Porcine Bone Marrow Mesenchymal Stem Cells In Vitro Experimental Study Of Myocardial Cell Differentiation

Objectives:Mesenchymal stem cells(MSCs) are a group of heterogenous stem cells with multipotency in growth and differentiation.The aim of this study was to explore the proliferative potential and cardiomyogenic phenotype development of porcine bone marrow- derived MSCs before or after 5-azacytidine treatment,and find out the regularity of MSCs differentiation in vitro.Methods:Bone marrow samples were aspirated from Chinese mini-swine.The mononuclear cells were isolated by density gradient centrifugation through 1.077g/ml Percoll and then cultured in high-glucose DMEM containing 10%fetal bovine serum.After primary culture,relatively purified MSCs were obtained by using the method of adherence screening.MSCs at passage9,10,11,12,15 and 20 were examined for their proliferation and differentiation ability before or after 5-azacytidine(10μM) treatment in vitro by FACS,immunocytochemistry, transmission electrical microscopy and real time-PCR.Results:In normal growth medium,porcine bone marrow-derived MSCs were positive for CD90 and CD29,negative for CD34 and CD45.The growth property analysis demenstrated P10 pMSCs showed a growth-arrest appearance,while other passages displayed an exponential growth pattern.MSCs at every passage in vitro expressed cardimyocyte-specific genes and proteins.However,only passage 10 took on the formation of myotube structure.After 5- azacytidine treatment,pMSCs at every passage in vitro took on cardiomyocyte -like phenotype and formation of myotube structure,and expressed cardimyocyte-specific genes and proteins.However,MSCs at passage 10 expressed cardimyocyte-specific genes higher than any other passages,especiallyα-SKA,P10 vs P9,P10vs P11,P10vs P12,P10vsP15,P10vs P20(P＜0.0001).Before or after 5- azacytidine treatment,MSCs at passage 9,11,12,15 and 20 all had cardiomyocyte-like ultrastructure such as irregular myofilaments in vitro. However,only MSCs at passage 10 took on relatively mature cardiomyocyte ultrastructure,including myofilaments.MSCs had no observation of sarcomeres, transverse striations,intercalated discs in vitro.Conclusion:Cardiomyogenic potential of porcine bone Marrow-derived MSCs spontaneously or in response to stimulation of 5-azacytidine in vitro is dependent upon the endogenous gene expression and passage-restricted pattern.These findings reveal a novel regulation on the transdifferentiation of MSCs and provide useful information for exploiting the clinical therapeutic potential of MSCs.