Efficient and Safe Method of Generating Induced Pluripotent Stem Cells from Human Skin Fibroblasts and Subsequent Differentiation into Functional Cardiomyocytes.

Abstract

Studies have shown that human-induced pluripotent stem cells (iPSCs) derived cardiomyocytes (iCMCs) would provide a limitless source of cells for regenerative therapy and drug discoveries. Similar to embryonic stem cells, iPSCs have the capability to differentiate into mature functional iCMCs. The objective of our study is to develop an animal-free and viral-free approach by using a highly efficient transfection method that utilizes a critical combination of DNAs and mRNAs of pluripotent genes to generate iPSCs from adult human skin fibroblasts (SF). Subsequently differentiated them into functional cardiomyocytes. We obtained 4% of SFs into iPSCs at Passage 0, which shows significantly higher efficiency of reprogramming when compared to the use of either DNA alone or mRNAs alone. These iPSCs cultured under cardiac culture conditions are capable of differentiating into iCMCs. Furthermore, >88% of iCMCs are positive for either cardiac troponin T (TNNT2) or GATA binding protein 4 (GATA4). The iCMCs produced from SFs have been used in our laboratory to demonstrate their in vitro and in vivo functional potentials. In this study, we present step-by-step procedures for the generation of iPSCs from SFs and further differentiate them toward functional iCMCs.