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Tomas Minelli, Michael J Young, Petr Baranov; Differentiation and transplantation of IPSc derived retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1373.
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© ARVO (1962-2015); The Authors (2016-present)
Optic neuropathies lead to irreversible retinal ganglion cell (RGC) death, followed byprogressive vision loss. The transplantation of donor RGCs into the retina may someday be a viable therapeutic option and proof of concept studies have demonstrated that it is possible to isolate RGCs from the developing retina and deliver them into the eye. However, translation of this approach to the clinic requires a more robust and scalable approach for cell manufacture. Here we have developed a rapid and scalable method for in vitro RGC differentiation from induced pluripotent stem cells (iPSC) using a 3D culture protocol.
Thy1-GFP IPSCs were cultured in conical low attachment 96 well plates to form anaggregate. Optic vesicle formation was induced with the addition of Matrigel® to the medium at day 2 and the culture was kept in low serum medium for 9 days. After day 9 the medium was replaced for a serum free medium containing NS21 that was changed every 3 days. Cells were collected at day 16 and sorted using fluorescence activated cell sorting. We compared the efficiency of differentiation starting with 1500 cells vs. 15000 cells. RT-PCR and flow cytometry was performed to study retinal and RGC marker expression at different timepoints of differentiation. For transplantation 10,000 cells were delivered intravitreally into normal B6 mice. Eyes were enucleated and analyzed 2 weeks after injection.
Aggregates formed optic vesicle/cup structures in both conditions and cells generatedafter 16 days of differentiation expressed markers specific for RGCs (Math5, Isl1, Brn3a). The yield of Thy1-GFP+ cells collected was not significantly different between two conditions (3.2% vs 1.5%, in 15000 cells/well vs. 1500 cells/well, p=0.33). Thy1-GFP cells replated after dissociation acquired neuronal morphology and grew processes. We were also able to identify donor Thy1-GFP cells for at least 2 weeks after the transplantation with characteristic neurite- like processes.
Here we show that RGCs can be differentiated and isolated from mouse inducedpluripotent stem cells. Our transplantation work warrants further studies in animal models of optic neuropathies including glaucoma and optic nerve damage.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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