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

Molecular Therapy: Nucleic Acids (Sep 2023)

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

Affiliations

Britt Hanson: Department of Paediatrics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK; Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK
Ioulia Vorobieva: Department of Paediatrics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK; Institute of Developmental and Regenerative Medicine, University of Oxford, IMS-Tetsuya Nakamura Building, Old Road Campus, Roosevelt Dr, Headington, Oxford OX3 7TY, UK
Wenyi Zheng: Department of Laboratory Medicine, Karolinska Institutet, Huddinge SE-141 86, Sweden
Mariana Conceição: Department of Paediatrics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK; Institute of Developmental and Regenerative Medicine, University of Oxford, IMS-Tetsuya Nakamura Building, Old Road Campus, Roosevelt Dr, Headington, Oxford OX3 7TY, UK
Yulia Lomonosova: Department of Paediatrics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK; Institute of Developmental and Regenerative Medicine, University of Oxford, IMS-Tetsuya Nakamura Building, Old Road Campus, Roosevelt Dr, Headington, Oxford OX3 7TY, UK
Imre Mäger: Department of Paediatrics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK; Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK
Pier Lorenzo Puri: Sanford Burnham Prebys Medical Discovery Institute, Development, Aging and Regeneration Program, La Jolla, CA 92037, USA
Samir El Andaloussi: Department of Laboratory Medicine, Karolinska Institutet, Huddinge SE-141 86, Sweden
Matthew J.A. Wood: Department of Paediatrics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK; Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK; Institute of Developmental and Regenerative Medicine, University of Oxford, IMS-Tetsuya Nakamura Building, Old Road Campus, Roosevelt Dr, Headington, Oxford OX3 7TY, UK; MDUK Oxford Neuromuscular Centre, South Parks Road, Oxford OX3 7TY, UK
Thomas C. Roberts: Department of Paediatrics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK; Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK; Institute of Developmental and Regenerative Medicine, University of Oxford, IMS-Tetsuya Nakamura Building, Old Road Campus, Roosevelt Dr, Headington, Oxford OX3 7TY, UK; MDUK Oxford Neuromuscular Centre, South Parks Road, Oxford OX3 7TY, UK; Corresponding author. Thomas C. Roberts, Institute of Developmental and Regenerative Medicine, University of Oxford, IMS-Tetsuya Nakamura Building, Old Road Campus, Roosevelt Dr, Headington, Oxford OX3 7TY, UK.

Journal volume & issue: Vol. 33
pp. 511 – 528

Abstract

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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.

Published in Molecular Therapy: Nucleic Acids

ISSN: 2162-2531 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Medicine: Therapeutics. Pharmacology
Website: https://www.cell.com/molecular-therapy-family/nucleic-acids/latest-content

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