Multicellular Perturb-seq Reveals Lineage-Specific Transcriptional Control During Human Cardiac Organoid Development

Heart development requires the coordinated differentiation of multiple cell lineages, including cardiomyocytes (CMs) and endothelial cells (ECs). Transcription factors (TFs) orchestrate lineage specification, morphogenesis, and maturation, yet how cardiac and vascular TFs shape multicellular organization and their interactions remain poorly understood. Many TFs exhibit pleiotropic and stage-dependent effects across cardiac lineages, particularly in cardiac progenitor cells, suggesting the presence of shared regulatory networks that synchronize cardiac and vascular programs during development and in response to stress. Human induced pluripotent stem cell (hiPSC) differentiated cardiac organoid provides a physiologically relevant 3D model to study these multicellular interactions, enabling simultaneous differentiation of ECs and CMs within a human genetic context. When combined with CRISPR interference (CRISPRi) based Perturb-seq, this system allows systematic, high-throughput dissection of TF-driven regulatory programs at single-cell resolution. In this study, we investigated how cardiac and vascular TFs coordinate lineage commitment and the cross-lineage signaling in human cardiovascular differentiation.