The Effect Of Vascular Endothelial Growth Factor On Differentiation Of Mouse Induced Pluripotent Stem Cells Into Cardiomyocytes

Objective To observe the effect of vascular endothelial growthfactor(VEGF) on differentiation of mouse induced pluripotent stem cells(iPScells) into cardiomyocytes.Methods Mouse iPS cells were cultured on mouse embryonicfibroblast (MEF) with medium containing leukemia inhibitory factor (LIF).Undifferentiated mouse iPS cells were identified by their morphologyunder microscope, expression of green fluorescent protein(GFP) promotedby stem cell genes Oct-4and alkaline phosphatase staining. Hanging dropmethod was used to make iPS cells differentiate.10ng/ml,20ng/ml,50ng/mlVEGF were respectively added to induce the differentiation of iPS cells.Natural differentiation without any inducer was negative group and1%dimethyl sulfoxide （DMSO）was added as positive control group. Cellswere observed under microscope, the time of beating embryonic bodies（EBs）appeared and numbers in each group were recorded and thedifferentiation rate of myocardial cells was calculated. The expression ofα-MHC and β-MHC mRNAin differentiated cells were detected by RT-PCRand the expression of cTnT was detected by immunofluorescence Results1. The mouse iPS cells cultured on MEF with mediumcontaining LIF were colony-like and could proliferate all the time. GFPpromoted by Oct-4were positive expressed and showed green fluorescence.iPS cells were stained as purple by alkaline phpsphatase staining.2.Spontaneously beating EBs could be observed in each group.Compared with the natural differentiation group, three concentrations ofVEGF all improved differentiation rate of myocardial cells(p<0.05),20ng/mlVEGF group had the highest rate which was53.17%±3.63%and had nosignificant difference compared to dimethyl sulfoxide group(P>0.05).3. Myocardial cells derived from mouse iPS cells in each groupexpressed cardiac genes α-MHC and β-MHC.VEGF could up-regulateα-MHC and β-MHC expression compared to natural differentiationgroup(P<0.05).This effect was most obvious at the concentration of20ng/ml(p<0.05).4. Myocardial cells derived from iPS cells in each group beatedspontaneously and expressed cardiac-specific protein cTnT。Conclusion1.Mouse iPS cells can maintain undifferentiated state inthe environment of LIF and MEF and begin to differentiate when removingthem through the formation of EBs.2. A certain concentration of VEGF can promote the differentiation ofmouse iPS cells into myocardial cells.