Oxidation of DJ-1 Cysteines in Retinal Pigment Epithelium Function.

The retina and RPE cells are regularly exposed to chronic oxidative stress as a tissue with high metabolic demand and ROS generation. DJ-1 is a multifunctional protein in the retina and RPE that has been shown to protect cells from oxidative stress in several cell types robustly. Oxidation of DJ-1 cysteine (C) residues is important for its function under oxidative conditions. The present study was conducted to analyze the impact of DJ-1 expression changes and oxidation of its C residues on RPE function. Monolayers of the ARPE-19 cell line and primary human fetal RPE (hfRPE) cultures were infected with replication-deficient adenoviruses to investigate the effects of increased levels of DJ-1 in these monolayers. Adenoviruses carried the full-length human DJ-1 cDNA (hDJ) and mutant constructs of DJ-1, which had all or each of its three C residues individually mutated to serine (S). Alternatively, endogenous DJ-1 levels were decreased by transfection and transduction with shPARK7 lentivirus. These monolayers were then assayed under baseline and low oxidative stress conditions. The results were analyzed by immunofluorescence, Western blot, RT-PCR, mitochondrial membrane potential, and viability assays. We determined that decreased levels of endogenous DJ-1 levels resulted in increased levels of ROS. Furthermore, we observed morphological changes in the mitochondria structure of all the RPE monolayers transduced with all the DJ-1 constructs. The mitochondrial membrane potential of ARPE-19 monolayers overexpressing all DJ-1 constructs displayed a significant decrease, while hfRPE monolayers only displayed a significant decrease in their ΔΨm when overexpressing the C2S mutation. Viability significantly decreased in ARPE-19 cells transduced with the C53S construct. Our data suggest that the oxidation of C53 is crucial for regulating endogenous levels of ROS and viability in RPE cells.

Regulation of phagolysosomal activity by miR-204 critically influences structure and function of retinal pigment epithelium/retina.

MicroRNA-204 (miR-204) is expressed in pulmonary, renal, mammary and eye tissue, and its reduction can result in multiple diseases including cancer. We first generated miR-204-/- mice to study the impact of miR-204 loss on retinal and retinal pigment epithelium (RPE) structure and function. The RPE is fundamentally important for maintaining the health and integrity of the retinal photoreceptors. miR-204-/- eyes evidenced areas of hyper-autofluorescence and defective photoreceptor digestion, along with increased microglia migration to the RPE. Migratory Iba1+ microglial cells were localized to the RPE apical surface where they participated in the phagocytosis of photoreceptor outer segments (POSs) and contributed to a persistent build-up of rhodopsin. These structural, molecular and cellular outcomes were accompanied by decreased light-evoked electrical responses from the retina and RPE. In parallel experiments, we suppressed miR-204 expression in primary cultures of human RPE using anti-miR-204. In vitro suppression of miR-204 in human RPE similarly showed abnormal POS clearance and altered expression of autophagy-related proteins and Rab22a, a regulator of endosome maturation. Together, these in vitro and in vivo experiments suggest that the normally high levels of miR-204 in RPE can mitigate disease onset by preventing generation of oxidative stress and inflammation originating from intracellular accumulation of undigested photoreactive POS lipids. More generally, these results implicate RPE miR-204-mediated regulation of autophagy and endolysosomal interaction as a critical determinant of normal RPE/retina structure and function.

Endogenous miR-204 Protects the Kidney against Chronic Injury in Hypertension and Diabetes.

