Abstract
Hair follicle stem cells (HFSCs) are an important source for skin tissue engineering studies and clinical applications. Here, we describe a differential enrichment approach to derive HFSCs from hair follicles of vibrissae and ear skin using the Rho-associated protein kinase (ROCK) inhibitor Y-27632. In the presence of Y-27632, primary cultured hair follicle cells grew in clustered colonies surrounded by keratinocyte-like cells and simultaneously expressed three HFSC markers: CD34, K15, and ITGB1. HFSCs cultured in medium containing Y-27632 were presented at a stable ratio of 30.7%, 34.1%, and 32.9% after passages 5, 10, and 15, respectively. By contrast, in medium containing epidermal growth factor, clustered HFSC colonies disappeared after 6 passages and lacked HFSC marker expression. After withdrawal of Y-27632 from the medium, HFSCs rapidly differentiated into keratinocyte-like cells. Furthermore, HFSCs derived with Y-27632 formed spherical clusters in collagen matrix in vitro, differentiated into keratinocytes and adipose cells under in vitro induction conditions, and cooperated with fetal dermal cells to regenerate hair follicles in vivo 6 weeks after their intracutaneous injection into immune-deficient mice. These findings suggest that Y-27632 maintains the self-renewal and stemness characteristics of HFSCs during primary skin tissue culture followed by enrichment passaging and that HFSCs derived with Y-27632 possess the differentiation potentials important for tissue engineering and other clinical applications.