Short-term rho-associated kinase inhibitor treatment promotes primary keratinocyte growth without affecting keratinocyte stem cell properties

Abstract Keratinocyte stem cells (KSCs) can be cultured as ex vivo epidermal sheets and are attractive for cell and gene therapy. However, the growth and expansion of primary keratinocytes (including KSCs) in in vitro culture can be inefficient as KSCs undergo apoptotic cell death, known as anoikis when isolated from skin biopsies and dissociated from their extracellular environment. This further affects the survival and stemness of KSCs in in vitro culture and hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle. However, it risks changing the characteristics of stem cells. In this study, we studied the effect of the Y-27632 ROCKi on cultured primary human keratinocytes. Following a short-term (6 days) ROCKi treatment, we assessed keratinocyte proliferation and differentiation potential and KSC stemness at the molecular-, cellular- and single-cell levels. A significant increase in colony-forming efficiency associated with increased expression of proliferation markers and mitochondrial mass was observed in treated cells vs. untreated cells. Similarly, treated cells showed a distinct transcriptional profile at the single-cell level, but these changes became indistinguishable after discontinuation of ROCKi treatment. Single-cell transcriptome analysis further confirmed that short-term ROCKi treatment did not affect stem cell characteristics. Finally, we showed that ROCKi rapidly activated Akt and upstream of ERK signalling pathways in primary human keratinocytes. In conclusion, rapid proliferation due to short-term ROCKi treatment was transient and reversible, and did not affect the KSC population or their characteristics. We thus present a safer approach for the use of ROCKi in ex vivo clinical applications.