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The choriocapillaris, a dense capillary network located at the posterior pole of the eye, is essential for supporting normal vision, supplying nutrients, and removing waste products from photoreceptor cells and the retinal pigment epithelium. The anatomical location, heterogeneity, and homeostatic interactions with surrounding cell types make the choroid complex to study both in vivo and in vitro. Recent advances in single-cell RNA sequencing, in vivo imaging, and in vitro cell modeling are vastly improving our knowledge of the choroid and its role in normal health and in age-related macular degeneration (AMD). Histologically, loss of endothelial cells (ECs) of the choriocapillaris occurs early in AMD concomitant with elevated formation of the membrane attack complex of complement. Advanced imaging has allowed us to visualize early choroidal blood flow changes in AMD in living patients, supporting histological findings of loss of choroidal ECs. Single-cell RNA sequencing is being used to characterize choroidal cell types transcriptionally and discover their altered patterns of gene expression in aging and disease. Advances in induced pluripotent stem cell protocols and 3D cultures will allow us to closely mimic the in vivo microenvironment of the choroid in vitro to better understand the mechanism leading to choriocapillaris loss in AMD.

The effects of radiation retinopathy on the retinal vasculature have been well established; however, the literature describing the pathologic changes in the choriocapillaris is relatively lacking. In this report, we describe the histologic findings of a donor eye with a choroidal melanoma with special attention to the choriocapillaris. Clinical and histological findings, including immunohistochemistry and transmission electron microscopy, are described for the retina and choroid of a donor eye affected by radiation retinopathy secondary to treatment of choroidal melanoma. Cells within the tumor exhibited an epithelioid structure and balloon melanosomes. Notable infiltration of macrophages with elongated morphology was also observed. Atrophy of photoreceptors, retinal pigmented epithelium, and choriocapillaris was observed on the inferior edge of the lesion and extending past the tumor. The choriocapillaris endothelium showed more severe dropout at the periphery of the lesion where loss of fenestration, thickened cytosol, and degenerated pericytes were observed. Morphologic analysis revealed choriocapillaris loss with pronounced degeneration of choroidal pericytes. Understanding the differences in sensitivity to radiation injury between different cell types and different patients will provide better insight into radiation retinopathy.

Prior to use, newly generated induced pluripotent stem cells (iPSC) should be thoroughly validated. While excellent validation and release testing assays designed to evaluate potency, genetic integrity, and sterility exist, they do not have the ability to predict cell type-specific differentiation capacity. Selection of iPSC lines that have limited capacity to produce high-quality transplantable cells, places significant strain on valuable clinical manufacturing resources. The purpose of this study was to determine the degree and root cause of variability in retinal differentiation capacity between cGMP-derived patient iPSC lines. In turn, our goal was to develop a release testing assay that could be used to augment the widely used ScoreCard panel. IPSCs were generated from 15 patients (14-76 years old), differentiated into retinal organoids, and scored based on their retinal differentiation capacity. Despite significant differences in retinal differentiation propensity, RNA-sequencing revealed remarkable similarity between patient-derived iPSC lines prior to differentiation. At 7 days of differentiation, significant differences in gene expression could be detected. Ingenuity pathway analysis revealed perturbations in pathways associated with pluripotency and early cell fate commitment. For example, good and poor producers had noticeably different expressions of OCT4 and SOX2 effector genes. QPCR assays targeting genes identified via RNA sequencing were developed and validated in a masked fashion using iPSCs from 8 independent patients. A subset of 14 genes, which include the retinal cell fate markers RAX, LHX2, VSX2, and SIX6 (all elevated in the good producers), were found to be predictive of retinal differentiation propensity.

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

The human choroid is a heterogeneous, highly vascular connective tissue that dysfunctions in age-related macular degeneration (AMD). In this study, we performed single-cell RNA sequencing on 21 human choroids, 11 of which were derived from donors with early atrophic or neovascular AMD. Using this large donor cohort, we identified new gene expression signatures and immunohistochemically characterized discrete populations of resident macrophages, monocytes/inflammatory macrophages and dendritic cells. These three immune populations demonstrated unique expression patterns for AMD genetic risk factors, with dendritic cells possessing the highest expression of the neovascular AMD-associated MMP9 gene. Additionally, we performed trajectory analysis to model transcriptomic changes across the choroidal vasculature, and we identified expression signatures for endothelial cells from choroidal arterioles and venules. Finally, we performed differential expression analysis between control, early atrophic AMD, and neovascular AMD samples, and we observed that early atrophic AMD samples had high expression of SPARCL1, a gene that has been shown to increase in response to endothelial damage. Likewise, neovascular endothelial cells harbored gene expression changes consistent with endothelial cell damage and demonstrated increased expression of the sialomucins CD34 and ENCM, which were also observed at the protein level within neovascular membranes. Overall, this study characterizes the molecular features of new populations of choroidal endothelial cells and mononuclear phagocytes in a large cohort of AMD and control human donors.

