Efficient establishment of an optimized culture condition for cashmere goat primary hair follicle stem cells

Hair follicle stem cells (HFSCs) are an important basis for hair follicle morphogenesis and hair cycle growth. This cell type also represents an excellent model for studying the gene function and molecular regulation of the hair growth cycle, including proliferation, differentiation, and apoptosis. Basically, the functional investigation of hair growth-regulating genes demands a sufficient amount of HFSCs. However, efficient propagation of HFSCs in goats is a challenging process under the current culture conditions. Here, we investigated the effect of four components, including the Rho-associated protein kinase (ROCK) inhibitor Y-27632, leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and vitamin C, on cell growth and pluripotency in the basal culture medium (DMEM/F12 supplemented with 2% fetal bovine serum). We found that adding Y-27632, LIF, and bFGF independently increased the proliferation and pluripotency of goat HFSCs (gHFSCs), with Y-27632 having the most significant effect (P < 0.001). Fluorescence-activated cell sorting of the cell cycle revealed that Y-27632 promoted gHFSC proliferation by inducing the cell cycle from S to G2/M phase (P < 0.05). We further demonstrated that gHFSCs displayed superior proliferative capacity, clone-forming ability, and differentiation potential in the combined presence of Y-27632 (10 μM) and bFGF (10 ng/mL). We termed this novel culture condition as gHFEM, which stands for goat Hair Follicle Enhanced Medium. Taken together, these results indicate that gHFEM is an optimal condition for in vitro culture of gHFSCs, which will subsequently facilitate the study of HF growth and biology.Hair follicle stem cells (HFSCs) are indispensable for skin repair, hair growth, development, and regeneration. One major challenge in primary cell culture is achieving efficient growth while maintaining stemness to achieve a high yield. Various factors, including the Rho-associated protein kinase inhibitor Y-27632, leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and vitamin C are known to regulate the growth of cells. Here, we investigated the optimal concentrations of Y-27632, LIF, bFGF, and vitamin C for promoting goat HFSCs (gHFSCs) proliferation and pluripotency. We further found that the combination of Y-27632 and bFGF exhibited optimal growth conditions. These findings offer valuable insights into the factors affecting gHFSC culture and potential applications for studying the cellular and molecular mechanisms underlying periodic HF growth and gene function associated with HF development.