cells
were confi ned to exactly the same areas as were X-gal–positive
cells in Wnt1-Cre/R26R and Ht-PA-Cre/R26R mice, including
nerves, melanocytes, and a domain consistently found below
the sebaceous gland in anagen and telogen stage that encom-
passes the hair follicle bulge with the niche for melanocyte stem
cells and nerve endings (Fig. S1). Importantly, in both Wnt1-
Cre/R26R and Ht-PA-Cre/R26R mice, X-gal–positive cells in
the region below the sebaceous gland coexpressed Sox10 and
p75 protein (Fig. 6, E–L). Thus, p75/Sox10-positive multipotent
neural crest–derived cells from the trunk skin (Figs. 1–4) are
connected to the glial or the melanocyte lineage or to both of
To elucidate whether p75/Sox10 expression and the ca-
pacity to form spheres are associated with glial cells from skin,
we made use of desert hedgehog (Dhh)-Cre mice that express
Cre recombinase in the peripheral glial lineage from early stages
onward, but not in migrating neural crest cells or in neural
crest–derived cells of other than glial lineages (Jaegle et al.,
2003). β-Galactosidase activity was detectable in nerves and
nerve endings in the back skin of adult Dhh-Cre/R26R mice
(Fig. 7 A). As predicted from the proposed location of glial cells
associated with nerve endings in the hair follicle (Botchkarev
et al., 1997), X-gal staining in pelage follicles of Dhh-Cre/R26R
mice was confi ned to a region around the bulge (Fig. 7 A),
corresponding to the area that also contains β-galactosidase–
expressing cells in Wnt1-Cre/R26R, Ht-PA-Cre/R26R, and
Sox10lacZ mice (Fig. 6, A and B; and Fig. S1). In Dhh-Cre/R26R
mice, X-gal–labeled cells of the bulge region were also labeled
with anti-Sox10 antibody (Fig. 7 C) and anti-p75 antibody (Fig.
7 E). Pigmented melanocytes in the hair follicle bulb were
X-gal negative, however, indicating that cells labeled in Dhh-Cre/
R26R mice do not give rise to melanocyte s and thus are not
related to the melanocyte lineage (Fig. 7 G).
Figure 5. Many mesenchymal structures of
whisker hair follicles derive from the neural
crest and harbor cells capable of generating
spheres. (A) Whisker follicles from Wnt1-
Cre/R26R mice were incubated in X-gal so-
lution to reveal neural crest–derived cells in
the dermal sheath (DS), capsula (C), dermal
papilla (DP), ringwulst (RW), nerve (N), and
regions above the sebaceous gland (SG). The
sebaceous gland itself was negative. Melano-
cytes (M) were not or only very weakly X-gal
positive, as reported previously (Fernandes
et al., 2004). (B) Whiskers dissected from
Ht-PA-Cre/R26R mice and treated with X-gal
solution. The same structures as in the Wnt1-
Cre/R26R follicles were shown to be derived
from the neural crest. (C) Whiskers dissected
from Sox10lacZ mice and incubated in X-gal
solution. Cells in which the Sox10 promoter
was active were identifi ed in dermal sheath,
nerve, and melanocytes. Capsula, dermal
papilla, ringwulst, and sebaceous gland were
negative. (D) Different neural crest–derived
structures were microdissected from Wnt1-
Cre/R26R whisker follicles (A) and cultured
in medium permissive for sphere formation.
All structures appear to contain cells with the
potential to form X-gal–positive spheres. The
ringwulst was isolated from rat whisker fol-
licles and therefore not stained with X-gal. Six
independent experiments were performed for
each structure. Bar, 50 μm.
on May 31, 2012
Published December 11, 2006
JCB • VOLUME 175 • NUMBER 6 • 2006 1010
To directly demonstrate that cells from the glial lineage
tracked by Dhh-Cre promoter activity possess sphere-forming
potential, these cells have to be prospectively identifi ed and
freshly isolated. One possibility to achieve this would be by us-
ing specifi c surface antigen markers. However, such markers for
the early glial lineage are currently unavailable. Furthermore,
nerves present in the skin cannot be isolated by microdissection.
Therefore, we used a genetic strategy to prospectively identify
and directly isolate cells associated with the glial lineage. Dhh-
Cre mice were mated with R26R-EYFP mice that express EYFP
upon Cre-mediated recombination (Srinivas et al., 2001). Cells
expressing EYFP in the trunk skin of Dhh-Cre/R26R-EYFP
double-transgenic mice were isolated by FACS and transferred
into medium permissive for sphere formation (Fig. 8, A and B).
Although from unselected skin samples >106 cells were used
to generate ?50 spheres (Fig. 1; see Materials and methods),
<10,000 cells from both the EYFP-positive and -negative cell
fraction were seeded in these experiments, to assess a possible
enrichment in the spherogenic potential of FACS-selected cells.
In two independent experiments, the EYFP-positive (Fig. 8 A,
green frame), but not the EYFP-negative (Fig. 8 A, blue frame),
cell population gave rise to spheres. Moreover, acutely fi xed
primary spheres of EYFP-positive cells were composed of cells
expressing both p75 and Sox10 (Fig. 8 C). Thus, p75/Sox10-
positive cells related to the glial lineage can be isolated from the
skin and form spheres.
We next asked whether sphere-forming potential is a
common feature of peripheral glia. Therefore, we investi-
gated whether sphere cultures can also be established from
adult peripheral nerves. In agreement with others (Toma et al.,
2001), we were unable to obtain spheres from cultures of
Figure 6. Localization of neural crest–derived and p75/Sox10-positive
cells in murine back skin. Back skin from Wnt1-Cre/R26R (A, C, E, G, I,
and K) and Ht-PA-Cre/R26R (B, D, F, H, J, and L) mice with pelage follicles
in anagen phase was stained with X-gal solution. In both transgenic mouse
models, positive cells were localized in nerves (N), in the hair follicle bulge
region below the sebaceous gland (arrows; A and B), and in the bulb con-
taining melanocytes (M; C and D). E, F, I, and J show pictures of back skin
sections stained with X-gal and either antibodies against p75 or against
Sox10 visualized by immunofl uorescence. Enlarged areas show X-gal–
positive cells in the bulge area coexpressing p75 (G and H) and Sox10
(K and L). The panels to the right represent overlays of X-gal and marker
stainings. Note that although p75/X-gal– and Sox10/X-gal–positive cells
in the bulge area point to the existence of NCSC-like cells in this location,
p75 and Sox10 are not specifi c for neural crest derivatives in the skin and
also label X-gal–negative structures (white arrows). Moreover, not all neu-
ral crest–derived cells coexpress p75 and Sox10 (arrowheads). The der-
mal papilla (DP) was X-gal negative in both mouse lines. Bars: (A, C, E,
and I) 20 μm; (G) 10 μm.
