Macrophages derived from human induced pluripotent stem cells (iPSCs) serve as a high-fidelity cellular model for investigating HIV-1, dengue, and influenza viruses.

Macrophages are important target cells for diverse viruses and thus represent a valuable system for studying virus biology. Isolation of primary human macrophages is done by culture of dissociated tissues or from differentiated blood monocytes, but these methods are both time consuming and result in low numbers of recovered macrophages. Here, we explore whether macrophages derived from human induced pluripotent stem cells (iPSCs)-which proliferate indefinitely and potentially provide unlimited starting material-could serve as a faithful model system for studying virus biology. Human iPSC-derived monocytes were differentiated into macrophages and then infected with HIV-1, dengue virus, or influenza virus as model human viruses. We show that iPSC-derived macrophages support the replication of these viruses with kinetics and phenotypes similar to human blood monocyte-derived macrophages. These iPSC-derived macrophages were virtually indistinguishable from human blood monocyte-derived macrophages based on surface marker expression (flow cytometry), transcriptomics (RNA sequencing), and chromatin accessibility profiling. iPSC lines were additionally generated from non-human primate (chimpanzee) fibroblasts. When challenged with dengue virus, human and chimpanzee iPSC-derived macrophages show differential susceptibility to infection, thus providing a valuable resource for studying the species-tropism of viruses. We also show that blood- and iPSC-derived macrophages both restrict influenza virus at a late stage of the virus lifecycle. Collectively, our results substantiate iPSC-derived macrophages as an alternative to blood monocyte-derived macrophages for the study of virus biology. IMPORTANCE: Macrophages have complex relationships with viruses: while macrophages aid in the removal of pathogenic viruses from the body, macrophages are also manipulated by some viruses to serve as vessels for viral replication, dissemination, and long-term persistence. Here, we show that iPSC-derived macrophages are an excellent model that can be exploited in virology.

Rapid and Quantitative Detection of Lung Cancer Biomarker ENOX2 Using a Novel Aptamer in an Electrochemical DNA-Based (E-DNA) Biosensor.

To overcome early cancer detection challenges, diagnostic tools enabling more sensitive, rapid, and noninvasive detection are necessary. An attractive cancer target for diagnostic blood tests is human Ecto-NOX disulfide-thiol exchanger 2 (ENOX2), expressed in most human cancer types and regularly shed into blood sera. Here, we developed an electrochemical DNA-based (E-DNA) biosensor that rapidly detects physiologically relevant levels of ENOX2. To identify ENOX2-binding aptamers that could potentially be used in a biosensor, recombinantly expressed ENOX2 was used as a binding target in an oligonucleotide library pull-down that generated a highly enriched ENOX2-binding aptamer. This candidate aptamer sensitively bound ENOX2 via gel mobility shift assays. To enable this aptamer to function in an ENOX2 E-DNA biosensor, the aptamer sequence was modified to adopt two conformations, one capable of ENOX2 binding, and one with disrupted ENOX2 binding. Upon ENOX2 introduction, a conformational shift to the ENOX2 binding state resulted in changed dynamics of a redox reporter molecule, which generated a rapid, significant, and target-specific electrical current readout change. ENOX2 biosensor sensitivity was at or below the diagnostic range. The ENOX2 E-DNA biosensor design presented here may enable the development of more sensitive, rapid, diagnostic tools for early cancer detection.

Differentiating Human Pluripotent Stem Cells to Cardiomyocytes Using Purified Extracellular Matrix Proteins.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into cardiomyocytes (hESC-CMs and iPSC-CMs, respectively), which hold great promise for cardiac regenerative medicine and disease modeling efforts. However, the most widely employed differentiation protocols require undefined substrates that are derived from xenogeneic (animal) products, contaminating resultant hESC- and iPSC-CM cultures with xenogeneic proteins and limiting their clinical applicability. Additionally, typical hESC- and iPSC-CM protocols produce CMs that are significantly contaminated by non-CMs and that are immature, requiring lengthy maturation procedures. In this review, we will summarize recent studies that have investigated the ability of purified extracellular matrix (ECM) proteins to support hESC- and iPSC-CM differentiation, with a focus on commercially available ECM proteins and coatings to make such protocols widely available to researchers. The most promising of the substrates reviewed here include laminin-521 with laminin-221 together or Synthemax (a synthetic vitronectin-based peptide coating), which both resulted in highly pure CM cultures. Future efforts are needed to determine whether combinations of specific purified ECM proteins or derived peptides could further improve CM maturation and culture times, and significantly improve hESC- and iPSC-CM differentiation protocols.

