GENERATION OF HEART ORGANOIDS MODELING EARLY HUMAN CARDIAC DEVELOPMENT UNDER DEFINED CONDITIONS
Cardiovascular-related disorders are a significant worldwide health problem. Cardiovascular disease (CVD) is the leading cause of death in developed countries, making up a third of the mortality rate in the US1.  Congenital heart defects (CHD) affect ~1% of all live births2, making it the common birth defect in humans. Current technologies provide some insight into how these disorders originate but are limited in their ability to provide a complete overview of disease pathogenesis and progression due to their lack of physiological complexity. There is a pressing need to develop more faithful organ-like platforms recapitulating complex in vivo phenotypes to study human development and disease in vitro. Here, we report the most faithful in vitro organoid model of human cardiovascular development to date using human pluripotent stem cells (hPSCs). Our protocol is highly efficient, scalable, shows high reproducibility and is compatible with high-throughput approaches. Furthermore, our hHOs showed very high similarity to human fetal hearts, both morphologically and in cell-type complexity. hHOs were differentiated using a two-step manipulation of Wnt signaling using chemical inhibitors and growth factors in completely defined media and culture conditions. Organoids were successfully derived from multiple independent hPSCs lines with very similar efficiency. hHOs started beating at ~6 days, were mostly spherical and grew up to ~1 mm in diameter by day 15 of differentiation. hHOs developed sophisticated, interconnected internal chambers and confocal analysis for cardiac markers revealed the presence of all major cardiac lineages, including cardiomyocytes (TNNT2+), epicardial cells (WT1+, TJP+), cardiac fibroblasts (THY1+, VIM+), endothelial cells (PECAM1+), and endocardial cells (NFATC1+). Morphologically, hHOs developed well-defined epicardial and adjacent myocardial regions and presented a distinct vascular plexus as well as endocardial-lined microchambers. RNA-seq time-course analysis of hHOs, monolayer differentiated iPSCs and fetal human hearts revealed that hHOs recapitulate human fetal heart tissue development better than previously described differentiation protocols.  hHOs allow higher-order interaction of distinct heart tissues for the first time, and display biologically relevant physical and topographical 3D cues that closely resemble the human fetal heart. Our model constitutes a powerful novel tool for discovery and translational studies in human cardiac development and disease. Overall design: RNA-seq was performed on human iPSC-derived heart organoids and monolayer differentiated cardiomyocytes to determine global gene expression changes and developmental committment