Background Glaucoma is a group of optic neuropathies that affects approximately 2.7 million people in the United States and 76 million people worldwide. Glaucoma is irreversible and can result in permanent blindness if it is not treated. The main risk factor is elevated intraocular pressure (IOP) which predominantly affects retinal ganglion cells (RGC), resulting in cell death and permanent vision loss. Current therapeutics for glaucoma involve reducing IOP and halting progression of disease, but no current treatments can revive degenerated RGCs. Our project aims to evaluate the therapeutic effect of a neurotrophic factor, human Neuritin 1 (NRN1), in regenerating and protecting RGCs lost due to glaucomatous disease progression. Human NRN1 has been proven to enhance plasticity and maturation of RGC axons and dendrites in an acute glaucoma mouse model. The present study utilized non-glaucomatous and glaucomatous induced pluripotent stem cells (iPSC) derived RGCs seeded within in vitro collagen scaffolds. By administering NRN1 to the scaffolds, we hope to elucidate the ability of the therapeutic to increase survival of glaucomatous RGCs.

Methods The iPSCs derived from non-glaucomatous and glaucomatous donor corneal fibroblasts were differentiated to retinal organoids (RO) to generate RGCs in vitro. After around 30 days of differentiation, the ROs were dissociated to isolate RGCs. The RGCs were seeded at one end of three different in vitro collagen scaffolds. The first scaffold received no NRN1 treatment, the second received NRN1 at the cell body, and the third received NRN1 at the opposite end of the cultured RGCs. Downstream immunofluorescence with the TRA-1-60 antibody was performed on iPSCs to confirm that they were successfully reprogrammed from donor fibroblasts. Additionally, gene expression experiments for iPSC markers were performed to further verify successful iPSC reprogramming. Karyotyping procedures were performed on iPSCs to ensure no reprogramming induced chromosomal aberrations were evident.

Results We successfully generated iPSC-derived RGCs from both non-glaucomatous and glaucomatous donor eyes. These RGCs effectively integrated within the three collagen scaffolds. After NRN1 treatment, both cell types demonstrated differential expression of apoptotic markers. Downstream iPSC experimentation confirmed that iPSCs were successfully reprogrammed from donor fibroblasts with no chromosomal aberrations.

Conclusion Our study demonstrates that NRN1 exhibits a therapeutic effect on glaucomatous RGCs. NRN1 could potentially be a useful intervention to restore vision in patients suffering from severe glaucoma. The results also indicate that iPSC-derived RGCs can successfully be obtained from donor eyes and used in collagen scaffolds for future research.