Electrochemical DNA Biosensor That Detects Early Celiac Disease Autoantibodies.

Although it is estimated that more than one million Americans have celiac disease (CD), it remains challenging to diagnose. CD, an autoimmune and inflammatory response following the ingestion of gluten-containing foods, has symptoms overlapping with other diseases and requires invasive diagnostics. The gold standard for CD diagnosis involves serologic blood tests followed by invasive confirmatory biopsies. Here, we propose a less invasive method using an electrochemical DNA (E-DNA) biosensor for CD-specific autoantibodies (AABs) circulating in blood. In our approach, CD-specific AABs bind a synthetic neoepitope, causing a conformational change in the biosensor, as well as a change in the environment of an attached redox reporter, producing a measurable current reduction. We assessed the biosensor's ability to detect CD-specific patient-derived AABs in physiological buffer as well as buffer supplemented with bovine serum. Our biosensor was able to detect AABs in a dose-dependent manner; increased signal change correlated with increased AAB concentration with an apparent dissociation constant of 0.09 ± 0.03 units/mL of AABs. Furthermore, we found our biosensor to be target-specific, with minimal off-target binding of multiple unrelated biomarkers. Future efforts aimed at increasing sensitivity in complex media may build upon the biosensor design presented here to further improve CD AAB detection and CD diagnostic tools.

Allele-specific proximal promoter hypomethylation of the telomerase reverse transcriptase gene (TERT) associates with TERT expression in multiple cancers.

Telomerase reverse transcriptase (TERT) is pathologically expressed in the vast majority of human cancers, but the epigenetic regulation of its expression is only beginning to be understood. In particular, the active TERT gene in cancer cells has been characterized as having a hypermethylated CpG island, opposite to the general association of DNA methylation with gene repression. Here, we analyzed TERT promoter CpG methylation in 833 human cancer cell lines representing 23 different tissue types and found hypermethylation of the upstream portion of the CpG island and more conserved hypomethylation of a region including the proximal TERT promoter and exon 1. In cell lines with monoallelic expression of TERT, we found allelic methylation of the proximal TERT promoter. This included cell lines with the -124 or -146 activating promoter mutation as well as wild-type TERT cancer lines. In these cell line types, decreased proximal promoter methylation is associated with the active allele. Compared to cells with monoallelic expression of TERT, lines with biallelic expression of TERT had even lower methylation in the proximal TERT promoter. Thus, in cell lines from cancers of many different tissues, the TERT proximal promoter has canonical DNA methylation, with low methylation correlating with increased TERT expression.

Danon Disease-Associated LAMP-2 Deficiency Drives Metabolic Signature Indicative of Mitochondrial Aging and Fibrosis in Cardiac Tissue and hiPSC-Derived Cardiomyocytes.

Danon disease is a severe X-linked disorder caused by deficiency of the lysosome-associated membrane protein-2 (LAMP-2). Clinical manifestations are phenotypically diverse and consist of hypertrophic and dilated cardiomyopathies, skeletal myopathy, retinopathy, and intellectual dysfunction. Here, we investigated the metabolic landscape of Danon disease by applying a multi-omics approach and combined structural and functional readouts provided by Raman and atomic force microscopy. Using these tools, Danon patient-derived cardiac tissue, primary fibroblasts, and human induced pluripotent stem cells differentiated into cardiomyocytes (hiPSC-CMs) were analyzed. Metabolic profiling indicated LAMP-2 deficiency promoted a switch toward glycolysis accompanied by rerouting of tryptophan metabolism. Cardiomyocytes' energetic balance and NAD+/NADH ratio appeared to be maintained despite mitochondrial aging. In turn, metabolic adaption was accompanied by a senescence-associated signature. Similarly, Danon fibroblasts appeared more stress prone and less biomechanically compliant. Overall, shaping of both morphology and metabolism contributed to the loss of cardiac biomechanical competence that characterizes the clinical progression of Danon disease.

