Generation of Retinal Ganglion Cells from Reprogrammed Keratocytes of Non‐Glaucoma and Glaucoma Donors

Human induced pluripotent stem cell (hiPSC)-based disease modeling can be successfully recapitulated to mimic disease characteristics across various human pathologies. Glaucoma, a progressive optic neuropathy, primarily affects the retinal ganglion cells (RGCs). While multiple groups have successfully generated RGCs from non-diseased hiPSCs, producing RGCs from glaucomatous human samples holds significant promise for understanding disease pathology by revealing patient-specific disease signatures. Given that keratocytes originate from the neural crest and previous reports suggest that ocular fibroblasts from glaucomatous donors carry pathogenic signatures, it is highly plausible that these signatures imprinted within the keratocytes will also be present in the derived RGCs. Thus, we aimed to generate RGCs from both glaucomatous and non-glaucomatous donor keratocytes and validate disease-specific signatures in 3D retinal organoids and in isolated RGCs. Our protocol describes the generation of iPSCs from keratocytes of both glaucomatous and non-glaucomatous donors, followed by their differentiation into retinal organoids. Subsequent isolation and culturing of RGCs were performed. Disease signatures in the RGCs were validated in both 3D retinal organoids (ROs) and 2D RGC cultures, and glaucomatous RGCs in 3D and 2D cultures demonstrated increased cleaved CASP3 and significant RGC loss, indicating disease imprints in the hiPSC-derived RGCs. This model offers a venue and high throughput platform for studying glaucomatous disease pathology and holds significant potential for drug discovery using RGCs derived from human donors. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing of keratocytes from human cadaveric donors Basic Protocol 2: Reprogramming donor keratocytes into iPSCs Basic Protocol 3: Evaluation of chromosomal loss during reprogramming in iPSCs by karyotyping Basic Protocol 4: Generation of 3D ROs Basic Protocol 5: Dissociation and culturing of RGCs from 3D ROs Support Protocol 1: Immunostaining for phenotypic characterization of cells Support Protocol 2: Sectioning of 3D ROs and immunostaining Support Protocol 3: Western blotting for cleaved CASP3 and THY1.