Fig 1: MSC and EV characterization. Cultured MSCs. (A) adhere to plastic and can be differentiated into adipogenic, chondrogenic and osteogenic lineages (scale bar 100 µm). They (B) express the essential surface markers CD73, CD90 and CD105 and lack CD11b, CD14, CD34 and CD45, all required to be absent from MSC, as assessed by flow cytometry (specific marker in blue, isotype control in red). Isolated sEVs are enriched in (C) Flotillin-1 and depleted of the nuclear marker protein Lamin A/C and the mitochondrial proteins ATP5a and TOM20. GAPDH was used as a loading control. The (D) modal size of sEVs is 149 nm, as demonstrated by NTA, and (E) sucrose density gradient analysis followed by immunoblotting of fractions indicates that sEVs have a density of 1.13–1.14 g/ml (density of each fraction and bottom fraction [Bott.] indicated).