A human chimeric retinal organoid model for studying donor-host cell interactions in cell therapies for USH2A-associated retinitis pigmentosa

Abstract

Purpose : There are currently no human models of retinal cell engraftment that enable the study of allogenic donor-host cell interactions between healthy and disease cells in a degenerating retina. Our lab has generated chimeric retinal organoids (“chimeroids”) from a mixture of pluripotent stem cell lineages. We hypothesize that our chimeroids represent a novel in vitro human model of retinal cell engraftment in a common USH2A mutant of autosomal recessive retinitis pigmentosa, and will provide a new way to study cell-cell interactions between healthy and disease human retinal cells.

Methods : Chimeroids were generated using patient-derived USH2A mutant iPSCs, which we edited with CRISPR/Cas9 to express nuclear H2B promoter-driven GFP (USH2A-GFP), and H9 ESCs that are lentiviral-transduced to express nuclear H2B promoter-driven RFP (H9-RFP). We combined cultures of undifferentiated H9-RFP and USH2A-GFP cells in various ratios in 2D, alongside single lineage controls. We then differentiated 3D retinal organoids following established protocols. USH2A has a role in photoreceptor (PR) outer segment (OS) maintenance, which is critical to PR cell survival. Chimeroids and control organoids were evaluated for markers of retinal cell fate specification, gene expression, and PR morphology at different stages of organoid development.

Results : USH2A diseased organoids show markedly shorter presumptive OS and lower Rhodopsin (Rho) expression in mature organoids (Week 24) compared with healthy H9 controls. In contrast, chimeroids display longer presumptive OS and Rho levels compared to diseased controls. Restored Rho-expressing USH2A-derived cells within chimeroids can be observed by immunohistochemistry in regions of high mosaicism as well as USH2A-GFP dominated regions. FAC sorting of dissociated chimeroids demonstrates increased Rho gene expression specifically in the USH2A-GFP fraction of chimeroids, relative to USH2A disease controls.

Conclusions : We have generated retinal chimeroids using a patient-derived diseased iPSC line. Ongoing small molecule testing and FAC sorting will allow us to examine the modified properties of healthy and diseased cells in our chimeroids. This may reveal new therapeutic targets. Our modular chimeroid platform may, in principle, be applied to any inherited retinal disease genotype for which human iPSCs and an organoid phenotype exist.

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