Abstract
Extracellular vesicles (EVs) have been implicated in the regulation of myogenic differentiation. We observed that treatment of C2C12 murine myoblasts with either GW4869 (to inhibit exosome biogenesis) or heparin (to inhibit EV uptake) reduced myogenic differentiation. Conversely, conditioned media collected from differentiated C2C12 myotubes enhanced myogenic differentiation. Ultrafiltration-size exclusion liquid chromatography (UF-SEC) was used to isolate pure EV preparations and extracellular protein from C2C12 myoblast- and myotube-conditioned media in parallel. UF-SEC purified EVs promoted myogenic differentiation at low doses (≤2×108 particles/ml), had no effect at 2×1010 particles/ml, and inhibited myo<genic differentiation at the highest dose tested (2×1011 particles/ml). Similar effects were observed with both myoblast- and myotube-derived EVs. Given that muscle-enriched miRNAs (myomiRs) are largely absent in myoblast cultures, these findings are indicative of a myomiR-independent mechanism underlying the observed pro-myogenic effects. Indeed, individual myomiRs were found to be scarce in EVs (e.g. the most abundant myomiR, miR-133a-3p, was present at 1 copy per 195 EVs). UF-SEC-purified extracellular protein had no effect on myogenic differentiation when collected in serum-free DMEM. However, a potent pro-myogenic effect was observed when Opti-MEM was used as EV harvest media. Opti-MEM contains insulin, which was sufficient to recapitulate the pro-myogenic effect. Similarly, when EVs were isolated by polymer-based precipitation, a pro-myogenic effect was observed, but only when Opti-MEM was used as a collection media. These findings highlight Opti-MEM as a potential confounding factor, and provide further evidence that polymer-based precipitation techniques should be avoided in EV research.
Competing Interest Statement
MJAW and SEA are founders, shareholders and consultants for Evox Therapeutics, a biotech company that aims to commercialise Extracellular Vesicles. All other authors declare no conflicts of interest
Footnotes
Author Contact Information
Britt Hanson britt.hanson{at}paediatrics.ox.ac.uk +44 1865 272167
Mariana Conceição mariana.conceicao{at}paediatrics.ox.ac.uk +44 1865 272159
Yulia Lomonsova yulia.lomonosova{at}paediatrics.ox.ac.uk +44 1865 272159
Imre Mäger imre.mager{at}gmail.com
Pier Lorenzo Puri lpuri{at}sbpdiscovery.org
Samir EL Andaloussi samir.el-andaloussi{at}ki.se
Matthew J. A. Wood matthew.wood{at}paediatrics.ox.ac.uk +44 1865 272169
Thomas C. Roberts thomas.roberts{at}paediatrics.ox.ac.uk +44 1865 272159