Size‐Modulated Mesoderm‐Endoderm Divergence and Myocardial Cavitation in Micropatterned Cardioids

ABSTRACT 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, while mesoderm‐endoderm crosstalk remain incompletely understood. 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. scRNA‐seq with 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. We found that micropattern sizes influenced cellular composition, cardioid cavitation, contractile functions, and mesoderm‐endoderm signaling crosstalk. The cardioids generated from 600 µm diameter circle patterns showed larger cavity formation resembling early heart chamber formation. Our findings establish micropatterned cardioids as a model for mesoderm‐endoderm co‐development, enhancing our understanding of heart‐foregut synergy during early embryogenesis.