Abstract
Purpose :
We have recently demonstrated that membrane-bound extracellular vesicles (EVs) released by Müller glia contain microRNA (miRNA) and proteins that may regulate anti-apoptotic processes and therefore be beneficial in providing neuroprotection in retinal degenerative conditions. Advances in the development of 3D retinal organoid formation from human stem cells has provided an in vitro model to examine specific cell types such as retinal ganglion cells that are affected in glaucoma. Therefore, the aim of this study was to examine the effect of EVs in human organoid derived RGC-like exposed to NMDA to induce cytotoxicity.
Methods :
Human embryonic stem cells were differentiated into 3D retinal organoids using protocols adapted from Nakano et al, 2012. Retinal organoids were grown up to 60 days after initiation of retinal organoid differentiation and retinal ganglion like cells were isolated and characterised using immunofluorescence staining of characteristic RGC markers (βIII-tubulin, Brn3, γ-synuclein, RBPMS, Thy1, PAX6). RGC induced cytotoxicity was induced by addition of NMDA (1mM), followed by treatment with a population of Müller cell derived EVs. Neurite features including number and length were then subsequently examined using Image J.
Results :
The expression of RGC markers during retinal organoid development supported previously published work, showing a peak in RGC marker expression (BRN3B, β3Tubulin, γ-synuclein) in organoids aged 40-50days. Isolated RGC-like cells expressed characteristic RGC markers such as βIII tubulin, Brn3b, RBPMS, γ-synuclein, Thy1 and NMDAR1. Exposure of this cell population to NMDA lead to a significant reduction in the average neurite length from clusters examined. The average neurite length from cell clusters was observed to increase in the NMDA-damaged cells after exposure to Müller cell derived EVs
Conclusions :
Isolated RGC-like cells may be used as effective in vitro models to investigate RGC degeneration. In addition, EVs derived from Müller glia support the survival of RGC-like cells. Further work is needed to identify the mechanisms by which Müller derived EVs exert therapeutic benefit to these cells. It is hoped that these studies will aid future investigations to assess new EV-related therapies to treat RGC damage occurring in retinal degenerative conditions such as glaucoma
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.