Figure 7. Lineage tracing of cells from the glial or the melanocyte lineage
in murine back skin. Back skin from Dhh-Cre/R26R (A and G) and Dct-
Cre/R26R (B and H) mice was stained with X-gal solution to identify pro-
genitors and differentiated cells from the glial and melanocyte lineage,
respectively, by lineage tracing. (A) X-gal–expressing cells on sections from
Dhh-Cre/R26R mice were localized inside hair follicles in the bulge region
below the sebaceous gland (arrow) and in glial cells (N) outside hair
follicles. (G) Melanocytes (M) in the hair follicle bulb were not stained.
(B) X-gal–expressing cells on Dct-Cre/R26R sections were localized inside
hair follicles in the bulge region (arrow). (H) Melanocytes also stained for
X-gal in Dct-Cre/R26R skin. Enlarged areas show X-gal–positive cells in the
bulge area costained for Sox10 (C and D) and p75 (E and F). Bars: (A and G)
20 μm; (C) 10 μm.
on May 31, 2012
Published December 11, 2006
MULTIPOTENT NEURAL CREST–DERIVED CELLS IN THE ADULT SKIN • WONG ET AL.1011
dissociated sciatic and trigeminal nerves from adult mice
(unpublished data). Thus, nerves or nerve endings in skin, but
not peripheral nerves in general, contain cells with sphere-
In cell preparations from the trunk skin of Dhh-Cre/R26R
mice, only a fraction of all p75/Sox10-positive cells also ex-
pressed β-galactosidase (unpublished data). This could point to
ineffi cient Cre-mediated recombination in Dhh-Cre/R26R mice.
Alternatively, sources in the skin other than the glial lineage
might yield sphere-forming neural crest–related cells. To ad-
dress whether spherogenic neural crest–derived cells might be
connected to the melanocyte lineage, we traced the fate of trunk
skin cells in Dct-Cre/R26R mice (Guyonneau et al., 2002). Dct
codes for the enzyme dopachrome tautomerase (also called Trp-2),
which is required for melanin synthesis and already expressed
in melanocyte stem cells (Nishimura et al., 2002). As expected,
β-galactosidase activity in the back skin of Dct-Cre/R26R mice
was detectable in melanocytes (Fig. 7 H) and in the hair follicle
bulge region corresponding to the location of melanocyte stem
cells (Fig. 7 B; Nishimura et al., 2002). Moreover, some X-gal–
positive cells in the bulge region also expressed Sox10 (Fig.
7 D) and p75 (Fig. 7 F).
To investigate whether, in addition to cells of the glial lin-
eage, the early melanocyte lineage also comprises undifferenti-
ated neural crest–derived cells with the capacity to generate
spheres, we isolated EYFP-expressing cells prospectively iden-
tifi ed in the skin of Dct-Cre/R26R-EYFP mice. Intriguingly, in
two independent experiments, FACS isolation and culturing of
<10,000 cells revealed that only EYFP-expressing (Fig. 8 E,
green frame), but not EYFP-negative, cells (Fig. 8 E, blue
frame) were able to form spheres (Fig. 8 H). Analysis of acutely
fi xed primary spheres revealed many cells coexpressing p75
and Sox10, whereas pigmented differentiated melanocytes were
absent (Fig. 8 G). These data indicate that the early melanocyte
lineage comprises p75/Sox10-positive cells that can be propa-
gated as spheres. Thus, as in whisker follicles of the face, the
trunk skin contains more than one source of sphere-forming
neural crest–derived cells, namely, cells of the glial and melano-
In the present study, we show that cells with NCSC features
can be isolated from the adult trunk skin of both mouse and
human. Like NCSCs from other embryonic and postnatal sources,
these neural crest–derived cells in the skin express p75 and
Sox10 and are multipotent, able to generate several neural and
nonneural lineages. Moreover, multipotent neural crest– derived
cells from the adult skin display a self-renewing capacity, in
that mouse and human skin–derived cells can be grown and
expanded for months in fl oating sphere cultures. Intriguingly,
in whisker follicles of facial skin, several structures of neural
crest origin appear to comprise cells with sphere-forming
capacity. In the trunk skin, however, genetic cell fate mapping,
p75/Sox10 expression analysis in vivo, and, importantly, pro-
spective identifi cation and direct isolation demonstrate that
cells displaying NCSC properties do not reside in mesenchymal
structures of hair follicles but, rather, are associated with
the melanocyte and glial lineages. Thus, self-renewing neural
crest–derived cells from the skin are not confi ned to a particu-
lar niche but can be attributed to distinct locations in face and
Sphere-forming neural crest–derived
cells reside in distinct structures
of the adult skin
Several reports have described the isolation of multipotent cells
from murine and human skin (Belicchi et al., 2004; Dyce et al.,
2004; Joannides et al., 2004; Sieber-Blum et al., 2004; Amoh
et al., 2005; Shih et al., 2005; Toma et al., 2005), raising the ques-
tion about the origin of the endogenous cells able to self-renew
and to generate multiple cell lineages, including cell types nor-
mally not found in the skin. Multipotent cells expressing GFP
under the control of a Nestin regulatory element have been iso-
lated from the hair follicle bulge area of transgenic mice and
reported to undergo neurogenesis in vivo upon transplantation
into the murine subcutis (Amoh et al., 2005). In these mice,
Nestin-GFP–expressing cells are associated with the outer root
Figure 8. Prospective identifi cation and direct isolation of sphere-forming
cells from glial and melanocyte lineages. Sorting of cells from the skin of
Dhh-Cre/R26R-EYFP (A) and Dct-Cre/R26R-EYFP mice (E) by FACS. Green
and blue regions indicate EYFP-positive and -negative cells, respectively.
