A single-cell transcriptome atlas of the adult human retina.

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.

A molecular and cellular analysis of human embryonic optic fissure closure related to the eye malformation coloboma.

Ocular coloboma is a congenital eye malformation, resulting from a failure in optic fissure closure (OFC) and causing visual impairment. There has been little study of the epithelial fusion process underlying closure in the human embryo and coloboma aetiology remains poorly understood. We performed RNAseq of cell populations isolated using laser capture microdissection to identify novel human OFC signature genes and probe the expression profile of known coloboma genes, along with a comparative murine analysis. Gene set enrichment patterns showed conservation between species. Expression of genes involved in epithelial-to-mesenchymal transition was transiently enriched in the human fissure margins during OFC at days 41-44. Electron microscopy and histological analyses showed that cells transiently delaminate at the point of closure, and produce cytoplasmic protrusions, before rearranging to form two continuous epithelial layers. Apoptosis was not observed in the human fissure margins. These analyses support a model of human OFC in which epithelial cells at the fissure margins undergo a transient epithelial-to-mesenchymal-like transition, facilitating cell rearrangement to form a complete optic cup.

Generation of a Cone Photoreceptor-specific GNGT2 Reporter Line in Human Pluripotent Stem Cells.

Fluorescent reporter lines generated in human pluripotent stem cells are a highly useful tool to track, isolate, and analyze cell types and lineages in live cultures. Here, we generate the first human cone photoreceptor reporter cell line by CRISPR/Cas9 genome editing of a human embryonic stem cell (hESC) line to tag both alleles of the Guanine nucleotide-binding protein subunit gamma-T2 (GNGT2) gene with a mCherry reporter cassette. Three-dimensional optic vesicle-like structures were produced to verify reporter fidelity and track cones throughout their development in culture. The GNGT2-T2A-mCherry hESC line faithfully and robustly labels GNGT2-expressing cones throughout the entirety of their differentiation in vitro, recapitulating normal fetal expression of this gene. Our observations indicate that human cones undergo significant migratory activity during the course of differentiation in vitro. Consistent with this, our analysis of human fetal retinae from different stages of development finds positional differences of the cone population depending on their state of maturation. This novel reporter line will provide a useful tool for investigating human cone development and disease.

Modeling Retinitis Pigmentosa with Patient-Derived iPSCs.

Retinitis pigmentosa (RP) causes blindness in 1 out of 3000-4000 individuals worldwide. Understanding the disease mechanism underlying the death of photoreceptors in RP patient is crucial for the discovery and development of therapies to prevent and stop the progression of retinal degeneration. Despite having provided valuable insight into RP pathology, several shortcomings of animal models warrant the need for a better modeling system. This review discusses the current use of patient-derived induced pluripotent stem cells (iPSCs) to model RP and its advantages over animal models. Further improvement to enhance the representativeness of iPSC RP models is also discussed.

NRL-/- gene edited human embryonic stem cells generate rod-deficient retinal organoids enriched in S-cone-like photoreceptors.

Organoid cultures represent a unique tool to investigate the developmental complexity of tissues like the human retina. NRL is a transcription factor required for the specification and homeostasis of mammalian rod photoreceptors. In Nrl-deficient mice, photoreceptor precursor cells do not differentiate into rods, and instead follow a default photoreceptor specification pathway to generate S-cone-like cells. To investigate whether this genetic switch mechanism is conserved in humans, we used CRISPR/Cas9 gene editing to engineer an NRL-deficient embryonic stem cell (ESC) line (NRL-/- ), and differentiated it into retinal organoids. Retinal organoids self-organize and resemble embryonic optic vesicles (OVs) that recapitulate the natural histogenesis of rods and cone photoreceptors. NRL-/- OVs develop comparably to controls, and exhibit a laminated, organized retinal structure with markers of photoreceptor synaptogenesis. Using immunohistochemistry and quantitative polymerase chain reaction (qPCR), we observed that NRL-/- OVs do not express NRL, or other rod photoreceptor markers directly or indirectly regulated by NRL. On the contrary, they show an abnormal number of photoreceptors positive for S-OPSIN, which define a primordial subtype of cone, and overexpress other cone genes indicating a conserved molecular switch in mammals. This study represents the first evidence in a human in vitro ESC-derived organoid system that NRL is required to define rod identity, and that in its absence S-cone-like cells develop as the default photoreceptor cell type. It shows how gene edited retinal organoids provide a useful system to investigate human photoreceptor specification, relevant for efforts to generate cells for transplantation in retinal degenerative diseases.

