Mapping Fusion-Driven Cell Reprogramming Through Integrative Single-Cell Computational Frameworks

Cell fusion, a naturally occurring phenomenon between similar or dissimilar cell types, can generate hybrid cells with unique traits. To understand the transcriptional and signaling alterations after fusion, we analyzed a previously published single-cell RNA-sequencing dataset of fused murine cardiomyocytes (mHL1) and mesenchymal stromal/stem cells (mMSC). Using advanced computational tools, we identified commonalities in fusion products. Our analysis showed that fused cells manage a rapid change to stabilize over time. Consistent with previous findings, we observed asymmetric plasticity. Initially, at Day 1, fusion hybrids had gene expression similar to the parental mMSC cells, suggesting dominant mesenchymal reprogramming. However, by Day 3, their gene expression shifted to resemble the parental mHL1 cells, indicating a later switch to myogenic reprogramming. Beyond previous findings, our analysis also identified distinct transcriptional subpopulations. We found a subset of cells enriched for tenascin, indicative of active extracellular matrix remodeling of hybrids. This was accompanied by dynamic changes in cell adhesion and intercellular communication. We also saw a significant shift in signaling pathways over time. At Day 1, fusion populations exhibited downregulation of Wnt and Melanogenesis signaling, implying suppression of regenerative and antioxidant responses. By Day 3, pathways associated with stress resistance and cellular adaptation became enriched, suggesting the emergence of new functional traits. Gene regulatory network analysis via pySCENIC revealed key changes in 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 drives a dynamic and coordinated reprogramming process, where evolving gene regulatory and signaling networks generate novel hybrid cell states, highlighting fusion as a regulated mechanism for creating cellular diversity.