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The retina is a specialized neural tissue that senses light and initiates image processing. Although the functional organization of specific retina cells has been well studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile the human retina, we performed single-cell RNA sequencing on 20,009 cells from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct cell populations representing all known neural retinal cells: rod photoreceptors, cone photoreceptors, Müller glia, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, astrocytes, and microglia. Our data captured molecular profiles for healthy and putative early degenerating rod photoreceptors, and revealed the loss of MALAT1 expression with longer post-mortem time, which potentially suggested a novel role of MALAT1 in rod photoreceptor degeneration. We have demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Müller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to understanding retinal biology and disease., (© 2019 The Authors.)

Single-cell RNA sequencing (scRNA-seq) has facilitated a deeper comprehension of the molecular mechanisms behind eye diseases and has prompted the selection of precise therapeutic targets by examining the cellular and molecular intricacies at the single-cell level. This review delineates the pivotal role of scRNA-seq in elucidating the functions of innate immune cells within the context of ocular pathologies. Recent advancements in scRNA-seq have revealed that innate immune cells, both from the periphery and resident in the retina, are actively engaged in various stages of multiple eye diseases. Notably, resident microglia and infiltrating neutrophils exhibit swift responses during the initial phase of injury, while peripheral monocyte-derived macrophages exhibit transcriptomic profiles akin to those of activated microglia, suggesting their potential for long-term residence within the retina. The scRNA-seq analyses have underscored the cellular heterogeneity and gene expression alterations within innate immune cells, which, while sharing commonalities, exhibit disease-specific variations. These insights have not only broadened our understanding of the cellular and molecular mechanisms in eye diseases but also paved the way for the identification of candidate targets for targeted therapeutic interventions. The application of scRNA-seq technology has heralded a new era in the study of ocular pathologies, enabling a more detailed appreciation of the roles that innate immune cells play across a spectrum of eye diseases. [ABSTRACT FROM AUTHOR]

The development of the retina is under tight temporal and spatial control. To gain insights into the molecular basis of this process, we generate a single-nuclei dual-omic atlas of the human developing retina with approximately 220,000 nuclei from 14 human embryos and fetuses aged between 8 and 23-weeks post-conception with matched macular and peripheral tissues. This atlas captures all major cell classes in the retina, along with a large proportion of progenitors and cell-type-specific precursors. Cell trajectory analysis reveals a transition from continuous progression in early progenitors to a hierarchical development during the later stages of cell type specification. Both known and unrecorded candidate transcription factors, along with gene regulatory networks that drive the transitions of various cell fates, are identified. Comparisons between the macular and peripheral retinae indicate a largely consistent yet distinct developmental pattern. This atlas offers unparalleled resolution into the transcriptional and chromatin accessibility landscapes during development, providing an invaluable resource for deeper insights into retinal development and associated diseases. The authors build an atlas of the human retinal development, using approximately 220,000 nuclei from 14 embryos and fetuses (8–23 weeks post-conception). The study reveals major cell classes, key transcription factors, and differences in the development of macular and peripheral retina. [ABSTRACT FROM AUTHOR]

The emergence of retinal progenitor cells and differentiation to various retinal cell types represent fundamental processes during retinal development. Herein, we provide a comprehensive single cell characterisation of transcriptional and chromatin accessibility changes that underline retinal progenitor cell specification and differentiation over the course of human retinal development up to midgestation. Our lineage trajectory data demonstrate the presence of early retinal progenitors, which transit to late, and further to transient neurogenic progenitors, that give rise to all the retinal neurons. Combining single cell RNA-Seq with spatial transcriptomics of early eye samples, we demonstrate the transient presence of early retinal progenitors in the ciliary margin zone with decreasing occurrence from 8 post-conception week of human development. In retinal progenitor cells, we identified a significant enrichment for transcriptional enhanced associate domain transcription factor binding motifs, which when inhibited led to loss of cycling progenitors and retinal identity in pluripotent stem cell derived organoids. Formation of the retina during development involves the coordinated action of retinal progenitor cells and their differentiated cell types, which is key for producing a functioning eye. Here the authors provide a detailed atlas of human retinal development, combining scRNA-seq and spatial transcriptomics, and identify key genetic factors that mediate retinal progenitor cell proliferation and differentiation. [ABSTRACT FROM AUTHOR]

