A universal and robust integrated platform for the scalable production of human cardiomyocytes from pluripotent stem cells

Recent advances in the generation of cardiomyocytes (CMs) fromhumanpluripotent stemcells (hPSCs), in conjunctionwith the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy.We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precisemodulation of the differentiation process by smallmolecule activation ofWNT signaling, followed by inactivation of transforming growth factor-b andWNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CMspheroids containing virtually pure (∼90%) CMs in 10 days.Moreover, the developed differentiation strategywas universal,asdemonstratedbytestingmultiplehPSClines (5humanembryonic stem celland4humaninduciblePSClines)without cell sortingor selection.Theproducedh PSC-CMssuccessfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays.