DNA damage accrued in induced pluripotent stem cell (iPSC)-derived cardiomyocytes during in vitro culture practices lessens their therapeutic potential. We determined whether DNA-damage-free iPSCs (DdF-iPSCs) can be selected using stabilization of p53, a transcription factor that promotes apoptosis in DNA-damaged cells, and differentiated them into functionally competent DdF cardiomyocytes (DdF-CMs). p53 was activated using Nutlin-3a in iPSCs to selectively kill the DNA-damaged cells, and the stable DdF cells were cultured further and differentiated into CMs. Both DdF-iPSCs and DdF-CMs were then characterized. We observed a significant decrease in the expression of reactive oxygen species and DNA damage in DdF-iPSCs compared with control (Ctrl) iPSCs. Next-generation RNA sequencing and Ingenuity Pathway Analysis revealed improved molecular, cellular, and physiological functions in DdF-iPSCs. The differentiated DdF-CMs had a compact beating frequency between 40 and 60 beats/min accompanied by increased cell surface area. Additionally, DdF-CMs were able to retain the improved molecular, cellular, and physiological functions after differentiation from iPSCs, and, interestingly, cardiac development network was prominent compared with Ctrl-CMs. Enhanced expression of various ion channel transcripts in DdF-CMs implies DdF-CMs are of ventricular CMs and mature compared with their counterparts. Our results indicated that DdF-iPSCs could be selected through p53 stabilization using a small-molecule inhibitor and differentiated into ventricular DdF-CMs with fine-tuned molecular signatures. These iPSC-derived DdF-CMs show immense clinical potential in repairing injured myocardium.NEW & NOTEWORTHY Culture-stress-induced DNA damage in stem cells lessens their performance. A robust small-molecule-based approach, by stabilizing/activating p53, to select functionally competent DNA-damage-free cells from a heterogeneous population of cells is demonstrated. This protocol can be adopted by clinics to select DNA-damage-free cells before transplanting them to the host myocardium. The intact DNA-damage-free cells exhibited with fine-tuned molecular signatures and improved cellular functions. DNA-damage-free cardiomyocytes compared with control expressed superior cardiomyocyte functional properties, including, but not limited to, enhanced ion channel signatures. These DNA-intact cells would better engraft, survive, and, importantly, improve the cardiac function of the injured myocardium.
Keywords: DNA damage; iPS-CM; iPSCs; p53.