OCT4 expression mediates partial cardiomyocyte reprogramming of mesenchymal stromal cells
Fig 5
OCT4 expression is required for partial cardiomyocyte differentiation of MSCs during co-culture with RECs.
BM-MSCs were incubated for 24 h with OCT4 siRNA (siOCT4) or a scrambled control siRNA (siScr) and transfected cells were then selected using puromycin. (A) Quantitative realtime PCR assay for expression of OCT4, SOX2 and NANOG in OCT4 siRNA silenced (siOCT4) and control siRNA MSCs (siScr). Individual PCR reactions were normalized against internal controls (GAPDH) and plotted relative to the expression level in untreated MSCs. Data represent mean±SD of four independent experiments. *p<0.01 and #p<0.001 between groups derived from unpaired t test. (B) Western blot analysis of the OCT4 protein in OCT4 siRNA silenced (siOCT4) and control siRNA MSCs (siScr) (n = 3). (C) Expression of cardiac specific genes in siOCT4 vs. siScr MSCs after co-culture with RECs for 5 days as assessed by qRT-PCR. Data were normalized against the reference gene GAPDH and presented as relative units taking the expression in mouse heart as 1 unit. Data represent mean±SD of four independent experiments. *p<0.05 and #p<0.0001 between siOCT4 and siScr MSCs groups derived from one-way ANOVA after Tukey's multiple comparisons test. (D) Cardiomyocyte differentiation frequency in siOCT4 vs. siScr MSCs after co-culture with RECs for 5 days. α-MHC promoter activity was calculated as the percentage of Col IV-positive cells expressing GFP. Data represent mean±SD of four independent experiments. *p<0.001 between groups derived from unpaired t test. Abbreviations: α-MHC, alpha-myosin heavy chain; ANF, atrial natriuretic factor; β-Tub, β-tubulin; CA, cardiac actin; CC, co-culture; Col IV, collagen type IV; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mHe, mouse heart; RECs, rat embryonic cardiomyocytes.