Self-organizing human heart organoids with an autologous tissue-resident macrophage immune niche
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Aitor Aguirre
PROVIDER: MSV000096319 | MassIVE | Wed Nov 06 10:45:00 GMT 2024
SECONDARY ACCESSION(S): PXD057582
REPOSITORIES: MassIVE
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Project description: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.
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Project description:Self-organizing human heart organoids with an autologous tissue-resident macrophage immune niche
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Project description:The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs. The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.
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