Local factor H production by human choroidal endothelial cells mitigates complement deposition: implications for macular degeneration.

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.

Stepwise differentiation and functional characterization of human induced pluripotent stem cell-derived choroidal endothelial cells.

BACKGROUND: Endothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically. METHODS: We transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function. RESULTS: We report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors. CONCLUSION: ECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.

Label-free microfluidic enrichment of photoreceptor cells.

Inherited retinal degenerative disorders such as retinitis pigmentosa and Usher syndrome are characterized by progressive death of photoreceptor cells. To restore vision to patients blinded by these diseases, a stem cell-based photoreceptor cell replacement strategy will likely be required. Although retinal stem cell differentiation protocols suitable for generating photoreceptor cells exist, they often yield a rather heterogenous mixture of cell types. To enrich the donor cell population for one or a few cell types, scientists have traditionally relied upon the use of antibody-based selection approaches. However, these strategies are quite labor intensive and require animal derived reagents and equipment that are not well suited to current good manufacturing practices (cGMP). The purpose of this study was to develop and evaluate a microfluidic cell sorting device capable of exploiting the physical and mechanical differences between retinal cell types to enrich specific donor cell populations such as Retinal Pigment Epithelial (RPE) cells and photoreceptor cells. Using this device, we were able to separate a mixture of RPE and iPSC-derived photoreceptor precursor cell lines into two substantially enriched fractions. The enrichment factor of the RPE fraction was 2 and that of the photoreceptor precursor cell fraction was 2.7. Similarly, when human retina, obtained from 3 independent donors, was dissociated and passed through the sorting device, the heterogeneous mixture could be reliably sorted into RPE and photoreceptor cell rich fractions. In summary, microfluidic cell sorting is a promising approach for antibody free enrichment of retinal cell populations.

Bulk and single-cell gene expression analyses reveal aging human choriocapillaris has pro-inflammatory phenotype.

The human choroidal vasculature is subject to age-related structural and gene expression changes implicated in age-related macular degeneration (AMD). In this study, we performed both bulk and single-cell RNA sequencing on infant (n = 4 for bulk experiments, n = 2 for single-cell experiments) and adult (n = 13 for bulk experiments, n = 6 for single-cell experiments) human donors to characterize how choroidal gene expression changes with age. Differential expression analysis revealed that aged choroidal samples were enriched in genes encoding pro-inflammatory transcription factors and leukocyte transendothelial cell migration adhesion proteins. Such genes were observed to be differentially expressed specifically within choroidal endothelial cells at the single-cell level. Immunohistochemistry experiments support transcriptional findings that CD34 is elevated in infant choriocapillaris endothelial cells while ICAM-1 is enriched in adults. These results suggest several potential drivers of the pro-inflammatory vascular phenotype observed with advancing age.

Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and 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.

Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-Associated X-Linked Retinitis Pigmentosa.

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.

Correction of NR2E3 Associated Enhanced S-cone Syndrome Patient-specific iPSCs using CRISPR-Cas9.

Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient's iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient's c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.

Disruption of RPGR protein interaction network is the common feature of RPGR missense variations that cause XLRP.

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease with severe vision impairment leading to blindness. About 10-15% of RP cases are caused by mutations in the RPGR gene, with RPGR mutations accounting for 70% of X-linked RP cases. The mechanism by which RPGR mutations cause photoreceptor cell dysfunction is not well understood. In this study, we show that the two isoforms of RPGR (RPGR1-19 and RPGRORF15) interact with endogenous PDE6D, INPP5E, and RPGRIP1L. The RPGR1-19 isoform contains two PDE6D binding sites with the C-terminal prenylation site being the predominant PDE6D binding site. The C terminus of RPGR1-19 that contains the prenylation site regulates its interaction with PDE6D, INPP5E, and RPGRIP1L. Only the RPGR1-19 isoform localizes to cilia in cultured RPE1 cells. Missense variations found in RPGR patients disrupt the interaction between RPGR isoforms and their endogenous interactors INPP5E, PDE6D, and RPGRIP1L. We evaluated a RPGR missense variation (M58K) found in a family with X-linked retinitis pigmentosa (XLRP) and show that this missense variation disrupts the interaction of RPGR isoforms with their endogenous interactors. The M58K variation also disrupts the ciliary localization of the RPGR1-19 isoform. Using this assay, we also show that some of the RPGR missense variants reported in the literature might not actually be disease causing. Our data establishes an in vitro assay that can be used to validate the potential pathogenicity of RPGR missense variants.

