Functional Screening in Human Cardiac Organoids Reveals a Metabolic Mechanism for Cardiomyocyte Cell Cycle Arrest

Abstract

Background: The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of the adult. However, the key drivers of cardiomyocyte maturation and cell cycle arrest remain poorly defined. Methods: We developed a 96-well device for functional screening of human cardiac organoids to systematically screen for drivers of maturation and cell cycle arrest. Through interrogation of >10,000 cardiac organoids derived from human pluripotent stem cells, we systematically optimised parameters, including extracellular matrix, metabolic substrate and growth factor conditions that enhance cardiac tissue viability, reproducibility, function and maturation. Results: Under optimised maturation conditions, functional and molecular characterisation revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Additionally, under maturation conditions cardiomyocytes were refractory to mitogenic stimuli. We subsequently performed screening using a boutique library of kinase inhibitors and identified a small molecule capable of inducing cardiomyocyte cell cycle re-entry in mature human cardiac tissues and also in the adult mouse heart in vivo. Conclusions: These studies highlight that human organoids coupled with higher throughput screening platforms have the potential to rapidly expand our knowledge of cardiac biology and potentially unlock novel therapeutic strategies for cardiovascular disease.