Project description:Background: Experimental studies have suggested that stem cells (SC) may exert their beneficial effects on the ischemic heart by paracrine activation of antiapoptotic pathways. In order to identify potential cardioprotective mediators, we performed a systematic analysis of the differential gene expression of lin-/c-kit+ SC after coculture with cardiomyocytes (CM). Methods: SC were separated from murine bone marrow and labeled with the green fluorescent CFDA. SC were then cocultivated with neonatal rat ventricular CM (NRVCM). After 48 h, we performed two cell sorting steps to generate a highly purified population of conditioned SC (>99%) and isolated the RNA. Next, we performed a genome-wide microarray analysis of cocultured vs. monocultured SC with Illumina Chips (Coculture n=3; Monoculture n=3). Our systematic analysis of differentially expressed genes focused on products that are secretable, membrane-bound and potentially involved in antiapoptotic signal transduction. Results: Our analysis resulted in 3 genes (out of more than 1000 differentially regulated genes) that met the criteria mentioned above and which could also be confirmed by RT-PCR. We found CCL-12 (12x, p<0.05), MIF (2x, p<0.05) and connexin 40 (4.5x; p<0.05) upregulated in our coculture model. An ELISA of cell culture supernatants was performed to proof secretability of candidate genes and showed that supernatants of coculture experiments have higher CCL-12 concentrations than monoculture experiments (20 pg/ml vs. 4 pg/ml; p<0.01). Next, we stimulated NRVCM with concentrated coculture supernatants which resulted in a significantly higher phosphorylation of AKT (p<0.01). Finally, NRVCM were labeled with Annexin-V and apoptosis of NRVCM in a co- and monoculture was measured by FACS. Thereby, we observed a reduction in apoptotic NRVCM in the coculture model (12% vs. 25%; p<0.05). Conclusion: Our results provide evidence that coculture of SC with NRVCM leads to an upregulation of antiapoptotic genes and a paracrine-mediated increase in the phosphorylation of AKT. This results in less apoptotis in cocultured NRVCM. We therefore conclude, that cell-cell interactions lead to a modified gene expression in SC that may in part explain the cardioprotective effects of stem cell therapy. We analysed six RNA samples extracted from three cocultures and three monocultures. Every sample represents an independant biological experiment, derived from individual preparations of cardiac myocytes and bone marrow stem cells.

Project description:Background: Experimental studies have suggested that stem cells (SC) may exert their beneficial effects on the ischemic heart by paracrine activation of antiapoptotic pathways. In order to identify potential cardioprotective mediators, we performed a systematic analysis of the differential gene expression of lin-/c-kit+ SC after coculture with cardiomyocytes (CM). Methods: SC were separated from murine bone marrow and labeled with the green fluorescent CFDA. SC were then cocultivated with neonatal rat ventricular CM (NRVCM). After 48 h, we performed two cell sorting steps to generate a highly purified population of conditioned SC (>99%) and isolated the RNA. Next, we performed a genome-wide microarray analysis of cocultured vs. monocultured SC with Illumina Chips (Coculture n=3; Monoculture n=3). Our systematic analysis of differentially expressed genes focused on products that are secretable, membrane-bound and potentially involved in antiapoptotic signal transduction. Results: Our analysis resulted in 3 genes (out of more than 1000 differentially regulated genes) that met the criteria mentioned above and which could also be confirmed by RT-PCR. We found CCL-12 (12x, p<0.05), MIF (2x, p<0.05) and connexin 40 (4.5x; p<0.05) upregulated in our coculture model. An ELISA of cell culture supernatants was performed to proof secretability of candidate genes and showed that supernatants of coculture experiments have higher CCL-12 concentrations than monoculture experiments (20 pg/ml vs. 4 pg/ml; p<0.01). Next, we stimulated NRVCM with concentrated coculture supernatants which resulted in a significantly higher phosphorylation of AKT (p<0.01). Finally, NRVCM were labeled with Annexin-V and apoptosis of NRVCM in a co- and monoculture was measured by FACS. Thereby, we observed a reduction in apoptotic NRVCM in the coculture model (12% vs. 25%; p<0.05). Conclusion: Our results provide evidence that coculture of SC with NRVCM leads to an upregulation of antiapoptotic genes and a paracrine-mediated increase in the phosphorylation of AKT. This results in less apoptotis in cocultured NRVCM. We therefore conclude, that cell-cell interactions lead to a modified gene expression in SC that may in part explain the cardioprotective effects of stem cell therapy.

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Expression of miRNA in Embryonic Stem Cells-Derived Cardiomyocytes after coculture with Endothelial Cells

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