Diseased Retinal Pigment Epithelium Research Articles

HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment.

Recent advances in cellular scaffolds for retinal pigment epithelium cell transplantation

Replacement of diseased retinal pigment epithelium (RPE) cells with healthy RPE cells by transplantation is one option to treat several retinal degenerative diseases including age-related macular degeneration, which are caused by RPE loss and dysfunction. A cellular scaffold as a carrier for transplanted cells, may hold immense promise for facilitating cell migration and promoting the integration of RPE cells into the host environment. Scaffolds can be prepared from a variety of natural and synthetic materials. Strategies, such as surface modification and structure adjustment, can improve the biomimetic properties of the scaffolds, optimize cell attachment and cellular function following transplantation and lay a foundation of clinical application in the future. Key words: Tissue scaffolds; Retinal pigment epithelium/transplantation; Macular degeneration/therapy; Review

Characterization of lincRNA expression in the human retinal pigment epithelium and differentiated induced pluripotent stem cells.

Long intervening non-coding RNAs (lincRNAs) are increasingly being implicated as important factors in many aspects of cellular development, function, and disease, but remain poorly understood. In this study, we examine the human retinal pigment epithelium (RPE) lincRNA transcriptome using RNA-Seq data generated from human fetal RPE (fRPE), RPE derived from human induced pluripotent stem cells (iPS-RPE), and undifferentiated iPS (iPS). In addition, we determine the suitability of iPS-RPE, from a transcriptome standpoint, as a model for use in future studies of lincRNA structure and function. A comparison of gene and isoform expression across the whole transcriptome shows only minimal differences between all sample types, though fRPE and iPS-RPE show higher concordance than either shows with iPS. Notably, RPE signature genes show the highest degree of fRPE to iPS-RPE concordance, indicating that iPS-RPE cells provide a suitable model for use in future studies. An analysis of lincRNAs demonstrates high concordance between fRPE and iPS-RPE, but low concordance between either RPE and iPS. While most lincRNAs are expressed at low levels (RPKM < 10), there is a high degree of concordance among replicates within each sample type, suggesting the expression is consistent, even at levels subject to high variability. Finally, we identified and annotated 180 putative novel genes in the fRPE samples, a majority of which are also expressed in the iPS-RPE. Overall, this study represents the first characterization of lincRNA expression in the human RPE, and provides a model for studying the role lincRNAs play in RPE development, function, and disease.

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.

Magnetic nanoparticles conjugated with "RPE cell -MCP-1 antibody -VEGF antibody" compounds for the targeted therapy of age-related macular degeneration: a hypothesis.

Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly throughout the world. Treatment of AMD utilizing retinal pigment epithelium (RPE) transplantation represents a promising therapy. However, simplex RPE transplantation can only replace the diseased RPE cells, but has no abilities to stop the development of AMD. It has been indicated that oxidization triggers the development of AMD by inducing the dysfunction and degeneration of RPE cells, which results in the upregulation of local monocyte chemotactic protein-1 (MCP-1) expression. MCP-1 induces macrophage recruiment which triggers local inflammation. As a result, the expression of vascular endothelial growth factor (VEGF) is upregulated by MCP-1 mediated inflammation and results in the formation of choroidal neovascularization (CNV). We accordingly propose a targeted therapy of AMD by subretinal transplanting the compound of RPE cell, MCP-1 antibody, and VEGF antibody and using a magnetic system to guide RPE cell compounds conjugated with superparamagnetic iron oxide nanoparticles (SPIONs). Furthermore, SPION-labelled RPE cells can be tracked and detected in vivo by non-invasive magnetic resonance imaging (MRI). This novel RPE cell transplantation methodology seems very promising to provide a new therapeutic approach for the treatment of AMD.

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.

Impact of retinal pigment epithelium pathology on spectral-domain optical coherence tomography-derived macular thickness and volume metrics and their intersession repeatability.

