Experimental Study Of Differentiation And Paracrine Action Of Porcine Bone Marrow Mesenchymal Stem Cell In Vitro

Objectives:The aim of this study was to explore the proliferative potential, cardiomyogenic phenotype development and the paracrine action of porcine bone marrow-derived MSCs cultured in normal growth medium and after other3different treatments in vitro.Methods:Bone marrow samples were aspirated from Chinese mini-swine. The mononuclear cells were isolated by density gradient centrifugation through1.077g/ml Percoll and then cultured in high-glucose DMEM containing10%fetal bovine serum. After primary culture, relatively purified MSCs were obtained by using the method of adherence screening. MSCs at passage1,2,3,4and5were examined for their proliferation and differentiation ability by immunocytochemistry, Real Time-PCR, ELISA and transmission electrical microscopy. MSCs at passage1were induced by5-aza, myocardium lysate,5-aza and myocardium lysate respectively. They were examined by the techniques above at1,2,3weeks after the treatments.Results:In normal growth medium, porcine bone marrow-derived MSCs were postivie for CD90and CD29, but negative for CD45. The growth property analysis demonstrated P1-P5MSCs showed fibroblast-like appearance. P4MSCs expressed cardiomyocyte-specific genes and VEGF, and higher than any other passage (P<0.001). The P1MSCs showed active proliferation with the treatment of myocardium lysate. After induced by5-aza and myocardium lysate2weeks, the PI MSCs showed the highest differentiation rate than any other group, and had the highest expression of cardiomyocyte-specific genes and VEGF. All of the3groups of PI MSCs showed senescent by the3weeks after the treatments.P1-P5MSCs cultured in normal growth medium had cardiomyocyte-like ultrastructure such as myofilaments, and no significant difference was found. P1MSCs induced by5-aza and myocardium lysate for2weeks had abundance myofilaments and non-specific junctions had formed. No transverse striations, intercalated discs were observed.Conclusion:Cardiomyogenic potential of porcine bone marrow-derived MSCs spontaneously in vitro is dependent upon the endogenous gene expression and passage-restricted pattern. The expression of VEGF may be associated to the differentiation and growth condition of MSCs. Myocardium lysate may be a better medium for the differentiation of MSCs into cardiomyocyte.