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Venkata R M Chavali, Vrathasha Vrathasha, Jie He, Yingrui Liu, Sergei S Nikonov, Brent A Bell, Katherine E. Uyhazi; Stem cell transplantation for treatment of retinal ganglion cell loss in Glaucoma. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1118.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is an optic neuropathy characterized by progressive death of retinal ganglion cells (RGCs) leading to permanent visual field loss. Existing treatments for glaucoma target lowering the intraocular pressure, laser trabeculoplasty, and surgical interventions. There are no treatments available once RGC death has occurred in the advanced stages of this disease. We investigated induced pluripotent stem cell derived RGC replacement as a therapy to treat for ganglion cell loss in glaucoma.
Human induced pluripotent stem cells (hiPSCs) were differentiated in vitro using a standardized two-step protocol in 36 days. iPSC-RGCs were characterized for RGC markers by FACS sorting with RGC specific antibodies. Mature iPSC-RGCs were labeled by transduction with AAV2.7m8 SNCG-eGFP. Purified and labeled iPSC-RGCs transplanted intravitreally (5 X 105 cells/2ul) into wild type 4-month-old C57BL/6J mice using a 33-guage needle (n=10). Survival of the transplanted hiPSC-RGCs was assessed at 2, 4 and 6-weeks post-transplantation using Micron III and confocal scanning laser ophthalmoscopy (cSLO). Histological studies were performed 2 and 5 months post-transplantation on retinal flatmounts, retinal and optic nerve sections to study localization and integration of the transplanted hiPSC-RGCs. Functional integration was assessed by electrophysiologic responses to full field photopic stimuli in voltage and current clamp modes under whole cell configuration.
hiPSC differentiation in vitro generates pure populations of iPSC-RGCs. Purified iPSC-RGCs are positive for RGC markers BRN3, SNCG, CD90, and RBPMS. iPSC-RGCs transduced with AAV2.7m8 express eGFP as early as 48-hour post-transduction. eGFP+ve hiPSC-RGCs were detected within the ganglion cell layer of the murine retina as early as 2-weeks post injection and seen as punctate hyperfluorescent foci in the cSLO and color fundus imaging. We detected hiPSC-RGCs integrated in the mouse retina with extensive arborization in 6 weeks following intravitreal injections. hiPSC-RGCs stained positive for BRN3 and RBPMS markers and produced spontaneous firing in response to depolarizing stimuli indicating that the integrated iPSC-RGCs are functional.
Our studies will provide key strategies to enhance the efficiency of stem cell replacement therapy and advance potential treatments for neurodegenerative diseases including glaucoma and optic neuritis.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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