Mice not carrying the corresponding Cre were used as negative controls
for EYFP fl uorescence (B and F). Primary spheres derived from EYFP-
positive cells sorted from Dhh-Cre/R26R-EYFP (C) or Dct-Cre/R26R-EYFP
(G) trunk skin contain cells positive for both Sox10 (red) and p75 (green).
(D and H) Quantifi cation (two independent experiments) of the number of
spheres formed from EYFP-positive and -negative cells sorted from Dhh-
Cre/R26R-EYFP and Dct-Cre/R26R-EYFP skin. Bars, 20 μm.
on May 31, 2012
Published December 11, 2006
JCB • VOLUME 175 • NUMBER 6 • 2006 1012
sheath (Li et al., 2003), which, however, does not originate from
the neural crest (Figs. 5 and 6). The nature of Nestin-expressing
cells in the skin remains to be determined, though, as Nestin-
GFP in another transgenic mouse line (Kawaguchi et al., 2001)
marks the inner but not outer root sheath (unpublished data).
Moreover, Nestin may be widely expressed in multiple struc-
tures of human skin (Wang et al., 2006).
The dermal papilla from whisker follicles has been re-
ported to be of neural crest origin and to harbor sphere-forming
SKPs that are p75/Sox10 negative (Fernandes et al., 2004).
However, many structures of the whisker follicle turned out to
be neural crest derived, including the dermal sheath, the ring-
wulst, and the capsula, apart from the dermal papilla (Fig. 5).
Intriguingly, upon microdissection, all these tissues appeared to
have cells with sphere-forming potential, although we cannot
formally exclude possible contamination of the microdissected
material by other structures. Thus, the capacity to self-renew
appears to be a widespread feature of neural crest–derived cells
in the adult skin of the face. Moreover, at least some of these
self-renewing cells are also multipotent, as both SKP spheres
from whisker pads and explant cultures of the upper part of
whisker follicles with dermal sheath and the bulge region con-
tain multipotent neural crest–derived cells (Fernandes et al.,
2004; Sieber-Blum et al., 2004).
It has been proposed that, similar to SKPs from face skin,
p75-negative cells isolated from the mouse back skin or the hu-
man foreskin are also neural crest derived, although this has not
been addressed yet (Fernandes et al., 2004; Toma et al., 2005).
Most likely, however, these latter sphere-forming cells are not
associated with the dermal papilla of hair follicles, because the
human foreskin is devoid of hair follicles. Moreover, using
in vivo cell fate mapping, we demonstrate that, unlike in whisker
follicles of the face, only few structures of pelage follicles in
the trunk skin are actually neural crest derived. Dermal papilla,
dermal sheath, and other supportive structures do not appear to
be neural crest derivatives (Figs. 5 and 6). This is consistent
with studies in chicken and mouse, which showed that mesen-
chymal tissues in the trunk are not neural crest derived (Santagati
and Rijli, 2003). These differences in facial versus trunk neural
crest contribution might also be relevant for the study of hair
follicle development, given that whisker follicles are a widely
used model system to investigate mechanisms regulating follic-
ular cell fates (Alonso and Fuchs, 2003; Gambardella and
Skin structures harboring multipotent neural crest–derived
cells are presumably marked by p75 and Sox10 expression
in vivo, given that cells displaying NCSC properties after isolation
from the adult trunk skin express these markers. Indeed, in
Wnt1-Cre/R26R, Ht-PA-Cre/R26R, Dhh-Cre/R26R, and Dct-
Cre/R26R mice, β-galactosidase–expressing cells positive for
p75 and Sox10 were found in the bulge region encompassing
melanocyte stem cells and glial cells in nerve endings (Botchkarev
et al., 1997; Nishimura et al., 2002; Figs. 6 and 7). However,
the only way to unambiguously demonstrate that these lineages
contain resident multipotent cells with the potential to self-
renew is by prospectively identifying such cells in the adult
skin and testing their potential upon acute isolation. Because of
the lack of specifi c surface markers, we were not able to use anti-
bodies to directly isolate multipotent cells from back skin. In
particular, p75 expression does not distinguish between glial
and melanocyte lineages (Fig. 7) and, in addition, is found in
regions of the skin that are not neural crest derived, such as the
outer root sheath of hair follicles, and, at early stages of hair
follicle morphogenesis, the dermal papilla (Fig. 6; Botchkareva
et al., 1999; Rendl et al., 2005). Similarly, the melanocyte
marker c-Kit is not suitable for isolation of prospective multipo-
tent cells from the melanocyte lineage because it is not expressed
in the bulge of anagen hair follicles and because in vivo it is
also found in epithelial skin cells not originating from the neu-
ral crest (Peters et al., 2002, 2003). Moreover, in preliminary
experiments, we failed to obtain p75/Sox10-positive spheres
from c-Kit–positive skin cells isolated by FACS (unpublished
data). Nonetheless, we were able to identify spherogenic neural
crest–derived cells in the skin by using a genetic approach.