BACKGROUND: Aberrant microRNA (miRNA) expression affects biologic processes and downstream genes that are crucial to CKD initiation or progression. The miRNA miR-204-5p is highly expressed in the kidney but whether miR-204-5p plays any role in the development of chronic renal injury is unknown. METHODS: We used real-time PCR to determine levels of miR-204 in human kidney biopsies and animal models. We generated Mir204 knockout mice and used locked nucleic acid-modified anti-miR to knock down miR-204-5p in mice and rats. We used a number of physiologic, histologic, and molecular techniques to analyze the potential role of miR-204-5p in three models of renal injury. RESULTS: Kidneys of patients with hypertension, hypertensive nephrosclerosis, or diabetic nephropathy exhibited a significant decrease in miR-204-5p compared with controls. Dahl salt-sensitive rats displayed lower levels of renal miR-204-5p compared with partially protected congenic SS.13BN26 rats. Administering anti-miR-204-5p to SS.13BN26 rats exacerbated interlobular artery thickening and renal interstitial fibrosis. In a mouse model of hypertensive renal injury induced by uninephrectomy, angiotensin II, and a high-salt diet, Mir204 gene knockout significantly exacerbated albuminuria, renal interstitial fibrosis, and interlobular artery thickening, despite attenuation of hypertension. In diabetic db/db mice, administering anti-miR-204-5p exacerbated albuminuria and cortical fibrosis without influencing blood glucose levels. In all three models, inhibiting miR-204-5p or deleting Mir204 led to upregulation of protein tyrosine phosphatase SHP2, a target gene of miR-204-5p, and increased phosphorylation of signal transducer and activator of transcription 3, or STAT3, which is an injury-promoting effector of SHP2. CONCLUSIONS: These findings indicate that the highly expressed miR-204-5p plays a prominent role in safeguarding the kidneys against common causes of chronic renal injury.

Validation of iPS Cell-Derived RPE Tissue in Animal Models.

Previous work suggests that replacing diseased Retinal Pigment Epithelium (RPE) with a healthy autologous RPE sheet can provide vision rescue for AMD patients. We differentiated iPSCs into RPE using a directed differentiation protocol. RPE cells at the immature RPE stage were purified and seeded onto either electrospun poly(lactic-co-glycolic acid) (PLGA) scaffolds or non-biodegradable polyester cell culture inserts and compared the two tissues. In vitro, PLGA and polyester substrates produced functionally similar results. Following in vitro evaluation, we tested RPE tissue in animal models for safety and function. Safety studies were conducted in RNU rats using an injection composed of intact cells and homogenized scaffolds. To assess function and develop surgical procedures, the tissues were implanted into an acute RPE injury model pig eye and evaluated using optical coherence tomography (OCT), multifocal ERG (mfERG), and histology. Subretinal injection studies in rats demonstrated safety of the implant. Biodegradability and biocompatibility data from a pig model demonstrated that PLGA scaffold is safe, with the added benefit of being resorbed by the body over time, leaving no foreign material in the eye. We confirmed that biodegradable substrates provide suitable support for RPE maturation and transplantation.

Polarized Desmosome and Hemidesmosome Shedding via Exosomes is an Early Indicator of Outer Blood-Retina Barrier Dysfunction.

The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of exosomes that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral exosomes from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of exosome release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in exosome content, including basal-side specific desmosome and hemidesmosome shedding via exosomes. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases, (e.g., AMD) and broadly from blood-CNS barriers in other neurodegenerative diseases.

Polarized Desmosome and Hemidesmosome Shedding via Small Extracellular Vesicles is an Early Indicator of Outer Blood-Retina Barrier Dysfunction.

The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD).

MicroRNA-204/211 alters epithelial physiology.

