Wen Lihong, Miao Yong, Fan Zhexiang, Zhang Jiarui, Guo Yixuan, Dai Damao, Huang Junfei, Liu Zhen, Chen Ruosi, Hu Zhiqi
Department of Plastic and Aesthetic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Department of Plastic and Burn Surgery, Shenzhen Hospital, Southern Medical University, Shenzhen, China.
Front Cell Dev Biol. 2021 Mar 5;9:632882. doi: 10.3389/fcell.2021.632882. eCollection 2021.
Hair follicle tissue engineering is a promising strategy for treating hair loss. Human hair follicle stem cells (hHFSCs), which play a key role in the hair cycle, have potential applications in regenerative medicine. However, previous studies did not achieve efficient hHFSC expansion using feeder cells. Therefore, there is a need to develop an efficient primary culture system for the expansion and maintenance of hHFSCs.
The hHFSCs were obtained by two-step proteolytic digestion combined with microscopy. The cell culture dishes were coated with human fibronectin and inoculated with hHFSCs. The hHFSCs were harvested using a differential enrichment procedure. The effect of Rho-associated protein kinase (ROCK) inhibitor Y-27632, supplemented in keratinocyte serum-free medium (K-SFM), on adhesion, proliferation, and stemness of hHFSCs and the underlying molecular mechanisms were evaluated.
The hHFSCs cultured in K-SFM, supplemented with Y-27632, exhibited enhanced adhesion and proliferation. Additionally, Y-27632 treatment maintained the stemness of hHFSCs and promoted the ability of hHFSCs to regenerate hair follicles . However, Y-27632-induced proliferation and stemness in hHFSCs were conditional and reversible. Furthermore, Y-27632 maintained propagation and stemness of hHFSCs through the ERK/MAPK pathway.
An efficient short-term culture system for primary hHFSCs was successfully established using human fibronectin and the ROCK inhibitor Y-27632, which promoted the proliferation, maintained the stemness of hHFSCs and promoted the ability to regenerate hair follicles . The xenofree culturing method used in this study provided a large number of high-quality seed cells, which have applications in hair follicle tissue engineering and stem cell therapy.
毛囊组织工程是治疗脱发的一种有前景的策略。人毛囊干细胞（hHFSCs）在毛发周期中起关键作用，在再生医学中有潜在应用。然而，以往研究使用饲养层细胞未能实现hHFSCs的高效扩增。因此，需要开发一种高效的原代培养系统来扩增和维持hHFSCs。
通过两步蛋白酶消化结合显微镜观察获得hHFSCs。细胞培养皿用人类纤连蛋白包被并接种hHFSCs。采用差异富集程序收获hHFSCs。评估在角质形成细胞无血清培养基（K-SFM）中添加的Rho相关蛋白激酶（ROCK）抑制剂Y-27632对hHFSCs黏附、增殖和干性的影响及其潜在分子机制。
在添加Y-27632的K-SFM中培养的hHFSCs表现出增强的黏附和增殖能力。此外，Y-27632处理维持了hHFSCs的干性并促进了hHFSCs再生毛囊的能力。然而，Y-27632诱导的hHFSCs增殖和干性是有条件的且可逆的。此外，Y-27632通过ERK/MAPK途径维持hHFSCs的增殖和干性。
使用人类纤连蛋白和ROCK抑制剂Y-27632成功建立了一种高效的hHFSCs原代短期培养系统，该系统促进了hHFSCs的增殖，维持了其干性并增强了其再生毛囊的能力。本研究中使用的无动物源培养方法提供了大量高质量的种子细胞，可应用于毛囊组织工程和干细胞治疗。