Alterations in γ-aminobutyric acid (GABA) have been implicated in sensory differences in individuals with autism spectrum disorder (ASD). Visual signals are initially processed in the retina, and in this study, we explored the hypotheses that the GABA-dependent retinal response to light is altered in individuals with ASD. Light-adapted electroretinograms were recorded from 61 adults (38 males and 23 females; n = 22 ASD) in response to three stimulus protocols: (1) the standard white flash, (2) the standard 30 Hz flickering protocol, and (3) the photopic negative response protocol. Participants were administered an oral dose of placebo, 15 or 30 mg of arbaclofen (STX209, GABAB agonist) in a randomized, double-blind, crossover order before the test. At baseline (placebo), the a-wave amplitudes in response to single white flashes were more prominent in ASD, relative to typically developed (TD) participants. Arbaclofen was associated with a decrease in the a-wave amplitude in ASD, but an increase in TD, eliminating the group difference observed at baseline. The extent of this arbaclofen-elicited shift significantly correlated with the arbaclofen-elicited shift in cortical responses to auditory stimuli as measured by using an electroencephalogram in our prior study and with broader autistic traits measured with the autism quotient across the whole cohort. Hence, GABA-dependent differences in retinal light processing in ASD appear to be an accessible component of a wider autistic difference in the central processing of sensory information, which may be upstream of more complex autistic phenotypes. [ABSTRACT FROM AUTHOR]

Astrocytes are a major category of glial support cell in the central nervous system and play a variety of essential roles in both health and disease. As our understanding of the diverse functions of these cells improves, the extent of heterogeneity between astrocyte populations has emerged as a key area of research. Retinal astrocytes, which form the direct cellular environment of retinal ganglion cells somas and axons, undergo a reactive response in both human glaucoma and animal models of the disease, yet their contributions to its pathology and progression remain relatively unknown. This gap in knowledge is largely a function of inadequate isolation techniques, driven in part by the sparseness of these cells and their similarities with the more abundant retinal Müller cells. Here, we present a novel method of isolating retinal astrocytes and enriching their RNA, tested in both normal and ocular hypertensive mice, a common model of experimental glaucoma. Our approach combines a novel enzyme assisted microdissection of retinal astrocytes with selective ribosome immunoprecipitation using the Ribotag method. Our microdissection method is rapid and preserves astrocyte morphology, resulting in a brief post-mortem interval and minimizing loss of RNA from distal regions of these cells. Both microdissection and Ribotag immunoprecipitation require a minimum of specialized equipment or reagents, and by using them in conjunction we are able to achieve > 100-fold enrichment of astrocyte RNA. [ABSTRACT FROM AUTHOR]

To expedite gene discovery in eye development and its associated defects, we previously developed a bioinformatics resource-tool iSyTE (integrated Systems Tool for Eye gene discovery). However, iSyTE is presently limited to lens tissue and is predominantly based on transcriptomics datasets. Therefore, to extend iSyTE to other eye tissues on the proteome level, we performed high-throughput tandem mass spectrometry (MS/MS) on mouse embryonic day (E)14.5 retina and retinal pigment epithelium combined tissue and identified an average of 3300 proteins per sample (n = 5). High-throughput expression profiling-based gene discovery approaches–involving either transcriptomics or proteomics—pose a key challenge of prioritizing candidates from thousands of RNA/proteins expressed. To address this, we used MS/MS proteome data from mouse whole embryonic body (WB) as a reference dataset and performed comparative analysis–termed "in silico WB-subtraction"—with the retina proteome dataset. In silico WB-subtraction identified 90 high-priority proteins with retina-enriched expression at stringency criteria of ≥ 2.5 average spectral counts, ≥ 2.0 fold-enrichment, false discovery rate < 0.01. These top candidates represent a pool of retina-enriched proteins, several of which are associated with retinal biology and/or defects (e.g., Aldh1a1, Ank2, Ank3, Dcn, Dync2h1, Egfr, Ephb2, Fbln5, Fbn2, Hras, Igf2bp1, Msi1, Rbp1, Rlbp1, Tenm3, Yap1, etc.), indicating the effectiveness of this approach. Importantly, in silico WB-subtraction also identified several new high-priority candidates with potential regulatory function in retina development. Finally, proteins exhibiting expression or enriched-expression in the retina are made accessible in a user-friendly manner at iSyTE (https://research.bioinformatics.udel.edu/iSyTE/), to allow effective visualization of this information and facilitate eye gene discovery. [ABSTRACT FROM AUTHOR]

