Impacts of manipulating cell sorting on in vitro hair follicle regeneration.

Hair follicle morphogenesis is triggered by epithelial-mesenchymal interactions. Several approaches have been developed for preparing hair follicle organoids using epithelial and mesenchymal cells; however, the current understanding of the relevance of in vitro spontaneous organization processes to hair regeneration is limited. In the present study, we used Y27632, a rho-associated kinase inhibitor, to investigate the effects of manipulation of cell sorting on hair regeneration in vitro. Dissociated hair follicle-inducible epithelial and mesenchymal cells were cultured in Y27632-containing media in 96-well plates or polydimethylsiloxane microarray plates. We found that Y27632 supplementation modulated the spatial distribution of epithelial and mesenchymal cells from a dumbbell shape to a core-shell configuration via a spontaneous organization process. New hair follicles with typical morphological features emerged in the Y27632-treated core-shell-shaped aggregates, and hair shafts sprouted with approximately 100% efficiency in vitro. Gene chip analysis and pathway-inhibition experiments revealed that the phosphatidylinositol-3-kinase/protein kinase B- and Ras-signaling pathways were involved in hair-like sprouting in the Y27632-treated hair follicle organoids. Our findings enhance the understanding of hair follicle organogenesis and the development of hair follicle organoids.