The Research Of The Combination Of ISL-1 And Tbx18 In Differentiation Of Adipose-derived Stem Cells Into Pacemaker-like Cells

Objective: After transfecting lentiviruses overexpressing ISL-1 and Tbx18 into ADSCs,we observed the change of the phenotype and function of pacemaker-like cells and explored the possibility of transcription factors ISL-1 and Tbx18 constructing biological pacing in vitro.Methods: The 3-5 generation of ADSCs were randomly divided into five groups and were transfected virus: Blank,GFP,ISL-1,Tbx18 and ISL-1+Tbx18.By the fluorescence intensity and flow analysis,Selecting the optimal MOI to make subsequent experiments.ADSCs were co-cultured with NRVMs in vitro.The morphological changes of ADSCs were observed under microscope under co-culture,meanwhile the beat frequency of cells were recorded.The m RNA and protein expression levels of pacemaker cells after co-cultured for 5-7 days,and the changes of intracellular electrophysiological were tested by patch clamp.Results: The most amount of ADSCs became adherent with long-spindle shape.The optimum MOI value of transfection was 50 or 80.The morphological of ADSCs transfected ISL-1 and Tbx18 was changed and protruded cardiomyocyte-like pseudopodia,which mainly existed the three shapes of pindle-shaped,triangular and irregular.In co-culture system,transduced adipose stem cells formed connection with neonatal rats ventricular myocytes and pulsated synchronously.The expression levels of the specific sinoatrial node specific genes of HCN4,Cx45 and Tbx3 were up-regulated in cells transfected with ISL-1 or Tbx18,meanwhile the working myocardium specific gene of Nkx2.5 and Cx43 were down-regulated.The expression level of group ISL-1+Tbx18 was significantly higher than group ISL-1 and group Tbx18.There was a statistical significance between every two groups(P < 0.05).The immunofuorescence staining results revealed that cells transduced ISL-1 + Tbx18 were positive for c Tn T and HCN4 low expression but ADSCs transfected with GFP almost had not expression.The indirect currents of hyperpolarization activation were recorded in the experimental group ADSCs,and the inversion currents of hyperpolarization activation were recorded in more ADSCs of group ISL-1+Tbx18.Conclusion: ADSCs are successfully transformed into spontaneously beating cells that exhibit behavior similar to the pacemaker cells.Transcription factors ISL-1 and Tbx18 can respectively induce the differentiation of adipose stem cells into pacemaker-like cells.The combination of ISL-1 and Tbx18 can improve the productiveness of cardiac pacemaker-like cells from ADSCs.