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Journal of Chinese Integrative Medicine ›› 2013, Vol. 11 ›› Issue (6): 389-396.doi: 10.3736/jintegrmed2013039

• Research Article • Previous Articles     Next Articles

Mechanism of resveratrol on the promotion of induced pluripotent stem cells

Dao-fang Dinga,b, Xiao-feng Lia,b, Hao Xua,b, Zhen Wangc, Qian-qian Lianga,b, Chen-guang Lia,b, Yong-jun Wanga,b()   

  1. a. Institute of Spine, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
    b. Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
    c. Laboratory of Cancer Hospital, Fudan University, Shanghai 200032, China
  • Received:2013-03-24 Accepted:2013-05-15 Online:2013-11-10 Published:2013-11-15

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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 μmol/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 μmol/L, respectively. The reprogramming efficiency was significantly decreased by treatment with 20 μmol/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 μmol/L RV, induced cell apoptosis and proliferation inhibition were more obvious than those of 5 and 10 μmol/L treatments. Clones were selected from the 10 μmol/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-1α 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 μmol/L). The effect of 10 μmol/L RV on reprogramming was much greater than that of 5 μmol/L RV. However, high concentration of RV (20 μmol/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.

Key words: Resveratrol, Plant extracts, Pluripotent stem cells, Hypoxia, Mitochondria cavitation, In vitro

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Figure 1

Transfection and infection of MSCV-Oct4/MSCV-Sox2 (Fluorescence microscopy, ×100)A: MSCV-Oct4 was expressed in GP2-293t cells 48 h post-transfection (bottom, phase contrast). B: MSCV-Sox2 was expressed in GP2-293t cells 48 h post-transfection (bottom, phase contrast). C: Sox2/Oct4 was expressed in mouse embryonic fibroblasts 72 h post-infection (bottom, phase contrast).MSCV: murine stem cell virus."

Figure 2

Comparison of the number of ESC-like colonies and apoptosis/cell proliferation analysis of MEFs or 2F-MEFs treated with RV A: The number of ESC-like colonies on day 17; error bars indicate standard deviation, n=3; *P<0.05, vs control. B: The expression levels of Bcl2 and Bax when cells were treated with RV. C: The expression of PCNA in MEFs and 2F-MEFs treated with RV.ESC: embryonic stem cell; MEFs: mouse embryonic fibroblasts; 2F-MEFs: 2 factors-MEFs; RV: resveratrol; PCNA: proliferating cell nuclear antigen."

Figure 3

Representative iPSC clone was picked out and analyzed for the expression of pluripotency-associated genes A: Phase contrast and fluorescence image of ESC-like clone generated from RV 10 μmol/L group (Fluorescence microscopy, ×100). B: iPSC clone was picked and GFP was silenced after being cultured for one month (Fluorescence microscopy, ×100). C: Immunostaining of Oct4 in iPSCs; nuclei were stained by DAPI (Fluorescence microscopy, ×100). D: Immunostaining of Sox2 in iPSCs; nuclei were stained by DAPI (Fluorescence microscopy, ×100). E: ALP staining of iPSCs (Fluorescence microscopy, ×100). F: Pluripotency marker genes (Ecat1, Fgf4, Gdf3, Nanog, and Foxd3) were reactivated in iPSCs.iPSCs: induced pluripotent stem cells; ESC: embryonic stem cell; RV: resveratrol; GFP: green fluorescent protein; DAPI: 4′,6-diamidino-2-phenylindole; ALP: alkaline phosphatase; MEFs: mouse embryonic fibroblasts."

Figure 4

Hypoxia changes in 2F-MEFs cultured by RV A: Mitochondria in control cells (0 μmol/L) was intact which was denoted by arrow (Electron transmission microscopy, ×10 000). There are increasing cavitations in mitochondria with RV treatment for 24 h. Cavitations were denoted by arrows. B: The expression level of HIF-1α was higher in high concentration group than in low concentration group.2F-MEFs: 2 factors-mouse embryonic fibroblasts; RV: resveratrol; HIF-1α: hypoxia-inducible factor-1α; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

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