Utilization of Pioglitazone as A Novel Approach to Increase The Colony Formation Efficiency of Individualized Human Pluripotent Stem Cells
31 August till 2 September 2016، Tehran - Iran
Presentation Type: Speech
Objective: One problem in the development of human pluripotent stem cells (hPSCs) cultures is the vulnerability of these cells to undergo apoptosis or anoikis upon cellular detachment and dissociation. These cells undergo massive cell death, particularly after complete dissociation. The Rho-associated kinase (ROCK) inhibitor Y-27632 permits hPSCs survival upon dissociation. However, cloning efficiency is often still low. As, our previous studies showed that PPARgamma activation significantly enhanced the proliferation and survival rate of mouse embryonic stem cells, therefore, we hypothesized that the PPARgamma agonist, pioglitazone, might positively affect survival of dissociated single hPSCs and increase colony formation. Material and Methods: We evaluated the effect of PPARgamma activation on cloning efficiency of single dissociated single hPSCs using pioglitazone. Flow cytometry analysis of cell cycle and apoptosis was performed on treated cells compare with the control. Gene expression analysis in dissociated single cells and colony of hPSCs was carried out. On the other hand Positive role of pioglitazone in colony formation was assessed by Western blotting and immunostaining and co-immunopercipitation of memberanous beta-catenin. The relationship between Rho/ROCK signaling pathway and PPARgamma expression was also examined in a different cell type. Finally Pioglitazone and ROCK inhibitor Y-27632 maintenance of the pluripotency of hPSCs was examined by assessment of the respective markers in treated cells. Results: Our data indicated that pioglitazone, a selective peroxisome proliferative-activated receptor-gamma agonist, along with Y-27632 synergistically diminished dissociation- induced apoptosis and increased cloning efficiency (2–3-fold versus Y-27632) without affecting pluripotency of hPSCs. Pioglitazone exerted its positive effect by inhibition of glycogen synthase kinase (GSK3) activity and enhancement of membranous beta-catenin and E-cadherin proteins. These effects were reversed by GW-9662, an irreversible peroxisome proliferative-activated receptorgamma antagonist. Conclusion: This novel setting provided a step toward hPSC manipulation and its biomedical applications.