Thereby, the lineage-specifi c activity of Dhh-Cre and of Dct-
Cre, respectively, combined with a Cre reporter allele (Srinivas
et al., 2001), led to EYFP expression in cells from either the
glial or the melanocyte lineage. Consistent with a dual origin
of multipotent neural crest–derived cells in the trunk skin,
EYFP-positive cells isolated from both Dhh-Cre/R26R-EYFP
and Dct-Cre/R26R-EYFP mice formed spheres of p75/Sox10-
positive cells (Fig. 8). Thus, we conclude that in the trunk
skin of mice, spherogenic neural crest–derived cells are asso-
ciated with the glial as well as the melanocyte lineage. Based
on their high similarities to mouse cells in terms of marker
expression and potential, and based on the fact that humans
do not have whisker follicles, we assume that the p75/Sox10-
positive multipotent cells obtained from human skin (Figs. 1
and 3) are also related to glial and melanocyte rather than
That cells of the bulge region marked in Dct-Cre/R26R
mice generate melanocytes is in agreement with earlier fi ndings
(Nishimura et al., 2002), which identifi ed DctlacZ-positive cells
of the bulge region as so-called melanocyte stem cells generat-
ing differentiated melanocytes in the lower part of the hair folli-
cle. Moreover, a considerable fraction of Dct-positive cells in
the bulge also expresses Sox10 (Osawa et al., 2005), in agree-
ment with our results (Fig. 7). Our analysis of neural crest–
derived cells isolated from Dct-Cre/R26R mice supports the
hypothesis that melanocyte progenitors in the bulge region are
not only self-renewing (Nishimura et al., 2002) but indeed rep-
resent multipotent cells (Sommer, 2005).
The combined data indicate that the adult skin is host
to sphere-forming cells with different identities. In addition
to multipotent, undifferentiated neural crest–related cells
(Fernandes et al., 2004; this study), the potential to self-renew has
also been attributed to pigment cells and possibly other devel-
opmentally restricted neural crest–derived cell types (Dupin
et al., 2000; Trentin et al., 2004). Similar to NCSCs isolated at
different time points and from different peripheral nervous sys-
tem regions (Kleber and Sommer, 2004), the multipotent neural
crest–derived skin cells described in this study display altered
responsiveness to instructive growth factors as compared with
migratory NCSCs (Fig. 4). Thus, multipotent, self-renewing
on May 31, 2012
Published December 11, 2006
MULTIPOTENT NEURAL CREST–DERIVED CELLS IN THE ADULT SKIN • WONG ET AL.1013
neural crest–derived cells change intrinsic properties with time
and location. This presumably refl ects their specifi c functional
requirements, although the physiological role of multipotent
neural crest–derived cells in the skin remains to be determined.
In particular, it is unclear whether these cells display properties
of NCSCs only upon transfer into culture or whether they are
multifated and self-renewing in vivo as well. Moreover, it will
be interesting to elucidate whether and how these cells func-
tionally interact with other, unrelated stem cell types of the skin,
such as epithelial stem cells also found in the bulge region of
hair follicles (Taylor et al., 2000; Oshima et al., 2001; Blanpain
et al., 2004; Claudinot et al., 2005). Finally, it should be ad-
dressed whether spherogenic neural crest–derived cells persist-
ing in the adult skin might represent a target for mutational
transformation leading to cancers such as melanoma (Pardal
et al., 2003).
Limited developmental and therapeutic
potential of multipotent neural crest–derived
cells from the adult skin
Adult stem cells as an alternative to embryonic stem cells are a
prime target of applied research that seeks to treat degenerative
diseases by cell replacement therapies (Wagers and Weissman,
2004). The skin might represent an ideal source for adult stem
cells in tissue repair because it is easily accessible. Because
skin-derived multipotent cells described here are of neural crest
origin, their capacity to give rise to neural and nonneural cell
types at clonal density (Fig. 4) does apparently not refl ect trans-
differentiation but, rather, the broad potential inherent to NCSCs.
The fact that these cells can be easily expanded in culture, even
when isolated from the skin of adult humans, might make them
a valuable source for cell replacement therapies because suf-
fi cient cell material could be obtained for such purposes. However,
in our hands, spherogenic neural crest–derived cells from the
adult mouse skin displayed a rather restricted potential in vivo
(unpublished data). In particular, skin-derived spheres of neu-
ral crest origin, when dissociated and injected into the lateral
ventricles of rat and chicken embryos or transplanted onto
hippocampal brain slices, remained largely undifferentiated in
cell aggregates close to the injection site and failed to integrate
into the host central nervous system (CNS) tissue (unpublished
data). This is in contrast to neural progenitors obtained from
embryonic stem cells or neural stem cells from the CNS assessed
in the same experimental paradigms (Brüstle et al., 1997; Benninger
et al., 2003; Wernig et al., 2004). Moreover, we did not observe
neural differentiation, tissue integration, or behavioral improve-
ment upon transplantation of skin-derived multipotent neural
crest–derived cells into the striatum of a 6-hydroxydopamine–
lesioned mouse model for Parkinson’s disease (Bjorklund et al.,
2002; unpublished data), or upon intravenous injection into mice
with experimental autoimmune encephalomyelitis, a model for
multiple sclerosis previously used to test the therapeutic poten-
tial of adult neural stem cells from the CNS (Pluchino et al.,
2003; unpublished data). These trials suggest that multipotent
cells of neural crest origin that are present in the adult skin can-
not transdifferentiate into neural cell types of the CNS. Likewise,
SKPs failed to generate CNS neurons upon transplantation
into hippocampal slices (Fernandes et al., 2006). This does not
exclude the possibility that adult neural crest–related cells with
stem cell properties might be of high value for the generation of
Schwann cells, cartilage, or other neural crest–derived tissues
potentially useful in clinical applications. Hence, our study
underlines the importance of choosing the appropriate stem
cell type for a given task.
Materials and methods
Skin sphere cultures
Human thigh skin from an adult man (?45 yr of age) and face skin from
an adult woman (?57 yr of age; provided by G. Beer, University Hospital
of Zurich, Zurich, Switzerland) were obtained in the frame of cosmetic
surgery according to the guidelines of the University Hospital of Zurich.
Murine skin was taken from adult C57/BL6 mice of at least 8 wk of age. Skin
samples (composed of both dermis and epidermis) were dissected, cut into
small pieces, and digested in 0.1% Trypsin-EDTA (Invitrogen) in HBSS with-
out Ca2+ and Mg2+ (Animed) and digested for 50 min at 37°C. Partially
digested skin pieces were dissociated mechanically and fi ltered through a
40-μm cell strainer (BD Biosciences). The cell suspension was centrifuged
and washed with medium, and the cell pellet was resuspended in growth
medium (GM) consisting of DME-F12 1:1 containing 1× B-27 supplement
(Invitrogen), 20 ng/ml FGF2 (PeproTech), 10 ng/ml EGF (PeproTech),
penicillin/streptomycin (P/S), and Fungizone. GM for human cells also con-
tained 10 ng/ml leukemia inhibitory factor (Sigma-Aldrich). 2.5–4 million
cells were seeded in GM into an uncoated T-25 cell culture fl ask (BD Bio-
sciences). After 4–7 d in culture, sphere formation was observed. For FACS
analysis, skin was taken from mice between 10 and 16 d of age. Samples
were incubated in 0.5 mg/ml Dispase (Roche) in HBSS for 30 min at 4°C.
