Porcine Marrow Mesenchymal Stem Cells Into Cardiomyocyte-like Cells Down Notch4 Differentiation

Objectives:To explore the effect of Notch4on the process of mesenchymal stem cells (MSCs) differentiation into cardiomyocyte by measuring the level of Notch4mRNA and Notch4protein changing trends during in vitro culture stimulated with or without5-azacytidine.Methods:Mononuclear cells were isolated from bone marrow aspirated from Chinese mini-swine by density gradient centrifugation through1.077g/ml Percoll. By in vitro culturing and adherence screening, MSCs can be enriched and continuously passaged. The cultured MSCs in vitro were sub-cultured every3days in a Split ratios of1:2after primary culture, and MSCs at passage1,2,3and4(P1, P2, P3and P4) were examined for their proliferation and differentiation stimulated with or without5-azacytidine (10μM) in vitro by immunocytochemistry, transmission electrical microscopy and RT-PCR. Meanwhile, Notch4was measured by RT-PCR, western-blot and immunocytochemistry.Result:The round MSCs adhered to culture flask became multi-horned and long-spingled by day5. The PO MSCs became80%confluent in about7-10days. The MSCs adhered to flask the second day of sub-culturing and had an appearance of whirled growth pattern when flourished. They were CD45(-),CD29(+),CD90(+) identified by immunocytochemistry.When cultured without any stimulation, both of the cell to cell communication (indicated by Cx43expression) and cardiomyocyte specific maker (indicated by cTn-I expression) of swine bone marrow MSC increased, meanwhile, the Notch4was downregulated; when cultured with5-azacytidine stimulation, all MSCs showed more cell to cell communication but no significant change of cardiomyocyte specific maker compared to those without5-azacytidine stimulation control cells, meanwhile, the Notch4mRNA showed no difference but Notch4positive cell decreased compared with control.Conclusion:Notch4is down-regulated when swine bone marrow MSCs differentiate into cardiomyocyte-like cells.