Mobilization and differentiation of mesenchymal stem cells (CMG cells) into cardiomyocytes in vitro and in vivo. (A) The experimental protocol is shown. The CMG-ME cells are CMG cells that have been permanently transfected with a plasmid-encoding EGFP driven by the myosin light chain promoter (pMLC2v-EGFP). (B,C) Cultured CMG-ME cells were treated with 5-azacytidine and immunoassayed with anti-GATA4 (red) antibody on the seventh day of culture. GATA4 was expressed in the CMG-ME cells. Some of the CMG-ME cells became EGFP⁺ (green). (D,E) At 3 weeks, CMG-ME cells that were positive for both EGFP and actinin were observed, indicating that the cells had differentiated into cardiomyocytes. (F) The engraftment of CMG-ME cells into the recipients' BM was confirmed by PCR. Representative results using BM samples collected from recipient mice (samples 1 and 2) are shown. The transgene was clearly detected in the 2 samples. P indicates positive control (CMG-ME cells); N, negative control (CMG cells); M, marker. P:N controls are shown as the percentage with respect to the positive control. (G,H) The myocardium of infarcted mice that received transplants of CMG-ME cells was analyzed using immunofluorescent microscopy. The green, red, and blue signals indicate EGFP, actinin, and nuclei, respectively. EGFP⁺ actinin⁺ CMG-ME cells were observed in the myocardium, indicating that the CMG-ME cells had been mobilized into the ischemic myocardium. Actinin and EGFP expression, driven by the MLC-2v promoter, was detected, indicating that the cells had differentiated into cardiomyocytes. Bars indicate 10 μm.