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. 2012 Apr 17;3:784. doi: 10.1038/ncomms1784

Figure 4. Rearrangement of follicular stem cells and their niches in the bioengineered pelage and vibrissa follicles. (a) Immunohistochemical analysis of the bulge region in a bioengineered pelage (upper) and vibrissa (lower) follicle.

Figure 4

Boxed areas in the left panels (H&E staining) are shown as immunofluorescent microscopy images in the right panels. The bulge region was analysed by immunostaining with specific antibodies for CD34, CD49f, NPNT and α8 integrin. The nuclei were stained using Hoechst 33258 (Nuc, blue). Broken lines indicate the outermost part of the hair follicle. Arrowheads indicate positively stained areas. Scale bars, 200 μm for H&E, 50 μm for immunofluorescent microscopy images, 1 mm for macroscopy. (b) Immunohistochemical analysis of the DP in a bioengineered pelage (upper) and vibrissa (lower) hair follicle with anti-Sox2 antibodies. Boxed areas in the left panel are shown at a higher magnification in the right panels. Nuclei were stained using Hoechst 33258 (Nuc, blue). Broken lines indicate the outermost limit of the hair follicle. Arrows indicate positively stained nuclei. Scale bars, 50 μm. (c) Analyses of hair pigmentation of the bioengineered vibrissae by various combinations with the bioengineered vibrissa follicle cell components. The bioengineered vibrissa follicle germ, which was reconstituted between the bulge region-derived epithelial stem cells and the primary cultured DP cells (upper), was combined with the sub-bulge (+SB; middle) or proximal region of the hair matrix (+PHM; lower). These bioengineered hair shafts were analysed by macroscopic (left), SEM (centre) and TEM (right 2 panels) observations. Scale bars, 1 mm for macroscopy, 20 μm for SEM and 2 μm for TEM. (d) The frequencies of pigmented hair shafts (left) and pigmented hairs with normal morphological features (right) were compared. The data are presented as the mean±s.e. from three individual experiments; n=5–12 and n=3–8 per experiment in the +SB and +PHM conditions, respectively. (e) In situ hybridization analysis of the sub-bulge region of natural and bioengineered hair follicles, using a specific antisense probe for Dct mRNA. The nuclei were stained using Hoechst 33258 (Nuc, blue). The arrowheads indicate Dct mRNA expression in the cells. The broken lines indicate the outermost limit of the hair follicle. Scale bars, 50 μm.