MicroRNA (miRNA) expression in fetal human retinal pigment epithelium (hfRPE), retina, and choroid were pairwise compared to determine those miRNAs that are enriched by 10-fold or more in each tissue compared with both of its neighbors. miRs-184, 187, 200a/200b, 204/211, and 221/222 are enriched in hfRPE by 10- to 754-fold compared with neuroretina or choroid (P<0.05). Five of these miRNAs are enriched in RPE compared with 20 tissues throughout the body and are 10- to 20,000-fold more highly expressed (P<0.005). miR-204 and 211 are the most highly expressed in the RPE. In addition, expression of miR-204/211 is significantly lower in the NCI60 tumor cell line panel compared with that in 13 normal tissues, suggesting the progressive disruption of epithelial barriers and increased proliferation. We demonstrated that TGF-beta receptor 2 (TGF-betaR2) and SNAIL2 are direct targets of miR-204 and that a reduction in miR-204 expression leads to reduced expression of claudins 10, 16, and 19 (message/protein) consistent with our observation that anti-miR-204/211 decreased transepithelial resistance by 80% and reduced cell membrane voltage and conductance. The anti-miR-204-induced decrease in Kir7.1 protein levels suggests a signaling pathway that connects TGF-betaR2 and maintenance of potassium homeostasis. Overall, these data indicate a critical role for miR-204/211 in maintaining epithelial barrier function and cell physiology.

AMPK modulation ameliorates dominant disease phenotypes of CTRP5 variant in retinal degeneration.

Late-onset retinal degeneration (L-ORD) is an autosomal dominant disorder caused by a missense substitution in CTRP5. Distinctive clinical features include sub-retinal pigment epithelium (RPE) deposits, choroidal neovascularization, and RPE atrophy. In induced pluripotent stem cells-derived RPE from L-ORD patients (L-ORD-iRPE), we show that the dominant pathogenic CTRP5 variant leads to reduced CTRP5 secretion. In silico modeling suggests lower binding of mutant CTRP5 to adiponectin receptor 1 (ADIPOR1). Downstream of ADIPOR1 sustained activation of AMPK renders it insensitive to changes in AMP/ATP ratio resulting in defective lipid metabolism, reduced Neuroprotectin D1(NPD1) secretion, lower mitochondrial respiration, and reduced ATP production. These metabolic defects result in accumulation of sub-RPE deposits and leave L-ORD-iRPE susceptible to dedifferentiation. Gene augmentation of L-ORD-iRPE with WT CTRP5 or modulation of AMPK, by metformin, re-sensitize L-ORD-iRPE to changes in cellular energy status alleviating the disease cellular phenotypes. Our data suggests a mechanism for the dominant behavior of CTRP5 mutation and provides potential treatment strategies for L-ORD patients.

Gene expression profiling in autoimmune noninfectious uveitis disease.

Noninfectious uveitis is a predominantly T cell-mediated autoimmune, intraocular inflammatory disease. To characterize the gene expression profile from patients with noninfectious uveitis, PBMCs were isolated from 50 patients with clinically characterized noninfectious uveitis syndrome. A pathway-specific cDNA microarray was used for gene expression profiling and real-time PCR array for further confirmation. Sixty-seven inflammation- and autoimmune-associated genes were found differentially expressed in uveitis patients, with 28 of those genes being validated by real-time PCR. Several genes previously unknown for autoimmune uveitis, including IL-22, IL-19, IL-20, and IL-25/IL-17E, were found to be highly expressed among uveitis patients compared with the normal subjects with IL-22 expression highly variable among the patients. Furthermore, we show that IL-22 can affect primary human retinal pigment epithelial cells by decreasing total tissue resistance and inducing apoptosis possibly by decreasing phospho-Bad level. In addition, the microarray data identified a possible uveitis-associated gene expression pattern, showed distinct gene expression profiles in patients during periods of clinical activity and quiescence, and demonstrated similar expression patterns in related patients with similar clinical phenotypes. Our data provide the first evidence that a subset of IL-10 family genes are implicated in noninfectious uveitis and that IL-22 can affect human retinal pigment epithelial cells. The results may facilitate further understanding of the molecular mechanisms of autoimmune uveitis and other autoimmune originated inflammatory diseases.

IFN{gamma} regulates retinal pigment epithelial fluid transport.

