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Single-cell RNA-seq is a powerful tool in decoding the heterogeneity in complex tissues by generating transcriptomic profiles of the individual cell. Here, we report a single-nuclei RNA-seq (snRNA-seq) transcriptomic study on human retinal tissue, which is composed of multiple cell types with distinct functions. Six samples from three healthy donors are profiled and high-quality RNA-seq data is obtained for 5873 single nuclei. All major retinal cell types are observed and marker genes for each cell type are identified. The gene expression of the macular and peripheral retina is compared to each other at cell-type level. Furthermore, our dataset shows an improved power for prioritizing genes associated with human retinal diseases compared to both mouse single-cell RNA-seq and human bulk RNA-seq results. In conclusion, we demonstrate that obtaining single cell transcriptomes from human frozen tissues can provide insight missed by either human bulk RNA-seq or animal models. The retina is a heterogeneous tissue composed of multiple cell types. Via single-nuclei RNA sequencing on human neural retinal tissue, the authors characterise the transcriptome profile for individual cell types in the human retina.

Purpose: Leber congenital amaurosis (LCA) is an early-onset form of retinal degeneration. Six of the 22 known LCA genes encode photoreceptor ciliary proteins. Despite the identification of 22 LCA genes, the genetic basis of ~30% of LCA patients remains unknown. We sought to investigate the cause of disease in the remaining 30% by examining cilia-associated genes. Methods: Whole-exome sequencing was performed on an LCA cohort of 212 unsolved probands previously screened for mutations in known retinal-disease genes. Immunohistochemistry using mouse retinas was used to confirm protein localization and zebrafish were used to perform rescue experiments. Results: A homozygous nonsynonymous mutation was found in a single proband in CLUAP1 , a gene required for ciliogenesis and cilia maintenance. Cluap1 knockout zebrafish exhibit photoreceptor cell death as early as 5 days after fertilization, and rescue experiments revealed that our proband’s mutation is significantly hypomorphic. Conclusion: Consistent with the knowledge that CLUAP1 plays an important role in cilia function and that cilia are critical to photoreceptor function, our results indicate that hypomorphic mutations in CLUAP1 can result in dysfunctional photoreceptors without systemic abnormalities. This is the first report linking mutations in CLUAP1 to human disease and establishes CLUAP1 as a candidate LCA gene. Genet Med 18 10, 1044–1051.

Rod and cone photoreceptors are light-sensing cells in the human retina. Rods are dominant in the peripheral retina, whereas cones are enriched in the macula, which is responsible for central vision and visual acuity. Macular degenerations affect vision the most and are currently incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids differentiated from hESCs using an improved retinal differentiation system. Induced by extracellular matrix, aggregates of hESCs formed single-lumen cysts composed of epithelial cells with anterior neuroectodermal/ectodermal fates, including retinal cell fate. Then, the cysts were en bloc-passaged, attached to culture surface, and grew, forming colonies in which retinal progenitor cell patches were found. Following gentle cell detachment, retinal progenitor cells self-assembled into retinal epithelium— retinal organoid—that differentiated into stratified cone-rich retinal tissue in agitated cultures. Electron microscopy revealed differentiating outer segments of photoreceptor cells. Bulk RNA-sequencing profiling of time-course retinal organoids demonstrated that retinal differentiation in vitro recapitulated in vivo retinogenesis in temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-sequencing profiling of 8-mo retinal organoids identified cone and rod cell clusters and confirmed the cone enrichment initially revealed by quantitative microscopy. Notably, cones from retinal organoids and human macula had similar single-cell transcriptomes, and so did rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-rich retinal organoids and a reference of transcriptomes that are valuable resources for retinal studies.

Neural degenerative diseases often display a progressive loss of cells as a stretched exponential distribution. The mechanisms underlying the survival of a subset of genetically identical cells in a population beyond what is expected by chance alone remains unknown. To gain mechanistic insights underlying prolonged cellular survival, we used Spata7 mutant mice as a model and performed single-cell transcriptomic profiling of retinal tissue along the time course of photoreceptor degeneration. Intriguingly, rod cells that survive beyond the initial rapid cell apoptosis phase progressively acquire a distinct transcriptome profile. In these rod cells, expression of photoreceptor-specific phototransduction pathway genes is downregulated while expression of other retinal cell type-specific marker genes is upregulated. These transcriptomic changes are achieved by modulation of the epigenome and changes of the chromatin state at these loci, as indicated by immunofluorescence staining and single-cell ATAC-seq. Consistent with this model, when induction of the repressive epigenetic state is blocked by in vivo histone deacetylase inhibition, all photoreceptors in the mutant retina undergo rapid degeneration, strongly curtailing the stretched exponential distribution. Our study reveals an intrinsic mechanism by which neural cells progressively adapt to genetic stress to achieve prolonged survival through epigenomic regulation and chromatin state modulation.

The retina is the innermost layer of tissue in the eyes of human and most other vertebrates. It receives the information of the visual images like the film of a camera, translates the images into neural signals, and transduces the signal to the brain. Three layers of neural cells (photoreceptor cells, bipolar cells, and ganglion cells) within the retina are comprised of seven major cell types; rod, cone, Muller glia cell, amacrine cell, horizontal cell, bipolar cell, and retinal ganglion cells, which create visual perception through functional cooperation. In all retinal diseases, it is the ultimate degeneration of the photoreceptors, the rods and cones, which results in blindness. Understanding all the individual cell types and how they contribute the basic neural circuitry in both the human and mouse retina will be key to elucidating the mechanism of retinal degenerations. Here, we report to the best of our knowledge the first single-nuclei RNA-seq based transcriptomic study on the human neural retinal tissue. We sequenced 6544 nuclei from six samples, and 4730 nuclei passed the quality filtering steps. The donor samples came from both the macular and peripheral regions, respectively, from three donor's retina. None of the donors had any evidence of any ocular disease including retinal diseases as evidenced by medical record and postmortem imaging and phenotyping (Owen et al., IOVS in press). Unsupervised clustering was able to identify all seven cell populations. Known markers of neural retinal cell types were used to assign the clusters to these seven major retinal cell types, and thus, gene expression profiles for each cell type were obtained. With this dataset, the similarities and differences in cell proportion and gene expression between human and mouse were assessed. We highlighted the genes that demonstrated statistically significant differential distribution between human and mouse photoreceptors. In addition, the gene expression profiles of the same cell types between the macular and peripheral regions between human and mouse were compared. Specifically, as a means of validation, the highly expressed genes in photoreceptor cells were found to be significantly enriched in known inherited retinal disease genes. Thus, we propose that this cell-specific gene list would serve as a prioritization during not only novel disease gene discovery, but cell-specific pathway gene analysis. This should facilitate a better understanding of retinal cell biology with the goal of cell-specific therapeutic interventions for retinal degenerations, as therapies are limited.