Fat tissue was removed with forceps, and the rest of the skin was cut into
small pieces and digested in 1 mg/ml collagenase (Worthington) in HBSS
for 45 min at 37°C. After a fi nal digestion with 0.1% Trypsin-EDTA in HBSS
for 5 min at 37°C, the partially digested skin pieces were dissociated me-
chanically and treated as described above. FACS was performed with a
FACS Aria (Becton Dickinson).
Skin sphere passaging
Once a week, the sphere suspension was transferred into a 15-ml Falcon
tube. Cells adhering to the fl ask bottom were discarded. Spheres were
centrifuged, and one third of the supernatant was transferred as condi-
tioned medium into a new T-25 fl ask. Spheres were incubated with 300 μl
Trypsin-EDTA solution (0.25%) for 3–5 min at RT. 400 μl of Ovomucoid
solution (1 mg/ml Trypsin inhibitor [Sigma-Aldrich] and 10 mg DNase
[Roche] in 25 ml medium) were added, and spheres were dissociated me-
chanically, centrifuged, resuspended in fresh GM, and seeded into a new
fl ask containing one third conditioned medium. After some passages,
spheres were cultured in fl asks coated with Poly(2-hydroxyethylmethacrylate)
(Poly-Hema; Sigma-Aldrich). Coating was performed at RT with a solution
of 16 mg/ml Poly-Hema in 95% ethanol.
Microdissection and sphere cultures of whisker follicles
Anagen-phase whisker follicles from mouse and rat were dissected out of
the whisker pad and microdissected as described previously (Kobayashi
et al., 1993). Follicle structures were incubated in 0.05% Trypsin-EDTA in
DME for 1.5–2 h at 37°C. Trypsin activity was stopped with DME contain-
ing 10% FCS. After two washing steps with GM, cells of each structure
were plated in a well of a 24-well dish. Sphere formation was observed
within 1–2 wk. Passaging was performed as described for skin spheres
using 0.05% Trypsin-EDTA for 2 min at RT.
All differentiation assays were performed using spheres plated on dishes
coated with fi bronectin (FN) or poly-D-lysine/FN as described previously
(Stemple and Anderson, 1992). Neurogenesis, gliogenesis, and smooth
muscle formation were observed after 3–7 d in GM. Chondrocyte forma-
tion was obtained after 9 d in DME containing 10% FCS, 50 μg/ml ascor-
bic acid 2-phosphate (Sigma-Aldrich), 10 ng/ml FGF2, and P/S, followed
by DME containing 10% FCS, 50 μg/ml ascorbic acid 2-phosphate, and
10 ng/ml BMP2 (PeproTech) for another 3 d. Adipocytes were occasionally
observed when spheres were cultured in DME-F12 containing B-27 supple-
ment and 10 ng/ml BMP2. Melanocytes were observed when cultured in
on May 31, 2012
Published December 11, 2006
JCB • VOLUME 175 • NUMBER 6 • 2006 1014
MEM containing 10% FCS, 50 ng/ml murine stem cell factor (PeproTech),
100 nM endothelin-3 (Sigma-Aldrich), and P/S for at least 10 d.
Clonal analysis of neural crest–derived stem cells
Murine skin–derived spheres passaged >17 times were dissociated with
Trypsin-EDTA as described and plated at clonal density on pDL/FN-coated
35-mm dishes (Corning) in standard medium prepared as reported previ-
ously (Stemple and Anderson, 1992). Single p75-positive cells were
labeled and mapped as described previously (Hagedorn et al., 1999) and
incubated in standard medium alone or supplemented with 100 ng/ml
BMP2, 1 nM NRG1 (R&D Systems), or 0.1 ng/ml TGFβ (R&D Systems).
After 10 d, the cells were fi xed and analyzed immunocytochemically.
Immunofl uorescence on cells and tissue sections
Anti-p75, anti-Sox10, anti-SMA, and anti-GFAP antibody stainings on cells
were done as described by Kleber et al. (2005). Anti-TuJ1 antibody
(1:200; Sigma-Aldrich) and anti-Keratin antibody (1:500; Abcam) were
used for 2 h at RT, whereas anti–β-galactosidase (1:100; Roche) and anti-
NG2 (1:200; Chemicon) antibodies were used with incubation overnight
at 4°C. The following secondary antibodies were used for 1 h at RT: Cy3-
conjugated goat anti-mouse (1:200; Jackson ImmunoResearch Laborato-
ries), Cy3-conjugated goat anti-rabbit (1:200; Jackson ImmunoResearch
Laboratories), Alexa 488–conjugated goat anti-mouse (1:100; Invitrogen),
and Alexa 488–conjugated goat anti-rabbit (1:100; Invitrogen). Cell nu-
clei were stained with DAPI. Paraffi n sections of X-gal–treated skin were
stained for Sox10 as described previously (Dutt et al., 2006) using a con-
trolled antigen-retrieval device (FSG 120-T/T; Milestone). Heat unmasking
for the p75 staining was done in 10 mM trisodium citrate, pH 6.0, using
the same antigen-retrieval device. The antibody (Chemicon) was used at a
dilution of 1:5,000. Alexa 594–conjugated goat anti-mouse and Alexa
488–conjugated goat anti-rabbit (1:200; Invitrogen) were used as second-
ary antibodies. Immunofl uorescence of cells was analyzed using a micro-
scope (Axiovert 100; Carl Zeiss MicroImaging, Inc.) and magnifi cations of
32×. Pictures were made with a camera (AxioCam MRm) and Axiovision
4.3 software (Carl Zeiss MicroImaging, Inc.). Immunofl uorescence of sec-
tions was analyzed using a microscope (Axioskop 2; Carl Zeiss Micro-
Imaging, Inc.) with 20×, 63×, and 100× magnifi cations. Pictures were
made with a camera (AxioCam HRc) and Axiovision 4.2 software.
