Enhancing human pluripotent stem cell differentiation to cardiomyocytes through cardiac progenitor reseeding and cryopreservation.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have the potential to transform the understanding of heart development and heart failure treatment. However, hPSC-CM differentiation efficiency is plagued by batch-to-batch and line-to-line variability. Here, we describe a method to improve CM purity by 10-20% (absolute) without negatively affecting contractility, sarcomere structure, multinucleation, junctional Cx43, or CM number by detaching and reseeding progenitors between the EOMES+ mesoderm and ISL1+/NKX2-5+ cardiac progenitor stages. Moreover, we demonstrate that EOMES+ mesoderm and ISL1+/NKX2-5+ cardiac progenitors are cryopreservable with similar improvements in CM purity after resuming differentiation, facilitating storage of large batches of hPSC-CM progenitors for on-demand CM production. Reseeding during differentiation also enables transition to defined extracellular matrices, including fibronectin, vitronectin, and laminin-111, which all supported hPSC-derived EOMES+ mesoderm and ISL1+/NKX2-5+ cardiac progenitor differentiation to CMs. In summary, we present a method to increase hPSC-CM differentiation purity and demonstrate that specific CM progenitors are amenable to cryopreservation.