To determine the impact of retinal pigment epithelium (RPE) pathology on intersession repeatability of retinal thickness and volume metrics derived from Spectralis spectral-domain optical coherence tomography (Heidelberg Engineering, Heidelberg, Germany). Prospective cross-sectional single centre study. A total of 56 eyes of 56 subjects were divided into three groups: (i) normal RPE band (25 eyes); (ii) RPE elevation: macular soft drusen (13 eyes); and (iii) RPE attenuation: geographic atrophy or inherited retinal diseases (18 eyes). Each subject underwent three consecutive follow-up macular raster scans (61 B-scans at 119μm separation) at 1-month intervals. Retinal thicknesses and volumes for each zone of the macular subfields before and after manual correction of segmentation error. Coefficients of repeatability (CR) were calculated. Mean (range) age was 57 (21-88) years. Mean central subfield thickness (CST) and total macular volume were 264 and 258μm (P=0.62), and 8.0 and 7.8mm3 (P=0.31), before and after manual correction. Intersession CR (95% confidence interval) for CST and total macular volume were reduced from 40 (38-41) to 8.3 (8.1-8.5) and 0.62 to 0.16mm3 after manual correction of segmentation lines. CR for CST were 7.4, 23.5 and 66.7μm before and 7.0, 10.9 and 7.6μm after manual correction in groups i, ii and iii. Segmentation error in eyes with RPE disease has a significant impact on intersession repeatability of Spectralis spectral-domain optical coherence tomography macular thickness and volume metrics. Careful examination of each B-scan and manual adjustment can enhance the utility of quantitative measurement. Improved automated segmentation algorithms are needed.

Membrana neovascular subretiniana idiopática em uma criança

A case of idiopathic choroidal neovascular membrane (CNVM) is described in a 12-year-old male patient. On initial examination, his vision was 20/100 in the left eye (OS). Fluorescein angiography and optical coherence tomography scan (OCT) showed a classic type 2 choroidal neovascular membrane. Intravitreal ranibizumab was injected into the OS, three times, with an interval of 30 days between injections. At a follow-up visit, 3 weeks after the third injection, his best corrected visual acuity in the left eye was 20/20, with complete resolution of fluid on the OCT. This case is unusual, in that the CNVM developed in a young patient with no significant past medical history and in the absence of a choroidal or retinal pigment epithelial disease process that may be associated with a CNVM.

Repressed SIRT1/PGC-1α pathway and mitochondrial disintegration in iPSC-derived RPE disease model of age-related macular degeneration.

BackgroundStudy of age related macular degeneration (AMD) has been hampered by lack of human models that represent the complexity of the disease. Here we have developed a human in vitro disease model of AMD to investigate the underlying AMD disease mechanisms.MethodsGeneration of iPSCs from retinal pigment epithelium (RPE) of AMD donors, age-matched normal donors, skin fibroblasts of a dry AMD patient, and differentiation of iPSCs into RPE (AMD RPE-iPSC-RPE, normal RPE-iPSC-RPE and AMD Skin-iPSC-RPE, respectively). Immunostaining, cell viability assay and reactive oxygen species (ROS) production under oxidative stress conditions, electron microscopy (EM) imaging, ATP production and glycogen concentration assays, quantitative real time PCR, western blot, karyotyping.ResultsThe AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE present functional impairment and exhibit distinct disease phenotypes compared to RPE-iPSC-RPE generated from normal donors (Normal RPE-iPSC-RPE). The AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE show increased susceptibility to oxidative stress and produced higher levels of reactive oxygen species (ROS) under stress in accordance with recent reports. The susceptibility to oxidative stress-induced cell death in AMD RPE-iPSC-RPE and Skin-iPSC-RPE was consistent with inability of the AMD RPE-iPSC-RPE and Skin-iPSC-RPE to increase SOD2 expression under oxidative stress. Phenotypic analysis revealed disintegrated mitochondria, accumulation of autophagosomes and lipid droplets in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE. Mitochondrial activity was significantly lower in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE compared to normal cells and glycogen concentration was significantly increased in the diseased cells. Furthermore, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a regulator of mitochondrial biogenesis and function was repressed, and lower expression levels of NAD-dependent deacetylase sirtuin1 (SIRT1) were found in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE as compared to normal RPE-iPSC-RPE.ConclusionsOur studies suggest SIRT1/PGC-1α as underlying pathways contributing to AMD pathophysiology, and open new avenues for development of targeted drugs for treatment of this devastating neurodegenerative disease of the visual system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1101-8) contains supplementary material, which is available to authorized users.

