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Weixin Wang, Karen Eastlake, Celia Murray-Dunning, Erika Aquino, Peter J Coffey, G. Astrid Limb; Müller glia isolated from human iPSC/ESC derived retinal organoids produce neuroprotective antioxidants against oxidative stress. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1495.
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© ARVO (1962-2015); The Authors (2016-present)
Intravitreal transplantation of Müller cells into animal models of retina degeneration has shown that these cells partially restore visual function without integration into the neural retina. This effect has been ascribed to the neuroprotective effects and metabolic support of Müller glia to retinal neurons. They produce antioxidant molecules to protect neurons against oxidative stress and glutamate toxicity. Because of the need to produce Müller cell populations that can be used therapeutically, we have isolated Müller glia from retinal organoids formed by human iPSC/ESC, examined the antioxidant properties of these cells and compared them with the human Müller cell line MIO-M1.
Retinal organoids were derived from human ESC line Shef6 and iPSC line BJ using slight modifications of method published by Nakano et al., 2012. Müller glia were isolated by papain dissociation at various time points after initiation of organoid differentiation and propagated in vitro on fibronectin coated plates. The identity of these cells was confirmed by mRNA and protein expression of known Müller glia markers using RT-PCR, Western blotting and immunocytochemistry. Gene expression of various antioxidants produced by Müller glia was examined by RT-PCR. Quantification of antioxidants PRD6, PON2 and HO1 released by cells in culture was performed by ELISA.
The results showed that purified populations of Müller glia could be obtained from human iPSC/ESC derived retina organoids. These cells expressed Müller markers including CRALBP, Nestin, Vimentin, Glutamine synthetase, Sox9, Pax6 and Notch1 as confirmed by RT-PCR, Western blotting and immunostaining. They consistently expressed mRNA encoding the atioxidants PRD6, FOHL1, CQ10A, GST, GSR, LIAS and HO1. SOD2 mRNA was not detected in organoid-derived Müller glia but was observed in MIO-M1 cells. Interestingly, ELISA data showed that organoid-derived Müller glia released high levels of the antioxidants PRD6 and HO1 into the culture supernatant, whilst PON2 was not detected.
Our findings demonstrate that retinal organoid derived Müller glia display potent antioxidant ability as judged by the gene expression of antioxidants and the release of antioxidants into culture supernatant. These cells hold promising potential for in vivo transplantation to repair the neural retina with the neuroprotective antioxidants.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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