FIGURE 1.

Basic characterization of human MSC-derived extracellular vesicles. (A) Dynamic Light Scattering (DLS) of cell-conditioned media (CCM) from murine MSCs revealed three sub-populations of EVs based on size: small, (∼5–10 nm) medium, (∼100–1000 nm) and large (5,000–10,000 nm), which likely represent exosomes, microvesicles, and apoptotic bodies, respectively. Thin lines represent individual trials (n = 3), thick lines represent averaged curves. (B) Analysis by one way ANOVA of average percent area under the curve on DLS data revealed a significantly smaller 1^st (exosome) peak and a significantly larger 2^nd (microvesicle) peak for both stimulated groups (* = p≤0.05). (C) Immunoblotting of whole cell lysate (WCL) and microvesicle (MV), exosome (EX), and vesicle free media (VFM) fractions of CCM. Ubiquitous marker HSP90 is present in WCL, EX, and VFM fractions. The EV marker flotillin is present in all fractions except VFM, and the cellular marker IκBα is absent from exosome (EX) and microvesicle (MV) fractions, as expected. MT protein ATP5A1 was found only in WCL, but COXIV (MT) was found in WCL and MVs. (D) Nanoparticle tracking analysis (NTA) of PBS (negative control), EX, and MV fractions indicates that EXs outnumber MVs by several orders of magnitude amongst particles less than 1μm, the maximum detection size of NTA.