Mesenchymal stem cells with modification of junctional adhesion molecule a induce hair formation

Abstract

The junctional adhesion molecule A (JAM-A) has been shown to serve a crucial role in the proliferation, differentiation, and tube-like formation of epithelial cells during angiogenesis. The role of JAM-A in hair follicle (HF) regeneration has not yet been reported. In this study, we used human JAM-A-modified human mesenchymal stem cells (MSCs) to repair HF abnormalities in BALB/c nu/nu mice. The JAM-A gene and JAM-A short hairpin RNA were transfected into cultured human MSCs to generate the JAM-A overexpression MSCs (JAM-A(ov) MSCs) and JAM-A knockdown MSCs (JAM-A(kd) MSCs), respectively. These cells were injected intradermally into the skin of nude mice during the first telogen phase of the HF that occurs 21 days postnatally. We found that JAM-A(ov) MSCs migrated into the HF sheath and remodeled HF structure effectively. The HF abnormalities such as HF curve and HF zigzag were remodeled, and hair formation was improved 7 days following injection in both the JAM-A(ov) MSC and MSC groups, compared with the JAM-A(kd) MSC group or negative control group. Furthermore, the JAM-A(ov) MSC group showed enhanced hair formation in contrast to the MSC group, and the number of curved and zigzagged HFs was reduced by 80% (p < .05). These results indicated that JAM-A(ov) MSCs improved hair formation in nude mice through HF structure remodeling.

Keywords: Cell adhesion molecules; Cell transplantation; Differentiation; Mesenchymal stem cells.

Figure 1.

Regulation of JAM-A expression in MSC and JAM-A overexpression promoted MSC migration in vitro. (A): A high fluorescence signal was observed after gene transduction. Bright punctiform green fluorescence was observed in JAM-A^ov MSCs. In all images, magnification is ×100. (C): JAM-A mRNA levels were confirmed by real-time polymerase chain reaction 48 hours after gene transduction. Marked upregulation of JAM-A expression was detected in JAM-A^ov MSCs, whereas low levels of JAM-A expression were observed in JAM-A^kd MSCs. ∗∗, p < .01. (B, D): In vitro migration results showed that overexpression of JAM-A enhanced the migration of MSCs. JAM-A^ov MSCs significantly increased the number of migrating cells. In all images, magnification is ×100. ∗∗, p < .01. Abbreviations: GFP, green fluorescent protein; JAM-A, junctional adhesion molecule A; MSC, mesenchymal stem cell.

Figure 2.

JAM-A overexpression promoted hair formation in the skin of nude mice. Left column: hair formation in the skin of nude mice. The hair quality in the JAM-A^ov MSC group of mice was better than the other groups. (A–C): The number of twisted hair follicles (HFs) in the JAM-A^ov MSC group was decreased compared with other groups. A positive signal of mitochondria was observed in three groups. Scale bars = 200 μm (A–C). (A1, B1, C1): Hair diameter in the JAM-A^ov MSC group was significantly greater than in the other groups. Meanwhile, on the horizontal plane of the HF in the JAM-A^ov MSC group, the hair root is organized into concentric circles of cells from outer to inner. In the JAM-A^kd MSC group, the graft could be observed clearly (C1, C2). (C): The funnel-shaped mouths of hair follicles in the skin of JAM-A^kd MSC injection mice were ampliate. (A1, A2): JAM-A^ov MSCs migrated to HF. (B1, B2): GFP MSCs migrated to HF. The graft could be observed clearly too. But the number of graft under the same amplification was more than in the JAM-A^ov MSC group. Scale bars = 50 μm (A1, A2, B1, B2, C1, C2). Abbreviations: GFP, green fluorescent protein; JAM-A, junctional adhesion molecule A; MSC, mesenchymal stem cell.

Figure 3.

Overexpression of junctional adhesion molecule A correlates with HF remodeled and hair formation. Hair density (A), hair length (D), the number of twisted HF (C) and hair diameter (B) in the three groups were dissected and assayed. Statistical results revealed a marked difference among the three groups. ∗∗, p < .01. Abbreviations: HF, hair follicle; KD, knockdown; MSC, mesenchymal stem cell; OV, overexpression.

Figure 4.

Overexpression of JAM-A correlates with maintenance of funnel tube shape. (A, C): In JAM-A^ov mesenchymal stem cell (MSC) injection mice, the funnel-shaped mouths of hair follicles were small and straight. Straight hair root and hair shaft had been seen clearly in the upright funnel tube in JAM-A^ov MSC injection mice. JAM-A expression was clearly increased in JAM-A^ov MSC injection mice. (B): JAM-A expression was weak in JAM-A^kd MSC injection mice (white arrow). (D): Twisted hair root was clearly observed in the ampliate funnel tube in JAM-A^kd MSC injection mice (white arrow). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; JAM-A, junctional adhesion molecule A; KD, knockdown; OV, overexpression.

Figure 5.

Overexpression of junctional adhesion molecule A (JAM-A) correlates with maintenance of root sheath shape in the skin of nude mice. The skin from experimental day 7 was paraffin embedded and sectioned along the cephalocaudal axis. Green signal represents the positive signal of JAM-A, K19, p-FAK, and p-PKCζ separately. Red signal represents the positive signal of mitochondria, indicating the localization of the engrafted MSCs, which were also 4′,6-diamidino-2-phenylindole stained. (A–D): The increased expression of JAM-A, K19, p-PKCζ, and p-FAK in JAM-A^ov MSC injection mice, associated with HF structure improvement. (A1, B1, C1, D1): The expression of JAM-A, K19, p-PKCζ, and p-FAK in MSC injection mice. Hair follicles in this group were smaller than in the JAM-A^ov MSC group. (A2, B2, C2, D2): The decreased expression of JAM-A, K19, p-PKCζ, and p-FAK in JAM-A^kd MSC injection mice. HF structure was frizzy in this group. Abbreviations: KD, knockdown; MSC, mesenchymal stem cell; OV, overexpression; PKC, protein kinase C.