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Sankarathi Balaiya, Dominic Didiano, Claudia Bertalocini, Scott Hinger, Edward M Levine, James Patton; In vivo screen for extracellular vesicles that promote Muller glia proliferation and neurogenic activity in zebrafish and mice. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3107.
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© ARVO (1962-2015); The Authors (2016-present)
Extracellular vesicles (EVs), including exosomes, are released from all cells and are capable of intercellular signaling through the action of RNA, protein and lipid cargo. In zebrafish, but not mice, the retina regenerates by dedifferentiation of Muller glia and production of proliferating neural progenitor cells. Here we conducted a screen of EVs from various sources to test their ability to induce proliferation and ultimately neurogenic activity from zebrafish and mouse Muller glia.
EVs enriched for exosomes were prepped from over forty cellular sources, including neuronal, glial, epithelial, iPS, and carcinogenic lines for their capacity to induce proliferation in the zebrafish and mouse retina through the direct intravitreal injection. Post injection, retinas were evaluated for proliferation, marker expression, and morphological changes.
The majority of EVs induced modest proliferative responses in fish. The most consistent source of EVs were derived from C6 glioma cells showing 22.7 PCNA+ cells per section compared to 8.3 for vehicle control (P<0.0001, One-way ANOVA). We subjected C6 EVs to more rigorous purification using density gradient purification and found that these consistently produced the highest levels of proliferation. These preparations are being analyzed by RNAseq and mass spectrometry.In mice, most EVs induced proliferation as assessed by EdU incorporation. EdU+ cells were consistently observed far from the injection site and distributed from the outer plexiform layer to the ganglion cell layer. EdU incorporation in Muller glia was rare. Rather, EdU incorporation was often observed in cells of non-retinal origin (i.e. microglia, endothelium, pericyte). Current work is aimed at determining if Muller glia are responding to EV treatment by becoming reactive or migratory, and EV testing is being combined with injury models and genetic activation of Muller glial proliferation.
The majority of EV preparations induced proliferative effects, and EVs derived from C6 glioma cells consistently induced Muller glia to dedifferentiate and proliferate after intravitreal injection in otherwise undamaged fish. In mice, EV injection into the vitreous induced proliferative responses in cells of non-retinal origin and only rarely in Muller glia. Further work is needed to determine if mouse Muller glia will respond to EV treatments.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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