Abstract
Purpose :
Within the last decade, a wide variety of protocols have emerged focused on the generation of retinal organoids. In addition to this, a number of studies have compared protocols based on physical features of the microenvironment, internal versus external signals, and starting cell conditions. Most of these comparisons have focused on the effects of molecules triggering certain pathways. In this study, our aim was to understand how the starting cell conditions affect the production of retinal organoids, in terms of quality and reproducibility. Wagstaff et al., (2021) found that one of the most used methods of generating retinal organoids is to start with a 3D floating culture method. In the current study we evaluate the effect of the starting stem cell conditions (stem cell clumps in suspension compared to single cell aggregation) on the retinal organoid development.
Methods :
Based on the review by Wagstaff et al., 2021, we selected protocols starting with either stem cell clumps (Ohlemacher et al., 2015) or single cells (Huang et al., 2019). Both protocols present similar timelines of the main events during the first few weeks of differentiation, and do not incorporate additional small molecules, making a comparison possible to understand the effect of the starting material. Retinal organoids were differentiated up to 40 days. Samples were collected at three stages: embryoid bodies (day 7), neurospheres (day 18), and retinal organoids (day 40). Gene and protein expression profiles were evaluated.
Results :
Embryoid bodies generated from stem cell clumps in suspension or single cell aggregation revealed morphological differences at day 7 of differentiation. Embryoid bodies derived from stem cell clumps presented a smaller diameter (<300 μm) compared to embryoid bodies generated from single cell aggregation (<600 μm). Early developmental genes showed similar expression between protocols, but highlighted structural changes between the two at embryoid body stage. Nevertheless, later on in development at retinal organoids stage, retinal lineage genes showed different expression.
Conclusions :
Our results indicated that differences during the embryoid body stage influence retinal organoid development. This study will not only allow us to investigate the mechanisms underlying the embryoid body formation, but also will help in the selection of the most suitable protocol in order to study hereditary eye diseases.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.