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) and fixed after 8 h. Immunocytochemical analysis revealed that both murine and human skin–derived spheres contain numerous cells expressing p75 (visualized by Alexa 488 fluorescence, green) and Sox10 (revealed by Cy3 fluorescence, red; E and F). The arrow shows a double-positive cell, and the arrowhead shows a negative cell. (C and D) Corresponding DAPI staining.

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 for cell differentiation. Marker expression indicated 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 counterstained with DAPI. Cells with features of chondrocytes, melanocytes, and adipocytes were identified by Alcian blue staining (D and J), DOPA reaction (E and K), or with oil red O (F and L), respectively. Bar, 20 μm.

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 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. Melanocytes (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 Sox10^lacZ mice and incubated in X-gal solution. Cells in which the Sox10 promoter was active were identified 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 follicles and therefore not stained with X-gal. Six independent experiments were performed for each structure. Bar, 50 μm.

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 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.

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 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.

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. Cells were prospectively identified with p75 staining and mapped on the plate. A clone was counted positive 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 identified. (B) Cultures containing BMP2 generated S-only clones, S/O, and N/S/O clones. Clone types containing glial cells were significantly decreased. (C) Cultures containing NRG1 also generated S/O and S-only clones. Clone types containing neuronal cells were decreased. (D) TGFβ addition increased the numbers of S/O clones and S-only clones. Each bar represents the mean ± SD of three independent experiments, counting at least 50 clones per experiment.

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 containing 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 immunofluorescence. 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 specific for neural crest derivatives in the skin and also label X-gal–negative structures (white arrows). Moreover, not all neural crest–derived cells coexpress p75 and Sox10 (arrowheads). The dermal papilla (DP) was X-gal negative in both mouse lines. Bars: (A, C, E, and I) 20 μm; (G) 10 μm.

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 -negative cells, respectively. Mice not carrying the corresponding Cre were used as negative controls for EYFP fluorescence (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) Quantification (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.