Fig 1. BM-MSCs acquire specific cardiomyocyte markers after co-culture with rat embryonic cardiomyocytes (RECs) but not when co-cultured with cells derived from other embryonic tissues.

(A) Schematic for experimental design to study cardiomyocyte differentiation of MSCs by co-culture with RECs. BM-MSCs were isolated from genetically modified mice that express α-MHC promoter-driven GFP. (B) RT-PCR primers were designed to specifically detect the expression of cardiac specific genes in mouse-derived mRNA (mHe) and do not amplify rat mRNA sequences (RECs). (C) Quantitative real time RT-PCR was performed to determine the expression of cardiac specific genes in mouse BM-MSCs after co-culture with RECs for 5 days. 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 five independent experiments. *p<0.0001 between CC and MSCs groups derived from one-way ANOVA after Tukey's multiple comparisons test. (D) Representative images of BM-MSCs at 5 days of co-culture (original magnification 200x). MSCs were identified in the co-culture by Collagen type IV (Col IV) immunostaining. Cells undergoing cardiomyocyte differentiation express GFP (green cells). (E) 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<0.0001 between groups derived from unpaired t test. (F) RT-PCR assay for the expression of cardiac specific genes in mice BM-MSCs after co-culture with rat embryonic cells from lung (RELu), kidney (REKi) and liver (RELi). The results of three independent co-culture experiments are shown. Abbreviations: α-MHC, alpha-myosin heavy chain; ANF, atrial natriuretic factor; CA, cardiac actin; CC, co-culture; Col IV, collagen type IV; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mHe, mouse heart; RECs, rat embryonic cardiomyocytes.