The present experiments show that IFNgamma receptors are mainly localized to the basolateral membrane of human retinal pigment epithelium (RPE). Activation of these receptors in primary cultures of human fetal RPE inhibited cell proliferation and migration, decreased RPE mitochondrial membrane potential, altered transepithelial potential and resistance, and significantly increased transepithelial fluid absorption. These effects are mediated through JAK-STAT and p38 MAPK signaling pathways. Second messenger signaling through cAMP-PKA pathway- and interferon regulatory factor-1-dependent production of nitric oxide/cGMP stimulated the CFTR at the basolateral membrane and increased transepithelial fluid absorption. In vivo experiments using a rat model of retinal reattachment showed that IFNgamma applied to the anterior surface of the eye can remove extra fluid deposited in the extracellular or subretinal space between the retinal photoreceptors and RPE. Removal of this extra fluid was blocked by a combination of PKA and JAK-STAT pathway inhibitors injected into the subretinal space. These results demonstrate a protective role for IFNgamma in regulating retinal hydration across the outer blood-retinal barrier in inflammatory disease processes and provide the basis for possible therapeutic interventions.

Clinical-grade stem cell-derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs.

Considerable progress has been made in testing stem cell-derived retinal pigment epithelium (RPE) as a potential therapy for age-related macular degeneration (AMD). However, the recent reports of oncogenic mutations in induced pluripotent stem cells (iPSCs) underlie the need for robust manufacturing and functional validation of clinical-grade iPSC-derived RPE before transplantation. Here, we developed oncogenic mutation-free clinical-grade iPSCs from three AMD patients and differentiated them into clinical-grade iPSC-RPE patches on biodegradable scaffolds. Functional validation of clinical-grade iPSC-RPE patches revealed specific features that distinguished transplantable from nontransplantable patches. Compared to RPE cells in suspension, our biodegradable scaffold approach improved integration and functionality of RPE patches in rats and in a porcine laser-induced RPE injury model that mimics AMD-like eye conditions. Our results suggest that the in vitro and in vivo preclinical functional validation of iPSC-RPE patches developed here might ultimately be useful for evaluation and optimization of autologous iPSC-based therapies.

Primary Cilium-Mediated Retinal Pigment Epithelium Maturation Is Disrupted in Ciliopathy Patient Cells.

Primary cilia are sensory organelles that protrude from the cell membrane. Defects in the primary cilium cause ciliopathy disorders, with retinal degeneration as a prominent phenotype. Here, we demonstrate that the retinal pigment epithelium (RPE), essential for photoreceptor development and function, requires a functional primary cilium for complete maturation and that RPE maturation defects in ciliopathies precede photoreceptor degeneration. Pharmacologically enhanced ciliogenesis in wild-type induced pluripotent stem cells (iPSC)-RPE leads to fully mature and functional cells. In contrast, ciliopathy patient-derived iPSC-RPE and iPSC-RPE with a knockdown of ciliary-trafficking protein remain immature, with defective apical processes, reduced functionality, and reduced adult-specific gene expression. Proteins of the primary cilium regulate RPE maturation by simultaneously suppressing canonical WNT and activating PKCδ pathways. A similar cilium-dependent maturation pathway exists in lung epithelium. Our results provide insights into ciliopathy-induced retinal degeneration, demonstrate a developmental role for primary cilia in epithelial maturation, and provide a method to mature iPSC epithelial cells for clinical applications.

PDGF-C and -D induced proliferation/migration of human RPE is abolished by inflammatory cytokines.