Background Inherited retinal degeneration is a leading cause of incurable vision loss in the developed world. While autologous iPSC mediated photoreceptor cell replacement is theoretically possible, the lack of commercially available technologies designed to enable high throughput parallel production of patient specific therapeutics has hindered clinical translation. Methods In this study, we describe the use of the Cell X precision robotic cell culture platform to enable parallel production of clinical grade patient specific iPSCs. The Cell X is housed within an ISO Class 5 cGMP compliant closed aseptic isolator (Biospherix XVivo X2), where all procedures from fibroblast culture to iPSC generation, clonal expansion and retinal differentiation were performed. Results Patient iPSCs generated using the Cell X platform were determined to be pluripotent via score card analysis and genetically stable via karyotyping. As determined via immunostaining and confocal microscopy, iPSCs generated using the Cell X platform gave rise to retinal organoids that were indistinguishable from organoids derived from manually generated iPSCs. In addition, at 120 days post-differentiation, single-cell RNA sequencing analysis revealed that cells generated using the Cell X platform were comparable to those generated under manual conditions in a separate laboratory. Conclusion We have successfully developed a robotic iPSC generation platform and standard operating procedures for production of high-quality photoreceptor precursor cells that are compatible with current good manufacturing practices. This system will enable clinical grade production of iPSCs for autologous retinal cell replacement.

Purpose: Despite numerous recent advances in retinal gene therapy using adeno-associated viruses (AAVs) as delivery vectors, there remains a crucial need to identify viral vectors with the ability to transduce specific retinal cell types and that have a larger carrying capacity than AAV. In this study, we evaluate the retinal tropism of 2 chimeric helper-dependent adenoviruses (HDAds), helper-dependent adenovirus serotype 5 (HDAd5)/3 and HDAd5/35, both ex vivo using human retinal explants and in vivo using rats. Methods: We transduced cultured human retinal explants with HDAd5/3 and HDAd5/35 carrying an eGFP vector and evaluated tropism and transduction efficiency using immunohistochemistry. To assess in vivo transduction efficiency, subretinal injections were performed in wild-type Sprague-Dawley rats. For both explants and subretinal injections, we delivered 10 μL (1 × 10(6) vector genomes/mL) and assessed tropism at 7- and 14-days post-transduction, respectively. Results: HDAd5/3 and HDAd5/35 both transduced human retinal ganglion cells (RGCs) and Müller cells, but not photoreceptors, in human retinal explants. However, subretinal injections in albino rats resulted in transduction of the retinal pigmented epithelium only, highlighting species-specific differences in retinal tropism and the value of a human explant model when testing vectors for eventual human gene therapy. Conclusions: Chimeric HDAds are promising candidates for the delivery of large genes, multiple genes, or neuroprotective factors to Müller cells and RGCs. These vectors may have utility for targeted therapy of neurodegenerative diseases primarily involving retinal ganglion or Müller cell types, such as glaucoma or macular telangiectasia type 2.

Autologous photoreceptor cell replacement is one of the most promising approaches currently under development for the treatment of inherited retinal degenerative blindness. Unlike endogenous stem cell populations, induced pluripotent stem cells (iPSCs) can be differentiated into both rod and cone photoreceptors in high numbers, making them ideal for this application. That said, in addition to photoreceptor cells, state of the art retinal differentiation protocols give rise to all of the different cell types of the normal retina, the majority of which are not required and may in fact hinder successful photoreceptor cell replacement. As such, following differentiation photoreceptor cell enrichment will likely be required. In addition, to prevent the newly generated photoreceptor cells from suffering the same fate as the patient's original cells, correction of the patient's disease‐causing genetic mutations will be necessary. In this review we discuss literature pertaining to the use of different cell sorting and transfection approaches with a focus on the development and use of novel next generation microfluidic devices. We will discuss how gold standard strategies have been used, the advantages and disadvantages of each, and how novel microfluidic platforms can be incorporated into the clinical manufacturing pipeline to reduce the complexity, cost, and regulatory burden associated with clinical grade production of photoreceptor cells for autologous cell replacement., Incorporation of novel microfluidic transfection and cell sorting strategies into an autologous photoreceptor cell replacement pipeline. A‐F, Autologous photoreceptor cell replacement for the treatment of inherited retinal degenerative blindness begins with induced pluripotent stem cell (iPSC) generation (A), followed by CRISPR correction of the patient's disease‐causing genetic mutations (B), generation of 3D retinal organoids (C), dissociation and enrichment of photoreceptor precursor cells (D), seeding of photoreceptor cell grafts (E), and subretinal transplantation (F).