Genes and pathways in optic fissure closure.

Embryonic development of the vertebrate eye begins with the formation of an optic vesicle which folds inwards to form a double-layered optic cup with a fissure on the ventral surface, known as the optic fissure. Closure of the optic fissure is essential for subsequent growth and development of the eye. A defect in this process can leave a gap in the iris, retina or optic nerve, known as a coloboma, which can lead to severe visual impairment. This review brings together current information about genes and pathways regulating fissure closure from human coloboma patients and animal models. It focuses especially on current understanding of the morphological changes and processes of epithelial remodelling occurring at the fissure margins.

Mutations in CPAMD8 Cause a Unique Form of Autosomal-Recessive Anterior Segment Dysgenesis.

Anterior segment dysgeneses (ASDs) comprise a spectrum of developmental disorders affecting the anterior segment of the eye. Here, we describe three unrelated families affected by a previously unclassified form of ASD. Shared ocular manifestations include bilateral iris hypoplasia, ectopia lentis, corectopia, ectropion uveae, and cataracts. Whole-exome sequencing and targeted Sanger sequencing identified mutations in CPAMD8 (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8) as the cause of recessive ASD in all three families. A homozygous missense mutation in the evolutionarily conserved alpha-2-macroglobulin (A2M) domain of CPAMD8, c.4351T>C (p. Ser1451Pro), was identified in family 1. In family 2, compound heterozygous frameshift, c.2352_2353insC (p.Arg785Glnfs∗23), and splice-site, c.4549-1G>A, mutations were identified. Two affected siblings in the third family were compound heterozygous for splice-site mutations c.700+1G>T and c.4002+1G>A. CPAMD8 splice-site mutations caused aberrant pre-mRNA splicing in vivo or in vitro. Intriguingly, our phylogenetic analysis revealed rodent lineage-specific CPAMD8 deletion, precluding a developmental expression study in mice. We therefore investigated the spatiotemporal expression of CPAMD8 in the developing human eye. RT-PCR and in situ hybridization revealed CPAMD8 expression in the lens, iris, cornea, and retina early in development, including strong expression in the distal tips of the retinal neuroepithelium that form the iris and ciliary body, thus correlating CPAMD8 expression with the affected tissues. Our study delineates a unique form of recessive ASD and defines a role for CPAMD8, a protein of unknown function, in anterior segment development, implying another pathway for the pathogenicity of ASD.

The Oculome Panel Test: Next-Generation Sequencing to Diagnose a Diverse Range of Genetic Developmental Eye Disorders.

PURPOSE: To develop a comprehensive next-generation sequencing panel assay that screens genes known to cause developmental eye disorders and inherited eye disease and to evaluate its diagnostic yield in a pediatric cohort with malformations of the globe, anterior segment anomalies, childhood glaucoma, or a combination thereof. DESIGN: Evaluation of diagnostic test. PARTICIPANTS: Two hundred seventy-seven children, 0 to 16 years of age, diagnosed with nonsyndromic or syndromic developmental eye defects without a genetic diagnosis. METHODS: We developed a new oculome panel using a custom-designed Agilent SureSelect QXT target capture method (Agilent Technologies, Santa Clara, CA) to capture and perform parallel high-throughput sequencing analysis of 429 genes associated with eye disorders. Bidirectional Sanger sequencing confirmed suspected pathogenic variants. MAIN OUTCOME MEASURES: Collated clinical details and oculome molecular genetic results. RESULTS: The oculome design covers 429 known eye disease genes; these are subdivided into 5 overlapping virtual subpanels for anterior segment developmental anomalies including glaucoma (ASDA; 59 genes), microphthalmia-anophthalmia-coloboma (MAC; 86 genes), congenital cataracts and lens-associated conditions (70 genes), retinal dystrophies (RET; 235 genes), and albinism (15 genes), as well as additional genes implicated in optic atrophy and complex strabismus (10 genes). Panel development and testing included analyzing 277 clinical samples and 3 positive control samples using Illumina sequencing platforms; more than 30× read depth was achieved for 99.5% of the targeted 1.77-Mb region. Bioinformatics analysis performed using a pipeline based on Freebayes and ExomeDepth to identify coding sequence and copy number variants, respectively, resulted in a definitive diagnosis in 68 of 277 samples, with variability in diagnostic yield between phenotypic subgroups: MAC, 8.2% (8 of 98 cases solved); ASDA, 24.8% (28 of 113 cases solved); other or syndromic, 37.5% (3 of 8 cases solved); RET, 42.8% (21 of 49 cases solved); and congenital cataracts and lens-associated conditions, 88.9% (8 of 9 cases solved). CONCLUSIONS: The oculome test diagnoses a comprehensive range of genetic conditions affecting the development of the eye, potentially replacing protracted and costly multidisciplinary assessments and allowing for faster targeted management. The oculome enabled molecular diagnosis of a significant number of cases in our sample cohort of varied ocular birth defects.

