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Background: Advancing structural and functional maturation of stem cell–derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions.Methods: We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell–derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM.Results: EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β₁- and β₂-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair.Conclusions: We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell–derived cardiomyocytes under defined, serum-free conditions.

Endoglin is a transmembrane accessory receptor for transforming growth factor‐β (TGF‐β) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo . Endoglin, as well as other TGF‐β signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor‐like kinase 1 (ALK1), an endothelial specific TGF‐β type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF‐β/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF‐β/ALK1 signalling, which indirectly inhibits TGF‐β/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF‐β/ALK1 signalling is reduced and TGF‐β/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF‐β‐induced growth arrest by indirectly reducing TGF‐β/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.

Deletion of the transforming growth factor β1 (TGF‐β1) gene in mice has previously suggested that it regulates both hematopoiesis and angiogenesis. To define the function of TGF‐β more precisely, we inactivated the TGF‐β type I receptor (TβRI) gene by gene targeting. Mice lacking TβRI die at midgestation, exhibiting severe defects in vascular development of the yolk sac and placenta, and an absence of circulating red blood cells. However, despite obvious anemia in the TβRI−/− yolk sacs, clonogenic assays on yolk sac‐derived hematopoietic precursors in vitro revealed that TβRI−/− mice exhibit normal hematopoietic potential compared with wild‐type and heterozygous siblings. Endothelial cells derived from TβRI‐deficient embryos show enhanced cell proliferation, improper migratory behavior and impaired fibronectin production in vitro , defects that are associated with the vascular defects seen in vivo . We thus demonstrate here that, while TβRI is crucial for the function of TGF‐β during vascular development and can not be compensated for by the activin receptor‐like kinase‐1 (ALK‐1), functional hematopoiesis and development of hematopoietic progenitors is not dependent on TGF‐β signaling via TβRI.