Background and Objectives Although application of newly developed drugs, heart failure is the only one disease of increasing mortality in cardiovascular disease. In this background, stem cell transplantation is hopeful alternative for failed heart. We tried to establish the MSC (mesenchymal stem cell) - CMC (cardiomyocyte) co-culture system as a more physiologic and effective MSC differentiation to CMC model.
Material and Method We used MSC originated from human umbilical cord blood and CMC obtained from 1-2 day old Fisher 344 rat. Dil labeled MSCs and rat CMCs were co-cultured during 2 weeks in low glucose DMEM. To verify differentiation to CMC of MSC, we have done RT-PCR for detecting CMC specific markers, for example, 慣-MHC, Csx/Nkx2.5, Troponin I, GATA-4, MEF-2C and ANP. Immunocytochemistry were performed for simultaneous colorization both Dil labeled MSC and CMC markers at 5 days and 2 weeks after co-culture. For RT-PCR analysis, we separated MSC from co-cultured CMC with FACS at 1 week after co-culture and purity of separation was verified with fluorescence microscopy. And in order to confirm engraftment, we observed rat myocardial tissue with fluorescent microscopy after injecting MSCs which were double stained with DiI and DAPI to rat myocardium at 1, 2 weeks.
Results In RT-PCR analysis, GATA-4 expression was highest at 5 days and regressed at 2 weeks, Csx/Nkx2.5 and Troponin I were expressed at day 5 and lasted till 2 weeks. Some Dil positive MSC co-expressed Troponin I from day 5. The result of RT-PCR and IF staining were concordant. Although expression of cardiac specific markers in MSC, we could not observe spontaneously beating DiI labeled MSC. In vivo model, DiI and DAPI double stained MSC which were injected to rat myocardium were viable and well intercalated between rat CMC until 2 weeks follow up period.
Conclusion This in vitro MSC-CMC co-culture system is a good explanation model of transplanted MSC evolutional change with host CMC and supplies us important theoretical bridge from MSC differentiation in vitro to clinical application of cellular cardiomyoplasty to failed human heart.