Analysis of extracellular vesicles from subpopulations of mesenchymal stromal cells

The scientific study of extracellular vesicles (EVs) frequently involves the analysis of EVs secreted from heterogeneous, non-clonal cell cultures. Therefore, within population, EV variability is not accounted for, restricting our understanding of EV biology and their potential therapeutic applications. Previous work identified two distinct subtypes of mesenchymal stromal cells (MSCs) following immortalisation and isolation of single cell-derived colonies; Y201 MSCs (differentiation- competent, CD317-negative) and Y202 MSCs (differentiation-incompetent, CD317-positive). Here, EVs derived from Y201 and Y202 MSCs were examined to identify cell subtype-specific EV identities. Y201 vs Y202 EVs had similar modal sizes and morphological features by nanoparticle tracking analysis, transmission electron microscopy and image analysis. Using western blotting, Y201 EVs were more abundant in Flotillin-1, Alix, CD63 and CD81 than Y202 EVs, and bioinformatic miRNA analyses identified 9 upregulated miRNAs in Y201 EVs versus 2 in Y202 EVs. Similar variations were also identified in different MSC clones (Y101 and Y101.5). Therefore, MSC subtypes secrete morphometrically similar EVs with different miRNA and protein profiles. Using fluorescent tracking, it was shown that CFSE-labelled EVs from Y201 MSCs were taken up by Y202 MSCs and stimulated proliferation and migration. Y201 EVs also significantly increased the proliferation of articular chondrocytes in a dose-dependent manner determined by CyQuant, live imaging and single cell tracking (ptychography; cell counts, confluency, dry mass and doubling times, p<0.0001, 25% increase compared to control). The uptake and the phenotypic changes induced by Y201 EVs were inhibited following exposure to RGD- blocking peptide, indicating that their bioactivity is dependent on RGD-integrin function. Conversely, Y202 EVs were without effect. These findings demonstrate the need to develop acellular regenerative therapies using defined EV populations and cell sources to optimise clinical outcome.