OBJECTIVE:To investigate the effects of resveratrol(RV)in reprogramming mouse embryonic fibroblasts(MEFs)into induced pluripotent stem cells(iPSCs)and the related mechanism.METHODS:Primary MEFs were isolated from E13.5 embryos and used within three passages.Retroviruses expressing Sox2 and Oct4 were produced by transfecting GP2-293t cells with recombinant plasmids murine stem cell virus(MSCV)-Sox2 and MSCV-Oct4.Supernatants containing retroviruses were obtained after 48-hour transfection and MEFs were then infected.Different concentrations(0,5,10 and 20μmol/L)of RV were added to embryonic stem cell(ESC)medium to culture MEFs 48 h post-infection.iPSC clones emerged and were further cultured.Expression of pluripotent markers of iPSCs was identified by cell immunofluorescence and reverse transcription-polymerase chain reaction.Both cytotoxicity and cell proliferation were assayed by Western blot analysis after RV was added into ESC medium.The ultrastructure change of mitochondria was observed by electron microscopy.RESULTS:More than 2.9-fold and 1.3-fold increases in colony number were observed by treatment with RV at 5 and 10μmol/L,respectively.The reprogramming efficiency was significantly decreased by treatment with 20μmol/L RV.The proliferation effect on MEFs or MEFs infected by two factors Sox2/Oct4(2 factors-MEFs,2F-MEFs)was investigated after RV treatment.At20μmol/L RV,induced cell apoptosis and proliferation inhibition were more obvious than those of 5 and 10μmol/L treatments.Clones were selected from the 10μmol/L RV-treated group and cultured.Green fluorescent protein expression from one typical clone was silenced one month later which expressed ESC-associated marker genes Gdf3,Nanog,Ecat1,Fgf4 and Foxd3.Electron transmission microscope showed obvious cavitations in mitochondria.The expression of hypoxia-inducible factor-1αwas up-regulated when 2F-MEFs were treated with RV compared to the control group.CONCLUSION:RV improved the efficiency of reprogramming 2F-MEFs into iPSCs at low and moderate conce
Dao-fang DingXiao-feng LiHao XuZhen WangQian-qian LiangChen-guang LiYong-jun Wang
