Transdifferentiation Of Bone Marrow Mesenchymal Stem Cells Into Cardiomyocyte-like Cells By Non-direct Contact With Cardiomyocytes And Temporal Expression Of Regulative Genes

Objective To investigate transdifferentiation of bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes and the temporal expression of regulative genes in the simulated myocardial micro-environment in which MSCs were co-cultured with cardiomyocytes by non– direct contact in vitro and screen some important genes in this differentiation.Methods MSCs isolated from adult rats were cocultured with CMs obtained from neonatal rat ventricles at 1:5 ratio in a dual chamber dish separated by a semipermeable membrane for 2 weeks. During this non-direct contact coculture procedure,cardiomyogenic differentiation was confirmed by phase contrast microscope and immunostaining againstα-actin and cardiac troponin-T(cTnT), and reverse transcriptase-polymerase chain reaction was performed for the temporal expression of regulative genes TGF-β,Nkx-2.5,GATA-4,MEF-2C and TEF-1.Results After coculture with CMs, MSCs showed a stick-like or elliptical morphology. Immunofluorescent stain results revealed thatα-actin and cardiac troponin T (cTnT) positive cells were found in MSCs at 5 days after coculture with CMs, and 9.20%,22.88%,28.89% of MSCs were positive forα-actin and 7.14%,22.43%,28.77% for cTnT at 5,7,14 days respectively. Results from RT-PCR demonstrated the expression level of TGF-β,Nkx-2.5,GATA-4 and MEF-2C genes began to increase at 1 day,reached the peak at 7 days after coculture and remained at relatively high afterwards, even though there was a slight drop; TEF-1 gene, on the other hand, had insignificant changes in expression level in our cocultures.Conclusion Non-direct contact with CMs (1:5) is conducive for MSCs to differentiate into CMs in vitro, and TGF-β, Nkx-2.5, GATA-4 and MEF-2C genes may play a crucial role during the transdifferentiation.