Human umbilical cord mesenchymal stem cell-derived small extracellular vesicles accelerate diabetic wound healing by reprogramming fibroblast subpopulations and delivering pro-regenerative cargos

Diabetic wound healing is complex and challenging. Human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (hUC-MSC-sEVs) play a crucial role in tissue repair, but their specific mechanisms in diabetic wounds remain unclear. hUC-MSC-sEVs were isolated via tangential flow filtration and size-exclusion chromatography. Fibroblast proliferation was assessed using the CCK-8 assay. In vivo, the therapeutic effectiveness of hUC-MSC-sEVs in diabetic wound healing was evaluated by measuring wound-closure rates and by conducting histologic analyses. Single-cell transcriptomic sequencing (scRNA-seq) and bulk RNA sequencing were exploited to elucidate the mechanisms by which hUC-MSC-sEVs mediated the healing of diabetic wounds. Finally, we implemented the Proximity Barcoding Assay (PBA) technology to characterize the sEV subpopulations. hUC-MSC-sEVs were isolated and shown to dose-dependently amplify fibroblast proliferation. In diabetic mice, topical application accelerated wound closure via expedited re-epithelialization, robust neovascularization, and immunomodulation. scRNA-seq revealed alterations in the skin microenvironment following sEVs treatment, identifying and validating via immunofluorescence the presence of four fibroblast subpopulations. Among these, Trps1 + fibroblasts were demonstrated to be the principal drivers of reparative lineage commitment through reprogrammed ligand–receptor crosstalk. PBA analysis resolved sEVs into 11 distinct subpopulations. Integrated bioinformatics highlighted a key ITGB1-enriched sEV subpopulation, whose interaction network was fibroblast-specific, with FLNA implicated as a key downstream signaling node in fibroblasts linking this sEV subpopulation to phenotypic modulation. Our study revealed that hUC-MSC-sEVs accelerated diabetic wound healing through a dual mechanism: by reprogramming fibroblast subpopulations and by delivering pro-regenerative cargos (via functionally distinct sEV subpopulations enriched with immunomodulatory and reparative factors). These findings elucidate the molecular and cellular basis for hUC-MSC-sEV efficacy and provide a novel theoretical foundation for EV-based therapies in diabetic wound repair. • hUC-MSC-sEVs promote diabetic wound healing by transforming fibroblast phenotypes. • Trps1 + fibroblasts promote wound repair and initiate cell fate differentiation. • PBA analysis reveals ITGB1+ sEV mediate fibroblast-specific regenerative signal.