Abstract Body: The human heart, originating from the splanchnic mesoderm, is the first functional organ to develop, co-evolving with the foregut endoderm through reciprocal signaling. Previously, cardioid models offered new insights on cardiovascular cell lineages and tissue morphogenesis during heart development. The presence of endodermal derivatives in cardioids further underscores the importance of mesoderm-endoderm interactions. However, precise lineage divergence of heart-foregut within cardioids has not been well established, underscoring the need for further research into their complex interplay during heart-foregut development. Here, we integrated micropatterned cardioids, CRISPR-engineered reporter hiPSCs, deep-tissue imaging, and single-cell RNA sequencing (scRNA-seq) to explore synergistic mesoderm-endoderm co-development.

Using surface micropatterning techniques, we generated cardioids from circular patterns with different sizes (200 µm, 600 µm, 1000 µm in diameters). Our scRNA-seq dataset contained 600µm micropatterned samples at seven timepoints spanning a 21-day period of cardiac differentiation. PHATE trajectory mapping reconstructed lineage bifurcations of mesoderm-heart and endoderm-foregut lineages, identifying key cell types in cardiac and hepatic development. Ligand-receptor interaction analysis highlighted mesodermal cells enriched in non-canonical WNT, NRG, and TGF-β signaling, while endodermal cells exhibited VEGF and Hedgehog activity. For deep tissue imaging inside the cardioids, we used two hiPSC lines with fluorescent reporters for cardiomyocytes (TNNI-GFP and ACTN-GFP) to generate the cardioids, which were then processed by tissue clearance procedures prior to immunostaining. The whole-mounted cleared cardioids were imaged intact using a two-photon microscope for deep tissue penetration. Z-projection of the resulting 3D images revealed a prominent population of cardiomyocytes, along with staining for foregut endodermal cells, epicardial cells, and endothelial cells. The cardioids generated from 600 µm diameter circle patterns showed larger cavity formation resembling early heart chamber formation.

We found that micropattern sizes influenced cellular composition, cardioid cavitation, contractile functions, and mesoderm-endoderm signaling crosstalk. Our findings establish micropatterned cardioids as a model for mesoderm-endoderm co-development, enhancing our understanding of heart-foregut synergy during early embryogenesis.