Self-organizing human heart organoids with an autologous tissue-resident macrophage immune niche

Congenital heart defects (CHDs) are the most common birth defect in humans, accounting for 1% of all births per year in the United States. The exact pathogenesis of CHDs remains largely unknown due to several reasons: complex interaction of genetic and environmental factors, lack of appropriate animal models, and the inability to directly study human heart development. Our group recently developed an advanced, three-dimensional (3D) human heart organoid (hHO) system derived from human pluripotent stem cells (hPSCs) (Lewis-Israeli et al., Nat Comm, 2021) to address this gap. The hHOs recapitulated human heart development to a significant extent and could be used for disease modeling maternal diabetes-induced CHDs. Although our current hHO system mimics many aspects of the developing human heart, it still lacks some key features, such as embryonic tissue-resident macrophages (MPs). In mice, MPs contribute to electrical conductance, lymphatic channel development, and endothelial network patterning, however their role in human heart development remains poorly understood. We hypothesize that MPs are important contributors to heart development in humans, as they are present in embryonic human hearts, and we investigated their role in a novel in vitro hHO with integrated MPs (hHMOs). We created a stepwise protocol to generate hHOs with integrated MPs from human pluripotent stem cells. We used immunofluorescence confocal microscopy (IF) to visualize MPs within the hHOs and flow cytometry to confirm their presence in the hHOs over 60 days. We also utilized single cell RNA sequencing (scRNAseq) data of hHMOs to identify cell population and gene expression changes over time. scRNAseq demonstrated expansion of cardiomyocyte populations and notable decreases in other cell populations in the hHMOs, such as conductance cells, stromal cells, and epicardial cells. The hHMOs recapitulate early aspects of immune cell integration in the developing heart and can be used to identify developmental changes induced by resident MPs. Overall design: Human heart organoids (hHOs) derived from H9-hESCs. hHOs were sampled based on inclusion (hHMOs) or exclusion of macrophages (hHOs) and the day of development: Day 15 hHO, Day 15 hHMO, Day 26 hHO, and Day 26 hHMO. Cells from these samples were dissociated using a previously described protol using cardiomyocyte dissociation medium and analyzed with scRNAseq.