Distribution Of Primary Cilia In hESC-Derived Retinal Organoid

Abstract

Purpose : Primary cilia are microtubule-based organelles that are found on differentiated cell types throughout the body. Proper cilia formation is critical during eye development in humans and rodents; ciliary dysfunction can result in wide-ranging ocular diseases. Our previous papers support both amacrine and retinal ganglion cells (RGC) harbor primary cilia in primate and mouse retina. However, whether primary cilia patterning changes during retinal development remains unknown. The purpose of this study is to describe the primary cilia distribution in hESC (human Embryonic Stem Cell)-derived retinal organoids.

Methods : Retinal organoids were differentiated from the Brn3b-Tdtomato hESC line and collected at different developmental stages (w6-w38) and immunostained for the presence of cilia. Three ciliary markers, including Arl13b, AC3 and Centrin3 were used to identify primary cilia. Chx10, PKC2a, Prox1, GFAP, AP2a GAD67, Calretinin antibodies were used to determine retinal cell types. Arl13b stained the axoneme while Centrin3 marked the basal body. Statistical analysis was performed using One-way ANOVA and Student t-test.

Results : A group of ciliated cells localized in the inner aspects of retinal organoids cultured w7 to w34 (n=3-5 per time point). At w38, the number of primary cilia among non-photoreceptor retinal cell was significantly decreased compared to w13 (n=5, p<0.001). Furthermore, there were very few ciliated Prox1-positive horizontal, GFAP-positive astrocytes and PKC2a-positive rod-bipolar cells at w38 or w25. For AP2a-positive amacrine cells, we found ciliation was stable among all stages of retinal organoids (n=3 per timepoint). Ciliation significantly decreased for Brn3b-positive RGCs at later timepoints (n=5). Additionally, AC3-positive ciliation in non-RGC retinal cells at w25 significantly decreased as compared to w9. AC3-positive ciliation in Brn3b-RGCs decreased by w25.

Conclusions : These novel findings indicate amacrine cells incorporate stable ciliation whereas the RGC ciliation decreased along retinal organoid development, suggesting primary cilia potentially play significant roles for RGC survival in the retinal organoid.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.