Treatment of MSCs with Wnt1a-conditioned medium activates DP cells and promotes hair follicle regrowth

Abstract

Hair loss (alopecia) is a common problem for people. The dermal papilla is the key signaling center that regulates hair growth and it engage in crosstalk with the microenvironment, including Wnt signaling and stem cells. In this study, we explored the effects of bone marrow mesenchymal stem cell overexpression of Wnt1a on mouse hair follicle regeneration. Wnt-CM accelerated hair follicle progression from telogen to anagen and enhanced the ALP expression in the DP area. Moreover, the hair induction-related genes were upregulated, as demonstrated by qRT-PCR. Wnt-CM treatment restored and increased DP cell expression of genes downregulated by dihydrotestosterone treatment, as demonstrated by qRT-PCR assays. Our study reveals that BM-MSC-generated Wnt1a promotes the DP's ability to induce hair cycling and regeneration.

Figure 1. Wnt1a expression in BM-MSCs.

(A), (C) RT-PCR analysis of Wnt1a expression and corresponding semi-quantitative analysis data. (B), (D) Wnt1a expression was validated by western blotting using anti-Wnt1a antibody and corresponding semi-quantitative analysis data. (E) Wnt1a expression levels of condition media (CM) from Wnt-MSCs and MSCs were measured by ELISA. The Wnt1a expression was up-regulated in Wnt-MSCs compared with MSCs. The data represents the means ± SEM, n = 3. *P < 0.05.

Figure 2. Wnt-CM promotes the hair follicle cycle in mice.

Telogen C57BL/6 mice were intradermally injected with Wnt-CM. Typical photos of dorsal skin reveal that the stage of the hair cycle by observed comparison of dorsal skin colors and hair growth (A, B). HE staining indicates a structural change of hair follicles (C–J). (E), (F), (G) represent different stages in the hair cycles at 14 days by HE stained. (H), (I), (J) show a magnification of the framed labeled area. Scale bar (E–G) = 50 μm, (C), (D), (H–J) = 20 μm.

Figure 3. Wnt-CM increases the number of hairs in mice.

(A) HE-stained dorsal skin sections at 14 days after depilation and (C) quantitative analysis of the number of hair follicles. (B) Typical photos of dorsal skin of C57BL/6 mice at 21 days after depilation and (D) quantitative analysis of the number of hair shafts. The data represent the means ± SEM, n = 3. *P < 0.05. Scale bar (A–C) = 100 μm.

Figure 4. Expression patterns of structural markers in Wnt-CM-injected skin.

(A) ALP activity was observed in the SG and DP at 3 and 5 days, Ki67 (B) at 3, 5days, and K15 (C) at 3 days. (D) shows a magnification of the framed labeled area. In each of the images, ALP (blue) is shown in blue, Hoechst (blue fluorescence), Ki67 (red fluorescence) and K15 (green fluorescence). The dashed line delineates the hair follicle structure. The arrowheads show the expression site of the DP (dermal papilla); SG, sebaceous gland; and EP, epidermis, scale bar (A–C) = 20 μm and (D) = 10 μm.

Figure 5. Hair follicle induction-related gene expression in the dorsal skin of mice.

qRT-PCR analysis revealed the expression levels of genes of Lef-1 (A), Versican (B), Ptc-1 (C), and Gli-1 (D) in the dorsal skin of mice. The data represent the means ± SEM, n = 3. Statistically significant at *P < 0.05 and **P < 0.01.

Figure 6. Wnt-CM enhances DP cell hair induction ability.

(A–D) The qRT-PCR results revealed that Lef-1, Versican, Ptc-1, and Gli-1 were significantly downregulated in DHT-treated. THE expression of hair induction-related genes was enhanced by MSC-CM and Wnt-CM treatment. (E) The migration ability of DP cells was impaired by DHT according to the scratch test. (F) Quantitative analysis the scratch closure rate of DP cells cultured for 24 hours. The data represent the means ± SEM, n = 3. *P < 0.05 and **P < 0.01. Scale bar = 500 μm.