Retinal Ganglion Cell Transplantation: Differentiation and Quantification Strategies.

Glaucoma is a leading cause of irreversible blindness worldwide. Current treatments focus on reducing intraocular pressure but cannot restore lost visual function once it is lost due to retinal ganglion cell (RGC) degeneration and death. Recent advances suggest that transplantation of stem cell-derived RGCs could offer new therapeutic approaches for glaucoma and vision restoration. Here, we present a detailed protocol for differentiating human RGCs from embryonic stem cells using both three-dimensional retinal organoid and two-dimensional culture approaches. Following differentiation, we describe methods for isolating and purifying retinal ganglion cells from these cultures and their subsequent transplantation into the mouse retina, with careful monitoring and postoperative care to ensure successful integration. Finally, we describe a quantitative method for assessing transplantation outcomes involving confocal imaging of retinal flat-mounts and custom ImageJ and MATLAB scripts to map and analyze the spatial distribution of donor RGCs within the host retina. Altogether, this approach provides a robust framework for exploring RGC transplantation as a potential therapy for vision loss in glaucoma and other optic neuropathies.