scholarly journals Functional Assessment of Patient-Derived Retinal Pigment Epithelial Cells Edited by CRISPR/Cas9

2018 ◽  
Vol 19 (12) ◽  
pp. 4127 ◽  
Author(s):  
Leah Foltz ◽  
Sara Howden ◽  
James Thomson ◽  
Dennis Clegg
Keyword(s):  
Retinitis Pigmentosa ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Splicing Factor ◽  
Wild Type ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Apicobasal Polarity ◽  
Gene And Protein Expression

Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the PRPF8 splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa.

scholarly journals Key Role for CRB2 in the Maintenance of Apicobasal Polarity in Retinal Pigment Epithelial Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Antonio E. Paniagua ◽  
Alicia Segurado ◽  
Jorge F. Dolón ◽  
Julián Esteve-Rudd ◽  
Almudena Velasco ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Cell Junction ◽  
Subretinal Space ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Apicobasal Polarity

Apicobasal polarity is essential for epithelial cell function, yet the roles of different proteins in its completion is not fully understood. Here, we have studied the role of the polarity protein, CRB2, in human retinal pigment epithelial (RPE) cells during polarization in vitro, and in mature murine RPE cells in vivo. After establishing a simplified protocol for the culture of human fetal RPE cells, we studied the temporal sequence of the expression and localization of polarity and cell junction proteins during polarization in these epithelial cells. We found that CRB2 plays a key role in tight junction maintenance as well as in cell cycle arrest. In addition, our studies in vivo show that the knockdown of CRB2 in the RPE affects to the distribution of different apical polarity proteins and results in perturbed retinal homeostasis, manifested by the invasion of activated microglial cells into the subretinal space. Together our results demonstrate that CRB2 is a key protein for the development and maintenance of a polarized epithelium.

Hesperidin Prevents High Glucose-Induced Damage of Retinal Pigment Epithelial Cells

Planta Medica ◽  
2018 ◽  
Vol 84 (14) ◽  
pp. 1030-1037 ◽  
Author(s):  
Wayne Liu ◽  
Shorong-Shii Liou ◽  
Tang-Yao Hong ◽  
I-Min Liu
Keyword(s):  
High Glucose ◽  
Retinal Pigment Epithelial ◽  
Colorimetric Assay ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Antioxidant Enzyme Activities ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Rpe Cells ◽  
Pigment Epithelial

AbstractThe present study aimed to determine whether hesperidin, a plant-based active flavanone found in citrus fruits, can prevent high glucose-induced retinal pigment epithelial (RPE) cell impairment. Cultured human RPE cells (ARPE-19) were exposed to a normal glucose concentration (5.5 mM) for 4 d and then soaked in either normal (5.5 mM) or high (33.3 mM) concentrations of D-glucose with or without different concentrations of hesperidin (10, 20, or 40 µM) for another 48 h. The survival rates of the cells were measured using a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay. With the help of a fluorescent probe, the intracellular production of reactive oxygen species (ROS) was evaluated. Colorimetric assay kits were used to assess the antioxidant enzyme activities, and western blotting was used to measure the expression of apoptosis-related protein. Hesperidin was effective in inhibiting high glucose-induced ROS production, preventing loss of cell viability, and promoting the endogenous antioxidant defense components, including glutathione peroxidase, superoxide dismutase, catalase, and glutathione, in a concentration-dependent manner. Furthermore, high glucose triggered cell apoptosis via the upregulation of caspase-9/3, enhancement of cytochrome c release into the cytosol, and subsequent interruption of the Bax/Bcl-2 balance. These detrimental effects were ameliorated by hesperidin in a concentration-dependent manner. We conclude that through the scavenging of ROS and modulation of the mitochondria-mediated apoptotic pathway, hesperidin may protect RPE cells from high glucose-induced injury and thus may be a candidate in preventing the visual impairment caused by diabetic retinopathy.

Orientation of actin filaments in teleost retinal pigment epithelial cells, and the effect of the lectin, Concanavalin A, on melanosome motility

2014 ◽  
Vol 31 (1) ◽  
pp. 1-10 ◽  
Author(s):  
CHRISTINA KING-SMITH ◽  
RONALD J. VAGNOZZI ◽  
NICOLE E. FISCHER ◽  
PATRICK GANNON ◽  
SATYA GUNNAM
Keyword(s):  
Epithelial Cells ◽  
Actin Filament ◽  
Concanavalin A ◽  
Actin Filaments ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Pigment Epithelial Cells

