Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro

Area of Science:

• Dermatology
• Cell Biology
• Regenerative Medicine

Background:

• Primary keratinocytes, including keratinocyte stem cells (KSCs), are crucial for skin regeneration and therapies for genetic skin disorders.
• Slow initial growth of primary keratinocytes limits their clinical application in cell and gene therapies.
• The effects of Rho-associated kinase inhibitor (ROCKi) on KSC characteristics and clinical safety are not well understood.

Purpose of the Study:

• To investigate the impact of short-term ROCKi treatment on primary keratinocyte expansion and KSC properties.
• To assess the safety and reversibility of ROCKi treatment for potential clinical use.

Main Methods:

• Primary keratinocytes were treated with ROCKi Y-27632 for six days.
• Assessed colony formation, cell proliferation, and differentiation potential using 3D-organotypic culture.
• Analyzed KSC numbers and activation of AKT and ERK pathways via single-cell transcriptomic analysis.

Main Results:

• Short-term ROCKi treatment significantly increased keratinocyte colony formation and proliferation.
• Treated keratinocytes retained their differentiation capacity.
• ROCKi treatment did not decrease KSC numbers, and its effects were reversible upon withdrawal.
• AKT and ERK pathways were rapidly activated by ROCKi.

Conclusions:

• Short-term ROCKi treatment offers a transient and reversible method to accelerate initial primary keratinocyte expansion.
• This approach preserves the holoclone-forming cell population (KSCs), indicating its safety for clinical applications in cell and gene therapies.