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Updated: Nov 4, 2025
An Ex Vivo Explant Model for Studying Glial Interactions in the Mouse Retina
Published on: July 15, 2025
Human primary retinal cells as an in-vitro model for investigating defective signalling caused by OPTN mutants
Zuberwasim Sayyad1, Sushma Vishwakarma2, Tarjani Vivek Dave2
1CSIR- Centre for Cellular and Molecular Biology, Hyderabad, 500007, India.
Abstract:
Studies carried out on the pathogenesis of glaucoma using murine cell lines and animal models require to be validated in human cells. Therefore, we explored the possibility of using human primary retinal cells (hPRCs) in culture as a model for molecular studies and testing of potential therapeutic drugs. For this purpose, central retinal tissue, obtained from the enucleated eyes of patients with anterior staphyloma, was digested with trypsin and grown in a medium containing supplements (basic fibroblast growth factor and fetal bovine serum). hPRCs at passage 1 and 2, show expression of either GFAP, a glial cell marker, or β-III tubulin, a retinal ganglion cell (RGC)-specific marker. But at passages 3-5 nearly all of hPRCs express several RGC-specific markers (Brn3 proteins, Thy-1, β-III tubulin, RBPMS and NeuN) but not GFAP. Expression of these markers indicated that these cells may have functional properties of RGCs. As RGCs are sensitive to glaucoma-associated mutants of OPTN, we analysed the survival of hPRCs upon overexpression of OPTN mutants. Glaucoma-associated mutants, E50K-OPTN and M98K-OPTN, induced significantly higher cell death in hPRCs compared to WT-OPTN, whereas an amyotrophic lateral sclerosis-associated mutant, E478G-OPTN, did not. TBK1 inhibitor Amlexanox protected hPRCs from E50K-OPTN and M98K-OPTN induced cell death. M98K-OPTN induced cell death was suppressed by inhibitors of CaMKKβ and AMPK in hPRCs as well as in 661W, a mouse cell line that expresses several markers of RGCs and RGC precursor cells. Our results suggest that hPRCs under appropriate culture condition show RGC-like properties. These cells can be used to explore the molecular mechanisms of cell death relevant for glaucoma pathogenesis and for testing of cytoprotective compounds.
Insights
Human primary retinal cells (hPRCs) cultured in vitro exhibit retinal ganglion cell (RGC) properties, proving useful for glaucoma research. These cells can model RGC death and test new glaucoma drug therapies.
Area of Science:
- Ophthalmology
- Neuroscience
- Cell Biology
Background:
- Glaucoma pathogenesis studies often use animal models, necessitating validation in human systems.
- Human primary retinal cells (hPRCs) offer a potential model for studying glaucoma molecular mechanisms and drug screening.
Purpose of the Study:
- To establish and characterize human primary retinal cells (hPRCs) in culture as a model for retinal ganglion cells (RGCs).
- To investigate the utility of hPRCs for studying glaucoma-associated cell death and evaluating potential therapeutic compounds.
Main Methods:
- Central retinal tissue from human eyes was processed to isolate and culture hPRCs.
- Immunocytochemistry was used to assess RGC-specific marker expression (Brn3, Thy-1, β-III tubulin, RBPMS, NeuN) at different passages.
- Overexpression of wild-type (WT) and mutant Optineurin (OPTN) was performed to induce cell death, followed by treatment with specific inhibitors (Amlexanox, CaMKKβ, AMPK inhibitors).
Main Results:
- hPRCs at passages 3-5 expressed multiple RGC markers and lacked glial markers, indicating RGC-like properties.
- Glaucoma-associated OPTN mutants (E50K and M98K) induced significantly higher hPRC death than WT-OPTN.
- Amlexanox protected hPRCs from OPTN mutant-induced cell death, and M98K-OPTN-induced death was suppressed by CaMKKβ and AMPK inhibitors.
Conclusions:
- Established hPRCs in culture exhibit RGC-like characteristics, making them a suitable model for glaucoma research.
- This hPRC model can be used to explore glaucoma's molecular mechanisms of cell death and screen for cytoprotective agents.