Genetic Risk of Arrhythmic Phenotypes in Patients With Dilated Cardiomyopathy.

BACKGROUND: Genotype-phenotype correlations in dilated cardiomyopathy (DCM) and, in particular, the effects of gene variants on clinical outcomes remain poorly understood. OBJECTIVES: The purpose of this study was to investigate the prognostic role of genetic variant carrier status in a large cohort of DCM patients. METHODS: A total of 487 DCM patients were analyzed by next-generation sequencing and categorized the disease genes into functional gene groups. The following composite outcome measures were assessed: 1) all-cause mortality; 2) heart failure-related death, heart transplantation, or destination left ventricular assist device implantation (DHF/HTx/VAD); and 3) sudden cardiac death/sustained ventricular tachycardia/ventricular fibrillation (SCD/VT/VF). RESULTS: A total of 183 pathogenic/likely pathogenic variants were found in 178 patients (37%): 54 (11%) Titin; 19 (4%) Lamin A/C (LMNA); 24 (5%) structural cytoskeleton-Z disk genes; 16 (3.5%) desmosomal genes; 46 (9.5%) sarcomeric genes; 8 (1.6%) ion channel genes; and 11 (2.5%) other genes. All-cause mortality was no different between variant carriers and noncarriers (p = 0.99). A trend toward worse SCD/VT/VF (p = 0.062) and DHF/HTx/VAD (p = 0.061) was found in carriers. Carriers of desmosomal and LMNA variants experienced the highest rate of SCD/VT/VF, which was independent of the left ventricular ejection fraction. CONCLUSIONS: Desmosomal and LMNA gene variants identify the subset of DCM patients who are at greatest risk for SCD and life-threatening ventricular arrhythmias, regardless of the left ventricular ejection fraction.

Single-cell imaging reveals unexpected heterogeneity of telomerase reverse transcriptase expression across human cancer cell lines.

Telomerase is pathologically reactivated in most human cancers, where it maintains chromosomal telomeres and allows immortalization. Because telomerase reverse transcriptase (TERT) is usually the limiting component for telomerase activation, numerous studies have measured TERT mRNA levels in populations of cells or in tissues. In comparison, little is known about TERT expression at the single-cell and single-molecule level. To address this, we analyzed TERT expression across 10 human cancer lines using single-molecule RNA fluorescent in situ hybridization (FISH) and made several unexpected findings. First, there was substantial cell-to-cell variation in number of transcription sites and ratio of transcription sites to gene copies. Second, previous classification of lines as having monoallelic or biallelic TERT expression was found to be inadequate for capturing the TERT gene expression patterns. Finally, spliced TERT mRNA had primarily nuclear localization in cancer cells and induced pluripotent stem cells (iPSCs), in stark contrast to the expectation that spliced mRNA should be predominantly cytoplasmic. These data reveal unappreciated heterogeneity, complexity, and unconventionality in TERT expression across human cancer cells.

Gold Nanoparticle-Functionalized Reverse Thermal Gel for Tissue Engineering Applications.

Utilizing polymers in cardiac tissue engineering holds promise for restoring function to the heart following myocardial infarction, which is associated with grave morbidity and mortality. To properly mimic native cardiac tissue, materials must not only support cardiac cell growth but also have inherent conductive properties. Here, we present an injectable reverse thermal gel (RTG)-based cardiac cell scaffold system that is both biocompatible and conductive. Following the synthesis of a highly functionalizable, biomimetic RTG backbone, gold nanoparticles (AuNPs) were chemically conjugated to the backbone to enhance the system's conductivity. The resulting RTG-AuNP hydrogel supported targeted survival of neonatal rat ventricular myocytes (NRVMs) for up to 21 days when cocultured with cardiac fibroblasts, leading to an increase in connexin 43 (Cx43) relative to control cultures (NRVMs cultured on traditional gelatin-coated dishes and RTG hydrogel without AuNPs). This biomimetic and conductive RTG-AuNP hydrogel holds promise for future cardiac tissue engineering applications.

Filamin C Truncation Mutations Are Associated With Arrhythmogenic Dilated Cardiomyopathy and Changes in the Cell-Cell Adhesion Structures.

