Rho-Associated Protein Kinase Inhibitor and Hypoxia Synergistically Enhance the Self-Renewal, Survival Rate, and Proliferation of Human Stem Cells

Abstract

Sarah Alsobaie,1 Tamador Alsobaie,2 Sakis Mantalaris3 1Department of Clinical Laboratory Science, King Saud University, Riyadh, 11451, Saudi Arabia; 2Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, SW7 2AZ, UK; 3Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, 30322, USACorrespondence: Sarah Alsobaie, Department of Clinical Laboratory Science, King Saud University, Prince Turki Alawal Street, Riyadh, 11451, Saudi Arabia, Tel [phone], Fax [phone], Email [email protected]: High-efficacy single-cell cloning of human-induced pluripotent cells (IPSCs) remains a major challenge. The development of a culture method that supports single-cell passaging while maintaining reproducibility, homogeneity, scalability, and cell expansion to clinically relevant numbers is necessary for clinical application.Methods: To address this issue, we combined the use of the rho-associated protein kinase (ROCK) inhibitor Y-27632 and hypoxic conditions in culture to produce a novel, efficient single-cell culture method for human IPSCs and embryonic stem cells.Results: Through immunocytochemistry, alkaline phosphatase assays, and flow cytometry, we demonstrated that our method enabled high single-cell proliferation while maintaining self-renewal and pluripotency abilities.Discussion: We showed the beneficial effect of the interaction between hypoxia and ROCK inhibition in regulating cell proliferation, pluripotency, and single-cell survival of pluripotent cells.Keywords: pluripotent cells, rho-associated protein kinase, stem cells

Keywords

• pluripotent cells
• rho-associated protein kinase
• stem cells