PURPOSE: The role of growth factors and inflammation in regulating retinal pigment epithelial (RPE) function is complex and still poorly understood. The present study investigated human RPE cell proliferation and migration mediated by platelet-derived growth factor (PDGF) and inflammatory cytokines. METHODS: Human fetal RPE (hfRPE) cells were obtained as previously described. Gene expressions of PDGF isoforms and their receptors were detected using real-time PCR. Protein expression, activity, and localization of PDGFR-alpha and -beta were analyzed by Western blot and immunohistochemistry. BrdU incorporation and wound healing assays were used to test the effects of different PDGF isoforms and inflammatory cytokines on hfRPE proliferation and migration. Annexin-V and phalloidin staining were used to detect apoptosis and the actin cytoskeleton, respectively. RESULTS: PDGF-C and PDGF-D proteins are expressed in native human adult RPE, and mRNA levels are up to 100-fold higher than PDGF-A and -B. PDGFR-alpha and -beta proteins are expressed in native adult RPE and hfRPE (mainly localized to the apical membrane). In hfRPE, these receptors can be activated by PDGF-CC and -DD. PDGF-CC, -DD, and -BB significantly increased hfRPE proliferation, whereas PDGF-DD, -BB, and -AB significantly increased cell migration. An inflammatory cytokine mixture (TNF-alpha/IL-1beta/IFN-gamma) completely inhibited the stimulatory effect of PDGF-BB, -CC, and -DD; in contrast, this mixture stimulated the proliferation of choroidal cells. This inflammatory cytokine mixture also induced apoptosis, significant disruption of actin filaments and zonula occludens (ZO-1), and a decrease in transepithelial resistance. CONCLUSIONS: These results suggest that proinflammatory cytokines in vivo can inhibit the proliferative effect of PDGF on human RPE and, at the same time, stimulate the proliferation of choroidal cells. They also suggest an important role of proinflammatory cytokines in overcoming local proliferative/wound-healing responses, thereby controlling the development of disease processes at the retina/RPE/choroid interface.

Acrolein, a toxicant in cigarette smoke, causes oxidative damage and mitochondrial dysfunction in RPE cells: protection by (R)-alpha-lipoic acid.

PURPOSE: To understand better the cell and molecular basis for the epidemiologic association between cigarette smoke, oxidant injury, and age-associated macular degeneration, the authors examined the effects of acrolein, a major toxicant in cigarette smoke, on oxidative mitochondrial damage in retinal pigment epithelial (RPE) cells and the reduction of this damage by lipoic acid. METHODS: Cultured human ARPE19 cells and primary cultures of human fetal (hf)RPE were treated with acrolein. The toxicity of acrolein and the protective effects of R-alpha-lipoic acid were examined with a variety of previously described techniques. RESULTS: Acute acrolein exposure exceeding 50 microM (24 hours) in ARPR19 cells caused toxicity, including decreases in cell viability, mitochondrial potential, GSH, antioxidant capacity, Nrf2 expression, enzyme activity (mitochondrial complexes I, II, III; superoxide dismutase; and glutathione peroxidase). Acute exposure also increased oxidant levels, protein carbonyls, and calcium. Continuous acrolein exposure over 8 or 32 days caused similar toxicity but from 10- to 100-fold lower doses (0.1-5 microM). Pretreatment with R-alpha-lipoic acid effectively protected ARPE-19 cells from acrolein toxicity. Primary hfRPE cells were comparable to the ARPE-19 cells in sensitivity to acrolein toxicity and lipoic acid protection. CONCLUSIONS: These results show that acrolein is a mitochondrial toxicant in RPE cells and that acrolein-induced oxidative mitochondrial dysfunction is reduced by lipoic acid. The similar sensitivity of the ARPE-19 and hfRPE cells suggests that both models are useful for studying RPE toxicity and protection. These experiments indicate that mitochondria-targeted antioxidants such as lipoic acid may be an effective strategy for reducing or preventing chronic oxidant-induced RPE degeneration in vivo from a variety of sources, including cigarette smoke.

Effect of simultaneous inhibition of epidermal growth factor receptor and cyclooxygenase-2 in HER-2/neu-positive breast cancer.

