Neural differentiation of hHFSCs. A: Real-time RT-PCR demonstrates expression of neuronal markers MAP2 and β3-tubulin (TUBB3) and loss of NANOG after neuronal differentiation. B: Differentiated cells become immunoreactive for MAP2, NFM, CGA, and neurotransmitter glutamate. MAP2 preferentially stains dendritic processes versus cell bodies (arrow). NFM, CGA, and glutamate stain cytoplasm of neuron-differentiated cells (arrows). Representative images were taken from at least three independent experiments. Scale bars in A = 100 μm.

Smooth muscle cell differentiation of hHFSCs. A: Real-time RT-PCR demonstrates expression of calponin (CNN3) and desmin (DES) and loss of NANOG after smooth muscle differentiation. B: Differentiated cells become immunoreactive for SMA. SMA stains cytoplasm of differentiated smooth muscle cells. C: Contraction assays show hHFSC contracted collagen gels. Epidermal keratinocytes (KC) and HUVSs serve as negative and positive controls, respectively. Black bars indicate collagen gel diameters. D: Quantification of collagen gel shrinkage shows that differentiated cells contract collagen gel similarly to HUVS control. Data shown are mean ± SD of contraction ratio relative to keratinocyte control from three independent experiments. Scale bars in A = 100 μm.

Oct4-positive cells are resided in the hair bulge. A: Oct4 and K15 immunohistochemical stains on consecutive normal human scalp tissues. Top: An anagen hair follicle; bottom: a catagen/telogen hair follicle. K15 highlights the bulge area, and a few Oct4-positive cells are present within K15-highlighted areas. B: Individual hair follicles were cultured in hESC medium for 6 days and then stained with Oct4 and Nanog. Immunostaining and differential interference contrast (DIC) images show that catagen/telogen bulge contains OCT4-positive cells (green, arrows) or NANOG-positive cells (red, arrows). C: Confocal images of catagen/telogen bulges reveal nuclear localization of OCT4 (green, arrow) and Nanog (red, arrow) in the bulge cells. Nuclei are counterstained with TO-PRO-3 (blue). Representative images were taken from at least three independent experiments. Scale bars in A = 30 μm; in B = 200 μm; and in C = 18.75 μm.

Melanocytic differentiation of hHFSCs. A: Dissociated, hair sphere-derived cells were treated in melanocyte differentiation medium for 14 days. We observed dendritic cells with typical melanocyte morphology (arrows). B: MITF and TYRP1 gene expression is detected after differentiation; however, expression of stemness gene NANOG is undetectable after differentiation. C: Fontana-Mason staining shows melanin pigment in a portion of differentiated cells (arrow and inset). D: Differentiated cells exhibit nuclear and cytoplasmic immunoreactivity for MITF and SILV/HMB45, respectively. E: After embedding in three-dimensional human skin reconstructs, differentiated cells homed to the dermal-epidermal junction (arrows and insets). They are readily identifiable by TYR immunoreactivity or by Fontana-Mason staining. Reconstructs embedded with undifferentiated hFSCs do not show TYR and melanin staining. Representative images were taken from two independent experiments. Scale bars in A and D (SILV) = 200 μm; in B, D (MITF), and E = 100 μm; and in inset in E = 30 μm.

Isolation and expansion of hair spheres in hESC medium. A: Morphology of a plucked catagen/telogen hair follicle. B: Morphology of a plucked anagen hair follicle. C: Histology of a plucked anagen hair follicle (H&E stain). D: Histology of a plucked catagen/telogen hair follicle (H&E). E: A suspension of single cells, obtained from trypsinized follicular epithelium, contains a heterogeneous cell population. Some cells display relatively large spindle morphology (arrows), whereas others are small round cells (arrowheads). F: Small round cells proliferate to form a cell cluster in suspension in hESC medium (arrowheads). G and H: Cell clusters become adherent to the culture plates and form sphere-like structures (arrowheads) with sprouting, adherent fibroblastic cells at the periphery (arrows). I–L: A single cell derived from a hair sphere recapitulates the similar process and re-forms a sphere with adherent fibroblastic cells at the periphery (arrowheads). Representative images were taken from at least three independent experiments. Scale bars in A, B, C, D, and G = 200 μm; in E, F, H, I, J, K, and L = 100 μm.

Human hair follicle-derived stem cells express various stem cell markers. A: Immunocytochemistry and confocal microscopy reveals expression of Nanog (red) and Oct4 (green) in a portion of heterogeneous populations within hair spheres. Their expression was typically localized to nuclei (stained blue with To Pro 3) (arrowheads). Nestin shows a cytoplasmic staining pattern. B: Spheres were dissociated into single cells and plated onto Matrigel-coated chamber slides for staining. Confocal images show that individual cells display nuclear staining pattern for Nanog (red) and Oct4 (green); cytoplasmic staining for Nestin. A characteristic punctate pattern of Oct4 staining is observed in both small round cells (top) and intermediate to large cells (bottom, arrows). The latter show a relatively weak staining intensity. Nanog immunoreactivity is observed in small round cells (bottom) and a portion of intermediate sized cells (top, arrows). Nestin also stains spindled cells (bottom). C: Real-time RT-PCR analysis shows that hHFSCs expressed SNAIL, SLUG, SOX9, TWIST, nestin (NES), and BMP4 genes. We used epidermal keratinocytes (KC) and embryonic bodies derived from hESCs as negative and positive controls, respectively. Representative images were taken from at least three independent experiments. Data shown are mean ± SD from three independent experiments. Scale bars in A for Oct4 = 18.75 μm and for Nanog and Nestin = 75 μm; and in B for Oct4: top = 3.75 μm, bottom = 30 μm; for Nanog: top = 7.5 μm, bottom = 30 μm; and for Nestin: top = 7.5 μm, bottom = 30 μm.