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

Britt Hanson; Ioulia Vorobieva; Wenyi Zheng; Mariana Conceição; Yulia Lomonosova; Imre Mäger; Pier Lorenzo Puri; Samir El Andaloussi; Matthew J A Wood; Thomas C Roberts

doi:10.1016/j.omtn.2023.07.005

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

Mol Ther Nucleic Acids. 2023 Jul 15:33:511-528. doi: 10.1016/j.omtn.2023.07.005. eCollection 2023 Sep 12.

Authors

Britt Hanson^{1

2}, Ioulia Vorobieva^{1

3}, Wenyi Zheng⁴, Mariana Conceição^{1

3}, Yulia Lomonosova^{1

3}, Imre Mäger^{1

2}, Pier Lorenzo Puri⁵, Samir El Andaloussi⁴, Matthew J A Wood^{1

2

3

6}, Thomas C Roberts^{1

2

3

6}

Affiliations

¹ Department of Paediatrics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK.
² Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK.
³ Institute of Developmental and Regenerative Medicine, University of Oxford, IMS-Tetsuya Nakamura Building, Old Road Campus, Roosevelt Dr, Headington, Oxford OX3 7TY, UK.
⁴ Department of Laboratory Medicine, Karolinska Institutet, Huddinge SE-141 86, Sweden.
⁵ Sanford Burnham Prebys Medical Discovery Institute, Development, Aging and Regeneration Program, La Jolla, CA 92037, USA.
⁶ MDUK Oxford Neuromuscular Centre, South Parks Road, Oxford OX3 7TY, UK.

Abstract

Extracellular vesicles (EVs) have been implicated in the regulation of myogenic differentiation. C2C12 murine myoblast differentiation was reduced following treatment with GW4869 or heparin (to inhibit exosome biogenesis and EV uptake, respectively). Conversely, treatment with C2C12 myotube-conditioned medium enhanced myogenic differentiation. Ultrafiltration-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 × 10⁸ particles/mL) and were inhibitory at the highest dose tested (2 × 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 insulin (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.

Keywords: MT: Exploiting Extracellular Vesicles as Therapeutic Agents Special Issue; Opti-MEM; exosomes; extracellular vesicles; insulin; myogenic differentiation.