Isolation of Human Photoreceptor Precursors via a Cell Surface Marker Panel from Stem Cell-Derived Retinal Organoids and Fetal Retinae.

Loss of photoreceptor cells due to retinal degeneration is one of the main causes of blindness in the developed world. Although there is currently no effective treatment, cell replacement therapy using stem-cell-derived photoreceptor cells may be a feasible future treatment option. In order to ensure safety and efficacy of this approach, robust cell isolation and purification protocols must be developed. To this end, we previously developed a biomarker panel for the isolation of mouse photoreceptor precursors from the developing mouse retina and mouse embryonic stem cell cultures. In the current study we applied this approach to the human pluripotent stem cell (hPSC) system, and identified novel biomarker combinations that can be leveraged for the isolation of human photoreceptors. Human retinal samples and hPSC-derived retinal organoid cultures were screened against 242 human monoclonal antibodies using a high through-put flow cytometry approach. We identified 46 biomarkers with significant expression levels in the human retina and hPSC differentiation cultures. Human retinal cell samples, either from fetal tissue or derived from embryonic and induced pluripotent stem cell cultures, were fluorescence-activated cell sorted (FACS) using selected candidate biomarkers that showed expression in discrete cell populations. Enrichment for photoreceptors and exclusion of mitotically active cells was demonstrated by immunocytochemical analysis with photoreceptor-specific antibodies and Ki-67. We established a biomarker combination, which enables the robust purification of viable human photoreceptors from both human retinae and hPSC-derived organoid cultures. Stem Cells 2018;36:709-722.

Restoring Vision Using Stem Cells and Transplantation.

The replacement of retinal cells, or the support of surviving retinal neurons, in a degenerated retina presents a significant challenge in the fields of ophthalmology and regenerative medicine. Stem cell-based therapies are being explored as an approach for treating retinal dystrophies, such as retinitis pigmentosa (RP), Stargardt's disease, and age-related macular degeneration (AMD). This review provides an update on the recent progress made toward the restoration of vision lost to degenerative disease using stem cell-based transplantation strategies and the challenges that need to be overcome. Both retinal pigmented epithelium (RPE) and photoreceptor replacement therapies are discussed.

Mutation of SALL2 causes recessive ocular coloboma in humans and mice.

Ocular coloboma is a congenital defect resulting from failure of normal closure of the optic fissure during embryonic eye development. This birth defect causes childhood blindness worldwide, yet the genetic etiology is poorly understood. Here, we identified a novel homozygous mutation in the SALL2 gene in members of a consanguineous family affected with non-syndromic ocular coloboma variably affecting the iris and retina. This mutation, c.85G>T, introduces a premature termination codon (p.Glu29*) predicted to truncate the SALL2 protein so that it lacks three clusters of zinc-finger motifs that are essential for DNA-binding activity. This discovery identifies SALL2 as the third member of the Drosophila homeotic Spalt-like family of developmental transcription factor genes implicated in human disease. SALL2 is expressed in the developing human retina at the time of, and subsequent to, optic fissure closure. Analysis of Sall2-deficient mouse embryos revealed delayed apposition of the optic fissure margins and the persistence of an anterior retinal coloboma phenotype after birth. Sall2-deficient embryos displayed correct posterior closure toward the optic nerve head, and upon contact of the fissure margins, dissolution of the basal lamina occurred and PAX2, known to be critical for this process, was expressed normally. Anterior closure was disrupted with the fissure margins failing to meet, or in some cases misaligning leading to a retinal lesion. These observations demonstrate, for the first time, a role for SALL2 in eye morphogenesis and that loss of function of the gene causes ocular coloboma in humans and mice.

Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel From Embryonic Stem Cell-Derived Self-Forming Retina.

Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell-derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post-mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(-) facilitates the isolation of photoreceptor precursors from three-dimensional self-forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell-derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation-competent cells for therapeutic application.

Repair of the degenerate retina by photoreceptor transplantation.

