Browsing by Subject "Retinal Ganglion Cells (RGCs)"

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    Determining the Therapeutic Effect of Human Neuritin 1 on the Restoration of Degenerated Retinal Ganglion Cells from Glaucoma Patients
    (2024-07-25) Hubbard, Nathan; Hameed, Shahna; Sharma, Tasneem
    BACKGROUND AND HYPOTHESIS: Glaucoma is a group of optic neuropathies that affects approximately 76 million people worldwide. 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 human Neuritin 1 (NRN1) in regenerating and protecting RGCs lost in glaucoma. By administering NRN1 to the RGCs in culture, we hope to elucidate the efficacy in helping glaucomatous RGCs recover from cell death. PROJECT METHODS: Immunofluorescence (IF), gene expression, and karyotyping experiments were performed on iPSCs to confirm they were successfully reprogrammed from donor keratocytes. The iPSCs 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 received no NRN1 treatment, the second received NRN1 at the cell body, and the third received NRN1 at the opposite end. IF was done on RGC-seeded scaffolds with RBPMS and NEFL antibodies to confirm RGC marker expression and neurite growth. RESULTS: The iPSCs were successfully reprogrammed from donor fibroblasts. We successfully generated RGCs from both non-glaucomatous and glaucomatous donor keratocytes. The RGCs effectively integrated within the collagen scaffolds. After NRN1 treatment, non-glaucomatous and glaucomatous RGCs demonstrated differential expression of RGC specific markers. CONCLUSIONS AND POTENTIAL IMPACT: Our study demonstrates that NRN1 exhibits a therapeutic effect on glaucomatous RGCs. This study lays the foundation that NRN1 could potentially restore vision in glaucoma patients. Additionally, iPSC-derived RGCs can successfully be obtained from human donor eyes and cultured for future research or testing therapeutics.
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    Differentiation and Three-dimensional Organization of Retinal Ganglion Cells using Human Induced Pluripotent Stem Cells
    (Office of the Vice Chancellor for Research, 2015-04-17) Ho-A-Lim, Kimberly T.; Ohlemacher, Sarah K.; Meyer, Jason S.
    Retinal Ganglion Cells (RGCs) are a type of neuron which function to relay visual messages between the retina and brain, and are characterized by their long axons which form part of the optic nerve. Dysfunction in this communication pathway is highly implicated in degenerative blinding disorders such as glaucoma. Unique applications using human induced pluripotent stem cells (hiPSCs) offer the ability to model human diseases, and potentially develop novel therapeutic approaches to rescue or replace damaged cells. In order to better understand the progression of degenerative eye diseases, a remaining challenge is to precisely identify the sequence of events which contribute to the diseased state, and how their features differ from non-diseased cells. Efforts were therefore undertaken to visually document the maturation of RGCs by analyzing their morphology and three-dimension organization at varying stages of development. Induced retinal cells were harvested at six different stages of development and fixed in 4% paraformaldehyde (PFA) solution to arrest their development. Cells were then cryoprotected in combinations of sucrose and Optimal Cutting Temperature (OCT) solutions, and frozen using powered dry ice. Following cryostat sectioning, samples were subject to immunocytochemistry staining to visualize for retinal-like organization of cells. Preliminary results have indicated the presence of the RGC marker Brn3, as well as markers for other retinal cell types. Future tests intend to characterize these retinal cell types according to their morphology and three-dimensional organization.