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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE280807 | GEO | 2025/11/14

REPOSITORIES: GEO

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Project description: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.

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

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