Establishment of an Efficient Primary Culture System for Human Hair Follicle Stem Cells Using the Rho-Associated Protein Kinase Inhibitor Y-27632

Abstract

BackgroundHair 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 in vitro using feeder cells. Therefore, there is a need to develop an efficient primary culture system for the expansion and maintenance of hHFSCs.MethodsThe 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.ResultsThe 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 in vivo. 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.ConclusionAn 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 in vivo. 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.

Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632

“…However, the exact mechanism by which Y-27632 induces the immortalization of keratinocytes remains elusive so far. Recently, one study suggested that Y-27632 maintained the proliferative capacity and stemness of human hair follicle stem cells through regulating the ERK/MAPK signaling pathway [45]. By contrast, another study also reported that the inhibition of PAK1-ROCK-Myosin II and TGF-β signaling pathways were crucial for the long-term proliferation of epithelial stem cells in a lowcalcium medium [46].…”

Dysregulated behaviour of hair follicle stem cells triggers alopecia and provides potential therapeutic targets

“…The specific culture method used in this study provides a direction to develop HF tissue engineering and stem cell therapy for alopecia. 57 EpSlKCs exhibit a phenotypic profile that is similar to that of follicular keratinocytes and can crosstalk with DPCs to promote the proliferation of DPCs, thus facilitating HF morphogenesis in vivo. 62 Exosomes can transmit information between cells, and previous work has shown that dermal papilla cell-derived exosomes (DPC-Exos) extend the anagen stage, slow the catagen stage of the HC, and stimulate ORS cells to proliferate and differentiate.…”

“…How to construct an efficient primary culture system for human HFSCs is a research focus 57 . Abreu et al showed that human interfollicular epidermal keratinocytes with stem‐like features (EpSlKCs) are ideal cells that can be used for hair regeneration 58 . Some specific microRNAs and dermal papilla cell‐derived exosomes (DPC‐Exos) could be potential therapeutic targets of HFSCs in hair loss 59‐61 …”

Editorial: Hair Follicle Stem Cell Regeneration in Aging

“…They identified that FOXA2 in ORS cells functions in regulating the trichogenicity of human ORS cells. Wen et al revealed that inhibition of Rho-Associated Protein Kinase by a specific inhibitor Y-27632 can increase the adhesion, proliferation, and stemness of the primary cultured human hair follicle stem cells through the ERK/MAPK pathway ( Wen et al, 2021 ). As two-dimensional (2D) cultured cells quickly lose their regenerative ability, most researches turned to modulate hair regeneration under 3D culture conditions.…”