Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging.

摘要: Optimization and development of better defined culture methods for human embryonic induced pluripotent stem cells (hESCs hiPSCs) will provide an invaluable contribution to the field regenerative medicine. However, one problem is vulnerability hESCs hiPSCs apoptosis that causes a low plating efficiency upon passaging. Herein, we have developed novel technique uses ROCK inhibitor (ROCKi) Y-27632 (10 microM) in Matrigel-coated dishes both serum- feeder-free conditions. This increases during enzymatic mechanical passaging as compared its presence solely medium. Under these conditions, (three lines) (two retain their typical morphology, stable karyotype, express pluripotency markers potential differentiate into derivatives all three germ layers after long-term culture. Real-time RT-PCR analysis stemness-related integrins (alphaV, alpha6, beta1) has demonstrated expression ROCKi. Similar efficiencies been obtained with lower concentration (800 nM) another ROCKi (HA-1077/Fasudil), thus ruling out non-specific effects Y-27632. These results show addition extracellular matrix can increase clusters. due not only anti-apoptotic effect, but also ECM-cells interaction. Therefore, believe this method be useful current future applications cells.