AbstractRetinal pigment epithelial cells of teleosts contain numerous melanosomes (pigment granules) that exhibit light-dependent motility. In light, melanosomes disperse out of the retinal pigment epithelium (RPE) cell body (CB) into long apical projections that interdigitate with rod photoreceptors, thus shielding the photoreceptors from bleaching. In darkness, melanosomes aggregate through the apical projections back into the CB. Previous research has demonstrated that melanosome motility in the RPE CB requires microtubules, but in the RPE apical projections, actin filaments are necessary and sufficient for motility. We used myosin S1 labeling and platinum replica shadowing of dissociated RPE cells to determine actin filament polarity in apical projections. Actin filament bundles within RPE apical projections are uniformly oriented with barbed ends toward the distal tips. Treatment of RPE cells with the tetravalent lectin, Concanavalin A, which has been shown to suppress cortical actin flow by crosslinking of cell-surface proteins, inhibited melanosome aggregation and stimulated ectopic filopodia formation but did not block melanosome dispersion. The polarity orientation of F-actin in apical projections suggests that a barbed-end directed myosin motor could effect dispersion of melanosomes from the CB into apical projections. Inhibition of aggregation, but not dispersion, by ConA confirms that different actin-dependent mechanisms control these two processes and suggests that melanosome aggregation is sensitive to treatments previously shown to disrupt actin cortical flow.

Carrier-dependent and carrier-independent uptake of myo-inositol in cultured retinal pigment epithelial cells: activation by heat and concentration

1988 ◽  
Vol 66 (9) ◽  
pp. 942-950 ◽  
Author(s):  
Mahin Khatami
Keyword(s):  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Primary Cultures ◽  
Retinal Pigment Epithelial Cells ◽  
Transport Systems ◽  
Rate Equations ◽  
Rpe Cells ◽  
Major Mechanism ◽  
Pigment Epithelial ◽  
Pigment Epithelial Cells

Transport of myo-inositol (MI) was studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. At low external concentrations (0.01–1 mM), uptake appeared to follow saturation kinetics, although the reciprocal forms of the rate equations did not fit either Lineweaver–Burk or Eadie–Hofstee plots. Increasing external concentrations dramatically changed the pattern of MI entry. At two to three orders of magnitude higher than physiological concentrations, a second saturation occurred (pseudo saturation). Cells incubated with 20 μM [3H]MI for 60 min had a ratio of intracellular to extracellular radioactivity ≥ 8, indicating active transport.MI transport reduction by Na+ replacement or inhibitors (phlorizin, ouabain, amiloride, KSCN, iodoacetamide, MI analogues) was greater when RPE cells were incubated with low (20–400 μM) than with high (10–20 mM) MI concentrations. Cells incubated with 20 μM MI at 53 or 65 °C showed increased transport (up to 560%) compared with cells at 22 °C. The effect on MI uptake (20 μM) of Na+ replacement also was reduced at 53 °C. The uptake of MI involved at least two transport systems. The major mechanism at low external MI concentrations (physiological levels) was a carrier-mediated active process. At high external MI levels, uptake occurred by a diffusion process. A lipotropic effect of MI may be responsible for this increased rate of diffusion.

Isolated bovine retinal pigment epithelial cells express delayed rectifier type and M-type K+ currents

1997 ◽  
Vol 273 (3) ◽  
pp. C790-C803 ◽  
Author(s):  
M. Takahira ◽  
B. A. Hughes
Keyword(s):  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Type K ◽  
K Current ◽  
K Currents

Outwardly rectifying K+ currents in freshly isolated bovine retinal pigment epithelial (RPE) cells were characterized using the whole cell and perforated-patch configurations of the patch-clamp technique. All cells exhibited a delayed rectifier type K+ current. This current had an activation threshold voltage of approximately -40 mV, activated with a sigmoidal trajectory, and inactivated completely over a period of several seconds. External tetraethylammonium (TEA) was an effective blocker of the delayed rectifier current [apparent dissociation constant (Kd) = 5.1 mM], but external Ba2+ was relatively ineffective. Approximately 24% of the cells also exhibited a sustained outwardly rectifying K+ current that became activated at voltages positive to approximately -80 mV. This current resembled the neuronal M-current. External Ba2+ was a potent blocker of this current (apparent Kd = 1.1 mM), but external TEA and Cs+ were relatively ineffective. These results indicate that freshly isolated bovine RPE cells express K+ currents of both the delayed rectifier and M types. The latter may contribute to the resting K+ conductances of the apical and basolateral membranes.

scholarly journals Immunochemical analysis of the integrin expression in retinal pigment epithelial cells

2020 ◽  
Author(s):  
Janosch P Heller ◽  
Tristan G Heintz ◽  
Jessica CF Kwok ◽  
Keith R Martin ◽  
James W Fawcett
Keyword(s):  
Retinal Pigment Epithelial ◽  
Expression Profiles ◽  
Retinal Pigment ◽  
Cell Types ◽  
Human Cell Line ◽  
Retinal Pigment Epithelial Cells ◽  
Proper Function ◽  
Integrin Expression ◽  
Rpe Cells ◽  
Pigment Epithelial