One hurdle in the development of zebrafish models of human disease is the presence of multiple zebrafish orthologs resulting from whole genome duplication in teleosts. Mutations in inositol polyphosphate 5-phosphatase K (INPP5K) lead to a syndrome characterized by variable presentation of intellectual disability, brain abnormalities, cataracts, muscle disease, and short stature. INPP5K is a phosphatase acting at position 5 of phosphoinositides to control their homeostasis and is involved in insulin signaling, cytoskeletal regulation, and protein trafficking. Previously, our group and others have replicated the human phenotypes in zebrafish knockdown models by targeting both INPP5K orthologs inpp5ka and inpp5kb. Here, we show that inpp5ka is the more closely related orthologue to human INPP5K. While both inpp5ka and inpp5kb mRNA expression levels follow a similar trend in the developing head, eyes, and tail, inpp5ka is much more abundantly expressed in these tissues than inpp5kb. In situ hybridization revealed a similar trend, also showing unique localization of inpp5kb in the pineal gland and retina indicating different transcriptional regulation. We also found that inpp5kb has lost its catalytic activity against its preferred substrate, PtdIns(4,5)P2. Since most human mutations are missense changes disrupting phosphatase activity, we propose that loss of inpp5ka alone can be targeted to recapitulate the human presentation. In addition, we show that the function of inpp5kb has diverged from inpp5ka and may play a novel role in the zebrafish. [ABSTRACT FROM AUTHOR]

Müller glia play very important and diverse roles in retinal homeostasis and disease. Although much is known of the physiological and morphological properties of mammalian Müller glia, there is still the need to further understand the profile of these cells during human retinal development. Using human embryonic stem cell-derived retinal organoids, we investigated the transcriptomic profiles of CD29+/CD44+ cells isolated from early and late stages of organoid development. Data showed that these cells express classic markers of retinal progenitors and Müller glia, including NFIX, RAX, PAX6, VSX2, HES1, WNT2B, SOX, NR2F1/2, ASCL1 and VIM, as early as days 10–20 after initiation of retinal differentiation. Expression of genes upregulated in CD29+/CD44+ cells isolated at later stages of organoid development (days 50–90), including NEUROG1, VSX2 and ASCL1 were gradually increased as retinal organoid maturation progressed. Based on the current observations that CD24+/CD44+ cells share the characteristics of early and late-stage retinal progenitors as well as of mature Müller glia, we propose that these cells constitute a single cell population that upon exposure to developmental cues regulates its gene expression to adapt to functions exerted by Müller glia in the postnatal and mature retina. [ABSTRACT FROM AUTHOR]

Microglia have been implicated in many degenerative eye disorders, including retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, uveitis, and retinal detachment. While the exact roles of microglia in these conditions are still being discovered, evidence from animal models suggests that they can modulate the course of disease. In this review, we highlight current strategies to target microglia in the eye and their potential as treatments for both rare and common ocular disorders. These approaches include depleting microglia with chemicals or radiation, reprogramming microglia using homeostatic signals or other small molecules, and inhibiting the downstream effects of microglia such as by blocking cytokine activity or phagocytosis. Finally, we describe areas of future research needed to fully exploit the therapeutic value of microglia in eye diseases. [ABSTRACT FROM AUTHOR]

Retinal degenerative diseases are a group of heterogeneous eye diseases that affect a significant percentage of the world's population, i.e., age-related macular degeneration (AMD), diabetic retinopathy, retinitis pigmentosa (RP), and glaucoma. Regenerative medicines look for novel therapies for severe injuries or chronic diseases, e.g., retina degeneration. Müller glia is the only retinal glia type with a common embryonic origin, with retinal neurons derived from the neural crest. Also, the lack of neurons in the retina does not automatically regenerate. Therefore, Müller glial cells, which make up about 5% of retinal cells, are a potent source for retinal regeneration. Following the retinal damage, Müller glial cells dedifferentiate and re-enter the cell cycle, producing multipotent progenitor cells. This feature leads to applying Müller glial cells in the regeneration of the retina. This study reviews this feature's molecular and clinical approaches, focusing on the critical signaling pathways, generation and transplantation methods, and clinical and sub-clinical challenges. [ABSTRACT FROM AUTHOR]