The human neural retina is a light sensitive tissue with remarkable spatial and cellular organization. Compared with the periphery, the central retina contains more densely packed cone photoreceptor cells with unique morphologies and synaptic wiring. Some regions of the central retina exhibit selective degeneration or preservation in response to retinal disease and the basis for this variation is unknown. In this study, we used both bulk and single-cell RNA sequencing to compare gene expression within concentric regions of the central retina. We identified unique gene expression patterns of foveal cone photoreceptor cells, including many foveal-enriched transcription factors. In addition, we found that the genes RORB1, PPFIA1 and KCNAB2 are differentially spliced in the foveal, parafoveal and macular regions. These results provide a highly detailed spatial characterization of the retinal transcriptome and highlight unique molecular features of different retinal regions., Graphical abstract Retinal diseases can selectively affect some regions and spare others. Voigt et al. used single-cell RNA sequencing to identify gene expression differences within concentric regions of the human retina. Within the fovea, they identify region-specific transcription factors, gene splicing and protein expression.

PURPOSE To investigate the reflectivity of the structural optical coherence tomography images of the inner choroid as it relates to potential structural composition. METHODS The reflectivity of slab images 29 to 49 µm and 45 to 65 µm below the retinal pigment epithelium, obtained with the Zeiss Plex Elite 9000, was evaluated. The mean and SD of a group of subjects with no ocular disorders were determined. Binarization of the images was performed and a threshold level established at the mean plus one SD for each slab depth. The proportion of area binarized was evaluated with generalized estimating equations. Representative histologic images obtained from autopsy donors were stained with Masson's trichrome, a staining method helpful in evaluating collagen and ground substance of tissue. RESULTS There were 67 eyes of 38 subjects with a mean age of 44.5 (range 22-82) years. Using generalized estimating equations, age was found to be a significant predictor for the proportion of binarized pixels in both the 29 to 49 µm (P = 0.034) and the 45 to 65 µm (P < 0.001) slabs. The histologic specimens illustrated the loss of ground substance with increasing compaction of collagen fibers in the choroidal stroma with advancing age. CONCLUSION The reflectivity from the inner choroid is not uniform and changes with age. As suggested by the histologic specimens, we propose the optical coherence tomography reflectance from the inner choroid is related, in part, to the packing density of collagen fibers present there.

Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multi-system disease. The wide range of manifestations observed in mitochondrial disease patients results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify non- random distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA sequencing, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profile the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and healthy control donors. Utilizing the retina as a model for complex multi-lineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm- derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm- derived lineage, namely the vasculature of the choroid, was near homoplasmic for the wildtype allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the non- random nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment.

Pentosan polysulfate (PPS) sodium (Elmiron) is the only Food and Drug Administration (FDA)-approved oral medication to treat interstitial cystitis, also known as bladder pain syndrome. A symptomatic pigmentary maculopathy associated with PPS was reported in 2018. Since then, recognition of this unique drug toxicity has increased rapidly. This potentially sight-threatening side effect prompted the FDA in June 2020 to update the label for PPS to warn about "retinal pigmentary changes." A challenging feature of pentosan maculopathy is its ability to mimic many other retinal conditions, including inherited retinal dystrophies such as pattern dystrophy, mitochondrially inherited diabetes and deafness, and Stargardt disease, and age-related macular degeneration. In this review, we discuss the history of PPS maculopathy and its implications for thousands of at-risk interstitial cystitis patients. We use published literature and an illustrative case from our institution to highlight the importance of diagnosing PPS maculopathy. We also compare PPS maculopathy to age-related macular degeneration, explain why differentiating between the 2 is clinically important, and highlight avenues for further research. Finally, we highlight the paucity of data on patients of color and why this lack of understanding may impact patient care.

Viral-mediated gene augmentation offers tremendous promise for the treatment of inherited retinal diseases. The development of effective gene therapy requires an understanding of the vector's tissue-specific behavior, which may vary depending on serotype, route of delivery, or target species. Using an ex vivo organotypic explant system, we previously demonstrated that retinal tropism and transduction of adeno-associated virus type 2 (AAV2) vary significantly depending on serotype in human eyes. However, the ex vivo system has limited ability to assess route of ocular delivery, and relatively little literature exists on tropic differences between serotypes and routes of delivery in vivo. In this study, we demonstrate that retinal tropism and transduction efficiency of five different AAV2 serotypes (AAV2/1, AAV2/2, AAV2/6, AAV2/8, and AAV2/9) expressing enhanced green fluorescent protein driven by a cytomegalovirus promoter vary greatly depending on serotype and route of delivery (intravitreal, subretinal, or suprachoroidal) in rats. With subretinal delivery, all serotypes successfully transduced the retinal pigmented epithelium and outer nuclear layer (ONL), with AAV2/1 displaying the highest transduction efficiency and AAV2/2 and AAV2/6 showing lower ONL transduction. There was minimal transduction of the inner retina through subretinal delivery for any serotype. Tropism by suprachoroidal delivery mirrored that of subretinal delivery for all AAV serotypes but resulted in a wider distribution and greater ONL transduction. With intravitreal delivery, retinal transduction was seen primarily in the inner retina (retinal nerve fiber, ganglion cell, and inner nuclear layers) for AAV2/1 and AAV2/6, with AAV2/6 showing the highest transduction. When compared with data from human explant models, there are substantial differences in tropism and transduction that are important to consider when using rats as preclinical models for the development of ocular gene therapies for humans.

