Generation, transcriptome profiling, and functional validation of cone-enriched human retinal organoids

The macula of the retina has a high ratio of cones to rods and is critical for central vision and visual acuity. Macula degenerations affect vision the most and are incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-enriched human retinal organoids differentiated from hESCs. Transcriptome profiling using bulk RNA-seq demonstrated that retinal differentiation in vitro recapitulated retinogenesis in vivo in the temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-seq of 8-month retinal organoids identified clusters of cone and rod photoreceptors and confirmed the cone enrichment initially revealed by immunostaining. Notably, comparisons of single-cell transcriptomes demonstrated the similarity between retinal organoids and human macula in cones and rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-enriched retinal organoids and a reference of transcriptomes that are rich resources for retinal studies. To globally characterize the transcriptomes during human retinal cell differentiation, we performed bulk RNA-seq of retinal organoids at multiple time points (15 days, 1, 3, 6.5, and 9 months, three replicates per time point, Supplemental Table S1), starting from the stage preceding retinal ganglion cell differentiation to the stage when photoreceptors display outer segments.