Abstract
Purpose :
Mutations in the Eyes Shut Drosophila homolog (EYS) gene are one of the major causes of autosomal recessive retinitis pigmentosa (RP). Despite the high prevalence of this disease, little is known about the role of EYS in the retina and the molecular mechanism of the EYS-associated disease, which are necessary for the development of genetic therapies. The purpose of this project is to study EYS in the human iPSC-derived retinal organoid (RO) model.
Methods :
We differentiated wild type IMR90 human iPSC cell line to retinal organoids until day (d)250 using 3D-2D-3D protocol described by Cowan et.al., 2021. Samples for immunohistochemistry were collected at days 50, 70, 100, 130, 150, 170, 200, 230, 250 and differentiation was characterized by immunostaining with early development markers (PAX6, OTX2, CRX, CHX10) and photoreceptor markers (RHO, ARR3, OPSIN R/G, CRALBP, RCVRN). EYS expression was analyzed with RT-PCR. EYS localization was studied by immunohistochemistry and co-staining with photoreceptor ciliary markers (Acetylated α-tubulin, RP1, Centrin, USH2A).
Results :
RT-PCR and immunofluorescence analysis showed EYS expression at d170, which was the earliest timepoint assayed with RT-PCR. EYS located to cones of the developing organoid (up to d150) and in cones and rods of the mature organoid (d170-d250). EYS located in the proximity of different photoreceptor cilia proteins. Co-staining with RP1 demonstrated likely localization of EYS near the transition zone. Co-staining with USH2A, revealed that EYS is localized in its proximity, but the two proteins do not co-localize. EYS was also observed in distal peripheral parts of the rod and cone outer segments.
Conclusions :
Our results show that EYS is expressed in mature retinal organoids at day 170 and beyond, proving, that Retinal Organoids can be used as a model for EYS associated disease studies.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.