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.
Highlights
Alopecia results from various factors that decrease the regeneration ability of hair follicles and disrupt the hair cycle (York et al, 2020)
The expression levels of CK15 and CK19 were upregulated (Figure 4F). These results suggest that Rho-associated protein kinase (ROCK) inhibition is necessary for the propagation and maintenance of Human hair follicle stem cells (hHFSCs) in vitro
Western blotting analysis revealed that the phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK) decreased upon treatment with 10 μM U0126 (Figure 5G). These results indicate that the ERK/mitogen-activated protein kinase (MAPK) signaling pathways mediate Y-27632-induced hHFSC proliferation and stemness maintenance
Summary
Alopecia results from various factors that decrease the regeneration ability of hair follicles and disrupt the hair cycle (York et al, 2020). Hair transplantation is one of the treatment strategies for hair loss. Hair transplantation does not provide satisfactory therapeutic outcomes for large-scale hair loss (Vasserot et al, 2019). Recent advances in hair tissue engineering have enabled effective treatment of hair loss, especially in cases of large-scale hair loss with insufficient donor hair follicles (Coelho et al, 2012; Owczarczyk-Saczonek et al, 2018). A prerequisite for efficient hair tissue engineering is the availability of seed cell source (Mistriotis and Andreadis, 2013). 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. Previous studies did not achieve efficient hHFSC expansion in vitro using feeder cells. There is a need to develop an efficient primary culture system for the expansion and maintenance of hHFSCs
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