The Research On The Differentiation Of Human Umbilical Cord Derived Mesenchymal Stem Cells Into Cardiomyogenic Cells

BackgroundStem cells have become the focus of attention in the treatment of heart diseases in the recent years. Stem cells application in heart diseases and its mechanism have still kept unkown. There are many reports in stem cell differentiation of cardiomyocytes, but mostly focus on animal-derived mesenchymal stem cells and embryonic stem cells. Because of abundant sources, proliferation and differentiation ability, low immunogenicity, no ethical issues, HUCMSCs become a hot spot for myocardial differentiation. But vaisartan and HFHTE induce HUCMSCs have still not to be reported.ObjectiveTo explore the feasibility of valsartan and HFHTE induce the differentiation of HUCMSCs into cardiomyogenic cells in vitro, and whether interaction exists between the two.Methods1. Cell culture:Cells were cultured in growth medium(low sugar DMEM with10%FBS,100U/mL penicillin and100μg/mL streptomycin)under the condition of saturated humidity,37℃and5%CO2.The medium was changed every three days. When the conflucency reaches to80～90%,0.25%trypsin(pH7.4) was used to digest cells. Cells were passaged according to the proportion of1:2.2. Cell induction and differenitiation:HUCMSCs were induced by the following three ways:(1)the cells were incubated in growth medium with valsartan for24h, then change the medium into growth medium and continue culture to1W,2W and3W respectively;(2) the cells were incubated in growth medium with Human fetal heart tissue extracts(HFHTE) for3d, then change the medium into growth medium and continue culture to1W,2W and3W respectively;(3) the cells were incubated in growth medium with valsartan and HFHTE for3d, then change the medium into growth medium and continue culture to1W,2W and3W respectively.3. The expressions of Tbx5, GATA4, Nkx2.5, cTnl and cTnT were detected by RT-PCR, Western blotting and Immunocytochemistry.Results1. RT-PCR, Western blotting and Immunochemical results showed that HUCMSCs cultured in growth medium can express cardiac-related transcription factor Tbx5, GATA4and Nkx2.5, but can’t express cardiac specific genes ANP,(3-MHC, cTnl and cTnT.2. After valsartan induced HUCMSCs, the expressions of Tbx5, GATA4and Nkx2.5increased, then gradually decline, the peak of three transcription factors were all at1W. The expressions of cardiac specific proteins cTnI and cTnT were detected at3W, but had no expressions of ANP and β-MHC.3. After HFHTE induced HUCMSCs, the expressions of Tbx5, GATA4and Nkx2.5increased, then gradually decline, the peak of three transcription factors were all at2W. The expressions of cardiac specific proteins cTnI and cTnT were detected at3W, but had no expressions of ANP and P-MHC.4. After valsartan+HFHTE induced HUCMSCs, the expressions of Tbx5, GATA4and Nkx2.5increased, then gradually decline, the peak of Tbx5and Nkx2.5were all at1W, the peak of GATA4was at2W. cTnI began to express at2W, then the expression increased and reached the peak at3W. The expression of cTnT were detected at3W, and ANP was weak express at2W and3W, but had no expression of β-MHC.ConclusionsHUCMSCs cultured in the growth medium can express cardiac-related transcription factor, but can not spontaneous differentiate into cardiomyogenic cells. Both valsartan and HFHTE can induce the differentiation of HUCMSCs into cardiomyogenic cells in vitro, and the interaction which exsits between the two can promote the differentiation process.