Dermal papilla cells (DPCs) play a pivotal role in the regulation of hair follicle (HF) growth, formation, and cycling, mainly through paracrine mechanisms. In the last decade, extracellular vesicles (EVs) have been recognized as a new paracrine mechanism that can modify the physiological state of recipient cells by transferring biological material. Herein, we investigated the effect of EVs isolated from stimulated human dermal fibroblasts (DFs) on DPC activation and HF growth. We found that these EVs (st-EVs) enhanced HF growth ex vivo. Comparative transcriptomic analysis on DPCs identified specific activation of the NDP gene, encoding the non-Wnt ligand Norrin. We found that Norrin was secreted by st-EVs-stimulated DPCs activating in a noncell autonomous manner β-catenin pathway in follicular keratinocytes (human HF keratinocyte [HHFK]) and hair growth ex vivo. Although Norrin-specific receptor Frizzled4 was barely detected in HHFK, we found its presence in DF-EVs. Accordingly, DF-EVs provided Frizzled4 to potentiate Norrin effects ex vivo. Our study identifies DF-EVs as efficient activators of DPCs and Norrin as a novel modulatory player in HF physiopathology. STEM CELLS 2019;37:1166-1175 SIGNIFICANCE STATEMENTDermal papilla cells (DPCs) control the growth and regeneration of hair follicles (HFs) via cellcell interactions and extracellular molecules. These data demonstrate that activated dermal fibroblasts (DFs) secrete specific extracellular vesicles (st-EVs) that enhance HF growth ex vivo. DPCs treated with st-EVs secrete Norrin, a non-Wnt ligand that activates the β-catenin pathway of recipient human hair follicular keratinocytes. DF-EVs contain Norrin-specific Frizzled4 receptor that is barely detected in human HF keratinocytes. Combinatory treatment of Norrin and DF-EVs is sufficient to recapitulate the stimulating effect ex vivo of st-EVs on HF growth. The study identifies Norrin as a novel modulatory player in HF physiopathology.

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Objectives: Hair loss and reduction in hair volume are hallmarks of hair disorders, such as telogen effluvium, or male or female pattern hair loss, and hair ageing, which can cause severe distress in both men and women. Common antihair loss drugs carry some side effects; therefore, novel, safer approaches targeting milder phenotypes are highly advocated. In this context, we investigated an extract of the alpine plant Edelweiss, Leontopodium alpinum var. Helvetia, for its ability to modulate hair follicle (HF) growth ex vivo and inhibit hair loss while increasing hair regeneration in vivo.Methods: Human amputated HFs were microdissected from three donors, two women and one man, and cultured ex vivo for 6 days. After treatment with 0.001% Edelweiss extract (EWDE), we investigated hair shaft production and anagen/catagen conversion, and measured known parameters associated with hair growth, that is hair matrix keratinocyte proliferation and apoptosis, dermal papilla inductivity, and growth factors, by quantitative (immuno)histomorphometry. To assess the anti-hair loss potential of the alpine plant compound, we performed a randomized, placebo-controlled human study enrolling Caucasian women and men, aged 18 to 65 years, with normal hair loss. After 5 months' daily use of an extract containing leave-on serum, we analysed hair density and anagen-to-catagen/telogen ratio by the Trichogram analysis. Results:Our results revealed a significant prolongation in the anagen phase in HFs treated with 0.001% Edelweiss, as indicated by an increase in HFs remaining in anagen and a significant decrease in hair cycle score. In line with this effect, EWDE significantly stimulated hair matrix (HM) keratinocyte proliferation, and dermal papilla inductivity, as shown by a significant up-regulation of versican expression and alkaline phosphatase activity, and a tendential increase in FGF7 immunoreactivity in the dermal papilla of all HFs or only anagen VI HFs.Corroborating the ex vivo results, we observed a significant increase in growingThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Vitiligo is a skin disease characterized by the destruction of epidermal melanocytes. Oxidative stress is closely related to the development of vitiligo. As the main constituent cells in the epidermis, keratinocytes regulates the viability and the function of melanocytes, but the underlying mechanism is still not clear. We hypothesized that exosomes derived from keratinocytes under oxidative stress induce the destuction of melanocytes. We treated human keratinocytes (HaCaT) with H 2 O 2 for 24h and extracted exosomes from culture media by ultracentrifugation. TEM images revealed that exosomes secreted by keratinocytes displayed double layer membranes and cup-like structure, and varied in size (30e200 nm), findings consistent with NTA result. The markers of exosomes, CD63 and Hsp70, can be detected by western blot. Interestingly, we found that the amount of exosomes secreted by keratinocytes was dramatically increased under H 2 O 2 treatment, with a concentration dependent. H 2 O 2 at 0.4mM enhanced exosomes secretion by 3-fold. Meanwhile, we found that exosomes secreted by keratinocytes were successfully intake by melanocytes after 24h co-culture. Importantly, exosomes derived from H 2 O 2-treated keratinocytes (Exo. KC-H 2 O 2) significantly suppressed the proliferation of melanocytes after 96h co-culture. Subsequently, micorRNAs (miRNAs)-seq data showed that the miRNAs expression profile of Exo.KC-H 2 O 2 had distinct characteristics. The qRT-PCR results showed that there are six miRNAs enriched in Exo.KC-H 2 O 2. Further, we proved that two of the six miRNAs inhibited proliferation through regulating MITF axis and induced caspase triggered cell apoptosis of melanocytes in both normal condition and oxidative stress. In conclusion, our data have proved that miRNAs enriched in exosomes derived from keratinocytes under oxidative stress contribute to melanocyte loss. The findings provide new insights for understanding the pathogenesis of vitiligo.

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Hair loss disorders, such as telogen effluvium or androgenetic alopecia, as well as reduction of hair volume during aging result mainly from the shortening of the phase during which the hair follicles (HFs) are producing the hair shaft (anagen). Therefore, novel anti hair loss strategies should aim at efficiently inhibiting the development of the regression phase (catagen). Here, we investigated the potential of a novel cosmetically applicable extract deriving from Leontopodium alpinum (Edelweiss), first ex vivo, in human microdissected anagen VI scalp hair follicles (HFs) from three individuals (two females, one male). After 5 days of treatment with the 0.001% edelweiss extract, significantly more HFs remained in anagen and showed increased hair matrix keratinocyte proliferation. Given that higher activity of alkaline phosphatase, and up-regulated expression of versican, and FGF7 was observed in the dermal papilla of female treated HFs, the anagen prolonging effect of the extract may be possibly mediated by stimulation of dermal papilla fibroblasts inductivity. To independently confirm these results, a 5 months double-blind, placebo controlled clinical study on Caucasian volunteers (both male and female, age 18-65, 30 volunteers per group) was conducted. Treatment with the edelweiss extract lead to a significant increase in hair density (Trichoscan) over time, as well as anagen to catagen/telogen ratio (Trichogram) compared to placebo. Therefore, our results provide evidence for a beneficial effect of the Leontopodium alpinum extract as an adjuvant anti-hair loss agent.

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