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.

Reprogramming of three-dimensional microenvironments for in vitro hair follicle induction.

During embryonic development, reciprocal interactions between epidermal and mesenchymal layers trigger hair follicle morphogenesis. This study revealed that microenvironmental reprogramming via control over these interactions enabled hair follicle induction in vitro. A key approach is to modulate spatial distributions of epithelial and mesenchymal cells in their spontaneous organization. The de novo hair follicles with typical morphological features emerged in aggregates of the two cell types, termed hair follicloids, and hair shafts sprouted with near 100% efficiency in vitro. The hair shaft length reached ~3 mm in culture. Typical trichogenic signaling pathways were up-regulated in hair follicloids. Owing to replication of hair follicle morphogenesis in vitro, melanosome production and transportation were also monitored in the hair bulb region. This in vitro hair follicle model might be valuable for better understanding hair follicle induction, evaluating hair growth and inhibition of hair growth by drugs, and modeling gray hairs in a well-defined environment.