NR2E3 loss disrupts photoreceptor cell maturation and fate in human organoid models of retinal development. NR2E3 loss disrupts photoreceptor cell maturation and fate in human organoid models of retinal development

While dysfunction and/or death of light-detecting photoreceptor cells underlies most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generate retinal organoids using induced pluripotent stem cells (iPSC) derived from a patient with genetic photoreceptor disease, an isogenic control, and an unrelated control. Organoids were sampled using single-cell RNA sequencing across the developmental window encompassing photoreceptor specification, emergence, and maturation; up to 260 days of in vitro differentiation. Using single-cell transcriptomics data, we reconstruct the rod photoreceptor developmental lineage and identify a branchpoint in development unique to the disease state that gives rise to a divergent rod photoreceptor cell population. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including expression of the light-sensitive protein rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes at both the transcript and protein level. Using joint multimodal single-cell sequencing on late-stage retinal organoids, we further identify the specific putative regulatory sites where rod-specific factors act to steer rod and cone photoreceptor cell development. Importantly, these findings are strikingly different than that observed in rodent models of disease. Together, these data provide a roadmap of human photoreceptor development and leverage patient iPSCs to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation. Overall design: Three iPSC lines (ND [no disease] Control, NR2E3-null, and Isogenic Control) were differentiated to retinal organoids for 260 days. Samples were assayed by 3' scRNAseq at D40, D80, D120, D160 and D260 from all three lines. In a separate differentiation batch, NR2E3-null and Isogenic Control organoids were assayed by joint (RNA & ATAC) single-nucleus multimodal sequencing at D160 and D280. Multimodal runs yield paired scRNAseq and scATACseq libraries. For AAV experiments, NR2E3-null organoids were cultured for 120 days. Organoids were treated with AAV carrying NR2E3-T2A-GFP or GFP or left untransduced as described. Organoids were harvested at D120 or following treatment at D160 and assayed by 3' scRNAseq. For ESCS Patient 2 experiment, an iPSC line from ESCS Patient 2 was differentiated to retinal organoids and assayed at D80 and D160 by 3' scRNAseq. >>>Raw data were not provided for ATAC-seq Samples due to patient privacy concerns<<<