A ROCK inhibitor permits survival of dissociated human embryonic stem cells

Abstract

Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research, hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from approximately 1% to approximately 27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1(+) cortical and basal telencephalic progenitors, as do SFEB-cultured mouse ES cells.

Rho/ROCK pathway is essential to the expansion, differentiation, and morphological rearrangements of human neural stem/progenitor cells induced by lysophosphatidic acid

“…The ROCK inhibitor Y27632 signifi cantly increases hESC single-cell survival ( 62,63 ) and NS/PCs following neurosphere dissociation ( 64 ). Further, upon dissociation to single cells, the Rho/ ROCK pathway is activated and responsible for modulating cell-cell contact-induced apoptosis in hESCs ( 65 ), NS/PCs ( 64 ), and neurons ( 66 ), an effect associated with MLC phosphorylation in hESCs ( 47 ).…”

High-Content Screening for Chemical Modulators of Embryonal Carcinoma Cell Differentiation and Survival

“…for example, a striking increase in eC cell numbers was caused by a group of compounds that are potent inhibitors of both roCK and prK2. interestingly, roCK and prK2 inhibitors also promote survival of human es cells, 26,27 indicating that common pathways are implicated in the survival of es and eC cells. roCK and prK2 are downstream effectors of rho gtpases that regulate cytoskeleton organization.…”

Chemical approaches to studying stem cell biology

“…Recently, Sasai's group screened a panel of growth factors and small molecule modulators of apoptosis-related enzymes and kinases in dissociated hESCs. Small molecule Y-27632, a selective Rho-associated kinase (ROCK) inhibitor was identified [15], which is able to promote survival of single-cell-dissociated hESCs without the loss of pluripotency. To gain a better understanding of the molecular mechanisms governing hESC survival, we performed a high-throughput phenotypic screening of 50 000 novel synthetic compounds to identify small molecules that allow hESC survival after trypsin dissociation [16].…”