OBJECTIVES: The purpose of this study was to assess the phenotype of Filamin C (FLNC) truncating variants in dilated cardiomyopathy (DCM) and understand the mechanism leading to an arrhythmogenic phenotype. BACKGROUND: Mutations in FLNC are known to lead to skeletal myopathies, which may have an associated cardiac component. Recently, the clinical spectrum of FLNC mutations has been recognized to include a cardiac-restricted presentation in the absence of skeletal muscle involvement. METHODS: A population of 319 U.S. and European DCM cardiomyopathy families was evaluated using whole-exome and targeted next-generation sequencing. FLNC truncation probands were identified and evaluated by clinical examination, histology, transmission electron microscopy, and immunohistochemistry. RESULTS: A total of 13 individuals in 7 families (2.2%) were found to harbor 6 different FLNC truncation variants (2 stopgain, 1 frameshift, and 3 splicing). Of the 13 FLNC truncation carriers, 11 (85%) had either ventricular arrhythmias or sudden cardiac death, and 5 (38%) presented with evidence of right ventricular dilation. Pathology analysis of 2 explanted hearts from affected FLNC truncation carriers showed interstitial fibrosis in the right ventricle and epicardial fibrofatty infiltration in the left ventricle. Ultrastructural findings included occasional disarray of Z-discs within the sarcomere. Immunohistochemistry showed normal plakoglobin signal at cell-cell junctions, but decreased signals for desmoplakin and synapse-associated protein 97 in the myocardium and buccal mucosa. CONCLUSIONS: We found FLNC truncating variants, present in 2.2% of DCM families, to be associated with a cardiac-restricted arrhythmogenic DCM phenotype characterized by a high risk of life-threatening ventricular arrhythmias and a pathological cellular phenotype partially overlapping with arrhythmogenic right ventricular cardiomyopathy.

Injectable Hydrogels for Cardiac Tissue Engineering.

In light of the limited efficacy of current treatments for cardiac regeneration, tissue engineering approaches have been explored for their potential to provide mechanical support to injured cardiac tissues, deliver cardio-protective molecules, and improve cell-based therapeutic techniques. Injectable hydrogels are a particularly appealing system as they hold promise as a minimally invasive therapeutic approach. Moreover, injectable acellular alginate-based hydrogels have been tested clinically in patients with myocardial infarction (MI) and show preservation of the left ventricular (LV) indices and left ventricular ejection fraction (LVEF). This review provides an overview of recent developments that have occurred in the design and engineering of various injectable hydrogel systems for cardiac tissue engineering efforts, including a comparison of natural versus synthetic systems with emphasis on the ideal characteristics for biomimetic cardiac materials.

Injectable Carbon Nanotube-Functionalized Reverse Thermal Gel Promotes Cardiomyocytes Survival and Maturation.

The ability of the adult heart to regenerate cardiomyocytes (CMs) lost after injury is limited, generating interest in developing efficient cell-based transplantation therapies. Rigid carbon nanotubes (CNTs) scaffolds have been used to improve CMs viability, proliferation, and maturation, but they require undesirable invasive surgeries for implantation. To overcome this limitation, we developed an injectable reverse thermal gel (RTG) functionalized with CNTs (RTG-CNT) that transitions from a solution at room temperature to a three-dimensional (3D) gel-based matrix shortly after reaching body temperature. Here we show experimental evidence that this 3D RTG-CNT system supports long-term CMs survival, promotes CMs alignment and proliferation, and improves CMs function when compared with traditional two-dimensional gelatin controls and 3D plain RTG system without CNTs. Therefore, our injectable RTG-CNT system could potentially be used as a minimally invasive tool for cardiac tissue engineering efforts.

Electrochemical Aptamer Scaffold Biosensors for Detection of Botulism and Ricin Proteins.

Electrochemical DNA (E-DNA) biosensors enable the detection and quantification of a variety of molecular targets, including oligonucleotides, small molecules, heavy metals, antibodies, and proteins. Here we describe the design, electrode preparation and sensor attachment, and voltammetry conditions needed to generate and perform measurements using E-DNA biosensors against two protein targets, the biological toxins ricin and botulinum neurotoxin. This method can be applied to generate E-DNA biosensors for the detection of many other protein targets, with potential advantages over other systems including sensitive detection limits typically in the nanomolar range, real-time monitoring, and reusable biosensors.

