July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Analysis of extracellular vesicles released by Müller glial cells in vitro.
Author Affiliations & Notes
  • William Lamb
    University College London, London, United Kingdom
    NIHR Biomedical Research Centre for Ophthalmology, United Kingdom
  • Karen Eastlake
    NIHR Biomedical Research Centre for Ophthalmology, United Kingdom
    University College London, London, United Kingdom
  • Gareth Williams
    School of Pharmacy, UCL, United Kingdom
  • Peng Tee Khaw
    NIHR Biomedical Research Centre for Ophthalmology, United Kingdom
  • G. Astrid Limb
    NIHR Biomedical Research Centre for Ophthalmology, United Kingdom
    University College London, London, United Kingdom
  • Footnotes
    Commercial Relationships   William Lamb, None; Karen Eastlake, None; Gareth Williams, None; Peng Khaw, None; G. Astrid Limb, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 621. doi:
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      William Lamb, Karen Eastlake, Gareth Williams, Peng Tee Khaw, G. Astrid Limb; Analysis of extracellular vesicles released by Müller glial cells in vitro.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):621.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Transplantation of Müller glia stem cells into animal models of retinal degeneration results in protection of neurons and restoration of retinal function, despite no integration into the neural retina, suggesting that visual improvements may be due to extracellular communications. A hitherto under-appreciated mechanism of cell-cell signalling is via transfer of nano-sized, extracellular vesicles (EVs); enriched with various bioactive cargos. The aim of this work was to identify and characterise Müller-glia derived EVs, for subsequent evaluation of the neuroprotective potential of these organelles.

Methods : After obtaining confluent monolayers of the human Müller glia cell line, MIO-M1, media containing FBS was removed, and flasks were washed 3 times, followed by 48-hour incubation in serum-free medium at 37. EVs were purified from cell supernatants through sequential rounds of differential-centrifugation, and particles were characterized by size, proteomic content, and morphology, using nanoparticle tracking, western blotting, and transmission electron microscopy respectively. Total RNA was isolated and assessed for quality and concentration.

Results : Nanoparticle tracking analysis and TEM imaging indicated that Müller glia secrete a diverse array of membrane-bound vesicles, sized 40 to >1000 nm in diameter. Variations to the centrifugation protocol allowed for selection of homogenous ‘small EV’ preparations (110 ± 55 nm), or a more heterogeneous vesicle population, enriched with vesicles sized 280 ± 93 nm. Preparations were positive for markers of endocytic membranes (CD9, CD63, and ALIX) whilst being negative for proteins associated with cytosolic debris (calnexin, calreticulin). Müller-derived vesicles were abundant in RNA, of which the majority (~60%) was under 200 nucleotides in length.

Conclusions : This work demonstrates that Müller glia cells readily secrete diverse populations of extracellular vesicles. Analyses indicates that these contain bioactive molecules, including small RNAs, which potentially represent a novel mechanism by which Müller glia may exert their neuroprotective influence in the retina. Further studies, including MicroRNA sequencing, will elucidate the neuroprotective potential of signals contained within these organelles.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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