For Alcian blue staining, cells fi xed with 4% formaldehyde were incubated
with a 3% solution of glacial acetic acid in distilled water for 3 min at RT
followed by a 1% Alcian blue solution (Chroma Gesellschaft) in 3% acetic
acid for 5 min at RT. For oil red O staining, fi xed cells were incubated in
60% isopropanol in water for 15 min at RT followed by another 15 min in
an oil red O mixture (0.35 g oil red O [Sigma-Aldrich] in 50 ml isopropa-
nol and water in a 3:2 dilution). For DOPA reaction, cells were incubated
in a 0.1% solution of 3-(3,4-Dihydroxyphenyl)-L-alanine (L-DOPA) in PBS for
5 h at 37°C.
Skin pieces from 12-d-old mice for anagen or 8-wk-old mice for telogen
stage and single whiskers from Wnt1-Cre/R26R, Ht-PA-Cre/R26R, and
Sox10lacZ mice were fi xed and incubated in X-gal solution (Paratore et al.,
2002). Samples were washed three times with PBS, incubated for 24 h at
RT in Bouin’s fi xative, washed twice for 15 min in H2O, and dehydrated in
80%, 95%, and three times 100% ethanol. Samples were transferred into
a 1:1 mixture of 100% ethanol and glycomethacrylate (Leica), left for infi l-
tration at 4°C for 1–2 wk, and embedded in glycomethacrylate resin.
4–5-μm sections were cut on a microtome using a glass knife. Counter-
stainings were performed with Fast red (Merck) for 30 min at 60°C.
Paraffi n sections
Skin from 12-d-old mice from Dhh-Cre/R26R and Dct-Cre/R26R mice was
stained with X-gal, embedded in paraffi n, and sectioned as described
previously (Wurdak et al., 2005).
Online supplemental material
Fig. S1 shows localization of neural crest–derived cells expressing p75/
Sox10 in the adult skin. Online supplemental material is available at
We thank Ned Mantei for critical reading of the manuscript; Andrew
McMahon, Philippe Soriano, Michael Wegner, and Frank Costantini for provid-
ing transgenic animals; and Michael Wegner for the anti-Sox10 antibody.
We thank Gertrude Beer for providing human skin samples, Liliane Schnell
for excellent technical assistance, and Eva Niederer and the Zentrallabor für
Zellsortierung ETH/UNI for support with the FACS. We are grateful to Oliver
Brüstle, Gudrun Gossrau, Alberto Perez-Bouza, Patrick Bargsten, Burkhard
Becher, Esther Stoeckli, and Boris Ferger for their support with in vivo trans-
This work was supported by the Swiss National Science Foundation
(grant PP00A-108344), the National Center of Competence in Research on
Neural Plasticity and Repair, the Neuroscience Center Zurich, the Bonizzi-
Theler Foundation, the Ecole Polytechnique Fédérale de Lausanne, and the
Lausanne University Hospital.
Submitted: 12 June 2006
Accepted: 9 November 2006
Alonso, L., and E. Fuchs. 2003. Stem cells of the skin epithelium. Proc. Natl.
Acad. Sci. USA. 100(Suppl. 1):11830–11835.
Amoh, Y., L. Li, K. Katsuoka, S. Penman, and R.M. Hoffman. 2005. Multipotent
nestin-positive, keratin-negative hair-follicle bulge stem cells can form
neurons. Proc. Natl. Acad. Sci. USA. 102:5530–5534.
Belicchi, M., F. Pisati, R. Lopa, L. Porretti, F. Fortunato, M. Sironi, M.
Scalamogna, E.A. Parati, N. Bresolin, and Y. Torrente. 2004. Human
skin-derived stem cells migrate throughout forebrain and differentiate
into astrocytes after injection into adult mouse brain. J. Neurosci. Res.
Benninger, F., H. Beck, M. Wernig, K.L. Tucker, O. Brüstle, and B. Scheffl er.
2003. Functional integration of embryonic stem cell-derived neurons in
hippocampal slice cultures. J. Neurosci. 23:7075–7083.
Bixby, S., G.M. Kruger, J.T. Mosher, N.M. Joseph, and S.J. Morrison. 2002.
Cell-intrinsic differences between stem cells from different regions of
the peripheral nervous system regulate the generation of neural diversity.
Bjorklund, L.M., R. Sanchez-Pernaute, S. Chung, T. Andersson, I.Y. Chen, K.S.
McNaught, A.L. Brownell, B.G. Jenkins, C. Wahlestedt, K.S. Kim, and
O. Isacson. 2002. Embryonic stem cells develop into functional dopami-
nergic neurons after transplantation in a Parkinson rat model. Proc. Natl.
Acad. Sci. USA. 99:2344–2349.
Blanpain, C., W.E. Lowry, A. Geoghegan, L. Polak, and E. Fuchs. 2004. Self-
renewal, multipotency, and the existence of two cell populations within
an epithelial stem cell niche. Cell. 118:635–648.
Botchkarev, V.A., S. Eichmuller, O. Johansson, and R. Paus. 1997. Hair cycle-
dependent plasticity of skin and hair follicle innervation in normal murine
skin. J. Comp. Neurol. 386:379–395.
Botchkareva, N.V., V.A. Botchkarev, L.H. Chen, G. Lindner, and R. Paus.
1999. A role for p75 neurotrophin receptor in the control of hair follicle
morphogenesis. Dev. Biol. 216:135–153.
Brault, V., R. Moore, S. Kutsch, M. Ishibashi, D.H. Rowitch, A.P. McMahon,
L. Sommer, O. Boussida, and R. Kemler. 2001. Inactivation of the β-
catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain
malformation and failure of craniofacial development. Development.
