The Effect of ROCK Inhibitor on Recovery and Proliferation of Thawed Pluripotent Stem Cells

Background and Aim: Both embryonic stem cells (ESCs) and inducedpluripotent stem cells (iPSCs) have significant obstacles for culture withinactivated feeder layer when defrosted from cryopreserved stocks.ROCK inhibitor treatment enhances stem cell survival efficiency andpoor recovery after thawing. In this study, we investigated the effectof different incubation times of a specific pyridine-based Rho kinase(ROCK) inhibitor on recovery enhancement and proliferation of thawedhESCs and iPSCs.Methods: The protocol of this study was approved by the local ethicscommittee (code IR.TBZMED.REC.1396.1031). Mouse embryonicfibroblasts (MEFs) were cultured in DMEM supplemented with 10% FBS.Before use as a feeder layer, their growth was arrested with Mitomycin C(2 mg/mL) for 3 hours in 37°C incubator. Inactivated cells were seededat 600 000 cell density on 6 cm culture plates. Pluripotent stem cellmedium containing DMEM/F-12 and Knockout serum replacement wasconditioned for 4 hours on inactivated MEFs. Cryovials of Royan H6 hESCand R1 hiPSC4 lines were thawed immediately into conditioned mediawith Y-27632 ROCK inhibitor at 10 μM concentration. To investigate theeffect of Y-27632, the medium was refreshed with basic medium withoutinhibitor at different time points after 8-24 hours. The number of coloniesand cell proliferation was assessed microscopically.Results: We observed that the number of adherent colonies wassignificantly higher when the medium supplemented with Y-27632removed after 8-12 h from culture plates (≥2-fold versus later time points). In addition, the rate of reaching confluency after 10 days washigher in the mentioned culture condition (80% versus <60% at latertime points), which was more prominent in iPSCs. But when we removedROCK inhibitor after 18 or 24 hours, both hESCs and iPSCs reached toconfluency later, after 12 days.Conclusion: We demonstrated that a short time treatment with ROCKinhibitor only for 8-12 hours is more appropriate for recovery andproliferation of thawed pluripotent stem cells, maybe due to the inhibitoryeffect of ROCK inhibitor on cell proliferation in long-term incubation.According to our results optimizing of pluripotent stem cell culturecondition with respect to ROCK inhibitor treatment time is essential.

کلیدواژه ها:

Pyridines ، Embryonic stem cells ، Induced pluripotent stem cells ، Cryopreservation