SUMMARY Retinal degeneration is a form of neurodegenerative disease and is the leading cause of vision loss globally. The Toll-like receptors (TLRs) are primary components of the innate immune system involved in signal transduction. Here we show that TLR2 induces complement factors C3 and CFB, the common and rate-limiting factors of the alternative pathway in both retinal pigment epithelial (RPE) cells and mononuclear phagocytes. Neutralization of TLR2 reduces opsonizing fragments of C3 in the outer retina and protects photoreceptor neurons from oxidative stress-induced degeneration. TLR2 deficiency also preserves tight junction expression and promotes RPE resistance to fragmentation. Finally, oxidative stress-induced formation of the terminal complement membrane attack complex and Iba1+ cell infiltration are strikingly inhibited in the TLR2-deficient retina. Our data directly implicate TLR2 as a mediator of retinal degeneration in response to oxidative stress and present TLR2 as a bridge between oxidative damage and complement-mediated retinal pathology., Graphical Abstract, In Brief Oxidative stress and complement deposition are common to many retinal degenerative diseases. Mulfaul et al. demonstrate that TLR2 blockade protects against photoreceptor neuronal cell death and RPE fragmentation in experimental models of oxidative stress-induced retinal degeneration and present TLR2 as a bridge between oxidative damage and complement-mediated retinal pathology.

PURPOSE: 1) To describe a case of autoimmune retinopathy mimicking heritable photoreceptor degeneration in a patient with common variable immune deficiency, and 2) to investigate the humoral and cell-mediated branches of the immune system in this patient to better understand the mechanism of immune-mediated photoreceptor damage in this disease. METHODS: Retrospective chart review with evaluation of multimodal imaging, genotype analysis, and investigation of circulating autoantibodies and T cell response to retinal antigens. RESULTS: A 40-year-old woman with bilateral, progressive vision loss was referred for evaluation of a possible inherited retinal degeneration. She was found to have asymmetric peripheral visual field constriction, cystoid macular edema, vitreous cells, and bone-spicule-like pigmentary changes in both eyes. An extensive workup for underlying infectious or inflammatory causes was unrevealing, and molecular analysis for heritable retinal degeneration failed to identify a plausible disease-causing genotype. Screening for anti-retinal antibodies showed the presence of multiple anti-retinal antibodies, consistent with a diagnosis of autoimmune retinopathy. Immunologic workup demonstrated markedly decreased levels of serum IgA and IgG, consistent with common variable immune deficiency. T-cells isolated from the patient showed increased proliferation when stimulated with human retinal proteins, supporting a role for both cell and humoral-mediated autoimmunity. Treatment with mycophenolate mofetil and intravenous immunoglobin therapy slowed the progression of disease and resulted in preservation of her central vision. CONCLUSIONS: Autoimmune retinopathy can be seen in common variable immune deficiency and have clinical findings similar to heritable photoreceptor degeneration. Both the humoral and cellular immune responses are involved in the pathophysiology. Immune modulatory therapy has stabilized the disease course in this patient and may play an important role in the management of autoimmune retinopathy.

Activation of the alternative complement pathway is an initiating event in the pathology of age-related macular degeneration (AMD). Unchecked complement activation leads to the formation of a pro-lytic pore, the membrane attack complex (MAC). MAC deposition is observed on the choriocapillaris of AMD patients and likely causes lysis of choroidal endothelial cells (CECs). Complement factor H (FH, encoded by the gene CFH) is an inhibitor of complement. Both loss of function of FH and reduced choroidal levels of FH have been reported in AMD. It is plausible that reduced local FH availability promotes MAC deposition on CECs. FH is produced primarily in the liver; however, cells including the retinal pigment epithelium can produce FH locally. We hypothesized that CECs produce FH locally to protect against MAC deposition. We aimed to investigate the effect of reduced FH levels in the choroid to determine whether increasing local FH could protect CECs from MAC deposition. We demonstrated that siRNA knockdown of FH (CFH) in human immortalized CECs results in increased MAC deposition. We generated AMD iPSC-derived CECs and found that overexpression of FH protects against MAC deposition. These results suggest that local CEC-produced FH protects against MAC deposition, and that increasing local FH protein may be beneficial in limiting MAC deposition in AMD. © 2022 The Pathological Society of Great Britain and Ireland.