Background: The development of highly scalable single-cell transcriptome technology has resulted in the creation of thousands of datasets, >30 in the retina alone. Analyzing the transcriptomes between different projects is highly desirable because this would allow for better assessment of which biological effects are consistent across independent studies. However it is difficult to compare and contrast data across different projects because there are substantial batch effects from computational processing, single-cell technology utilized, and the natural biological variation. While many single-cell transcriptome-specific batch correction methods purport to remove the technical noise, it is difficult to ascertain which method functions best. Results: We developed a lightweight R package (scPOP, single-cell Pick Optimal Parameters) that brings in batch integration methods and uses a simple heuristic to balance batch merging and cell type/cluster purity. We use this package along with a Snakefile-based workflow system to demonstrate how to optimally merge 766,615 cells from 33 retina datsets and 3 species to create a massive ocular single-cell transcriptome meta-atlas. Conclusions: This provides a model for how to efficiently create meta-atlases for tissues and cells of interest. [ABSTRACT FROM AUTHOR]

The human retina is a complex neural tissue that detects light and sends visual information to the brain. However, the molecular and cellular processes that underlie aging primate retina remain unclear. Here, we provide a comprehensive transcriptomic atlas based on 119 520 single cells of the foveal and peripheral retina of humans and macaques covering different ages. The molecular features of retinal cells differed between the two species, suggesting distinct regional and species specializations of the human and macaque retinae. In addition, human retinal aging occurred in a region- and cell-type-specific manner. Aging of human retina exhibited a foveal to peripheral gradient. MYO9A− rods and a horizontal cell subtype were greatly reduced in aging retina, indicating their vulnerability to aging. Moreover, we generated a dataset showing the cell-type- and region-specific gene expression associated with 55 types of human retinal disease, which provides a foundation to understanding of the molecular and cellular mechanisms underlying human retinal diseases. Such datasets are valuable to understanding of the molecular characteristics of primate retina, as well as molecular regulation of aging progression and related diseases. [ABSTRACT FROM AUTHOR]

Retinal diseases constitute a genetically and phenotypically diverse group of clinical conditions leading to vision impairment or blindness with limited treatment options. Advances in reprogramming of somatic cells to induced pluripotent stem cells and generation of three‐dimensional organoids resembling the native retina offer promising tools to interrogate disease mechanisms and evaluate potential therapies for currently incurable retinal neurodegeneration. Next‐generation sequencing, single‐cell analysis, advanced electrophysiology, and high‐throughput screening approaches are expected to greatly expand the utility of stem cell‐derived retinal cells and organoids for developing personalized treatments. In this review, we discuss the current status and future potential of combining retinal organoids as human models with recent technologies to advance the development of gene, cell, and drug therapies for retinopathies. [ABSTRACT FROM AUTHOR]

Single-cell sequencing technologies have emerged as a revolutionary tool with transformative new methods to profile genetic, epigenetic, spatial, and lineage information in individual cells. Single-cell RNA sequencing (scRNA-Seq) allows researchers to collect large datasets detailing the transcriptomes of individual cells in space and time and is increasingly being applied to reveal cellular heterogeneity in retinal development, normal physiology, and disease, and provide new insights into cell-type specific markers and signaling pathways. In recent years, scRNA-Seq datasets have been generated from retinal tissue and pluripotent stem cell-derived retinal organoids. Their cross-comparison enables staging of retinal organoids, identification of specific cells in developing and adult human neural retina and provides deeper insights into cell-type sub-specification and geographical differences. In this article, we review the recent rapid progress in scRNA-Seq analyses of retina and retinal organoids, the questions that remain unanswered and the technical challenges that need to be overcome to achieve consistent results that reflect the complexity, functionality, and interactions of all retinal cell types. [ABSTRACT FROM AUTHOR]