Mechanism of resveratrol on the promotion of induced pluripotent stem cells

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 20 mu mol/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 10 mu mol/L, respectively. The reprogramming efficiency was significantly decreased by treatment with 20 mu mol/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. At 20 mu mol/L RV, induced cell apoptosis and proliferation inhibition were more obvious than those of 5 and 10 mu mol/L treatments. Clones were selected from the 10 mu mol/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-1a was 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 concentrations (5 and 10 mu mol/L). The effect of 10 mu mol/L RV on reprogramming was much greater than that of 5 mu mol/L RV. However, high concentration of RV (20 mu mol/L) led to more severe cavitations in mitochondria and caused cytotoxic effects. Taken together, these findings suggest that RV mimics hypoxia in cells and promotes reprogramming at a low concentration.