Gene Therapy and Stem Cell Transplantation in Retinal Disease: The New Frontier

Gene and cell therapies have the potential to prevent, halt, or reverse diseases of the retina in patients with currently incurable blinding conditions. Over the past 2 decades, major advances in our understanding of the pathobiologic basis of retinal diseases, coupled with growth of gene transfer and cell transplantation biotechnologies, have created optimism that previously blinding retinal conditions may be treatable. It is now possible to deliver cloned genes safely and stably to specific retinal cell types in humans. Preliminary results testing gene augmentation strategies in human recessive diseases suggest promising safety and efficacy profiles, including improved visual function outcomes over extended periods. Additional gene-based strategies under development include approaches to autosomal dominant disease ("gain of function"), attempts to deliver genes encoding therapeutic proteins with proven mechanisms of action interfering with specific disease pathways, and approaches that could be used to render retinal cells other than atrophied photoreceptors light sensitive. In the programs that are the furthest along-pivotal regulatory safety and efficacy trials studying individuals with retinal degeneration resulting from RPE65 mutations-initial results reveal a robust safety profile and clinically significant improvements in visual function, thereby making this program a frontrunner for the first approved gene therapy product in the United States. Similar to gene therapy, progress in regenerative or stem cell-based transplantation strategies has been substantial. It is now possible to deliver safely stem cell-derived, terminally differentiated, biologically and genetically defined retinal pigment epithelium (RPE) to the diseased human eye. Although demonstration of clinical efficacy is still well behind the gene therapy field, multiple programs investigating regenerative strategies in RPE disease are beginning to enroll subjects, and initial results suggest possible signs of efficacy. Stem cells capable of becoming other retinal cell types, such as photoreceptors, are on the cusp of clinical trials. Stem cell-derived transplants can be delivered to precise target locations in the eye, and their ability to ameliorate, reverse, regenerate, or neuroprotect against disease processes can be assessed. Results from these studies will provide foundational knowledge that may lead to clinically significant therapies for currently untreatable retinal disease.

Matrigel and Activin A promote cell-cell contact and anti-apoptotic activity in cultured human retinal pigment epithelium cells

Age-related macular degeneration (AMD) is a leading cause of blindness among the aging population. Currently, replacement of diseased retinal pigment epithelium (RPE) cells with transplanted healthy RPE cells could be a feasible approach for AMD therapy. However, maintaining cell-cell contact and good viability of RPE cells cultured in vitro is difficult and fundamentally determines the success of RPE cell transplantation. This study was conducted to examine the role of Matrigel and Activin A (MA) in regulating cell-cell contact and anti-apoptotic activity in human RPE (hRPE) cells, as assessed by atomic force microscopy (AFM), scanning electron microscope (SEM), immunofluorescence staining, quantitative polymerase chain reaction (qPCR) analysis, Annexin V/propidium iodide (PI) analysis, mitochondrial membrane potential (△Ψ m) assays, intracellular reactive oxygen species (ROS) assays and Western blotting. hRPE cells cultured in vitro could maintain their epithelioid morphology after MA treatment over at least 4 passages. The contact of N-cadherin to the lateral cell border was promoted in hRPE cells at P2 by MA. MA treatment also enhanced the expression of tight junction-associated genes and proteins, such as Claudin-1, Claudin-3, Occludin and ZO-1, as well as polarized ZO-1 protein distribution and barrier function, in cultured hRPE cells. Moreover, MA treatment decreased apoptotic cells, ROS and Bax and increased △Ψ m and Bcl2 in hRPE cells under serum withdrawal-induced apoptosis. In addition, MA treatment elevated the protein expression levels of β-catenin and its target proteins, including Cyclin D1, c-Myc and Survivin, as well as the gene expression levels of ZO-1, β-catenin, Survivin and TCF-4, all of which could be down-regulated by the Wnt/β-catenin pathway inhibitor XAV-939. Taken together, MA treatment could effectively promote cell-cell contact and anti-apoptotic activity in hRPE cells, partly involving the Wnt/β-catenin pathway. This study will benefit the understanding of hRPE cells and future cell therapy.

Targeting the cAMP and Transforming Growth Factor-β Pathway Increases Proliferation to Promote Re-Epithelialization of Human Stem Cell-Derived Retinal Pigment Epithelium.

Retinal pigment epithelium (RPE) cell integrity is critical to the maintenance of retinal function. Many retinopathies such as age-related macular degeneration (AMD) are caused by the degeneration or malfunction of the RPE cell layer. Replacement of diseased RPE with healthy, stem cell-derived RPE is a potential therapeutic strategy for treating AMD. Human embryonic stem cells (hESCs) differentiated into RPE progeny have the potential to provide an unlimited supply of cells for transplantation, but challenges around scalability and efficiency of the differentiation process still remain. Using hESC-derived RPE as a cellular model, we sought to understand mechanisms that could be modulated to increase RPE yield after differentiation. We show that RPE epithelialization is a density-dependent process, and cells seeded at low density fail to epithelialize. We demonstrate that activation of the cAMP pathway increases proliferation of dissociated RPE in culture, in part through inhibition of transforming growth factor-β (TGF-β) signaling. This results in enhanced uptake of epithelial identity, even in cultures seeded at low density. In line with these findings, targeted manipulation of the TGF-β pathway with small molecules produces an increase in efficiency of RPE re-epithelialization. Taken together, these data highlight mechanisms that promote epithelial fate acquisition in stem cell-derived RPE. Modulation of these pathways has the potential to favorably impact scalability and clinical translation of hESC-derived RPE as a cell therapy. Stem cell-derived retinal pigment epithelium (RPE) is currently being evaluated as a cell-replacement therapy for macular degeneration. This work shows that the process of generating RPE in vitro is regulated by the cAMP and transforming growth factor-β signaling pathway. Modulation of these pathways by small molecules, as identified by phenotypic screening, leads to an increased efficiency of generating RPE cells with a higher yield. This can have a potential impact on manufacturing transplantation-ready cells at large scale and is advantageous for clinical studies using this approach in the future.

