Neural potential of a stem cell population in the hair follicle

Abstract

The bulge region of the hair follicle serves as a repository for epithelial stem cells that can regenerate the follicle in each hair growth cycle and contribute to epidermis regeneration upon injury. Here we describe a population of multipotential stem cells in the hair follicle bulge region; these cells can be identified by fluorescence in transgenic nestin-GFP mice. The morphological features of these cells suggest that they maintain close associations with each other and with the surrounding niche. Upon explantation, these cells can give rise to neurosphere-like structures in vitro. When these cells are permitted to differentiate, they produce several cell types, including cells with neuronal, astrocytic, oligodendrocytic, smooth muscle, adipocytic, and other phenotypes. Furthermore, upon implantation into the developing nervous system of chick, these cells generate neuronal cells in vivo. We used transcriptional profiling to assess the relationship between these cells and embryonic and postnatal neural stem cells and to compare them with other stem cell populations of the bulge. Our results show that nestin-expressing cells in the bulge region of the hair follicle have stem cell-like properties, are multipotent, and can effectively generate cells of neural lineage in vitro and in vivo.

Figure 1

Nestin-GFP cells in the hair follicle. (A) GFP-expressing cells in the bulge region of the telogen hair follicle in the dorsal skin of nestin-GFP transgenic mice. Note fine processes running along and across the rows of cells. Inset shows a lower magnification view of the tissue. (B) GFP expressing cells in the follicle are connected to a network of GFP-expressing vessels located closer to the skin surface. (C) Colabeling of nestin-GFP cells in the bulge with antibodies (red) to (a) nestin, (b) S100a, (c) peripherin, (d) keratin, (e) β-tubulin and (f) GFAP. Scale bar is 15 μm in (A), 10 μm in (B), and 10 μm in all six images in (C).

Figure 2

Nestin-GFP cells from the hair follicle generate multiple cell types. The bulge area was microdissected from the hair follicle and cells were plated into media containing FGF and EGF under conditions for growing neurospheres. After the neurosphere-like colonies were formed, they were plated onto laminin- and polyornithine-coated plates in the presence of FBS and later examined by immunochemistry. (a–c) and (e–g) neurosphere-like colonies grown from the hair follicle bulge explants (hairspheres); (a–c) phase contrast, (e–g) fluorescence. (a and e) colonies 12 days after cultivation, (b and f) 20 days, (c and g) 24 days. (d and h) two days after plating onto coated plates, hairspheres adhere to the surface; cells start to migrate away from the sphere and lose their GFP fluorescence; (d) phase contrast, (h) fluorescence. (i) one week after plating cells start to express neuronal marker β-tubulin (red); some cells still express low levels of GFP (green). (j) two weeks after plating GABA-positive neuronal cells are evident. (k) one week after plating cells start to express astrocytic marker GFAP (red); some cells still express low levels of GFP (green). (l) two weeks after plating adipocyte-like cells with large vacuole-like inclusions are evident. (m) two weeks after plating some cells express smooth muscle actin. Scale bar is 50 μm in (a–h), 25 μm in (i–l) and 10 μm in (m).

Figure 3

Clonally derived hairspheres generate multiple cell types. Nestin-GFP cells were isolated from the bulge of the hair follicle, grown under clonal conditions in 1.2% methylcellulose, transferred to regular media for further growth, and then plated onto laminin- and polyornithine-coated plates in the presence of FBS with or without added growth factors and later examined by immunochemistry with antibodies to β-tubulin (a), GABA (b), GFAP (c), CNPase (d), keratin (e), and smooth muscle actin (f). Scale bar is 20 μm in (a–d), and 10 μm in (e and f).

Figure 4

Hairspheres generate neuronal cells upon transplantation into developing chick embryo. Hairspheres and neurospheres generated from actin-GFP mice were transplanted into developing two day chick embryo. (a) hairsphere cells implanted into the ventral part of the chick neural tube (two days) and analyzed 4 days later. Here and in all other images (except for m) - green - GFP, red - TuJ1, blue - DAPI. (b–e) many transplanted cells start to express β-tubulin. (f) hairsphere cells implanted into the dorsal part of the chick neural tube (two days) and analyzed two days later. (g and h) higher magnification of implanted cells shown in (f); cells start to express β-tubulin. (i and j) four days after implantation cells express higher levels of β-tubulin and extend long processes. (k–m) hairsphere cells implanted into the otic placode of the chick embryo and analyzed two days later. Cells are found in association with cranial ganglia; note, however, that although they mingle with β-tubulin-expressing ganglia cells, very few of them express β-tubulin (l), and none of the cells expressed Islet-1 (red) (m). (n and o) neurosphere cells implanted into the dorsal part of the chick neural tube and analyzed two days later. Many cells start to incorporate into the neural tube and many of them express β-tubulin (higher magnification in l). (p) neurosphere cells implanted into the otic placode migrate towards the perimeter of the placode. Scale bar is 100 μm in (a), 20 μm in (b–e), 100 μm in (f), 20 μm in (g and h), 20 μm in (i and j), 400 μm in (k), 50 μm in (l), 50 μm in (m), 200 μm in (n), 50 μm in (o), 200 μm in (p).

Figure 5

Transcription profiling of hairsphere and neurosphere derived cells. (A) Clustering of the genes expressed in hairspheres isolated from p4 and p49 animals and neurospheres isolated from 14 day embryo (e14) or postnatal brain (p4). The relations between the samples are presented as correlation coefficients. The data were generated using Affymetrix gene arrays. (B) Expression of selected markers, determined by Q-PCR; the samples include, in addition to those analyzed in (A), undifferentiated and differentiated keratinocytes. The relations between the samples are presented as correlation coefficients.

Figure 5

Transcription profiling of hairsphere and neurosphere derived cells. (A) Clustering of the genes expressed in hairspheres isolated from p4 and p49 animals and neurospheres isolated from 14 day embryo (e14) or postnatal brain (p4). The relations between the samples are presented as correlation coefficients. The data were generated using Affymetrix gene arrays. (B) Expression of selected markers, determined by Q-PCR; the samples include, in addition to those analyzed in (A), undifferentiated and differentiated keratinocytes. The relations between the samples are presented as correlation coefficients.