ObjectiveTo correlate structural features seen on optical coherence tomography (OCT) with best-corrected visual acuity (BCVA) and Gass lesion type in patients with Best vitelliform macular dystrophy (BVMD).Methods and analysisThis is a retrospective case series of consecutive patients with molecularly confirmed BEST1-associated BVMD. OCT scans were reviewed for lesion status and presence of subretinal pillar, focal choroidal excavation (FCE), intraretinal fluid or atrophy. Available OCT angiography images were used to evaluate for the presence of choroidal neovascularisation (CNV). These features were then correlated with BCVA and Gass lesion type.Results95 eyes from 48 patients (mean age 38.9 years, range 4–87) were included. The presence of a pillar (24.2%), FCE (20.0%) and atrophy (7.4%) were associated with poor BCVA (p0.4). Among 65 eyes with longitudinal data (mean follow-up 5.1 years), 7 eyes (10.8%) reverted from higher to lower Gass lesion type; of these, 4 eyes (57.1%) had CNV responsive to intravitreal anti-vascular endothelial growth factor treatment.ConclusionOCT-based structural features are readily identifiable in patients with BVMD and have prognostic importance due to their correlation with BCVA.

Genomic studies in age-related macular degeneration (AMD) have identified genetic variants that account for the majority of AMD risk. An important next step is to understand the functional consequences and downstream effects of the identified AMD-associated genetic variants. Instrumental for this next step are ‘omics’ technologies, which enable high-throughput characterization and quantification of biological molecules, and subsequent integration of genomics with these omics datasets, a field referred to as systems genomics. Single cell sequencing studies of the retina and choroid demonstrated that the majority of candidate AMD genes identified through genomic studies are expressed in non-neuronal cells, such as the retinal pigment epithelium (RPE), glia, myeloid and choroidal cells, highlighting that many different retinal and choroidal cell types contribute to the pathogenesis of AMD. Expression quantitative trait locus (eQTL) studies in retinal tissue have identified putative causal genes by demonstrating a genetic overlap between gene regulation and AMD risk. Linking genetic data to complement measurements in the systemic circulation has aided in understanding the effect of AMD-associated genetic variants in the complement system, and supports that protein QTL (pQTL) studies in plasma or serum samples may aid in understanding the effect of genetic variants and pinpointing causal genes in AMD. A recent epigenomic study fine-mapped AMD causal variants by determing regulatory regions in RPE cells differentiated from induced pluripotent stem cells (iPSC-RPE). Another approach that is being employed to pinpoint causal AMD genes is to produce synthetic DNA assemblons representing risk and protective haplotypes, which are then delivered to cellular or animal model systems. Pinpointing causal genes and understanding disease mechanisms is crucial for the next step towards clinical translation. Clinical trials targeting proteins encoded by the AMD-associated genomic loci C3, CFB, CFI, CFH, and ARMS2/HTRA1 are currently ongoing, and a phase III clinical trial for C3 inhibition recently showed a modest reduction of lesion growth in geographic atrophy. The EYERISK consortium recently developed a genetic test for AMD that allows genotyping of common and rare variants in AMD-associated genes. Polygenic risk scores (PRS) were applied to quantify AMD genetic risk, and may aid in predicting AMD progression. In conclusion, genomic studies represent a turning point in our exploration of AMD. The results of those studies now serve as a driving force for several clinical trials. Expanding to omics and systems genomics will further decipher function and causality from the associations that have been reported, and will enable the development of therapies that will lessen the burden of AMD.

Severe burns on the posterior trunk present a treatment challenge in that these surfaces bear the major portion of body weight, with shearing forces exerted when changing the patient from supine to prone position. In their high-volume center at Burn and Reconstructive Centers of America, the authors developed protocols for use of cultured epidermal autografts (CEAs) for coverage of large burns, including those specific to posterior burns. This paper describes techniques and approaches, including milestone timelines, to treat and manage these patients. Key factors for successful treatment begin with early development of a detailed surgical plan. Members of the trained team participate in the plan and understand standard procedures and any deviation. Patients are identified early for treatment with CEA so that a full thickness skin biopsy can be sent to the manufacturer for processing. Patients with >30% total body surface area (TBSA) burns are considered for CEA burn wound coverage due to the potential for conversion of superficial partial thickness to deep partial thickness or full thickness burns over hospitalization time. We also present the outcomes in patients with posterior trunk burns treated with CEA from 2016 to 2019 in three participating centers within our network. Data in 40 patients with mean TBSA of 56% demonstrated a high rate of successful CEA engraftment (83%), and overall survival rate (90%) following one or two applications with CEA and/or CEA + split thickness skin graft (STSG). Development of standard treatment protocols and surgical plans has enabled positive outcomes with CEA in severe burns including posterior burns.

