Absence of pilosebaceous units in chimeric ESS.

<p>Nile red and ALP stainings were performed on epidermis and dermis of ESS controls and ESS with mDPC-GFP (a). In ESS controls, epithelium was thin enough to visualize the skin surface lipids on the serosal side of the epidermis (a). Contrary to host skin containing sebaceous glands (arrowheads), no sebaceous glands were detected in ESS controls and ESS with mDPC-GFP. ALP in the dermis corresponded to the area where the hair follicles were situated (a, arrows). Close examination confirmed that neofollicles were deficient of sebaceous glands (b). On the other hand, sebaceous glands (white arrowheads) were observed above the bulge regions of pelage hairs. Immunohistochemistry confirmed the human origin (HuNu) and demonstrated no co-localization between Mel-5 and GFP in the bulb (c). Skin barrier integrity was evaluated by TEWL (d). Significantly higher TEWL was observed in ESS with mDPC-GFP compared to host skin, but not different from human volunteers (NHS). Scale bars in (a) = 500 µm; (b) = 100 µm and (c) = 50 µm.</p

Comparison of ESS models.

<p>Gene expression analyses of selected genes involved in Wnt/β-catenin pathway, fatty acid metabolism and skin cornification, including <i>LEF1, WNT10B, LOR, INV, SOX9, PRDM1, SCD</i> and <i>FABP3</i>, were compared between ESS controls and ESS with mDPC-GFP after normalization to NHS (a). Asterisks represent statistically significant differences between the ESS groups (<i>p</i><0.05). Morphological comparison of regenerated hair follicles in ESS with mDPC-GFP (b–d) and in newborn murine ESS was demonstrated (e–h). Scale bars = 50 µm, except in (b, e) = 100 µm and in (f) = 1 mm.</p

Immunostaining of regenerated hair follicles.

<p>Indirect immunohistochemistry was performed on frozen sections of microdissected hair follicles using antibodies against (a) CD34, (b) SOX9, (c) LHX2, (d) CD200 and (e) K15, respectively. Alexa Fluor 488 (green) was used to localize selected molecular markers before counterstaining of nuclei with DAPI (blue). Scale bars = 50 µm.</p

Characteristic features of hair induction <i>in vivo</i>.

<p>Compared to epithelium of ESS controls, which has fewer Ki67+ cells and no K6 staining (a), epithelium of ESS with mDPC-GFP contains numerous Ki67 with K6 expression (b). K10 stained positively in the suprabasal layer of ESS controls (c) and ESS with mDPC-GFP (d), but not the invaginating epidermis in ESS with mDPC-GFP (d). Similarly to embryonic development, LEF1 was restricted to the actively growing hair bulb (f, arrowhead) and placode (g, arrowheads). No nuclear LEF1 was observed in ESS controls (e). Corroborating these results, GFP+ dermal condensation was detected beneath the developing placode (h, arrowhead) and within the bulb (i, arrowhead). Dotted lines represent dermal-epidermal junctions. Scale bars = 100 µm.</p