PURPOSE: HER-2/erbB2/neu is overexpressed in 25% to 30% of all invasive breast cancers and is associated with an aggressive course and reduced survival. HER-2/erbB2/neu breast tumors are frequently associated with up-regulation of cyclooxygenase (COX)-2 and activation of the epidermal growth factor receptor (EGFR) pathway, which promote enhanced cell growth and resistance to apoptosis. This study investigated whether simultaneously blocking both EGFR and COX-2 pathways with ZD1839 and celecoxib, respectively, would be more effective in inhibiting cell growth and inducing apoptosis than either agent alone. EXPERIMENTAL DESIGN: The effects of ZD1839, celecoxib, and their combination on cell survival, cell cycle progression, and apoptosis were determined in NMF11.2 mouse mammary tumor cells. We also investigated the potential downstream EGFR-activated pathways that are altered by simultaneous inhibition of COX-2 and EGFR. RESULTS: Celecoxib alone and ZD1839 alone produced a concentration- and time-dependent inhibition of cell survival. Both agents combined produced supra-additive inhibitory effects on cell survival and apoptosis compared with either agent alone. This effect was associated with increased cleaved poly(ADP-ribose)polymerase and reduced protein expression of bcl-2. Phosphorylation of extracellular signal-regulated kinase 1/2 was partially blocked by ZD1839 and celecoxib alone and was completely blocked by the combination of both agents. The enhanced proapoptotic effects of the combined agents were also associated with decreased phosphorylation of Akt and increased phosphorylation of p38. CONCLUSIONS: These findings show that both COX-2 and EGFR are important targets for inhibiting survival and inducing apoptosis in breast cancer. The data suggest a potential cross-talk between COX-2 and EGFR signaling in breast cancer cells overexpressing HER-2/erbB2/neu.

A basis for comparison: sensitive authentication of stem cell derived RPE using physiological responses of intact RPE monolayers.

The retinal pigment epithelium (RPE) is a monolayer of highly specialized cells that help maintain the chemical composition of its surrounding subretinal and choroidal extracellular spaces. Retinal cells (photoreceptors in particular), RPE, and choroidal endothelial cells together help ensure a homeostatically stable metabolic environment with exquisitely sensitive functional responses to light. Aging and disease of the RPE impairs its supportive functions contributing to the progressive loss of photoreceptors and vision. The prevalence of RPE associated retinal degenerations has prompted researchers to develop new therapies aimed at replacing the affected RPE with induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) derived RPE. Despite recent attempts to characterize stem cell derived RPE and to truly authenticate RPE for clinical applications, there remains a significant unmet need to explore the heterogeneity resulting from donor to donor variation as well as the variations inherent in the current processes of cell manufacture. Additionally, it remains unknown whether the starting cell type influences the resulting RPE phenotype following reprogramming and differentiation. To address these questions, we performed a comprehensive evaluation (genomic, structural, and functional) of 15 iPSC derived RPE originating from different donors and tissues and compiled a reference data set for the authentication of iPSC-derived RPE and RPE derived from other stem cell sources.

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors.

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease.

Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.

PURPOSE: Provide a reproducible method for culturing confluent monolayers of hfRPE cells that exhibit morphology, physiology, polarity, and protein expression patterns similar to native tissue. METHODS: Human fetal eyes were dissected on arrival, and RPE cell sheets were mechanically separated from the choroid and cultured in a specifically designed medium comprised entirely of commercially available components. Physiology experiments were performed with previously described techniques. Standard techniques were used for immunohistochemistry, electron microscopy, and cytokine measurement by ELISA. RESULTS: Confluent monolayers of RPE cell cultures exhibited epithelial morphology and heavy pigmentation, and electron microscopy showed extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. The mean transepithelial potential (TEP) was 2.6 +/- 0.8 mV, apical positive, and the mean transepithelial resistance (R(T)) was 501 +/- 138 Omega . cm(2) (mean +/- SD; n = 35). Addition of 100 microM adenosine triphosphate (ATP) to the apical bath increased net fluid absorption from 13.6 +/- 2.6 to 18.8 +/- 4.6 microL . cm(-2) per hour (mean +/- SD; n = 4). In other experiments, VEGF was mainly secreted into the basal bath (n = 10), whereas PEDF was mainly secreted into the apical bath (n = 10). CONCLUSIONS: A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.