The m.3243A>G mutation in the mitochondrial genome commonly causes retinal degeneration in patients with maternally inherited diabetes and deafness and mitochondrial encephalopathy, lactic acidosis and stroke-like episodes. Like other mitochondrial mutations, m.3243A>G is inherited from the mother with a variable proportion of wild type and mutant mitochondrial genomes in different cells. The mechanism by which the m.3243A>G variant in each tissue relates to the manifestation of disease phenotype is not fully understood. Using a digital PCR assay, we found that the % m.3243G in skin derived dermal fibroblasts was positively correlated with that of blood from the same individual. The % m.3243G detected in fibroblast cultures remained constant over multiple passages and was negatively correlated with mtDNA copy number. Although the % m.3243G present in blood was not correlated with severity of vision loss, as quantified by Goldmann visual field, a significant negative correlation between % m.3243G and the age of onset of visual symptoms was detected. Altogether, these results indicate that precise measurement of % m.3243G in clinically accessible tissues such as skin and blood may yield information relevant to the management of retinal m.3243A>G-associated disease.

Significance The retinal pigment epithelium and the choroid are complex tissues whose dysfunction can lead to irreversible visual loss. In this study, single-cell RNA sequencing of both of these tissues was performed to characterize gene expression patterns specific to the retinal pigment epithelium and all major choroidal cell populations. Unique gene expression signatures of arterial, venous, and choriocapillaris vascular beds within the choroid were identified. RGCC, a gene that responds to complement and has been shown to induce endothelial apoptosis, was specifically expressed in choriocapillaris endothelial cells. This study provides potential insight into the molecular mechanisms of choroidal vascular disease and its contribution to age-related macular degeneration., The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene (RGCC), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

The identification of >100 genes causing inherited retinal degeneration and the promising results of recent gene augmentation trials have led to an increase in the number of studies investigating the preclinical efficacy of viral-mediated gene transfer. Despite success using adeno-associated viruses, many disease-causing genes, such as ABCA4 or USH2A, are too large to fit into these vectors. One option for large gene delivery is the family of integration-deficient helper-dependent adenoviruses (HDAds), which efficiently transduce postmitotic neurons. However, HDAds have been shown in other organ systems to elicit an immune response, and the immunogenicity of HDAds in the retina has not been characterized. In this study, HDAd serotype 5 (HDAd5) was found to successfully transduce rod and cone photoreceptors in ex vivo human retinal organ cultures. The ocular inflammatory response to subretinal injection of the HDAd5 was evaluated using a rat model. Subretinal injection of HDAd5 carrying cytomegalovirus promoter-driven enhanced green fluorescent protein (HDAd5-CMVp-eGFP) elicited a robust inflammatory response by 3 days postinjection. This reaction included vitreous infiltration of ionized calcium-binding adapter molecule 1 (Iba1)-positive monocytes and increased expression of the proinflammatory protein, intercellular adhesion molecule 1 (ICAM-1). By 7 days postinjection, most Iba1-positive infiltrates migrated into the neural retina and ICAM-1 expression was significantly increased compared with buffer-injected control eyes. At 14 days postinjection, Iba1-positive cells persisted in the retinas of HDAd5-injected eyes, and there was thinning of the outer nuclear layer. Subretinal injection of an empty HDAd5 virus was used to confirm that the inflammatory response was in response to the HDAd5 vector and not due to eGFP-induced overexpression cytotoxicity. Subretinal injection of lower doses of HDAd5 dampened the inflammatory response, but also eGFP expression. Despite their larger carrying capacity, further work is needed to elucidate the inflammatory pathways involved and to identify an immunomodulation paradigm sufficient for safe and effective transfer of large genes to the retina using HDAd5.

In a screen of 1,000 consecutively ascertained families, we recently found that mutations in the gene RPGR are the third most common cause of all inherited retinal disease. As the two most frequent disease-causing genes, ABCA4 and USH2A, are far too large to fit into clinically relevant adeno-associated virus (AAV) vectors, RPGR is an obvious early target for AAV-based ocular gene therapy. In generating plasmids for this application, we discovered that those containing wild-type RPGR sequence, which includes the highly repetitive low complexity region ORF15, were extremely unstable (i.e., they showed consistent accumulation of genomic changes during plasmid propagation). To develop a stable RPGR gene transfer vector, we used a bioinformatics approach to identify predicted regions of genomic instability within ORF15 (i.e., potential non-B DNA conformations). Synonymous substitutions were made in these regions to reduce the repetitiveness and increase the molecular stability while leaving the encoded amino acid sequence unchanged. The resulting construct was subsequently packaged into AAV serotype 5, and the ability to drive transcript expression and functional protein production was demonstrated via subretinal injection in rat and pull-down assays, respectively. By making synonymous substitutions within the repetitive region of RPGR, we were able to stabilize the plasmid and subsequently generate a clinical-grade gene transfer vector (IA-RPGR). Following subretinal injection in rat, we demonstrated that the augmented transcript was expressed at levels similar to wild-type constructs. By performing in vitro pull-down experiments, we were able to show that IA-RPGR protein product retained normal protein binding properties (i.e., analysis revealed normal binding to PDE6D, INPP5E, and RPGRIP1L). In summary, we have generated a stable RPGR gene transfer vector capable of producing functional RPGR protein, which will facilitate safety and toxicity studies required for progression to an Investigational New Drug application.

