Effected of Rho-associated Protein Kinase Inhibitor Y-27632 on Improving the Cryopreserved Survival and Passage of Porcine(Sus scrofa) Induced Pluripotent Stem Cells
Abstract：Pig is used as an important model animal, and induced pluripotent stem cells (iPS cells) have been established in pig now. However, the efficiency of cryopreserved survival and passage is very low. Rho-associated protein kinase(ROCK) inhibitor Y-27632 enhanced the survival of cryopreserved of human embryonic stem (ES) cells, and improved the ES colony formation. In this study, we used Y-27632 for porcine(Sus scrofa) induced pluripotent stem cells. 5 and 10 μmol/L Y-27632 was used for cryopreservation and passage of porcine iPS cells, and it suggested that Y-27632 could greatly suppress cryopreserved-induced apoptosis and increase thaw-survival rates of porcine iPS cells. Meanwhile, it was able to help the adhesion of porcine iPS cell in passage when 5 and 10 μmol/L Y-27632 was mixed into culture medium, and it also contributed to porcine iPS colonies formation in 24 h. Furthermore, 10 μmol/L Y-27632 would change the morphology of porcine iPS cells, and made the colonies become flat and loose, but it did not affect alkaline phosphatase (AP) activity and the expression level of pluripotenct genes, octamer-binding transcription factor-4 (Oct4), SRY-related high-mobility-group (HMG)-box protein-2 (Sox2) and homeobox transcription factor (Nanog) in porcine iPS cells treated with Y-27632. Besides, PB[Act-RFP] DS, the transposon reporter construct, was introduced into porcine iPS cells through electric transfected, and RFP positive cells were sorted by flow-cytometry. After treated with 10 μmol/L Y-27632 in culture medium, these sorted cells were easier to grow. After these sorting porcine iPS cells injected into porcine early embryos through micromanipulation, the cells treated with Y-27632 were easier to integrate into porcine parthenogenetic embryos than that without treated cells. Above all Y-27632 were able to improve the cryopreserved survival and passage of porcine iPS cells, and suppress the apoptosis induced by single cell dissociated and fluorescence-activated cell sorting, and enhance the integration of RFP carrying porcine iPS cells in porcine embryos. This study contributes to cryopreservation and associated research for porcine and other species pluripotenct stem cells
