Differentiation of human umbilical cord mesenchymal stem cells using 5-azacytidine to generate a cardiac patch on thermo-responsive polymer

摘要

Introduction Human heart, the non-tirelessly working; pumping organ. Incidence and prevalence of cardiovascular diseases (CVDs) are increasing worldwide. Current treatment strategies mitigate only the symptoms, with no role in damaged tissue regeneration. This calls for myocardial tissue engineering (MTE) with scaffolds and cells. A bioengineered cardiac patch with native extracellular matrix can overrule these limitations. Cell sheet technology thus helps in developing transplantable scaffold and suture free in vitro tissue constructs. For this, choice of cell source is also important. Hence hUMSCs which are least researched upon for MTE, but reported to be better source of seed cells for generating cardiomyocytes were used (1). Materials and Methods HiFi Wharton's jellyMesenchymalStemCellswere used for the study. Cells were cultured, characterised by immunostaining and flow cytometry for CD90, CD105, Stro-1 and CD34. Cells were grown on NGMA, the in-house developed thermo-responsive polymer and differentiated using 5μM 5-azacytidine. After 24 hours, the cells were washed twice with PBS and the media was changed to normal LG-DMEM and studied for 21 days with media change at alternate days. The differentiated cells were characterised for cardiac markers Troponin T, Myosin heavy chain, Alpha cardiac sarcomeric actinin and Connexin 43 by immunostaining and by RT PCR for Connexin-43, Cardiac myosin heavy chain, Cardiac troponin-T, GATA4, Nkx2.5 and house keeping gene GAPDH. The sheet was retrieved by lowering the temperature. FDA - PI live dead staining was performed to ensure the viability of the retrieved cell sheet Results and Discussion The hUCMSCs are being looked upon with great enthusiasm, as they have proved to be highly competent to beat the bone marrow derived MSCs; one of the most extensively researched cell source; In availability, proliferation and even immuno-modulation. The risk of viral contamination is also lower for hUCMSCs (2). In spite of being better seed cells for for generating cardiomyocytes, they have never been advocated as a potential cardiac patch differentiated using 5-azacytidine alone as inducer, except for the study by Zhao et al., 2011 which depicts sphingosine -1 - phosphate in combination with cardiac myocytes conditioned medium for cardiac differentiation(3). Cells were found to express CD90 (Fig. 1), CD105 and Stro-1 (Fig 2) by immunostaining and flow cytometry, confirming sternness and homogeneity. Differentiated cells aligned parallel to one another and showed morphological features similar to cardiomyocytes. Molecular expression of sarcomeric-alpha-actinin, Nkx 2.5 and Connexin 43 (Fig 3), the molecules which are major to cardiac cells for cell contraction and electrical impulse propagation to other myocardial cells in the syncytium, when transplanted were observed. Cells differentiated to myocardial lineage were also retrieved as intact viable cell sheet which is highly recommended for suture free transplantations. Conclusion Differentiated hUCMSCs cells have been retrieved as a viable cardiac patch for suture free transplantation with minimal cell loss and effective nutrient diffusion. Also, our study, illustrates more economical method by adding small amount of inducer to cells, for a short period.