Early age-related macular degeneration (AMD) is characterized by degeneration of the choriocapillaris, the vascular supply of retinal photoreceptor cells. We assessed vascular loss during disease progression in the choriocapillaris and larger vessels in the deeper choroid. Human donor maculae from controls (n = 99), early AMD (n = 35), or clinically diagnosed with geographic atrophy (GA; n = 9, collected from outside the zone of retinal pigment epithelium degeneration) were evaluated using Ulex europaeus agglutinin-I labeling to discriminate between vessels with intact endothelial cells and ghost vessels. Morphometric analyses of choriocapillaris density (cross-sectional area of capillary lumens divided by length) and of vascular lumen/stroma ratio in the outer choroid were performed. Choriocapillaris loss was observed in early AMD (Bonferroni-corrected P = 0.024) with greater loss in GA (Bonferroni-corrected P < 10(−9)), even in areas of intact retinal pigment epithelium. In contrast, changes in lumen/stroma ratio in the outer choroid were not found to differ between controls and AMD or GA eyes (P > 0.05), suggesting choriocapillaris changes are more prevalent in AMD than those in the outer choroid. In addition, vascular endothelial growth factor-A levels were negatively correlated with choriocapillaris vascular density. These findings support the concept that choroidal vascular degeneration, predominantly in the microvasculature, contributes to dry AMD progression. Addressing capillary loss in AMD remains an important translational target.

Age-related macular degeneration (AMD) is a common cause of blindness worldwide. While recent studies have revealed that the loss of choroidal endothelial cells (ChECs) is critical to the disease pathogenesis of dry AMD, in vitro studies are needed to fully elucidate the disease mechanism. However, these studies remain hindered due to the lack of publically available human ChEC lines. To address this need, ChECs were harvested form donor tissue and enriched for by using magnetic cell separation using anti-CD31 conjugated microbeads. Next, lenti-viral vectors with endothelial-specific promoters driving genes necessary for immortalization, CDH5p-hTERT and CDH5p TAg, were generated. Stable integration of both gene cassettes allowed cells to maintain their proliferative state and yielded an immortalized cell line (iChEC-1). Immunocytochemical analysis of iChEC-1 confirmed the expression of important ChEC markers such as CA4, a marker of choriocapillaris endothelial cells, CDH5, and CD34, pan-endothelial cell markers. qRT-PCR analysis of expanded clones from iChEC-1 further showed that the line maintained expression of other important endothelial markers, vWF, PECAM1, and PLVAP, similar to primary cells. Functional responses were characterized by tube-forming assays and repopulation of decellularized choroid with the immortalized cell line. In conclusion, the iChEC-1 line presents a suitable immortalized human ChEC line for future in vitro studies of AMD.

Subretinal delivery of stem cell-derived retinal cells as a strategy to treat retinal degenerative blindness holds great promise. Currently, two clinical trials are underway in which human fetal retinal progenitor cells (RPCs) are being delivered to patients by intravitreal or subretinal injection to preserve or restore vision, respectively. With the advent of the induced pluripotent stem cell (iPSC), and in turn three-dimensional derivation of retinal tissue, it is now possible to generate autologous RPCs for cell replacement. The purpose of this study was to evaluate the effect of commonly used cell isolation and surgical manipulation strategies on donor cell viability. iPSC-RPCs were subjected to various conditions, including different dissociation and isolation methods, injection cannula sizes, and preinjection storage temperatures and times. The effects of commonly used surgical techniques on both host and donor cell viability were evaluated in Yucatan mini-pigs (n = 61 eyes). We found a significant increase in cell viability when papain was used for RPC isolation. In addition, a significant decrease in cell viability was detected when using the 41G cannula compared with 31G and at storage times of 4 hours compared with 30 minutes. Although 96.4% of all eyes demonstrated spontaneous retinal reattachment following injection, retinal pigment epithelium (RPE) abnormalities were seen more frequently in eyes receiving injections via a 31G cannula; interestingly, eyes that received cell suspensions were relatively protected against such RPE changes. These findings indicate that optimization of donor cell isolation and delivery parameters should be considered when developing a subretinal cell replacement strategy. Stem Cells Translational Medicine 2019;8:797–809

