Small extracellular vesicle-enriched preparations derived from genetically modified multipotent mesenchymal stromal cells promote fibroblast proliferation and extracellular matrix remodeling in vitro

Impaired skin repair is associated with insufficient fibroblast activity and dysregulated extracellular matrix turnover. Small extracellular vesicles (sEVs) derived from multipotent mesenchymal stromal cells (MMSCs) may provide a cell-free approach to modulate these processes; however, the effects of sEVs from genetically modified producer cells remain incompletely characterized. We evaluated sEV preparations obtained from native MMSCs and from MMSCs separately transfected with OCT4/SOX2, HIF-1alpha, or Klotho expression plasmids. A 1:1:1 mixture of sEV preparations from the three modified cell populations was examined as a combined treatment. Rat skin fibroblasts were exposed to the preparations for 24, 48, or 72 h. Fibroblast density and concentrations of matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), collagen type I, and collagen type III were assessed. The combined sEV preparation produced the largest observed increase in fibroblast density, reaching 112.63 +/- 5.55 x 10^3 cells/cm2 at 72 h, compared with 40.60 +/- 1.12 x 10^3 cells/cm2 for native MMSC-derived sEVs and 37.10 +/- 3.01 x 10^3 cells/cm2 in the untreated control. At 24 h, MMP-1 and MMP-3 concentrations in the combined group were 140.0 +/- 4.77 ng/mL and 144.0 +/- 4.74 ng/mL, respectively, compared with 283.1 +/- 25.2 ng/mL and 208.1 +/- 39.2 ng/mL in controls. Collagen type III concentration increased to 1.95 +/- 0.25 ng/mL at 72 h versus 1.15 +/- 0.15 ng/mL in controls. These findings indicate that the combined sEV preparation was associated with enhanced fibroblast proliferation and altered extracellular matrix remodeling in vitro. Further work is required to define sEV composition, dose-response relationships, and in vivo efficacy and safety.