Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells.

The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFβ signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders

The Role of Neprilysin in Regulating the Hair Cycle

<div><p>In most mammals, each hair follicle undergoes a cyclic process of growing, regressing and resting phases (anagen, catagen, telogen, respectively) called the hair cycle. Various biological factors have been reported to regulate or to synchronize with the hair cycle. Some factors involved in the extracellular matrix, which is a major component of skin tissue, are also thought to regulate the hair cycle. We have focused on an enzyme that degrades elastin, which is associated with skin elasticity. Since our previous study identified skin fibroblast elastase as neprilysin (NEP), we examined the fluctuation of NEP enzyme activity and its expression during the synchronized hair cycle of rats. NEP activity in the skin was elevated at early anagen, and decreased during catagen to telogen. The expression of NEP mRNA and protein levels was modulated similarly. Immunostaining showed changes in NEP localization throughout the hair cycle, from the follicular epithelium during early anagen to the dermal papilla during catagen. To determine whether NEP plays an important role in regulating the hair cycle, we used a specific inhibitor of NEP (NPLT). NPLT was applied topically daily to the dorsal skin of C3H mice, which had been depilated in advance. Mice treated with NPLT had significantly suppressed hair growth. These data suggest that NEP plays an important role in regulating the hair cycle by its increased expression and activity in the follicular epithelium during early anagen.</p> </div

Image analysis of mice hair.

<p>Eight-week old C3H mice were treated topically with NPLT or with the vehicle control (80% ethanol) twice a day after chemical depilation. Rate of hair re-growth measured by image analysis at 10, 12, 14, 17, 21, 24 and 28 days after depilation (A). Growing hair follicles (Anagen III to Anagen VI) were classified according to accepted morphological guidelines in HE-stained skin sections, and were counted using a microscope (B). Lengths of hair follicles were measured by image analysis using Image J software (C). NPLT (•) and vehicle control (△). *: P<0.05, **: P<0.01 (vs vehicle control, t test). Data represent means ± SD (n = 5).</p

Localization of NEP at each stage of the hair cycle in rats.

<p>Immunostaining of rat skin sections including hair follicles was performed with an anti-NEP antibody. In early anagen hair follicles, the follicular epithelium showed strong NEP immunoreactivity (A). Further positive staining existed in the dermis in a direction toward the follicular extension in early anagen (B). Positive staining for NEP appeared at the inner root sheath and the basal plate in anagen (C, D), at the dermal papillae and connective tissue sheath in catagen (E), and weak signals in the fibroblast sheath around club hair were evident in telogen (F). A negative control treated with normal IgG showed no specific staining in any stage of hair cycle or any site of skin (G). Bars = 10 µm (A-F) or 50 µm (G).</p

Proteinase activities at each stage of the rat hair cycle.

<p>Dorsal skins of SD rats at different hair cycle stages were homogenized and measured for Type I (△) and Type IV (□) collagenases and elastase (•) activities (A). Values represent means ± S.D. from 5 independent experiments. Typical histologic sections of the rat hair cycle (B). Skin specimens (4 mm diameter) biopsied from the dorsal skin at the same time periods used to measure enzyme activities were fixed with formalin and embedded in paraffin. Sections were stained with Hematoxylin and Eosin. Images at early anagen (5 weeks), anagen (6 weeks) and telogen (8 weeks) are shown, as noted. Bar 0.1 mm.</p

Effect of topical application of the NEP inhibitor on hair re-growth in mice.

<p>Eight-week old C3H mice were treated topically with NPLT or with the vehicle control (80% ethanol) twice a day after chemical depilation. Typical photos of dorsal skins (A–F) or HE-stained skin sections (G–L) of C3H mice: left side is vehicle-treated, right side is NPLT-treated mice, upper column 12 days, middle column 17 days, lower column 24 days after depilation.</p

Effect of the NEP inhibitor on human scalp hair follicles in culture.

<p>Human hair follicles were isolated from occipital scalp specimens, and were cultivated for 10 days. Lengths of hair shafts were measured by Image analysis using Image J software and the growth rate per day (A) was calculated. Data represent means ± SE (n = 6). When the shape of the anagen hair follicle changed to catagen (dermal papillae separated from the follicular epithelium), the follicle was removed from the length measurement and the number of catagen hair follicles was counted (B). Typical photos of cultured human hair follicles are shown in C–H (C–E: vehicle treated, F–H: NPLT treated). Human scalp skin immunostained with an anti-NEP antibody showed similar positive staining as rat (the follicular epithelium, the dermis intrusive in a direction toward the follicular extension and the connective tissue sheath at early anagen (I), IRS and connective tissue sheath at anagen (J)).</p

NEP activity and expression at each stage of the rat hair cycle.

<p>Dorsal skins of SD rats at different hair cycle stages were homogenized and NEP activity was measured (A). Values represent means ± S.D. from 5 independent experiments. Dorsal skins of SD rats at different hair cycle stages were homogenized and solubilized in RIPA buffer. After immunoprecipitation with an anti-NEP antibody, western blotting was performed. Blots were visualized using ECL detection reagents (C) and chemiluminescence was determined using a Molecular Imager System (B). The loading control is shown by CBB staining (D). Northern blotting was performed with a specific probe for rat NEP labeled with ³²P-dCTP, radioactivity was measured using a BAS2000 bio-image analyzer (E, F).</p

hiPSCs-derived SKPs display multipotency similar to traditionally isolated SKPs.

<p>(A) Adipogenic differentiation of hiPSC-SKPs was confirmed by Oil Red-O staining and the up-regulation of PPARγ gene expression by qPCR. (B) Osteogenic differentiation was confirmed by ALP staining and the up-regulation of RUNX2 and COL1A1 gene expression by qPCR. (C) Differentiated Schwann cell lineages were detected by S100β staining. Scale bars, 50 μm. UD: undifferentiated hiPSC-SKPs, Dif: differentiated hiPSC-SKPs.</p