Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro
Abstract
:1. Introduction
2. Methods
2.1. Skin Samples and Primary Keratinocyte Culture
2.2. ROCK Inhibitor Y-27632, Treatment Regimen and Cell Sampling
2.3. Keratinocyte Growth Rate
2.4. Colony-Formation Assay
2.5. Immunoblotting
2.6. MitoTracker Green Staining
2.7. 3D Organotypic Culture
2.8. Cryosectioning and Immunostaining
2.9. Single-Cell Library Preparation and RNA Sequencing
2.10. Generation of Gene Expression Matrix of Counts and Quality Control for scRNAseq
2.11. Normalisation and Cell Cycle Removal
2.12. Dimensionality Reduction and Donor Effect Removal
2.13. Differential Expression and Differential Abundance Analyses
2.14. Cell Type Proportion Comparison
2.15. Trajectory Analysis
2.16. Statistics
3. Results
3.1. Accelerated Colony Formation in the Culture with Six-Day ROCKi Treatment Stopped after ROCKi Withdrawal
3.2. Keratinocytes Treated with Short-Term ROCKi Maintained Differentiation Ability
3.3. Transient and Reversible Changes in Transcriptome in Keratinocytes following Withdrawal of ROCKi Treatment
3.4. The Proportion of Holoclone-Forming Cells Was Reduced after 6-day ROCKi Treatment but Recovered following Its Withdrawal
3.5. ROCKi Treatment Rapidly Activated AKT1/2 and ERK1/2 Signalling
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Jayarajan, V.; Hall, G.T.; Xenakis, T.; Bulstrode, N.; Moulding, D.; Castellano, S.; Di, W.-L. Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro. Cells 2023, 12, 346. https://doi.org/10.3390/cells12030346
Jayarajan V, Hall GT, Xenakis T, Bulstrode N, Moulding D, Castellano S, Di W-L. Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro. Cells. 2023; 12(3):346. https://doi.org/10.3390/cells12030346
Chicago/Turabian StyleJayarajan, Vignesh, George T. Hall, Theodoros Xenakis, Neil Bulstrode, Dale Moulding, Sergi Castellano, and Wei-Li Di. 2023. "Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro" Cells 12, no. 3: 346. https://doi.org/10.3390/cells12030346