Despite different aetiologies, age-related macular degeneration and most inherited retinal disorders culminate in the same final common pathway, the loss of photoreceptors. There are few treatments and none reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy applicable to all degenerations. Recently, we demonstrated restoration of vision following rod-photoreceptor transplantation into a mouse model of stationary night-blindness, raising the critical question of whether photoreceptor replacement is equally effective in different types and stages of degeneration. We present a comprehensive assessment of rod-photoreceptor transplantation across six murine models of inherited photoreceptor degeneration. Transplantation is feasible in all models examined but disease type has a major impact on outcome, as assessed both by the morphology and number of integrated rod-photoreceptors. Integration can increase (Prph2(+/Δ307)), decrease (Crb1(rd8/rd8), Gnat1(-/-), Rho(-/-)), or remain constant (PDE6ß(rd1/rd1), Prph2(rd2/rd2)) with disease progression, depending upon the gene defect, with no correlation with severity. Robust integration is possible even in late-stage disease. Glial scarring and outer limiting membrane integrity, features that change with degeneration, significantly affect transplanted photoreceptor integration. Combined breakdown of these barriers markedly increases integration in a model with an intact outer limiting membrane, strong gliotic response, and otherwise poor transplantation outcome (Rho(-/-)), leading to an eightfold increase in integration and restoration of visual function. Thus, it is possible to achieve robust integration across a broad range of inherited retinopathies. Moreover, transplantation outcome can be improved by administering appropriate, tailored manipulations of the recipient environment.

Functional analysis of FOXE3 mutations causing dominant and recessive ocular anterior segment disease.

Mutations in FOXE3 are associated with both recessive and dominant inheritance of severe anterior ocular malformations and glaucoma. However, functional analyses of putative pathogenic mutations have not been performed. We tested the hypothesis that variations in FOXE3 activity underlie the different modes of inheritance and disease phenotype. In band shift assays, three recessive mutants showed loss-of-function, one retained DNA binding activity, whereas two dominant mutants showed altered activity. All six mutants showed reduced transactivation function compared with wild-type, and modeling the heterozygous state resulted in an intermediate level of activity providing no evidence for dominant negative action. Our in vitro data are consistent with loss-of-function below a dosage sensitive threshold as a mechanism of action for recessive mutations, but indicate an altered mutant protein function rather than a haploinsufficient mechanism for dominant mutations. This study provides the first functional evidence demonstrating that FOXE3 mutations identified in patients impair protein function with differential effects.

X-linked megalocornea caused by mutations in CHRDL1 identifies an essential role for ventroptin in anterior segment development.

X-linked megalocornea (MGC1) is an ocular anterior segment disorder characterized by an increased cornea diameter and deep anterior chamber evident at birth and later onset of mosaic corneal degeneration (shagreen), arcus juvenilis, and presenile cataracts. We identified copy-number variation, frameshift, missense, splice-site and nonsense mutations in the Chordin-like 1 gene (CHRDL1) on Xq23 as the cause of the condition in seven MGC1 families. CHRDL1 encodes ventroptin, a bone morphogenic protein antagonist with a proposed role in specification of topographic retinotectal projections. Electrophysiological evaluation revealed mild generalized cone system dysfunction and, in one patient, an interhemispheric asymmetry in visual evoked potentials. We show that CHRDL1 is expressed in the developing human cornea and anterior segment in addition to the retina. We explored the impact of loss of ventroptin function on brain function and morphology in vivo. CHRDL1 is differentially expressed in the human fetal brain, and there is high expression in cerebellum and neocortex. We show that MGC1 patients have a superior cognitive ability despite a striking focal loss of myelination of white matter. Our findings reveal an unexpected requirement for ventroptin during anterior segment development and the consequences of a lack of function in the retina and brain.

Von Hippel-Lindau protein in the RPE is essential for normal ocular growth and vascular development.

