Unveiling fusion between bone marrow-derived cells and Purkinje cells: Patch-Seq analysis in a mouse model of multiple sclerosis

Neurodegenerative diseases pose significant challenges for society due to limited treatments. Among the potential therapies, cell therapy stands out as a particularly promising one. We know that bone marrow-derived cells (BMDCs) can infiltrate the brain and transdifferentiate to different cell types, or fuse, for example, with Purkinje cells. These fusion events are unusual, but they increase in some pathologies, such as multiple sclerosis. In the Experimental Autoimmune Encephalomyelitis (EAE), a mouse model of multiple sclerosis, fused cells are functional and maintain two transcriptionally independent nuclei. This suggests that BMDCs might have a therapeutic potential, e.g. as vehicles for the expression of therapeutic agents. Nevertheless, fusion mechanisms and the characteristics of fused cells are still unclear.We developed a protocol to study cell fusion events in EAE mice. We first transplanted mice with GFP-positive BMDCs to identify fused cells. Then, we optimized the time window between the transplant, EAE induction, and sacrifice. Once optimized, we used Patch-Seq technology to compare individual fused Purkinje cells and non-fused neighbours. Cells were recorded, filled with biocytin for morphological studies, and their cytoplasm was collected for single-cell RNASeq analysis.Using this multimodal approach, we could assess functional, morphological, and gene expression differences in fused and non-fused cells to unravel the cell type responsible for these events and possible fusion mechanisms.Support: MIU (FPU2020); MICINN (PID2022-140456NB-I00) (PID2022-140525NB-I00); USAL, FENS/IBRO-PERC Exchange Fellowship, Programa Interreg VI-AEspaña-Portugal (POCTEP) 2021-2027 (Cross-3DTool-4ALS).Contact: pabgonses@usal.es, jorgevalero@usal.es.