A human chimeric retinal organoid model for studying donor-host cell interactions in cell therapies of inherited retinal disease.

Abstract

Purpose : There are no human models of cell engraftment to study allogeneic donor-host cell interactions in a degenerating retina. We have generated chimeric retinal organoids (“chimeroids”) from a mixture of healthy human embryonic stem cell (hESC) and patient-derived, diseased induced pluripotent stem cell (iPSC) lineages. We hypothesize that our chimeroids provide a novel in vitro human model of retinal cell engraftment in a common genetic mutant of inherited retinal disease (IRD).

Methods : Our initial chimeroids were developed with an iPSC line derived from a male patient with USH2A-associated retinitis pigmentosa (RP), and a healthy female H9 reporter hESC line (H9-RFP). The most common causes of autosomal recessive RP are USH2A pathogenic variants. We combined H9-RFP and USH2A stem cells in a 1:1 ratio and co-cultured in 2D, alongside age-matched single lineage controls. We induced differentiation of mixed cultures into 3D retinal organoids following established protocols. We analyzed chimeroids by examining H9-RFP+ nuclei and USH2A Y-chromosome-FITC+ nuclei.

Results : We evaluated chimeroids and control organoids over time in culture for retinal lamination and markers of retinal cell specification. USH2A control organoids showed disorganized lamination and a thin, sparsely-labelled presumptive outer nuclear layer, examined by progenitor and photoreceptor marker expression (Otx2, Recoverin) at Week 8 of differentiation. By comparison, chimeroids show re-established retinal lamination and expression patterns similar to H9 controls. We engineered an USH2A reporter line using CRISPR/Cas9, where H2B promoter-driven GFP results in indelibly-tagged diseased cell nuclei in ongoing chimeroid lineage analysis and cell sorting experiments.

Conclusions : We can successfully generate retinal chimeroids using a patient-derived diseased iPSC line. Our preliminary results suggest that the presence of healthy cells engrafted with diseased cells permits the reestablishment of normal retinal lamination. Further investigation will focus on determining a minimum number of healthy cells required to rescue a diseased organoid, as well as using isogenic mutation-corrected healthy control cells. Our chimeroid platform will facilitate high-throughput analysis of donor-host cell interactions in potentially any retinal dystrophy for which human iPSC lines and an organoid phenotype exist.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.