Human Cardiac Organoids Reveal HAND1 Loss-Associated Alterations in Cell Composition During Early Cardiac Differentiation

Understanding early cardiac developmental abnormalities requires linking tissue phenotypes to the cellular populations produced during differentiation. We established a cardiac organoid platform derived from human induced pluripotent stem cells (hiPSCs) and combined phenotypic assays, time-course and day-9 single-cell transcriptomics, and cleavage under targets and tagmentation (CUT&Tag) to investigate developmental perturbations. Organoids contained multiple cell populations and exhibited stage-dependent cardiac transcriptional programs. Thalidomide exposure altered morphology and reduced TNNT2-positive tissue area, with lower calcium transient amplitude at 0.01 μg/mL. RNA sequencing at 10 μg/mL identified downregulation of heart and neural crest derivatives expressed 1 (HAND1), prompting a separate genetic investigation. Across three paired differentiation experiments using one characterized HAND1-knockout clone, reduced cardiomyocyte representation accompanied relative enrichment of mesodermal/mesenchymal-like cells. Selected cardiac-associated expression abnormalities persisted at day 15. Expression and chromatin occupancy analyses identified candidate regulatory associations, with locus-specific quantitative PCR supporting HAND1 occupancy at selected cardiac-associated regions. Independent genetic and functional validation is needed to test causal relationships. Together, these findings place HAND1 loss-associated tissue abnormalities in their cellular context and identify candidate occupied regions for investigating early cardiac differentiation in a human three-dimensional model.