The optimized method for mesenchymal stem cell differentiation into pacemaker-like cell

Background: Mesenchymal stem cells (MSCs) differentiate into pacemaker-like cells by activating TBX3 and downregulating Pitx2c by inhibiting the nodal signaling pathway. This optimization study aimed to determine the optimal conditions for differentiating MSCs into pacemaker-like cells. Methods: The differentiation of MSCs into pacemaker-like cells was carried out in three ways: transfecting TBX3 pcDNA into MSCs, inhibiting the nodal signaling pathway using the small molecule SB431542, and a combination of both methods. This optimization study was conducted in two batches. The first batch was an experiment with transfection carried out on the 5th day of differentiation and addition of 20 μM/mL of the small molecule SB431542. The second batch was an experiment with transfection carried out on the 3rd day of differentiation and the addition of 2 μM/mL of the small molecule SB431542. From the two batches, the expression patterns of TBX3, Cx30.2, Cx40, Cx43, HCN4, HCN1, HCN3, and KCNN4 genes were analyzed using the qRT-PCR method. Results: The results showed that the second batch had a better gene expression pattern in pacemaker-like cells than the first batch. These results were indicated by the increased expression of the TBX3, Cx30.2, HCN4, HCN1, HCN3, and KCNN4 genes. Furthermore, there was a decrease in Cx40 and Cx43 expression levels. Conclusion: The results showed that TBX3 transfection on the third day and a smaller dose (2 μM/mL) of small molecules and combination treatment induced differentiation from the initial MSC, leading to the expression of pacemaker-like cells.