The ROCK Inhibitor Y-26732 Enhances the Survival and Proliferation of Human Embryonic Stem Cell-Derived Neural Progenitor Cells upon Dissociation

Abstract

Human neural progenitor cells (hNPCs) are the starting material required for neuronal subtype differentiation. Proliferation of hNPCs allows researchers to study the mechanistic complexities and microenvironments present during neural differentiation and to explore potential applications for hNPCs in cell therapies. The use of enzymatic dissociation during hNPC proliferation causes dissociation-induced apoptosis; therefore, in the present study, we examined the effect of the p-160-Rho-associated coiled-coil kinase (ROCK) inhibitor Y-26732 on dissociation-induced apoptosis of hNPCs. We generated hNPCs via embryoid body formation using serum-free culture medium supplemented with noggin. The established hNPCs were characterized and the effect of the ROCK inhibitor on hNPC dissociation was studied. We demonstrated that supplementation of the culture media with 10 μM Y-26732 efficiently reduced apoptosis of dissociated hNPCs; this supplementation was effective when the inhibitor was applied either at (i) 24 h before dissociation of the cells and at 24 h after plating the cells or (ii) at 24 h after plating of the cells only. In addition to reducing apoptosis, both supplementation conditions with Y-26732 enhanced the proliferation of dissociated hNPCs. Our findings provide the optimal time window for ROCK treatment of hNPC dissociation in respect to apoptosis and cell proliferation.