Purpose To examine the association between sequence variants in genetic risk factors for age-related macular degeneration (AMD) and delayed rod-mediated dark adaptation (RMDA), the first functional biomarker for incident AMD, in older adults with normal macular health and early AMD. Design Cross-sectional. Participants Adults 60 years of age or older showing normal macular health (defined as both eyes at step 1 on the Age-Related Eye Disease Study 9-step AMD classification system) and those with AMD in one or both eyes (defined as steps 2–9). Methods Single nucleotide polymorphisms were genotyped in the complement factor H (CFH) and ARMS2 genes using a Taqman assay. Rod-mediated dark adaptation was assessed in 1 eye after photobleach with targets centered at 5° on the inferior vertical meridian. Rate of dark adaptation was defined by rod intercept time (RIT), duration (in minutes) required for sensitivity to reach a criterion sensitivity level in the latter half of the second component of rod recovery. Associations between CFH and ARMS2 polymorphisms and RMDA were adjusted for age and smoking. Main Outcome Measure Rod intercept time. Results The sample consisted of 543 participants having both genotype and RIT determination; 408 showed normal macular health and 135 demonstrated AMD, most having early AMD (124 of 135). For the combined sample, higher RIT (slower RMDA) was observed for both the A69S variant in ARMS2 and the Y402H variant in CFH (adjusted P = 0.0001 and P = 0.0023, respectively). For healthy participants, the A69S variant in ARMS2 was associated with higher RIT (adjusted P = 0.0011), whereas the Y402H variant in CFH was not (adjusted P = 0.2175). For AMD patients, the A69S variant of ARMS2 and the Y402H variant of CFH were associated with higher RIT (adjusted P = 0.0182 and P = 0.0222, respectively). Those with a larger number of high-risk ARMS2 and CFH alleles showed higher RIT, in both healthy and AMD groups (adjusted P = 0.0002 and P Conclusions We report a novel association wherein older adults with high-risk ARMS2 and CFH genotypes are more likely to demonstrate delayed RMDA, the first functional biomarker for incident early AMD. Before the AMD clinical phenotype is present, those showing normal macular health with the ARMS2 A69S allele demonstrate delayed RMDA. Understanding ARMS2 function is a research priority.

SUMMARY Toolsets available for in-depth analysis of scRNA-seq datasets by biologists with little informatics experience is limited. Here, we describe an informatics tool (PyMINEr) that fully automates cell type identification, cell type-specific pathway analyses, graph theory-based analysis of gene regulation, and detection of autocrine-paracrine signaling networks in silico. We applied PyMINEr to interrogate human pancreatic islet scRNA-seq datasets and discovered several features of co-expression graphs, including concordance of scRNA-seq-graph structure with both protein-protein interactions and 3D genomic architecture, association of high-connectivity and low-expression genes with cell type enrichment, and potential for the graph structure to clarify potential etiologies of enigmatic disease-associated variants. We further created a consensus co-expression network and autocrine-paracrine signaling networks within and across islet cell types from seven datasets. PyMINEr correctly identified changes in BMP-WNT signaling associated with cystic fibrosis pancreatic acinar cell loss. This proof-of-principle study demonstrates that the PyMINEr framework will be a valuable resource for scRNA-seq analyses., Graphical Abstract, In Brief Tyler et al. create PyMINEr, an open-source program (https://www.sciencescott.com/pyminer) that automates analyses of expression datasets without coding. These analyses include clustering, differential expression, pathway analyses, co-expression networks, marker gene identification, and autocrine-paracrine signaling prediction. Integration of seven datasets shows elevated BMP-WNT signaling in cystic fibrosis pancreata.

We sought to characterize the angiofibrotic and apoptotic effects of vascular endothelial growth factor (VEGF)-inhibition on fibrovascular epiretinal membranes in eyes with traction retinal detachment because of proliferative diabetic retinopathy.Membranes were excised from 20 eyes of 19 patients (10 randomized to intravitreal bevacizumab, 10 controls) at vitrectomy. Membranes were stained with antibodies targeting connective tissue growth factor (CTGF) or VEGF and colabeled with antibodies directed against endothelial cells (CD31), myofibroblasts, or retinal pigment epithelium markers. Quantitative and colocalization analyses of antibody labeling were obtained through immunofluorescence confocal microscopy. Masson trichrome staining, cell counting of hematoxylin and eosin sections, and terminal dUTP nick-end labeling staining were performed.High levels of fibrosis were observed in both groups. Cell apoptosis was higher (P = 0.05) in bevacizumab-treated membranes compared with controls. The bevacizumab group had a nonsignificant reduction in colocalization in CD31-CTGF and cytokeratin-VEGF studies compared with controls. Vascular endothelial growth factor in extracted membranes was positively correlated with vitreous levels of VEGF; CTGF in extracted membranes was negatively correlated with vitreous levels of CTGF.Bevacizumab suppresses vitreous VEGF levels, but does not significantly alter VEGF or CTGF in diabetic membranes that may be explained by high baseline levels of fibrosis. Bevacizumab may cause apoptosis within fibrovascular membranes.