EV-mediated promotion of myogenic differentiation is dependent on dose , collection medium, and isolation method

Extracellular vesicles (EVs) have been implicated in the regula-tion of myogenic differentiation. C2C12 murine myoblast dif-ferentiation was reduced following treatment with GW4869 or heparin (to inhibit exosome biogenesis and EV uptake, respec-tively). Conversely, treatment with C2C12 myotube-condi-tioned medium enhanced myogenic differentiation. Ultrafiltra-tion-size exclusion liquid chromatography (UF-SEC) was used to isolate EVs and non-EV extracellular protein in parallel from C2C12 myoblast-and myotube-conditioned medium. UF-SEC-purified EVs promoted myogenic differentiation at low doses (%2 x 108 particles/mL) and were inhibitory at the highest dose tested (2 x 1011 particles/mL). Conversely, extracellular protein fractions had no effect on myogenic differentiation. While the transfer of muscle-enriched miRNAs (myomiRs) has been proposed to mediate the pro-myogenic effects of EVs, we observed that they are scarce in EVs (e.g., 1 copy of miR-133a-3p per 195 EVs). Furthermore, we observed pro-myogenic effects with undifferentiated myoblast-derived EVs, in which myomiR concentrations are even lower, suggestive of a myomiR-independent mechanism underlying the observed pro-myogenic effects. During these investigations we identified technical factors with profound confounding effects on myogenic differentiation. Specifically, co-purification of insu-lin (a component of Opti-MEM) in non-EV LC fractions and polymer precipitated EV preparations. These findings provide further evidence that polymer-based precipitation techniques should be avoided in EV research.