Abstract
Purpose :
Within the last decade, a wide variety of protocols have emerged for the generation of retinal organoids. A subset of studies compared protocols based on stem cell source, the physical features of the microenvironment, and both intra- and extra-cellular signals. Most of these comparisons were focused on the effect of signalling pathways on retinal organoid development. Our aim in this study is to understand whether embryoid body formation methods affect the development of retinal organoids in terms of differentiation capacity and reproducibility.
Methods :
We used the popular 3D floating culture method to generate retinal organoids from stem cells. This method starts with either small clumps of stem cells generated from larger clones (clumps protocol), or with an aggregation of single cells (single cells protocol). Both protocols present similar differentiation timelines during the first weeks of differentiation. Samples were collected at embryoid bodies (day 4 and 7), neurospheres (day 18), and retinal organoids (day 43 and 120).
Results :
The developmental stage of embryoid bodies (EBs) was assessed by examining genes associated with the epithelial-to-mesenchymal transition (CDH1 and CDH2). In EBs generated through the clumps protocol, we observed a downregulation of CDH1 and an upregulation of CDH2 on day 7. In contrast, in EBs produced with the single cells protocol, we found an upregulated expression of CDH1 while CDH2 was absent on day 7. Consistently, EBs from the clumps protocol displayed early expression of eye field transcription factors RAX and PAX6 on both day 4 and 7, whereas no expression of these genes was detected in EBs from the single cells protocol. Nonetheless, the final organization of retinal organoids does not appear to be significantly impacted. This suggests the existence of a compensatory mechanism during the neurosphere stage by day 18, as suggested by differential gene expression analysis (RAX, PAX6, CDH1, CDH2 and VSX2).
Conclusions :
We found a retention of the pluripotency capacity in EBs produced through the single cells protocol compared to the clumps, while final retinal organoid development was indistinguishable between protocols. This study not only facilitates an in-depth exploration of EB development but also provides valuable insights for the selection of the most suitable protocol to study retinal development and to model inherited retinal disorders in vitro.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.