Multi-modal refinement of the human heart atlas during the first gestational trimester

Abstract

Understanding human cardiac development is essential to improving the diagnosis and treatment of congenital heart defects. Here, we present a multi-modal atlas of the developing human fetal heart during the critical first trimester. Using single-nucleus RNA sequencing, we sampled nearly 50,000 cardiac nuclei from three human fetuses at 8.6, 9.0, and 10.7 post-conceptional weeks (pcw). This dataset enabled distinction of 21 cell types, including novel contractile, conductive, and stromal cells. Lymphatic endothelial, epicardial and autonomic neural and glial cells were among the new, smaller populations for which we established high-resolution transcriptional profiles. We further integrated the snRNAseq data with published single-cell RNAseq data from hearts between 5 to 7 pcw. Combined trajectory analysis allowed us to identify a new human cardiomyofibroblast progenitor, preceding the segregation and diversification of cardiomyocyte and many stromal lineages. To refine cell-type annotations, we turned to spatial transcriptomics. Analysis of six Visium sections from two additional hearts was aided by deconvolution of spots with our snRNAseq data. We further confirmed key markers using in situ hybridization or immunofluorescence on sectioned or whole hearts, followed by standard or light-sheet confocal microscopy. Altogether, these complementary approaches permitted us to add anatomical-positional features such as chamber specificities, innervation and conduction system components, or subdomains of the atrioventricular septation complex. Each translates cell identity into specialized cardiac functions. A total of forty first-trimester hearts were studied to discover and validate cellular characteristics across a wide range of scales, from the individual nucleus to the entire organ. Our results offer a comprehensive resource for understanding the cellular dynamics of human cardiac development and lay the groundwork for further studies into the molecular mechanisms underlying congenital heart malformations. This atlas adds unprecedented spatial and temporal resolution to the characterization of human-specific aspects of early human heart formation.