Abstract
Purpose :
There is an unmet need for in vitro RGC models to better understand the underlying cellular pathogenesis of glaucoma. While there has been success with enriching for RGCs from intact retinas, the procedure is technically complex and the resulting RGCs are viable only for a short period of time (roughly 2 weeks). We hypothesized that Lentiviral transduction of R28 retinal precursor cells with Pou4f2, a transcription factor associated with RGCs, would drive R28 cells toward an RGC fate. The goal of this study was to generate an in vitro RGC model using R28 retinal precursor cells that constitutively express Pou4f2.
Methods :
R28 cells were transduced using a Lentivirus carrying the Pou4f2 gene fused to green fluorescent protein or mCherry. After antibiotic selection using puromycin, the Pou4f2-transduced R28 cells were cultured for 14 days in Neurobasal Plus Medium containing B27 supplement. The cells were observed using confocal microscopy to verify Pou4f2 expression and neuronal morphology. Immunocytochemistry was performed to detect RGC markers. Non-transduced R28 cells cultured in supplemented Dulbecco’s Modified Eagle Medium served as the control for all experiments.
Results :
Confocal microscopy of Pou4f2-transduced R28 cells confirmed the presence of the fluorescent fusion protein,the presence of axonal projections, and the formation of synaptic junctions. The expression and localization of at least 10 RGC and neuronal markers were confirmed in the transduced R28 cells including: β-tubulin 3 (TUBB3); POU domain, class 4, transcription factor 1 (POU4F1); Islet 1 transcription factor (ISL1); very long chain enoyl-CoA reductase (TECR); microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2); RNA binding protein with multiple splicing (RBPMS); synaptosomal-associated protein, 25kDa (SNAP25); synaptotagmin 11 (SYT11); and Thy 1.1 cell surface antigen.
Conclusions :
Pou4f2-expressing R28 cells demonstrate neuronal morphology and protein expression patterns similar to that of in situ RGCs, indicating that these cells can differentiate into RGC-like neurons. Thus, the Pou4f2 R28 cells show strong potential as a new in vitro RGC model.
This is a 2021 ARVO Annual Meeting abstract.