AbstractRetinal pigment epithelial (RPE) cells have been used in disease modelling and transplantation studies in retinal diseases. Several types of RPE cells have been trialed, ranging from primary cells and immortalized cell lines to stem cell-derived RPE cells. During aging and in disease, the extracellular environment of the RPE cells changes, interfering with RPE cell adhesion. We hypothesize that this could be a key problem in transplantation studies that have reported lack of adhesion and survival of the transplanted RPE cells. Integrins are essential for the proper function of the RPE, mediating adhesion to Bruch’s membrane, and the binding and subsequent phagocytosis of photoreceptor outer segments. Variability that has been found in clinical trials might be due to the variability of cell types used and their expression profiles of surface molecules. Here, we set out to analyze integrin expression in primary rat RPE cells and in the human cell line ARPE-19 using immunochemistry. We found that both cell types express integrins to varying degrees. After long-term culturing, ARPE-19 cells resemble mature RPE cells, and increase integrin expression. We hence argue that it is important to test the properties of these cells prior to transplantation to avoid failure of adhesion and to facilitate correct function.

scholarly journals Deletion of TSPO Resulted in Change of Metabolomic Profile in Retinal Pigment Epithelial Cells

2019 ◽  
Vol 20 (6) ◽  
pp. 1387 ◽  
Author(s):  
Abdulwahab Alamri ◽  
Lincoln Biswas ◽  
David Watson ◽  
Xinhua Shu
Keyword(s):  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Nucleotide Metabolism ◽  
Retinal Pigment Epithelial Cells ◽  
Age Related Macular Degeneration ◽  
Clinical Feature ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Age Related ◽  
The Impact

Age-related macular degeneration is the main cause of vision loss in the aged population worldwide. Drusen, extracellular lesions formed underneath the retinal pigment epithelial (RPE) cells, are a clinical feature of AMD and associated with AMD progression. RPE cells support photoreceptor function by providing nutrition, phagocytosing outer segments and removing metabolic waste. Dysfunction and death of RPE cells are early features of AMD. The translocator protein, TSPO, plays an important role in RPE cholesterol efflux and loss of TSPO results in increased intracellular lipid accumulation and reactive oxygen species (ROS) production. This study aimed to investigate the impact of TSPO knockout on RPE cellular metabolism by identifying the metabolic differences between wildtype and knockout RPE cells, with or without treatment with oxidized low density lipoprotein (oxLDL). Using liquid chromatography mass spectrometry (LC/MS), we differentiated several metabolic pathways among wildtype and knockout cells. Lipids amongst other intracellular metabolites were the most influenced by loss of TSPO and/or oxLDL treatment. Glucose, amino acid and nucleotide metabolism was also affected. TSPO deletion led to up-regulation of fatty acids and glycerophospholipids, which in turn possibly affected the cell membrane fluidity and stability. Higher levels of glutathione disulphide (GSSG) were found in TSPO knockout RPE cells, suggesting TSPO regulates mitochondrial-mediated oxidative stress. These data provide biochemical insights into TSPO-associated function in RPE cells and may shed light on disease mechanisms in AMD.

scholarly journals Depletion of Mitochondrial DNA in Differentiated Retinal Pigment Epithelial Cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Xinqian Hu ◽  
Melissa A. Calton ◽  
Shibo Tang ◽  
Douglas Vollrath
Keyword(s):  
Mitochondrial Dna ◽  
Retinal Pigment Epithelial ◽  
Aerobic Glycolysis ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Response To Treatment ◽  
Flux Analysis ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Mitochondrial Dna Depletion

Abstract We investigated the effects of treating differentiated retinal pigment epithelial (RPE) cells with didanosine (ddI), which is associated with retinopathy in individuals with HIV/AIDS. We hypothesized that such treatment would cause depletion of mitochondrial DNA and provide insight into the consequences of degradation of RPE mitochondrial function in aging and disease. Treatment of differentiated ARPE-19 or human primary RPE cells with 200 µM ddI for 6–24 days was not cytotoxic but caused up to 60% depletion of mitochondrial DNA, and a similar reduction in mitochondrial membrane potential and NDUFA9 protein abundance. Mitochondrial DNA-depleted RPE cells demonstrated enhanced aerobic glycolysis by extracellular flux analysis, increased AMP kinase activation, reduced mTOR activity, and increased resistance to cell death in response to treatment with the oxidant, sodium iodate. We conclude that ddI-mediated mitochondrial DNA depletion promotes a glycolytic shift in differentiated RPE cells and enhances resistance to oxidative damage. Our use of ddI treatment to induce progressive depletion of mitochondrial DNA in differentiated human RPE cells should be widely applicable for other studies aimed at understanding RPE mitochondrial dysfunction in aging and disease.

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