Mapping fusion-driven cell reprogramming through integrative single-cell computational frameworks.

Cell fusion generates hybrid cells with unique traits. To understand the transcriptional and signaling alterations after fusion, we analyzed a published single-cell RNA-sequencing dataset of fused murine cardiomyocytes (mHL1) and mesenchymal stromal/stem cells (mMSC). Our analysis showed that fused cells exhibit a transcriptional trajectory suggesting a rapid change that stabilizes over time. We observed asymmetric plasticity. Initially, at Day 1, fusion hybrids resembled mMSCs (mesenchymal reprogramming), but by Day 3, their gene expression shifted to resemble mHL1 cells (myogenic reprogramming). Our analysis also identified distinct transcriptional subpopulations, including a subset enriched for tenascin (extracellular matrix remodeling), accompanied by dynamic changes in cell adhesion and intercellular communication. We also saw a significant shift in signaling pathways over time. At Day 1, Wnt and Melanogenesis (regenerative/antioxidant) signaling were downregulated. By Day 3, stress resistance and cellular adaptation pathways became enriched. Gene regulatory network analysis revealed key changes in master regulators; genes associated with chromatin remodeling (Hmga2), circadian rhythm (Arntl), and mesenchymal identity (Prrx1) became more active by Day 3. Collectively, our findings demonstrate that cell fusion is a dynamic reprogramming process, where evolving gene regulatory and signaling networks generate novel hybrid cell states, creating cellular diversity.