Impaired mitophagy facilitates mitochondrial damage in Danon disease.

RATIONALE: Lysosomal associated membrane protein type-2 (LAMP-2) is a highly conserved, ubiquitous protein that is critical for autophagic flux. Loss of function mutations in the LAMP-2 gene cause Danon disease, a rare X-linked disorder characterized by developmental delay, skeletal muscle weakness, and severe cardiomyopathy. We previously found that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from Danon patients exhibited significant mitochondrial oxidative stress and apoptosis. Understanding how loss of LAMP-2 expression leads to cardiomyocyte dysfunction and heart failure has important implications for the treatment of Danon disease as well as a variety of other cardiac disorders associated with impaired autophagy. OBJECTIVE: Elucidate the pathophysiology of cardiac dysfunction in Danon disease. METHODS AND RESULTS: We created hiPSCs from two patients with Danon disease and differentiated those cells into hiPSC-CMs using well-established protocols. Danon hiPSC-CMs demonstrated an accumulation of damaged mitochondria, disrupted mitophagic flux, depressed mitochondrial respiratory capacity, and abnormal gene expression of key mitochondrial pathways. Restoring the expression of LAMP-2B, the most abundant LAMP-2 isoform in the heart, rescued mitophagic flux as well as mitochondrial health and bioenergetics. To confirm our findings in vivo, we evaluated Lamp-2 knockout (KO) mice. Impaired autophagic flux was noted in the Lamp-2 KO mice compared to WT reporter mice, as well as an increased number of abnormal mitochondria, evidence of incomplete mitophagy, and impaired mitochondrial respiration. Physiologically, Lamp-2 KO mice demonstrated early features of contractile dysfunction without overt heart failure, indicating that the metabolic abnormalities associated with Danon disease precede the development of end-stage disease and are not merely part of the secondary changes associated with heart failure. CONCLUSIONS: Incomplete mitophagic flux and mitochondrial dysfunction are noted in both in vitro and in vivo models of Danon disease, and proceed overt cardiac contractile dysfunction. This suggests that impaired mitochondrial clearance may be central to the pathogenesis of disease and a potential target for therapeutic intervention.

FLNC Gene Splice Mutations Cause Dilated Cardiomyopathy.

OBJECTIVE: To identify novel dilated cardiomyopathy (DCM) causing genes, and to elucidate the pathological mechanism leading to DCM by utilizing zebrafish as a model organism. BACKGROUND: DCM, a major cause of heart failure, is frequently familial and caused by a genetic defect. However, only 50% of DCM cases can be attributed to a known DCM gene variant, motivating the ongoing search for novel disease genes. METHODS: We performed whole exome sequencing (WES) in two multigenerational Italian families and one US family with arrhythmogenic DCM without skeletal muscle defects, in whom prior genetic testing had been unrevealing. Pathogenic variants were sought by a combination of bioinformatic filtering and cosegregation testing among affected individuals within the families. We performed function assays and generated a zebrafish morpholino knockdown model. RESULTS: A novel filamin C gene splicing variant (FLNC c.7251+1 G>A) was identified by WES in all affected family members in the two Italian families. A separate novel splicing mutation (FLNC c.5669-1delG) was identified in the US family. Western blot analysis of cardiac heart tissue from an affected individual showed decreased FLNC protein, supporting a haploinsufficiency model of pathogenesis. To further analyze this model, a morpholino knockdown of the ortholog filamin Cb in zebrafish was created which resulted in abnormal cardiac function and ultrastructure. CONCLUSIONS: Using WES, we identified two novel FLNC splicing variants as the likely cause of DCM in three families. We provided protein expression and in vivo zebrafish data supporting haploinsufficiency as the pathogenic mechanism leading to DCM.

Natural History of Dilated Cardiomyopathy in Children.

