Transitioning from animal to human cell sources represents a critical milestone in cardiac tissue engineering and biomedical research. Neonatal rat ventricular myocytes (NRVMs) have long served as the functional benchmark for engineered cardiac tissues; however, their rodent origin limits clinical relevance. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer a renewable, species-specific alternative but remain restricted by immature structure and function, small-scale yield, and high batch variability by conventional two-dimensional monolayer (2D-Mono) differentiation. Here, we systematically evaluated hiPSC-CMs generated by 2D-Mono and three-dimensional embryoid-body (3D-EB) differentiation using identical 15-day Wnt-modulated protocols without additional maturation steps. The 3D-EB method yielded 181 × 106 cells per 100 ml, approximately 2.7-fold higher than the 2D-Mono, while maintaining >80% cTnT+ purity and reduced batch variability. Structural analyses revealed improved sarcomeric organization in 3D-EB tissues compared with 2D-Mono, although both remained less organized than NRVMs, while other morphological parameters were comparable between groups. Functionally, 3D-EB tissues exhibited faster calcium conduction (33.7 cm/s), indicating enhanced electrical coupling relative to 2D-Mono. Although contractile performance remained similar between differentiation formats and below NRVM levels, 3D-EB tissues exhibited consistent structural and functional improvement in calcium wave velocity and contractility over time. These results show that, even without external maturation cues, 3D-EB differentiation yields reproducible, scalable, and human-relevant cardiomyocytes.

Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and regeneration. Challenges with quality, inter-batch consistency, cryopreservation and scale remain, reducing experimental reproducibility and clinical translation. Here, we report a robust stirred suspension cardiac differentiation protocol, and we perform extensive morphological and functional characterization of the resulting bioreactor-differentiated iPSC-CMs (bCMs). Across multiple different iPSC lines, the protocol produces 1.2E6/mL bCMs with ~94% purity. bCMs have high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs, bCMs are more reproducible across batches and have more mature functional properties. The protocol also works with magnetically stirred spinner flasks, which are more economical and scalable than bioreactors. Minor protocol modifications generate cardiac organoids fully in suspension culture. These reproducible, scalable, and resource-efficient approaches to generate iPSC-CMs and organoids will expand their applications, and our benchmark data will enable comparison to cells produced by other cardiac differentiation protocols. Subject terms: Cardiovascular biology, Induced pluripotent stem cells, Cardiovascular models