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
; Belicchi et al., 2004
; Fernandes et al., 2004
; Joannides et al., 2004
). To further characterize sphere-forming cells derived from the trunk skin of adult mice, dorsal and ventral skin biopsies comprising both dermis and epidermis were dissociated 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 (), 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-affinity neurotrophin receptor p75 and the transcription factor Sox10, both markers for NCSCs (; 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 heterogeneity 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.
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 allowed to spread on FN-coated plates (C–F) (more ...)
Similarly, spheres readily formed from dissociated surgical samples of adult human thigh and face skin (). 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 (). However, other markers for premigratory or migratory NCSCs, such as Sox9 and HNK-1, were not expressed.
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 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 ( 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.
Figure 2. Skin-derived spheres are of neural crest origin. Neural crest–derived cells were identified 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 (more ...)
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 fibrillary 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 (), 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 (), whereas treatment with stem cell factor and endothelin-3 resulted in formation of a few melanocytes (). Finally, occasional adipocytes were detected (). However, we never observed 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.
Figure 3. Cells from skin-derived spheres differentiate 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 (more ...)
The aforementioned data are consistent with the idea that skin-derived spheres contain multipotent cells capable of generating neural and nonneural cell types. In analogy to NCSCs isolated 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 identified and mapped single undifferentiated, 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 nonneural cell types (). Virtually no p75-positive cell was restricted to a single cell lineage. Thus, p75/Sox10-positive neural crest–derived cells prepared from the adult trunk skin are multipotent and can be expanded in culture. Upon isolation, these cells therefore exhibit properties of NCSCs.
Figure 4. Clonal analysis of multipotent neural crest–derived cells from adult skin. Spheres derived from mouse skin were dissociated after passage 17, and single cells were plated at clonal density with or without the addition of instructive growth factors. (more ...)
Several instructive growth factors, including Wnt, BMP, neuregulin (NRG), and TGFβ, have been shown to promote specific fate decisions in NCSCs at the expense of other possible fates. Although Wnt responsiveness is lost at later developmental 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
; Kruger et al., 2002
). Similarly, single prospectively identified p75-positive neural crest cells isolated from the adult back skin were sensitive to BMP2, NRG1, and TGFβ (). All three instructive growth factors suppressed multipotency without affecting survival of founder cells and promoted the generation 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 neural 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 ( and ), the neural crest origin of sphere-forming cells in the adult skin has been demonstrated for whisker follicle–derived SKPs, which, however, are negative for the NCSC markers p75 and Sox10 (Fernandes et al., 2004
). This could either reflect differential regulation of NCSC markers in the same cell type because of varying culture conditions or indicate sphere-forming capacity of skin cells from different neural crest derivatives. To address this issue, we first mapped neural 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 identified 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 (). 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 confirmed by fate mapping experiments performed in human tissue plasminogen activator
mice, in which Cre recombinase is expressed in neural crest cells independently from Wnt1
promoter activity (Pietri et al., 2003
; ). As revealed by X-gal staining of whisker follicles isolated from Sox10lacZ
mice (that express β-galactosidase 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 (). Thus, the whisker follicle comprises various Sox10-positive and -negative tissues of neural crest origin.
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 solution to reveal neural crest–derived cells (more ...)
To investigate which of these neural crest derivatives contain cells with sphere-forming potential, dermal papilla, capsula, 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 microdissection, 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 appear to harbor cells with the capacity to generate spheres (). X-gal staining of Wnt1-Cre/R26R mouse cell cultures confirmed that the spheres were neural crest derived. Therefore, neural crest cells with sphere-forming potential are not confined 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 back skin
Unlike in the head, the mesenchyme in the trunk is not derived 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 (). The same structures were also labeled in the back skin of Ht-PA-Cre/R26R
mice (). In particular, both in the anagen and telogen stage, X-gal staining was found in the permanent part of the pelage follicle, including the bulge region below the sebaceous gland (; 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 addition, pigmented melanocytes in the bulb region (the lower part of the hair follicles; ) 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 ().
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 (more ...)
To determine the potential origin of Sox10-positive sphere-forming cells in the skin (), we assessed Sox10 expression by virtue of β-galactosidase activity in the back skin of Sox10lacZ mice in vivo. Interestingly, Sox10-expressing cells were confined 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 encompasses 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 (). Thus, p75/Sox10-positive multipotent neural crest–derived cells from the trunk skin (–) are connected to the glial or the melanocyte lineage or to both of these lineages.
To elucidate whether p75/Sox10 expression and the capacity to form spheres are associated with glial cells from skin, we made use of desert hedgehog
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 (). 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 confined to a region around the bulge (), corresponding to the area that also contains β-galactosidase–expressing cells in Wnt1-Cre/R26R
, and Sox10lacZ
mice (; and Fig. S1). In Dhh-Cre/R26R
mice, X-gal–labeled cells of the bulge region were also labeled with anti-Sox10 antibody () and anti-p75 antibody (). 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 ().
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 progenitors and differentiated cells from the glial (more ...)
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 identified and freshly isolated. One possibility to achieve this would be by using specific 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 (). Although from unselected skin samples >106
cells were used to generate ~50 spheres (; 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 (, green frame), but not the EYFP-negative (, blue frame), cell population gave rise to spheres. Moreover, acutely fixed primary spheres of EYFP-positive cells were composed of cells expressing both p75 and Sox10 (). Thus, p75/Sox10-positive cells related to the glial lineage can be isolated from the skin and form spheres.
Figure 8. Prospective identification 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 (more ...)
We next asked whether sphere-forming potential is a common feature of peripheral glia. Therefore, we investigated 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 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-forming potential.
In cell preparations from the trunk skin of Dhh-Cre/R26R
mice, only a fraction of all p75/Sox10-positive cells also expressed β-galactosidase (unpublished data). This could point to inefficient 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 address 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
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 () and in the hair follicle bulge region corresponding to the location of melanocyte stem cells (; Nishimura et al., 2002
). Moreover, some X-gal–positive cells in the bulge region also expressed Sox10 () and p75 ().
To investigate whether, in addition to cells of the glial lineage, the early melanocyte lineage also comprises undifferentiated neural crest–derived cells with the capacity to generate spheres, we isolated EYFP-expressing cells prospectively identified 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 (, green frame), but not EYFP-negative, cells (, blue frame) were able to form spheres (). Analysis of acutely fixed primary spheres revealed many cells coexpressing p75 and Sox10, whereas pigmented differentiated melanocytes were absent (). These data indicate that the early melanocyte lineage comprises p75/Sox10-positive cells that can be propagated 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 melanocyte lineages.