Cell autonomous RPE dysfunction is sufficient to promote retina degeneration in an induced pluripotent stem cell model of CLN3 disease

Abstract

Purpose : Progressive vision loss in early childhood is a clinical feature of CLN3 disease. Our previous data has shown that CLN3 disease patient-derived induced pluripotent stem cell (iPSC)-retinal pigment epithelium (CLN3-RPE) shows defect in a key RPE cell function, phagocytosis of shed photoreceptor outer segments (POSs). However, the precise role of RPE cell dysfunction in promoting retinal degeneration is not known. To address this knowledge gap, the goal of this study was to i) develop an iPSC-derived retinal organoid (RO)-RPE co-culture model and ii) use this RO-RPE co-culture model to determine the independent role of RPE dysfunction in promoting retinal degeneration in CLN3 disease.

Methods : Control and CLN3 disease iPSCs (harboring the common 966 bp deletion spanning exon 7 and exon 8) were differentiated to obtain RO and RPE. To develop the RO-RPE co-culture model, RPE cells were seeded on transwell membranes and allowed to form a monolayer. Subsequently, control stage 2 and stage 3 ROs encapsulated in a poly(ethylene glycol)-hyaluronic acid (PEG-HA) hydrogel were placed on top of the RPE monolayer in transwells and cultured for 7 days. iPSC-derived RO-RPE co-cultures were maintained for 7 days and analyzed by immunocytochemistry and Western blotting for retinal cell markers including photoreceptors (rhodopsin/RHO, recoverin/RCVRN, and M/L opsin). Additionally, cell death was monitored by TUNEL staining.

Results : Consistent with the known role of RPE in photoreceptors maturation and homeostasis, control ROs co-cultured with RPE showed increased levels of photoreceptor proteins (e.g., RCVRN) compared to parallel monocultures of control ROs. Furthermore, showing an independent role of RPE dysfunction for retina degeneration in CLN3 disease, control ROs co-cultured with CLN3-RPE showed i) reduced number of RHO and M/L opsin-positive cells and ii) increased number of TUNEL-positive cells when compared to parallel cultures of control ROs.

Conclusions : Presence of RPE promotes photoreceptor maturation in an iPSC-derived RO-RPE model. Furthermore, cell-autonomous RPE dysfunction is sufficient for promoting a disease-associated retina degeneration phenotype in a CLN3 disease iPSC model.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.