Simultaneous determination of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in human retinal DNA by liquid chromatography nanoelectrospray-tandem mass spectrometry.

Age-related macular degeneration (AMD) is the leading cause of blindness among older adults in the developed world. Oxidative damage to mitochondrial DNA (mtDNA) in the retinal pigment epithelium (RPE) may play a key role in AMD. Measurement of oxidative DNA lesions such as 8-oxo-2’-deoxyguanosine (8-oxo-dG) and 8-oxo-2’-deoxyadenosine (8-oxo-dA) in diseased RPE could provide important insights into the mechanism of AMD development. We have developed a liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry method for simultaneous analysis of 8-oxo-dG and 8-oxo-dA in human retinal DNA. The developed method was applied to the analysis of retinal DNA from 5 donors with AMD and 5 control donors without AMD. In mtDNA, the levels of 8-oxo-dG in controls and AMD donors averaged 170 and 188, and 8-oxo-dA averaged 11 and 17 adducts per 106 bases, respectively. In nuclear DNA, the levels of 8-oxo-dG in controls and AMD donors averaged 0.54 and 0.96, and 8-oxo-dA averaged 0.04 and 0.05 adducts per 106 bases, respectively. This highly sensitive method allows for the measurement of both adducts in very small amounts of DNA and can be used in future studies investigating the pathophysiological role of 8-oxo-dG and 8-oxo-dA in AMD and other oxidative damage-related diseases in humans.

Cone and rod loss in Stargardt disease revealed by adaptive optics scanning light ophthalmoscopy.

Stargardt disease (STGD1) is characterized by macular atrophy and flecks in the retinal pigment epithelium. The causative ABCA4 gene encodes a protein localizing to photoreceptor outer segments. The pathologic steps by which ABCA4 mutations lead to clinically detectable retinal pigment epithelium changes remain unclear. We investigated early STGD1 using adaptive optics scanning light ophthalmoscopy. Adaptive optics scanning light ophthalmoscopy imaging of 2 brothers with early STGD1 and their unaffected parents was compared with conventional imaging. Cone and rod spacing were increased in both patients (P < .001) with a dark cone appearance. No foveal cones were detected in the older brother. In the younger brother, foveal cones were enlarged with low density (peak cone density, 48.3 × 103 cones/mm2). The ratio of cone to rod spacing was increased in both patients, with greater divergence from normal approaching the foveal center, indicating that cone loss predominates centrally and rod loss increases peripherally. Both parents had normal photoreceptor mosaics. Genetic testing revealed 3 disease-causing mutations. This study provides in vivo images of rods and cones in STGD1. Although the primary clinical features of STGD1 are retinal pigment epithelial lesions, adaptive optics scanning light ophthalmoscopy reveals increased cone and rod spacing in areas that appear normal in conventional images, suggesting that photoreceptor loss precedes clinically detectable retinal pigment epithelial disease in STGD1.

Regenerating Retinal Pigment Epithelial Cells to Cure Blindness: A Road Towards Personalized Artificial Tissue.

Retinal pigment epithelium (RPE) is a polarized monolayer tissue that functions to support the health and integrity of retinal photoreceptors (PRs). RPE atrophy has been linked to pathogenesis of age-related macular degeneration (AMD), a leading cause of blindness in elderly in the USA. RPE atrophy in AMD leads to the PR cell death and vision loss. It is thought that replacing diseased RPE with healthy RPE tissue can prevent PR cell death. Retinal surgical innovations have provided proof-of-principle data that autologous RPE tissue can replace diseased macular RPE and provide visual rescue in AMD patients. Current efforts are focused on developing an in vitro tissue using natural and synthetic scaffolds to generate a polarized functional RPE monolayer. In the future, these tissue-engineering approaches combined with pluripotent stem cell technology will lead to the development of personalized and "off-the-shelf" cell therapies for AMD patients. This review summarizes the historical development and ongoing efforts in surgical and in vitro tissue engineering techniques to develop a three-dimensional therapeutic native RPE tissue substitute.

Efficient derivation of retinal pigment epithelium cells from stem cells.

