In search for the RGC lineage: characterization of R28 cells and the expression of RGC markers

Abstract

Purpose : The degeneration of retinal cells, especially retinal ganglion cells (RGC) is noted in the underlying pathology in many retinal neurodegenerative diseases including diabetic retinopathy (DR) and glaucoma. Still, the mechanisms controlling the plasticity of retinal precursor cells into RGC are not completely understood. There is an unmet need of new in vitro models being the generation of the R28 cell line one of the tools posed to fill this void. In this study we characterized the R28 cell line and measured common markers to RGCs.

Methods : To investigate heterogeneity of the cell line, R28 cells were cultured in vitro in DMEM/10% calf serum media at 37C/5%CO_2, as detailed in the Kerafast website. Cells displaying a phenotype of Live Thy1^loCD48^negCD15^negCD57^neg underwent cell FACS-based cell sorting. Phenoptypic alterations were measured 6-days after sorting by flow cytometry and qPCR. To investigate if R28 could be poised to a RGC program, the transcription factor Pou4f2, which is required for embryonic differentiation and development of RGCs, was overexpressed using Lipofectamine 3000™ and confirmed by immunofluorescence microscopy.

Results : R28 cells are a heterogeneous population, which express the intracellular markers Thy1, RBPMS, Tubb3, and Brn3a, shared by RGCs. Genetic analyses demonstrated expression of other retinal markers associated with photoreceptor cells. We successfully transduced Pou4f2 into R28 cells and cells survived in vitro for a week.

Conclusions : Further studies characterizing Pou4f2-R28 cells will help to understand the mechanisms of retinal cell differentiation and will aid in our quest to understand RGC biology due to their relevance in retinal neurodegenerative diseases.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.