E-Mail karger@karger.com Original Paper Cells Tissues Organs 2013;198:127–138 DOI: 10.1159/000354031 The ROCK Inhibitor Y-26732 Enhances the Survival and Proliferation of Human Embryonic Stem Cell-Derived Neural Progenitor Cells upon Dissociation Ruttachuk Rungsiwiwut a, b Chirawattana Manolertthewan c Pranee Numchaisrika a Vichuda Ahnonkitpanit a Pramuan Virutamasen a Mongkol Techakumphu d Kamthorn Pruksananonda a a Human Embryonic Stem Cell Research Center, Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, b King Chulalongkorn Memorial Hospital, c Faculty of Medicine, Vajira Hospital, University of Bangkok Metropolis, and d Department of Obstetrics, Gynecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand 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 supple- mentation conditions with Y-26732 enhanced the prolifera- tion of dissociated hNPCs. Our findings provide the optimal time window for ROCK treatment of hNPC dissociation in re- spect to apoptosis and cell proliferation. © 2013 S. Karger AG, Basel Key Words Apoptosis · Human embryonic stem cells · Nestin · Neural differentiation · ROCK inhibitor Abstract Human neural progenitor cells (hNPCs) are the starting mate- rial required for neuronal subtype differentiation. Prolifera- tion 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 apop- tosis; therefore, in the present study, we examined the effect of the p-160-Rho-associated coiled-coil kinase (ROCK) inhibi- tor Y-26732 on dissociation-induced apoptosis of hNPCs. We generated hNPCs via embryoid body formation using serum- free culture medium supplemented with noggin. The estab- lished hNPCs were characterized and the effect of the ROCK inhibitor on hNPC dissociation was studied. We demonstrat- ed that supplementation of the culture media with 10 μ M Y-26732 efficiently reduced apoptosis of dissociated hNPCs; this supplementation was effective when the inhibitor was Accepted after revision: June 25, 2013 Published online: October 19, 2013 Assoc. Prof. Kamthorn Pruksananonda Human Embryonic Stem Cell Research Center Reproductive Medicine Unit, Department of Obstetrics and Gynecology Faculty of Medicine, Chulalongkorn University, Bangkok 10330 (Thailand) E-Mail pkamthorn  @  yahoo.com © 2013 S. Karger AG, Basel 1422–6405/13/1982–0127$38.00/0 www.karger.com/cto Abbreviations used in this paper ANOVA analysis of variance bFGF basic fibroblast growth factor CNS central nervous system EB embryoid body GFAP glial fibrillary acid protein hESCs human embryonic stem cells hiPSCs human induced pluripotent stem cells hNPCs human neural progenitor cells PBS phosphate-buffered saline PCR polymerase chain reaction PI propidium iodine ROCK p-160-Rho-associated coiled-coil kinase Downloaded by: Chulalongkorn University 161.200.98.189 - 10/21/2013 10:05:58 AM