Generation of an immortalized human choroid endothelial cell line (iChEC-1) using an endothelial cell specific promoter.

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.

CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease-causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR-Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome. CRISPR-based genome editing of patient-specific iPSCs shows great promise for future autologous cell replacement therapies. One caveat, however, is that hiPSCs are notoriously difficult to transfect, and optimized experimental design considerations are often necessary. This unit describes design strategies and methods for efficient CRISPR-based genome editing of patient- specific iPSCs. Additionally, it details a flexible approach that utilizes positive selection to generate clones with a desired genomic modification, Cre-lox recombination to remove the integrated selection cassette, and negative selection to eliminate residual hiPSCs with intact selection cassettes. © 2018 by John Wiley & Sons, Inc.

CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration.

Gene correction is a valuable strategy for treating inherited retinal degenerative diseases, a major cause of irreversible blindness worldwide. Single gene defects cause the majority of these retinal dystrophies. Gene augmentation holds great promise if delivered early in the course of the disease, however, many patients carry mutations in genes too large to be packaged into adeno-associated viral vectors and some, when overexpressed via heterologous promoters, induce retinal toxicity. In addition to the aforementioned challenges, some patients have sustained significant photoreceptor cell loss at the time of diagnosis, rendering gene replacement therapy insufficient to treat the disease. These patients will require cell replacement to restore useful vision. Fortunately, the advent of induced pluripotent stem cell and CRISPR-Cas9 gene editing technologies affords researchers and clinicians a powerful means by which to develop strategies to treat patients with inherited retinal dystrophies. In this review we will discuss the current developments in CRISPR-Cas9 gene editing in vivo in animal models and in vitro in patient-derived cells to study and treat inherited retinal degenerative diseases.

CRISPR-Cas9-Mediated Correction of the 1.02 kb Common Deletion in CLN3 in Induced Pluripotent Stem Cells from Patients with Batten Disease.

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a rare progressive neurodegenerative disorder caused by mutations in CLN3. Patients present with early-onset retinal degeneration, followed by epilepsy, progressive motor deficits, cognitive decline, and premature death. Approximately 85% of individuals with Batten disease harbor at least one allele containing a 1.02 kb genomic deletion spanning exons 7 and 8. This study demonstrates CRISPR-Cas9-based homology-dependent repair of this mutation in induced pluripotent stem cells generated from two independent patients: one homozygous and one compound heterozygous for the 1.02 kb deletion. Our strategy included delivery of a construct that carried >3 kb of DNA: wild-type CLN3 sequence and a LoxP-flanked, puromycin resistance cassette for positive selection. This strategy resulted in correction at the genomic DNA and mRNA levels in the two independent patient lines. These CRISPR-corrected isogenic cell lines will be a valuable tool for disease modeling and autologous retinal cell replacement.

Clinically Focused Molecular Investigation of 1000 Consecutive Families with Inherited Retinal Disease.

PURPOSE: To devise a comprehensive multiplatform genetic testing strategy for inherited retinal disease and to describe its performance in 1000 consecutive families seen by a single clinician. DESIGN: Retrospective series. PARTICIPANTS: One thousand consecutive families seen by a single clinician. METHODS: The clinical records of all patients seen by a single retina specialist between January 2010 and June 2016 were reviewed, and all patients who met the clinical criteria for a diagnosis of inherited retinal disease were included in the study. Each patient was assigned to 1 of 62 diagnostic categories, and this clinical diagnosis was used to define the scope and order of the molecular investigations that were performed. The number of nucleotides evaluated in a given subject ranged from 2 to nearly 900 000. MAIN OUTCOME MEASURES: Sensitivity and false genotype rate. RESULTS: Disease-causing genotypes were identified in 760 families (76%). These genotypes were distributed across 104 different genes. More than 75% of these 104 genes have coding sequences small enough to be packaged efficiently into an adeno-associated virus. Mutations in ABCA4 were the most common cause of disease in this cohort (173 families), whereas mutations in 80 genes caused disease in 5 or fewer families (i.e., 0.5% or less). Disease-causing genotypes were identified in 576 of the families without next-generation sequencing (NGS). This included 23 families with mutations in the repetitive region of RPGR exon 15 that would have been missed by NGS. Whole-exome sequencing of the remaining 424 families revealed mutations in an additional 182 families, and whole-genome sequencing of 4 of the remaining 242 families revealed 2 additional genotypes that were invisible by the other methods. Performing the testing in a clinically focused tiered fashion would be 6.1% more sensitive and 17.7% less expensive and would have a significantly lower average false genotype rate than using whole-exome sequencing to assess more than 300 genes in all patients (7.1% vs. 128%; P < 0.001). CONCLUSIONS: Genetic testing for inherited retinal disease is now more than 75% sensitive. A clinically directed tiered testing strategy can increase sensitivity and improve statistical significance without increasing cost.