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

The role of fundus autofluorescence imaging in the study of the course of posterior uveitis disorders.

Background. To evaluate the correlation of fundus autofluorescence (FAF) with indocyanine green angiography (ICGA) in patients with various posterior uveitis disorders. Methods. Interventional case series including 23 eyes of 15 patients with diagnosis of a specific type of retinochoroiditis, such as acute posterior multifocal placoid pigment epitheliopathy (APMPPE), serpiginous-like choroiditis, multifocal choroiditis (MFC), Harada disease, and syphilitic retinochoroiditis. Also, some cases with undefined retinochoroiditis were included. FAF and ICGA were performed and correlated at baseline and during follow-up after treatment. Results. In ICGA, early hypofluorescence was found to be the hallmark of acute choroidal inflammation, resolving in later stages and remaining in the late phase in areas with retinal pigment epithelium (RPE) damage. Poorly defined hyperautofluorescent areas correlated with acute choroidal lesions. Hypoautofluorescent delineation suggested the initiation of RPE healing processes, correlating well with the late phase of ICGA and delineating the RPE damage. Early hyperautofluorescence with late hypofluorescence in ICGA indicated the presence of primary RPE involvement. Conclusion. FAF contributes to the interpretation of RPE disease and may be a useful tool for the follow-up of progressive inflammatory disorders. Comparative evaluation of FAF and ICGA allows a characterization of the sequence of inflammatory events and the level of tissue affected.

Effect of Storage Temperature on Key Functions of Cultured Retinal Pigment Epithelial Cells.

Purpose. Replacement of the diseased retinal pigment epithelium (RPE) with cells capable of performing the specialized functions of the RPE is the aim of cell replacement therapy for treatment of macular degenerative diseases. A storage method for RPE is likely to become a prerequisite for the establishment of such treatment. Herein, we analyze the effect of storage temperature on key functions of cultured RPE cells. Methods. Cultured ARPE-19 cells were stored in Minimum Essential Medium at 4°C, 16°C, and 37°C for seven days. Total RNA was isolated and the gene expression profile was determined using DNA microarrays. Comparison of the microarray expression values with qRT-PCR analysis of selected genes validated the results. Results. Expression levels of several key genes involved in phagocytosis, pigment synthesis, the visual cycle, adherens, and tight junctions, and glucose and ion transport were maintained close to control levels in cultures stored at 4°C and 16°C. Cultures stored at 37°C displayed regulational changes in a larger subset of genes related to phagocytosis, adherens, and tight junctions. Conclusion. RPE cultures stored at 4°C and 16°C for one week are capable of maintaining the expression levels of genes important for key RPE functions close to control levels.

In situ cell surface proteomics reveals differentially expressed membrane proteins in retinal pigment epithelial cells during autoimmune uveitis.

We applied an innovative and successful method to detect changes in the plasma cell surface proteome of RPE cells in a spontaneous inflammatory eye disease, serving as a valuable model for human autoimmune uveitis. We were able to identify 27 differentially expressed plasma cell membrane proteins, including synaptotagmin 1, basigin and collectrin, which play important roles in cell adhesion, transport and cell communication.

Effects of small intestinal submucosa content on the adhesion and proliferation of retinal pigment epithelial cells on SIS-PLGA films.

The retinal pigment epithelium (RPE) plays a critical role in the maintenance of the normal functions of the retina, particularly the photoreceptors. RPE dysfunction, vision loss and degeneration have been implicated as the cause of many retinal diseases, including retinitis pigmentosa and age-related macular degeneration (AMD). To overcome such disorders, tissue engineering could offer useful strategies, using biodegradable polymeric films to replace diseased or lost RPE. Synthetic/natural hybrid films have been studied as a temporary substrate for growing RPEs in biological implantations. In this study, we prepared small intestinal submucosa (SIS)-poly(lactic-co-glycolic) (PLGA) hybrid films and seeded human RPE cells (ARPE-19 cells) onto the film surface. We investigated the film suitability for RPE cell proliferation by MTT assay. The morphology of cellular adhesion on the film was confirmed by scanning electron microscopy (SEM). Reverse transcription-polymerase chain reaction (RT-PCR) and 3-amino-9-ethylcarbazole (AEC) staining were performed to examine mRNA expression and to compare cell proliferation on the films, using cytokeratin as a marker of RPE. Conclusively, we confirmed the higher cell survival rate and much stronger phenotype expression of RPEs on SIS-PLGA films compared to pure PLGA films. These results demonstrated the potential application of SIS-PLGA films in tissue-engineering strategies. Copyright © 2014 John Wiley & Sons, Ltd.