Single-cell multimodal analysis reveals enhancer and transcription factor regulatory dynamics during human iPSC-derived cardiomyocyte differentiation.

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes are widely used to model cardiac development and disease, but directed differentiation produces heterogeneous cellular states whose regulatory mechanisms remain incompletely defined. Here, we used simultaneous high-throughput ATAC and RNA Expression with sequencing (SHARE-seq) to profile gene expression and chromatin accessibility during human iPSC-derived cardiomyocyte differentiation. Weighted nearest neighbor integration resolved sequential developmental cell states, including iPSCs, mesodermal cells, progenitor populations, cardiomyocytes, and off-target, mixed, or partially differentiated populations. Peak-to-gene linkage analysis nominated cell state-associated putative enhancer-gene relationships, including a cardiac-related regulatory linkage at the MYH6/MYH7 locus. Pseudotime analysis revealed progressive remodeling of transcriptional and chromatin-associated regulatory programs. In silico perturbation modeling further prioritized transcription factors (TFs) predicted to influence differentiation trajectory progression, and gene regulatory network inference identified candidate TF-centered modules associated with cardiac lineage progression. Comparative pseudotime analysis of these modules showed that motif-containing regulatory element accessibility appeared to emerge earlier than, or in parallel with, TF expression and downstream target module activation. Together, these findings provide a single-cell multimodal resource and analytical framework for linking cellular identity, chromatin accessibility, putative enhancer-gene relationships, and candidate transcription factor regulatory programs during human cardiomyocyte differentiation.