Immunofluorescence, flow cytometry, and capillary-like tube formation analyses of cardiac mesenchymal stem cells (CMSCs) induced for cardiac and endothelial cell differentiation. (A) Flow cytometry analysis of CD31 expression was evaluated in cells cultured or not with EGM-2 medium. An increase in the percentage of CD31 expression was observed in EGM-2 cell cultures. (B) Tubular-like structures of induced CMSCs were evaluated following culture on Matrigel for 24 hours induced by EGM-2 medium (magnification: 20×). (C) Confocal microscopy analysis of CMSCs after 10 days of endothelial induction by EGM-2 medium reveals positive staining for CD31 (red) in green fluorescent protein-positive (GFP+) CMSCs (green). (D, E) Four weeks after 5′ azacitidine induction, GFP+ CMSCs (green) began to express the cardiac transcription factor GATA-4 (red) (D) and the gap junction protein connexin 43 (red) (E). Sections were co-stained with 4,6-diamidino-2-phenylindole (DAPI) (blue) for nuclei visualization. Bars represent 30 μm in (B) and 50 μm in (D) and (E). *** p < 0.001.