Britsch, S., D.E. Goerich, D. Riethmacher, R.I. Peirano, M. Rossner, K.A. Nave,
C. Birchmeier, and M. Wegner. 2001. The transcription factor Sox10 is a
key regulator of peripheral glial development. Genes Dev. 15:66–78.
Brüstle, O., A.C. Spiro, K. Karram, K. Choudhary, S. Okabe, and R.D. McKay.
1997. In vitro-generated neural precursors participate in mammalian
brain development. Proc. Natl. Acad. Sci. USA. 94:14809–14814.
Claudinot, S., M. Nicolas, H. Oshima, A. Rochat, and Y. Barrandon. 2005. Long-
term renewal of hair follicles from clonogenic multipotent stem cells.
Proc. Natl. Acad. Sci. USA. 102:14677–14682.
Dupin, E., C. Glavieux, P. Vaigot, and N.M. Le Douarin. 2000. Endothelin 3 in-
duces the reversion of melanocytes to glia through a neural crest-derived
glial-melanocytic progenitor. Proc. Natl. Acad. Sci. USA. 97:7882–7887.
Dutt, S., M. Kleber, M. Matasci, L. Sommer, and D.R. Zimmermann. 2006.
Versican V0 and V1 guide migratory neural crest cells. J. Biol. Chem.
Dyce, P.W., H. Zhu, J. Craig, and J. Li. 2004. Stem cells with multilineage
potential derived from porcine skin. Biochem. Biophys. Res. Commun.
Fernandes, K.J., I.A. McKenzie, P. Mill, K.M. Smith, M. Akhavan, F. Barnabe-
Heider, J. Biernaskie, A. Junek, N.R. Kobayashi, J.G. Toma, et al. 2004.
A dermal niche for multipotent adult skin-derived precursor cells.
Nat. Cell Biol. 6:1082–1093.
on May 31, 2012
Published December 11, 2006
MULTIPOTENT NEURAL CREST–DERIVED CELLS IN THE ADULT SKIN • WONG ET AL.1015
Fernandes, K.J., N.R. Kobayashi, C.J. Gallagher, F. Barnabe-Heider, A. Aumont,
D.R. Kaplan, and F.D. Miller. 2006. Analysis of the neurogenic potential
of multipotent skin-derived precursors. Exp. Neurol. 201:32–48.
Gambardella, L., and Y. Barrandon. 2003. The multifaceted adult epidermal stem
cell. Curr. Opin. Cell Biol. 15:771–777.
Guyonneau, L., A. Rossier, C. Richard, E. Hummler, and F. Beermann. 2002.
Expression of Cre recombinase in pigment cells. Pigment Cell Res.
Hagedorn, L., U. Suter, and L. Sommer. 1999. P0 and PMP22 mark a multipotent
neural crest-derived cell type that displays community effects in response
to TGF-β family factors. Development. 126:3781–3794.
Jaegle, M., M. Ghazvini, W. Mandemakers, M. Piirsoo, S. Driegen, F.
Levavasseur, S. Raghoenath, F. Grosveld, and D. Meijer. 2003. The POU
proteins Brn-2 and Oct-6 share important functions in Schwann cell
development. Genes Dev. 17:1380–1391.
Jiang, X., D.H. Rowitch, P. Soriano, A.P. McMahon, and H.M. Sucov. 2000. Fate
of the mammalian cardiac neural crest. Development. 127:1607–1616.
Joannides, A., P. Gaughwin, C. Schwiening, H. Majed, J. Sterling, A. Compston,
and S. Chandran. 2004. Effi cient generation of neural precursors from
adult human skin: astrocytes promote neurogenesis from skin-derived
stem cells. Lancet. 364:172–178.
Kawaguchi, A., T. Miyata, K. Sawamoto, N. Takashita, A. Murayama, W.
Akamatsu, M. Ogawa, M. Okabe, Y. Tano, S.A. Goldman, and H. Okano.
2001. Nestin-EGFP transgenic mice: visualization of the self-renewal and
multipotency of CNS stem cells. Mol. Cell. Neurosci. 17:259–273.
Kleber, M., and L. Sommer. 2004. Wnt signaling and the regulation of stem cell
function. Curr. Opin. Cell Biol. 16:681–687.
Kleber, M., H.Y. Lee, H. Wurdak, J. Buchstaller, M.M. Riccomagno, L.M. Ittner,
U. Suter, D.J. Epstein, and L. Sommer. 2005. Neural crest stem cell mainte-
nance by combinatorial Wnt and BMP signaling. J. Cell Biol. 169:309–320.
Kobayashi, K., A. Rochat, and Y. Barrandon. 1993. Segregation of keratinocyte
colony-forming cells in the bulge of the rat vibrissa. Proc. Natl. Acad. Sci.
Kruger, G.M., J.T. Mosher, S. Bixby, N. Joseph, T. Iwashita, and S.J. Morrison.
2002. Neural crest stem cells persist in the adult gut but undergo changes
in self-renewal, neuronal subtype potential, and factor responsiveness.
Le Douarin, N.M., and E. Dupin. 2003. Multipotentiality of the neural crest.
Curr. Opin. Genet. Dev. 13:529–536.
Lee, H.Y., M. Kleber, L. Hari, V. Brault, U. Suter, M.M. Taketo, R. Kemler,
and L. Sommer. 2004. Instructive role of Wnt/beta-catenin in sensory fate
specifi cation in neural crest stem cells. Science. 303:1020–1023.
Li, L., J. Mignone, M. Yang, M. Matic, S. Penman, G. Enikolopov, and R.M.
Hoffman. 2003. Nestin expression in hair follicle sheath progenitor cells.
Proc. Natl. Acad. Sci. USA. 100:9958–9961.
Lutolf, S., F. Radtke, M. Aguet, U. Suter, and V. Taylor. 2002. Notch1 is required
for neuronal and glial differentiation in the cerebellum. Development.
Nishimura, E.K., S.A. Jordan, H. Oshima, H. Yoshida, M. Osawa, M. Moriyama,
I.J. Jackson, Y. Barrandon, Y. Miyachi, and S. Nishikawa. 2002. Dominant
role of the niche in melanocyte stem-cell fate determination. Nature.