BACKGROUND: The long-term progression of idiopathic dilated cardiomyopathy (DCM) in pediatric patients compared with adult patients has not been previously characterized. In this study, we compared outcome and long-term progression of pediatric and adult DCM populations. METHODS AND RESULTS: Between 1988 and 2014, 927 DCM patients were consecutively enrolled. The pediatric population (aged <18 years at enrollment) included 47 participants (5.1%). At presentation, the pediatric population compared with adult patients had a significantly increased occurrence of familial forms (P=0.03), shorter duration of heart failure (P=0.04), lower systolic blood pressure (P=0.01), decreased presence of left bundle-branch block (P=0.001), and increased left ventricular ejection fraction (P=0.03). Despite these baseline differences, long-term longitudinal trends of New York Heart Association class III to IV, left ventricular dimensions, left ventricular ejection fraction, and restrictive filling pattern were similar between the 2 populations. Regarding survival analysis, because of the size difference between the 2 populations, we compared the pediatric population with a sample of adult patients randomly matched using the above-mentioned baseline differences in a 3:1 ratio (141 adult versus 47 pediatric patients). During a median follow-up of 110 months, survival free from heart transplantation was significantly lower among pediatric patients compared with adults (P<0.001). Furthermore, pediatric age (ie, <18 years) was found to be associated with an increasing risk of both death from pump failure and life-threatening arrhythmias. CONCLUSIONS: Despite the pediatric DCM population having higher baseline left ventricular ejection fraction and similar long-term echocardiographic progression compared with the adult DCM population, the pediatric DCM patients had worse cardiovascular prognosis.

Danon disease - dysregulation of autophagy in a multisystem disorder with cardiomyopathy.

Danon disease is a rare, severe X-linked form of cardiomyopathy caused by deficiency of lysosome-associated membrane protein 2 (LAMP-2). Other clinical manifestations include skeletal myopathy, cognitive defects and visual problems. Although individuals with Danon disease have been clinically described since the early 1980s, the underlying molecular mechanisms involved in pathological progression remain poorly understood. LAMP-2 is known to be involved in autophagy, and a characteristic accumulation of autophagic vacuoles in the affected tissues further supports the idea that autophagy is disrupted in this disease. The LAMP2 gene is alternatively spliced to form three splice isoforms, which are thought to play different autophagy-related cellular roles. This Commentary explores findings from genetic, histological, functional and tissue expression studies that suggest that the specific loss of the LAMP-2B isoform, which is likely to be involved in macroautophagy, plays a crucial role in causing the Danon phenotype. We also compare findings from mouse and cellular models, which have allowed for further molecular characterization but have also shown phenotypic differences that warrant attention. Overall, there is a need to better functionally characterize the LAMP-2B isoform in order to rationally explore more effective therapeutic options for individuals with Danon disease.

Electrochemical aptamer scaffold biosensors for detection of botulism and ricin toxins.

Protein toxins present considerable health risks, but detection often requires laborious analysis. Here, we developed electrochemical aptamer biosensors for ricin and botulinum neurotoxins, which display robust and specific signal at nanomolar concentrations and function in dilute serum. These biosensors may aid future efforts for the rapid diagnosis of toxins.

Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins.

A potential application of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is the generation of retinal pigmented epithelium (RPE) to treat age-related macular degeneration (AMD), a common but incurable retinal disease. RPE cells derived from hESCs (hESC-RPEs) and iPSCs (iPSC-RPEs) express essential RPE markers and can rescue visual function in animal models. However, standard differentiation protocols yield RPE cells at low frequency, especially from iPSC lines, and the common use of Matrigel and xenogeneic feeder cells is not compatible with clinical applications. The extracellular matrix (ECM) can affect differentiation, and therefore changes in ECM composition may improve the frequency of stem cell-RPE differentiation. We selected several purified ECM proteins and substrates, based on the in vivo RPE ECM environment, and tested their ability to support iPSC-RPE differentiation and maintenance. iPSCs differentiated on nearly all tested substrates developed pigmented regions, with Matrigel and mouse laminin-111 supporting the highest pigmentation frequencies. Although iPSC-RPEs cultured on the majority of the tested substrates expressed key RPE genes, only six substrates supported development of confluent monolayers with normal RPE morphology, including Matrigel and mouse laminin-111. iPSCs differentiated on mouse laminin-111 produced iPSC-RPEs expressing RPE proteins, and hESCs differentiated on mouse laminin-111 resulted in high yields of functional hESC-RPEs. This identification of key ECM proteins may assist with future scaffold designs and provide peptide sequences for use in synthetic, xeno-free, GMP-compliant generation of RPE from human pluripotent stem cells relevant to clinical translation.