HaCaT-conditioned medium supplemented with the small molecule inhibitors SB431542 and CHIR99021 and the growth factor PDGF-AA prevents the dedifferentiation of dermal papilla cells in vitro

Hair loss, including alopecia, is a common and distressing problem for men and women, and as a result, there is considerable interest in developing treatments that can prevent or reverse hair loss. Dermal papillae closely interact with epidermal cells and play a key role during hair follicle induction and hair morphogenesis. As dermal papilla cells (DPCs) lose their hair-inducing ability in monolayer cultures in vitro, it is difficult to obtain de novo hair follicle structures following DPC transplantation in vivo. The present study aimed to explore culture conditions to maintain DPC characteristics using conditioned media (CM) from the supernatant of cultured HaCaT keratinocyte cells supplemented with other components. Initially, it was observed that during passaging of in vitro monolayer DPC cultures, the Wnt/β-catenin pathway was repressed, while the TGF-β/Smad pathway was activated, and that HaCaT cells cultivated in 1% fetal bovine serum had higher levels of expression of Wnt3a and Wnt10b compared with normal keratinocytes. Culturing of high-passage (P7) DPCs in CM from HaCaT cells (HaCaT-CM) actively stimulated cell proliferation and maintained Sox2 and Versican expression levels. Supplementation of HaCaT-CM with SB431542 (SB, a TGF-β receptor inhibitor), CHIR99021, (CHIR, a GSK3α/β inhibitor and activator of Wnt signaling) and platelet-derived growth factor (PDGF)-AA further increased the expression levels of Sox2, Versican and alkaline phosphatase (ALP) in P7 DPCs. Three-dimensional culture of P7 DPCs using hanging drop cultures in HaCaT-CM supplemented with SB, CHIR and PDGF-AA resulted in larger cell aggregates and a further significant upregulation of Sox2, ALP and Versican expression levels. Taken together, these findings demonstrated that HaCaT-CM supplemented with SB, CHIR and PDGF-AA may preserve the hair-inducing ability of high-passage DPCs and may therefore be useful in reconstructing new hair follicles in vivo.