Osawa, M., G. Egawa, S.S. Mak, M. Moriyama, R. Freter, S. Yonetani, F.
Beermann, and S. Nishikawa. 2005. Molecular characterization of mela-
nocyte stem cells in their niche. Development. 132:5589–5599.
Oshima, H., A. Rochat, C. Kedzia, K. Kobayashi, and Y. Barrandon. 2001.
Morphogenesis and renewal of hair follicles from adult multipotent stem
cells. Cell. 104:233–245.
Paratore, C., D.E. Goerich, U. Suter, M. Wegner, and L. Sommer. 2001. Survival
and glial fate acquisition of neural crest cells are regulated by an inter-
play between the transcription factor Sox10 and extrinsic combinatorial
signaling. Development. 128:3949–3961.
Paratore, C., C. Eichenberger, U. Suter, and L. Sommer. 2002. Sox10 haploin-
suffi ciency affects maintenance of progenitor cells in a mouse model of
Hirschsprung disease. Hum. Mol. Genet. 11:3075–3085.
Pardal, R., M.F. Clarke, and S.J. Morrison. 2003. Applying the principles of
stem-cell biology to cancer. Nat. Rev. Cancer. 3:895–902.
Peters, E.M., D.J. Tobin, N. Botchkareva, M. Maurer, and R. Paus. 2002. Migration
of melanoblasts into the developing murine hair follicle is accompanied by
transient c-Kit expression. J. Histochem. Cytochem. 50:751–766.
Peters, E.M., M. Maurer, V.A. Botchkarev, K. Jensen, P. Welker, G.A. Scott,
and R. Paus. 2003. Kit is expressed by epithelial cells in vivo. J. Invest.
Pietri, T., O. Eder, M. Blanche, J.P. Thiery, and S. Dufour. 2003. The human tissue
plasminogen activator-Cre mouse: a new tool for targeting specifi cally
neural crest cells and their derivatives in vivo. Dev. Biol. 259:176–187.
Pluchino, S., A. Quattrini, E. Brambilla, A. Gritti, G. Salani, G. Dina, R. Galli,
U. Del Carro, S. Amadio, A. Bergami, et al. 2003. Injection of adult
neurospheres induces recovery in a chronic model of multiple sclerosis.
Rendl, M., L. Lewis, and E. Fuchs. 2005. Molecular dissection of mesenchymal-
epithelial interactions in the hair follicle. PLoS Biol. 3:e331.
Reynolds, B.A., and R.L. Rietze. 2005. Neural stem cells and neurospheres—
re-evaluating the relationship. Nat. Methods. 2:333–336.
Santagati, F., and F.M. Rijli. 2003. Cranial neural crest and the building of the
vertebrate head. Nat. Rev. Neurosci. 4:806–818.
Shih, D.T., D.C. Lee, S.C. Chen, R.Y. Tsai, C.T. Huang, C.C. Tsai, E.Y. Shen,
and W.T. Chiu. 2005. Isolation and characterization of neurogenic mesen-
chymal stem cells in human scalp tissue. Stem Cells. 23:1012–1020.
Sieber-Blum, M., and M. Grim. 2004. The adult hair follicle: cradle for plu-
ripotent neural crest stem cells. Birth Defects Res. C. Embryo Today.
Sieber-Blum, M., M. Grim, Y.F. Hu, and V. Szeder. 2004. Pluripotent neural crest
stem cells in the adult hair follicle. Dev. Dyn. 231:258–269.
Slack, J. 2001. Skinny dipping for stem cells. Nat. Cell Biol. 3:E205–E206.
Sommer, L. 2001. Context-dependent regulation of fate decisions in multipo-
tent progenitor cells of the peripheral nervous system. Cell Tissue Res.
Sommer, L. 2005. Checkpoints of melanocyte stem cell development. Sci. STKE.
Srinivas, S., T. Watanabe, C.S. Lin, C.M. William, Y. Tanabe, T.M. Jessell, and
F. Costantini. 2001. Cre reporter strains produced by targeted insertion of
EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1:4.
Stemple, D.L., and D.J. Anderson. 1992. Isolation of a stem cell for neurons and
glia from the mammalian neural crest. Cell. 71:973–985.
Taylor, G., M.S. Lehrer, P.J. Jensen, T.T. Sun, and R.M. Lavker. 2000.
Involvement of follicular stem cells in forming not only the follicle but
also the epidermis. Cell. 102:451–461.
Toma, J.G., M. Akhavan, K.J. Fernandes, F. Barnabe-Heider, A. Sadikot, D.R.
Kaplan, and F.D. Miller. 2001. Isolation of multipotent adult stem cells
from the dermis of mammalian skin. Nat. Cell Biol. 3:778–784.
Toma, J.G., I.A. McKenzie, D. Bagli, and F.D. Miller. 2005. Isolation and char-
acterization of multipotent skin-derived precursors from human skin.
Stem Cells. 23:727–737.
Trentin, A., C. Glavieux-Pardanaud, N.M. Le Douarin, and E. Dupin. 2004. Self-
renewal capacity is a widespread property of various types of neural crest
precursor cells. Proc. Natl. Acad. Sci. USA. 101:4495–4500.
Wagers, A.J., and I.L. Weissman. 2004. Plasticity of adult stem cells. Cell.
Wang, Y., Y. Zhang, Y. Zeng, Y. Zheng, G. Fu, Z. Cui, and T. Yang. 2006. Patterns
of nestin expression in human skin. Cell Biol. Int. 30:144–148.
Wernig, M., F. Benninger, T. Schmandt, M. Rade, K.L. Tucker, H. Bussow, H.
Beck, and O. Brüstle. 2004. Functional integration of embryonic stem
cell-derived neurons in vivo. J. Neurosci. 24:5258–5268.
Wurdak, H., L.M. Ittner, K.S. Lang, P. Leveen, U. Suter, J.A. Fischer, S. Karlsson,
W. Born, and L. Sommer. 2005. Inactivation of TGFbeta signaling in
neural crest stem cells leads to multiple defects reminiscent of DiGeorge
syndrome. Genes Dev. 19:530–535.
on May 31, 2012
Published December 11, 2006