Promotion of Differentiating Bone Marrow Mesenchymal Stromal Cells (BMSCs) into Cardiomyocytes via HCN2 and HCN4 Cotransfection.

Abstract

Aim Investigation of the influences HCN2 and HCN4 has on bone marrow mesenchymal stromal cells (BMSCs) on cardiomyocyte differentiation. Methods Miniature adult pigs were used for bone marrow extraction and isolation of BMSCs. The identification of these BMSCs was done by using flow cytometry for the detection of expressed surface antigens CD45, CD11B, CD44, and CD90. Using HCN2 and HCN4 genes cotransfected into BMSCs as group HCN2+HCN4 while myocardial induction solution was used to induced BMSC differentiation in the BMSC induction group. Myocardial marker proteins α-actin and cTnT were detected by immunofluorescence staining, while α-actin, cTnT, and Desmin myocardial marker proteins expressed were detected by Western blot. The whole-cell patch-clamp technique was used to identify and detect cellular HCN2 channels, HCN4 channel current activation curve, and the inhibitory effect of CsCl on heterologous expression currents. Results Flow cytometry results showed that CD45 and CD11B were expressed negatively while CD90 and CD44 were positive. Post HCN2 and HCN4 gene transfection, immunofluorescence staining, and Western blot showed significantly increased HCN2, HCN4, α-actin, and cTnT expressed in group HCN2+HCN4 were, which could be compared to the expression levels in the BMSC-induced group. The HCN2+HCN4 group was able to document cell membrane channel ion currents that were similar to If properties. Conclusion HCN2 and HCN4 overexpression can considerably enhance the MSC ability to differentiate into cardiomyocytes in vitro and restore the ionic current.