Minimal Component, Protein‐Free, and Cost‐effective Human Pluripotent Stem Cell Cardiomyocyte Differentiation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become a powerful source for the in vitro modeling of cardiac diseases and various other essential applications, including cardiotoxicity screening and regenerative cell replacement therapies. Although many differentiation protocols have been developed to generate cardiomyocytes from human pluripotent stem cells, these protocols are costly and complex, requiring expensive and often unnecessary components (e.g., B27 medium supplement). In addition, the use of animal-derived growth factors limits their use for regenerative medicine purposes. To address these issues, herein, we have developed an efficient, cost-effective, and protein-free hPSC-CM protocol using only two components: DMEM/F12 basal medium and l-ascorbic acid 2-phosphate. By eliminating xenobiotic and complex components, the efficiency of directed differentiations is increased, the variability between cardiac differentiations is decreased, and the scalability of cell production is enhanced. Adaptation of this efficient, low-cost, and user-friendly cardiac differentiation protocol will enrich the utility and applicability of hPSC-CMs in drug discovery, cell therapies, tissue engineering, disease modeling, precision medicine, and cardiac regenerative medicine. © 2025 Wiley Periodicals LLC. Basic Protocol 1: hPSC cell culture Basic Protocol 2: hPSC-CM differentiation Basic Protocol 3: Characterization of hPSC-CMs by immunofluorescence (IF) imaging.