Anatomical and Gene Expression Changes in the Retinal Pigmented Epithelium Atrophy 1 (rpea1) Mouse: A Potential Model of Serous Retinal Detachment.

PURPOSE: The purpose of this study was to examine the rpea1 mouse whose retina spontaneously detaches from the underlying RPE as a potential model for studying the cellular effects of serous retinal detachment (SRD). METHODS: Optical coherence tomography (OCT) was performed immediately prior to euthanasia; retinal tissue was subsequently prepared for Western blotting, microarray analysis, immunocytochemistry, and light and electron microscopy (LM, EM). RESULTS: By postnatal day (P) 30, OCT, LM, and EM revealed the presence of small shallow detachments that increased in number and size over time. By P60 in regions of detachment, there was a dramatic loss of PNA binding around cones in the interphotoreceptor matrix and a concomitant increase in labeling of the outer nuclear layer and rod synaptic terminals. Retinal pigment epithelium wholemounts revealed a patchy loss in immunolabeling for both ezrin and aquaporin 1. Anti-ezrin labeling was lost from small regions of the RPE apical surface underlying detachments at P30. Labeling for tight-junction proteins provided a regular array of profiles outlining the periphery of RPE cells in wild-type tissue, however, this pattern was disrupted in the mutant as early as P30. Microarray analysis revealed a broad range of changes in genes involved in metabolism, signaling, cell polarity, and tight-junction organization. CONCLUSIONS: These data indicate changes in this mutant mouse that may provide clues to the underlying mechanisms of SRD in humans. Importantly, these changes include the production of multiple spontaneous detachments without the presence of a retinal tear or significant degeneration of outer segments, changes in the expression of proteins involved in adhesion and fluid transport, and a disrupted organization of RPE tight junctions that may contribute to the formation of focal detachments.

In Pursuit of Authenticity: Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium for Clinical Applications.

: Induced pluripotent stem cells (iPSCs) can be efficiently differentiated into retinal pigment epithelium (RPE), offering the possibility of autologous cell replacement therapy for retinal degeneration stemming from RPE loss. The generation and maintenance of epithelial apical-basolateral polarity is fundamental for iPSC-derived RPE (iPSC-RPE) to recapitulate native RPE structure and function. Presently, no criteria have been established to determine clonal or donor based heterogeneity in the polarization and maturation state of iPSC-RPE. We provide an unbiased structural, molecular, and physiological evaluation of 15 iPSC-RPE that have been derived from distinct tissues from several different donors. We assessed the intact RPE monolayer in terms of an ATP-dependent signaling pathway that drives critical aspects of RPE function, including calcium and electrophysiological responses, as well as steady-state fluid transport. These responses have key in vivo counterparts that together help determine the homeostasis of the distal retina. We characterized the donor and clonal variation and found that iPSC-RPE function was more significantly affected by the genetic differences between different donors than the epigenetic differences associated with different starting tissues. This study provides a reference dataset to authenticate genetically diverse iPSC-RPE derived for clinical applications. SIGNIFICANCE: The retinal pigment epithelium (RPE) is essential for maintaining visual function. RPE derived from human induced pluripotent stem cells (iPSC-RPE) offer a promising cell-based transplantation therapy for slowing or rescuing RPE-induced visual function loss. For effective treatment, iPSC-RPE must recapitulate the physiology of native human RPE. A set of physiologically relevant functional assays are provided that assess the polarized functional activity and maturation state of the intact RPE monolayer. The present data show that donor-to-donor variability exceeds the tissue-to-tissue variability for a given donor and provides, for the first time, criteria necessary to identify iPSC-RPE most suitable for clinical application.