biostudies-literatureJayarajan VMedical Research CouncilNational Institute for Health Research (NIHR)346https://www.ebi.ac.uk/biostudies/studies/S-EPMC9913223biostudies-literatureUnknown12(3)Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets <i>in vitro</i> and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treaCellsShort-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro.PMC9913223MR/S036989/1Bulstrode NJayarajan VMoulding DXenakis TCastellano SHall GTDi WLfalseShort-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro.Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets <i>in vitro</i> and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-trea2023-01-01T00:00:00Z2023 Jan2026-04-08T13:27:56.904Z2025-04-07T13:01:02.417ZS-EPMC99132233676668810.3390/cells12030346