September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
The effect of human embryonic stem cell-derived extracellular vesicles (hESEVs) on Müller cells and their potential use for retinal regeneration
Author Affiliations & Notes
  • Debora B Farber
    Ophthalmology, Stein Eye Institute/UCLA, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Debora Farber, None
  • Footnotes
    Support  Grant from The Vision of Children Foundation
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Debora B Farber; The effect of human embryonic stem cell-derived extracellular vesicles (hESEVs) on Müller cells and their potential use for retinal regeneration
      . Invest. Ophthalmol. Vis. Sci. 201657(12):.

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

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Abstract

Purpose : Human embryonic stem cells (hESCs) release into their culture medium heterogeneous populations of hESEVs that contain mRNAs, miRNAs and proteins. As important components of cell-cell communication, hESEVs transfer their contents to many different cell types, including Müller glial retinal progenitors cells. Our purpose is to demonstrate that hESEVs can be the signals that lead Müller cells to de-differentiate and re-entry the cell cycle, followed by differentiation into different retinal cell lineages.

Methods : Human H9 ESCs were cultured in serum- and feeder-free conditions in mTeSR™1 medium and human Müller cells in DME/F-12 1:1 medium supplemented with 10% FBS. hESEVs released from H9 ESCs were fractionated into microvesicles (MVs) and exosomes (EXOs) by differential ultracentrifugation, and vesicle size of each group was determined by dynamic light scattering and electron microscopy. Total RNA from MVs, EXOs and hESEVs were hybridized to human mRNA microarrays. Total RNA from human Müller cell cultures treated and non-treated with hESEVs or with MVs or EXOs was isolated and expression of mRNAs was compared by qRT-PCR. Proteins were analyzed by Westerns and immunocytochemistry.

Results : hESEVs are a heterogenous population of nanoparticles that range in size from 30 nm to 1 mm. Following fractionation, the MVs diameter varied from ~ 110 nm to 1mm and that of EXOs from 30 to 100 nm. Hybridization of total RNA from MVs, EXOs and hESEVs on mRNA microarrays showed very different profiles. MVs carry more mRNA than EXOs. hESEV treatment changed the level of Müller cell transcripts and corresponding proteins: i.e., pluripotency (Oct4 and Sox2) and early retinal genes (Pax6 and Rax). MV treatment of Müller cells showed similar results, but EXOs did not produce any change. After several treatments with hESEVs, cultured Müller cells decreased their level of vimentin and glutamine synthetase and increased those of OCT4 and PAX6. In addition, some Müller cells expressed marker proteins for ganglion (Brn3a) and amacrine (Calbindin) cells. Non-treated Müller cells show none of these proteins.

Conclusions : Only exposure to MVs changes the levels of some Müller cell mRNAs and induces the cells to de-differentiate and trans-differentiate into other retinal lineages. In vivo studies will establish whether MVs can regenerate damaged retinas.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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