Connective Tissue Growth Factor Promotes Efficient Generation of Human Induced Pluripotent Stem Cell-Derived Choroidal Endothelium.

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of retinal pigment epithelial (RPE) and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be lost in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient-specific, stem cell-derived CECs will be required. In this study, we report for the first time a stepwise differentiation protocol suitable for generating human iPSC-derived CEC-like cells. RNA-seq analysis of the monkey CEC line, RF/6A, combined with two statistical screens allowed us to develop media comprised of various protein combinations. In both screens, connective tissue growth factor (CTGF) was identified as the key component required for driving CEC development. A second factor tumor necrosis factor (TNF)-related weak inducer of apoptosis receptor was also found to promote iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF-driven iPSC-derived CEC-like cells formed capillary tube-like vascular networks, and expressed the EC-specific markers CD31, ICAM1, PLVAP, vWF, and the CEC-restricted marker CA4. In combination with RPE and photoreceptor cells, patient-specific iPSC derived CEC-like cells will enable scientists to accurately evaluate AMD pathophysiology and develop effective cell replacement therapies. Stem Cells Translational Medicine 2017;6:1533-1546.

Choosing surgery as a career: Early results of a longitudinal study of medical students.

BACKGROUND: Few studies have explored the factors associated with the preference of medical students to pursue a specific specialty, and even fewer have observed how these preferences and factors change over time. METHODS: A longitudinal survey of medical students was administered at the beginning of first year, second year, and clerkships from 2013-2016. Surveys included demographics and factors associated with their choice of specialty. RESULTS: Response rates were 78-94%. Students with mentors and research experience in any specialty were 3.4 times (P < .001) more likely to choose surgery by their third year of medical school. Surgical research experience on the first- and second-year surveys was associated with 39 (P < .001) and 10 times (P < .001) greater odds of preferring surgical specialties on their third-year survey. Medical students who had a surgery mentor during the first and second years were associated with 4 (P = .024) and 13 times (P < .001) greater odds of preferring surgical specialties on their third-year survey. CONCLUSION: Students who begin surgical research during their first year and develop relationships with surgeon mentors during their second year are significantly more likely to maintain an interest in surgical specialties.

Concise Review: Patient-Specific Stem Cells to Interrogate Inherited Eye Disease.

Whether we are driving to work or spending time with loved ones, we depend on our sense of vision to interact with the world around us. Therefore, it is understandable why blindness for many is feared above death itself. Heritable diseases of the retina, such as glaucoma, age-related macular degeneration, and retinitis pigmentosa, are major causes of blindness worldwide. The recent success of gene augmentation trials for the treatment of RPE65-associated Leber congenital amaurosis has underscored the need for model systems that accurately recapitulate disease. With the advent of patient-specific induced pluripotent stem cells (iPSCs), researchers are now able to obtain disease-specific cell types that would otherwise be unavailable for molecular analysis. In the present review, we discuss how the iPSC technology is being used to confirm the pathogenesis of novel genetic variants, interrogate the pathophysiology of disease, and accelerate the development of patient-centered treatments. Significance: Stem cell technology has created the opportunity to advance treatments for multiple forms of blindness. Researchers are now able to use a person's cells to generate tissues found in the eye. This technology can be used to elucidate the genetic causes of disease and develop treatment strategies. In the present review, how stem cell technology is being used to interrogate the pathophysiology of eye disease and accelerate the development of patient-centered treatments is discussed.