Molecular oxygen is essential for the development, growth and survival of multicellular organisms. Hypoxic microenvironments and oxygen gradients are generated physiologically during embryogenesis and organogenesis. In the eye, oxygen plays a crucial role in both physiological vascular development and common blinding diseases. The retinal pigment epithelium (RPE) is a monolayer of cells essential for normal ocular development and in the mature retina provides support for overlying photoreceptors and their vascular supply. Hypoxia at the level of the RPE is closely implicated in pathogenesis of age-related macular degeneration. Adaptive tissue responses to hypoxia are orchestrated by sophisticated oxygen sensing mechanisms. In particular, the von Hippel-Lindau tumour suppressor protein (pVhl) controls hypoxia-inducible transcription factor (HIF)-mediated adaptation. However, the role of Vhl/Hif1a in the RPE in the development of the eye and its vasculature is unknown. In this study we explored the function of Vhl and Hif1a in the developing RPE using a tissue-specific conditional-knockout approach. We found that deletion of Vhl in the RPE results in RPE apoptosis, aniridia and microphthalmia. Increased levels of Hif1a, Hif2a, Epo and Vegf are associated with a highly disorganised retinal vasculature, chorioretinal anastomoses and the persistence of embryonic vascular structures into adulthood. Additional inactivation of Hif1a in the RPE rescues the RPE morphology, aniridia, microphthalmia and anterior vasoproliferation, but does not rescue retinal vasoproliferation. These data demonstrate that Vhl-dependent regulation of Hif1a in the RPE is essential for normal RPE and iris development, ocular growth and vascular development in the anterior chamber, whereas Vhl-dependent regulation of other downstream pathways is crucial for normal development and maintenance of the retinal vasculature.

ATOH7 mutations cause autosomal recessive persistent hyperplasia of the primary vitreous.

The vertebrate basic helix-loop-helix (bHLH) transcription factor ATOH7 (Math5) is specifically expressed in the embryonic neural retina and is required for the genesis of retinal ganglion cells (RGCs) and optic nerves. In Atoh7 mutant mice, the absence of trophic factors secreted by RGCs prevents the development of the intrinsic retinal vasculature and the regression of fetal blood vessels, causing persistent hyperplasia of the primary vitreous (PHPV). We therefore screened patients with hereditary PHPV, as well as bilateral optic nerve aplasia (ONA) or hypoplasia (ONH), for mutations in ATOH7. We identified a homozygous ATOH7 mutation (N46H) in a large family with an autosomal recessive PHPV disease trait linked to 10q21, and a heterozygous variant (R65G, p.Arg65Gly) in one of five sporadic ONA patients. High-density single-nucleotide polymorphism analysis also revealed a CNTN4 duplication and an OTX2 deletion in the ONA cohort. Functional analysis of ATOH7 bHLH domain substitutions, by electrophoretic mobility shift and luciferase cotransfection assays, revealed that the N46H variant cannot bind DNA or activate transcription, consistent with structural modeling. The N46H variant also failed to rescue RGC development in mouse Atoh7-/- retinal explants. The R65G variant retains all of these activities, similar to wild-type human ATOH7. Our results strongly suggest that autosomal recessive persistent hyperplastic primary vitreous is caused by N46H and is etiologically related to nonsyndromic congenital retinal nonattachment. The R65G allele, however, cannot explain the ONA phenotype. Our study firmly establishes ATOH7 as a retinal disease gene and provides a functional basis to analyze new coding variants.

Brief report: self-organizing neuroepithelium from human pluripotent stem cells facilitates derivation of photoreceptors.

Retinitis pigmentosa, other inherited retinal diseases, and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells. Current methods for photoreceptor derivation from hPS cells require long periods of culture and are rather inefficient. Here, we report that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix is sufficient to induce a rapid conversion into retinal progenitors in 5 days. These retinal progenitors have the ability to differentiate very efficiently into Crx(+) photoreceptor precursors after only 10 days and subsequently acquire rod photoreceptor identity within 4 weeks. Directed differentiation into photoreceptors using this protocol is also possible with human-induced pluripotent stem (hiPS) cells, facilitating the use of patient-specific hiPS cell lines for regenerative medicine and disease modeling.

ARNT2 mutation causes hypopituitarism, post-natal microcephaly, visual and renal anomalies.

We describe a previously unreported syndrome characterized by secondary (post-natal) microcephaly with fronto-temporal lobe hypoplasia, multiple pituitary hormone deficiency, seizures, severe visual impairment and abnormalities of the kidneys and urinary tract in a highly consanguineous family with six affected children. Homozygosity mapping and exome sequencing revealed a novel homozygous frameshift mutation in the basic helix-loop-helix transcription factor gene ARNT2 (c.1373_1374dupTC) in affected individuals. This mutation results in absence of detectable levels of ARNT2 transcript and protein from patient fibroblasts compared with controls, consistent with nonsense-mediated decay of the mutant transcript and loss of ARNT2 function. We also show expression of ARNT2 within the central nervous system, including the hypothalamus, as well as the renal tract during human embryonic development. The progressive neurological abnormalities, congenital hypopituitarism and post-retinal visual pathway dysfunction in affected individuals demonstrates for the first time the essential role of ARNT2 in the development of the hypothalamo-pituitary axis, post-natal brain growth, and visual and renal function in humans.