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div>As expected, progenitor-like GATA-4+ cells expressed only mouse Lamin A/C, as shown by the negative stain with human-specific AZD-5363 antibody (Fig. 3C). In agreement with this finding, cardiac progenitor-like cells were GFP+, when coculture was performed with mouse GFP+ cardiomyocytes (Fig. 3D). The mouse phenotype of hybrid cells highlighted that cellular fusion between stem cells and cardiomyocytes did not trigger cardiomyogenic conversion of stem cells but, rather, promoted somatic reprogramming of cardiomyocytes toward a progenitor-like state. To show that somatic reprogramming resulted from cell fusion, cocultures were performed with (i) a cell-culture insert to impede physical interactions between the two cell types without affecting the diffusion of soluble factors or (ii) living cardiomyocytes and hMADS cells previously killed with PFA to inhibit cellular fusion . Indeed, PFA prefixation prevented fusion of live sperm cells or neural www.selleckchem.com stem cells with fixed sea urchin eggs or endothelial cells, respectively, but did not disrupt receptor-mediated recognition and association of the cocultivated cell types [41, 42]. Reprogramming did not occur in either condition, which indicates that soluble signaling molecules or cell-to-cell interaction through a receptor had little or no effect on the generation of mouse cardiac progenitor-like cells (Fig. 3E, 3F and not shown). Finally, we addressed the frequency of cardiomyocyte reprogramming by counting the number of immunostained cardiac ��gal+/GATA-4+ hybrid cells resulting from cocultures rendered mitotically inactive by mitomycin C treatment. We detected a mean of 10 hybrid cells per 100,000 cardiomyocytes initially seeded. However, because propidium iodide staining and flow cytometry revealed that more than 90% of cardiomyocytes were suffering before coculture (supporting information Fig. 2), the rate of cardiomyocytes efficiently reprogrammed might be about 0.1% when considering the fraction of healthy cardiomyocytes at the start of coculture. In addition, we tried to improve reprogramming SB431542 mw efficiency by increasing cardiomyocyte survival in vitro because living cardiomyocytes were found to have limited lifespan (<24 hours) under our coculture conditions. We performed cocultures on laminin-coated dishes in the presence of medium containing insulin-transferrin-selenium and 2,3butanedione monoxime as reported previously . Nevertheless, under these conditions, the yield of reprogrammed cardiomyocytes seemed not affected. To determine whether reprogramming of cardiomyocytes was specific to hMADS cells, we created cocultures with hBMS cells as another model of multipotent stem cells and with human fibroblasts (MRC5 cell line) as an example of proliferative cells devoid of plasticity potential.</div>