DNA damage-free iPS cells exhibit potential to yield competent cardiomyocytes

Abstract

DNA damage accrued in iPS-derived cardiomyocytes (iPS-CMs) during in vitro culture practices lessens their clinical potentials for therapy. We determined whether DNA damage-free iPSCs (DdF-iPS) can be selected using stabilization of p53, transcription factor that promotes apoptosis in DNA damaged cells, and differentiate them into functionally competent DdF-cardiomyocytes (DdF-CM). 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 CM. Both DdF-iPSCs and DdF-CMs were then characterized. We observed a significant decrease in the expression of reactive oxygen species (ROS) and DNA damage in DdF-iPSCs compared to 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 to 60 beats per minute 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 to Ctrl-CMs. Enhanced expression of various ion channel transcripts in DdF-CMs implies DdF-CMs is of ventricular CMs and matured compared to its counterpart. Our results indicated that DdF-iPSCs could be selected through p53 stabilization using small molecule and differentiated into ventricular DdF-CMS with fine-tuned molecular signatures. These iPS cells derived DdF-CMs shows immense clinical potential in repairing injured myocardium.