Abstract
Purpose :
There is a lack of in vitro models to study retinal degenerative conditions characterised by retinal ganglion cell (RGC) death such as glaucoma, which are needed to aid in the development of novel therapies. Advances in the development of 3D retinal organoid formation from iPSC/ESC cells has provided a useful platform to undertake in vitro studies on the human retina whilst reducing the use of animals, which may not accurately resemble human disease. Therefore, the aim of this study was to examine the use of human ESC derived retinal organoids as a model for future investigations of new therapies to treat retinal degeneration.
Methods :
Human embryonic stem cells were differentiated into 3D retinal organoids using protocols adapted from Nakano et al, 2012. Retinal organoids were grown for up to 70 days after initiation of retinal organoid differentiation and examined for the presence of RGC at various developmental stages. RGC induced cytotoxicity on these organoids was induced by different concentrations of N-methyl-D-Aspartate (NMDA; 10-50 μM), followed by examination of genes (qPCR) and proteins (immunostaining) characteristic of these cells. These included BRN3B, β3Tubulin (TUBB3), γ-synuclein (SNCG), PAX6 and ISL-1. Preliminary functional data were also used to assess cell activity by intracellular calcium imaging techniques.
Results :
The expression of RGC markers during retinal organoid development supported previously published work, showing a peak in RGC markers expression (BRN3B, β3Tubulin, γ-synuclein) in organoids aged 40-50 days. A decline in the number of positive cells was observed in organoids of increasing maturity. Addition of NMDA to retinal organoids in culture resulted in a significant reduction of BRN3B staining and significant increase in the number of TUNEL positive cells after 24hrs. Functional calcium imaging data showed lower spontaneous activity in organoids incubated with NMDA for 24hrs. Gene expression of RGC markers within retinal organoids showed a marked fold-downregulation in TUBB3 and PAX6 expression after 24hr NMDA treatment.
Conclusions :
Current results suggest that human ESC derived 3D retinal organoids may be used as effective models of RGC degeneration in vitro. It is hoped that this will aid future investigations to assess new therapies to treat RGC damage occurring in retinal degenerative conditions such as glaucoma.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.