CD47-mediated purification of human ventricular cardiomyocytes derived from pluripotent stem cells

Derivation of electrophysiologically mature cardiomyocytes from human pluripotent stem cells (hPSCs) remains a prerequisite for effective cardiac modeling and preclinical drug evaluation. However, current differentiation protocols often produce heterogeneous cell populations with limited maturity. In this study, we developed a CD47-based fluorescence-activated cell sorting strategy to purify ventricular-like cardiomyocytes from three-dimensionally differentiated hPSC-derived spheroids. CD47⁺ cardiomyocytes exhibited highly consistent contractile behavior and enhanced structural maturation, as confirmed using immunocytochemistry for myosin regulatory light chain 2 ventricular/cardiac muscle isoform and α-actinin. Electrophysiological analysis using a patch clamp revealed that the majority of CD47⁺ cells displayed ventricular-like action potential waveforms, characterized by prolonged repolarization duration, elevated amplitude, and a reduced negative maximum diastolic potential. Drug responsiveness was assessed using multi-electrode array recordings. CD47-enriched cardiomyocytes demonstrated reproducible and dose-dependent field potential alterations in response to known cardiotoxic agents, including remdesivir and quinidine, and outperformed metabolically purified controls in sensitivity and inter-replicate consistency analysis. These results establish CD47 as a reliable surface marker for isolating mature ventricular-like cardiomyocytes from hPSCs. The method enables the generation of functionally robust cardiomyocyte populations suitable for in vitro cardiac research and pharmacological testing.