RNAseq analysis of in vitro retinal organoids shows comparable pathway expression to developing eye in vivo

Abstract

Purpose : Retinal diseases, like glaucoma and age-related macular degeneration, form a major global health problem. There is no effective cure for these diseases, largely due to the lack of suitable models for these disorders. Lately, a lot of research is focused on the development of a new type of in vitro model, representing all retinal cell types: retinal organoids. It is important when considering the use of these organoids for disease models and treatments that they are comparable to the in vivo situation, both in the expression of important retinal genes, and the developmental timeline. To confirm this, we performed an in-depth RNAseq analysis to look at the developmental pathways compared to the developing eye.

Methods : Human-derived stem cells (hESCs or iPSCs) were encased in 3D Matrigel drops for 4 days in a mixture of iPSC medium and neural induction medium, before being plated on matrigel coated 6 well plates for 7 days, and lifted off to form retinal organoids. These organoids were grown for a total of 160 days, being dissociated and grown as single cells at specific intervals. Organoid samples were taken at important stages during the differentiation process, up to the formation of ganglion cells, and taken forward for RNAseq analysis.

Results : We found that the initial encasement of embryoid bodies in matrigel boosted the growth of our organoids. We could generate retinal ganglion cells within 28 days without the addition of other external factors to direct cells towards this fate. We have also been able to generate all other retinal cell types, confirmed by sq-PCR and immunohistochemistry using both developmental and cell specific markers. These cells include retinal pigmented epithelium and photoreceptors. The RNAseq showed similar developmental pathways were upregulated at certain timepoints, comparable to the in vivo developing eye.

Conclusions : Our results present a developmentally similar method of generating retinal ganglion cells in a shorter timeframe compared to already published protocols using RNAseq analysis. This will not only allow us to study the development and morphology of the emerging eye with different degenerative disease models, but also gives us the opportunity to generate multiple different cell types that can be used for transplantations in a variety of animal disease models and ultimately, human transplantations in the future.

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