A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells

Sonoda, Shozo; Spee, Christine; Barrón, Ernesto; Ryan, Stephen J.; Kannan, Ram; Hinton, David R.

doi:10.1038/nprot.2009.33

Cited by 237 publications

(279 citation statements)

References 22 publications

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“…23 Physiological RPE cells are mitotically quiescent, with cell-cell contact inhibition mediated by the homotypic adhesion of cadherins on adjacent cells. 52 In the late phase of AMD, RPE dissociation can occur because of RPE and retinal detachment subsequent to CNV formation.…”

Section: Discussionmentioning

confidence: 99%

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αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition

Ishikawa

Sreekumar

Spee

et al. 2016

The American Journal of Pathology

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Subretinal fibrosis is an end stage of neovascular age-related macular degeneration, characterized by fibrous membrane formation after choroidal neovascularization. An initial step of the pathogenesis is an epithelial-mesenchymal transition (EMT) of retinal pigment epithelium cells. aB-crystallin plays multiple roles in age-related macular degeneration, including cytoprotection and angiogenesis. However, the role of aB-crystallin in subretinal EMT and fibrosis is unknown. Herein, we showed attenuation of subretinal fibrosis after regression of laser-induced choroidal neovascularization and a decrease in mesenchymal retinal pigment epithelium cells in aB-crystallin knockout mice compared with wild-type mice. aB-crystallin was prominently expressed in subretinal fibrotic lesions in mice. In vitro, overexpression of aB-crystallin induced EMT, whereas suppression of aB-crystallin induced a mesenchymal-epithelial transition. Transforming growth factor-b2einduced EMT was further enhanced by overexpression of aB-crystallin but was inhibited by suppression of aB-crystallin. Silencing of aB-crystallin inhibited multiple fibrotic processes, including cell proliferation, migration, and fibronectin production. Bone morphogenetic protein 4 upregulated aB-crystallin, and its EMT induction was inhibited by knockdown of aB-crystallin. Furthermore, inhibition of aB-crystallin enhanced monotetraubiquitination of SMAD4, which can impair its nuclear localization. Overexpression of aB-crystallin enhanced nuclear translocation and accumulation of SMAD4 and SMAD5. Thus, aB-crystallin is an important regulator of EMT, acting as a molecular chaperone for SMAD4 and as its potential therapeutic target for preventing subretinal fibrosis development in neovascular age-related macular degeneration. (Am J Pathol 2016, 186: 859e873; http:// dx

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Section: Discussionmentioning

confidence: 99%

“…Primary cultures of RPE cells were established, as described previously. 23 The experiments used cultured RPE cells at two to four passages with normal morphology.…”

Section: Rpe Cell Culturementioning

confidence: 99%

αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition

Ishikawa

Sreekumar

Spee

et al. 2016

The American Journal of Pathology

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“…The TER assays were performed with cells cultured in Transwell® insert chambers and using an epithelial voltohmmeter (EVOM) according to the protocol described by Sonoda et al [4]. Briefly, measurements with the electrodes of the EVOM were made at RT after …”

Section: Transepithelial Resistance (Ter)mentioning

confidence: 99%

“…This monolayer is maintained and stabilized by the cytoskeleton of individual cells and their interactions at the basolateral junctional complexes [2][3][4]. These junctional complexes or tight junctions are composed of zonular occludens-1 (ZO-1), occludin, claudins 1, 2, 5, 12 and AL [1].…”

Section: Introductionmentioning

confidence: 99%

“…Finally, the expression of specific RPE markers and the development of cell polarity exhibiting transepithelial resistance and polygonal shaped-cells with microvilli, which are indicators of an adequate RPE cell morphology, were evaluated. The markers studied were the junctional ZO-1 protein and the retinal pigment epithelium-65 (RPE65), which is a RPE specific protein involved in the process of the visual cycle of retinal [1,4]. To the authors knowledge this is the first time such a specific study is conducted using BC as a substrate for RPE cells adhesion and proliferation.…”

Section: Introductionmentioning

confidence: 99%

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Acetylated bacterial cellulose coated with urinary bladder matrix as a substrate for retinal pigment epithelium

Gonçalves

Rodrigues

Padrão

et al. 2016

Colloids and Surfaces B: Biointerfaces

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This work evaluated the effect of acetylated bacterial cellulose (ABC) substrates coated with urinary bladder matrix (UBM) on the behavior of retinal pigment epithelium (RPE), as assessed by cell adhesion, proliferation and development of cell polarity exhibiting transepithelial resistance and polygonal shaped-cells with microvilli. Acetylation of bacterial cellulose (BC) generated a moderate hydrophobic surface (around 65°) while the adsorption of UBM onto these acetylated substrates did not affect significantly the surface hydrophobicity. The ABS substrates coated with UBM enabled the development of a cell phenotype closer to that of native RPE cells. These cells were able to express proteins essential for their cytoskeletal organization and metabolic function (ZO-1 and RPE65), while showing a polygonal shaped morphology with microvilli and a monolayer configuration. The coated ABC substrates were also characterized, exhibiting low swelling effect (between 1.5-2.0 swelling/mm3), high mechanical strength (2048MPa) and non-pyrogenicity (2.12EU/L). Therefore, the ABC substrates coated with UBM exhibit interesting features as potential cell carriers in RPE transplantation that ought to be further explored. Therefore, the ABC substrates coated with UBM showed a great potential to be applied as cell carriers in RPE transplantation.

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Replication ofMycobacterium Tuberculosisin Retinal Pigment Epithelium

2014

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uberculosis (TB) remains a global health problem, with almost one-third of the world's population being infected. 1 The disease presents primarily as pulmonary TB and, in approximately 20% of patients, as extrapulmonary disease. After Mycobacterium tuberculosis has been inhaled, the organisms undergo phagocytosis by alveolar macrophages, resulting in bacterial containment. 2 In some patients, the bacilli may disseminate systemically, establishing latent infection at extrapulmonary sites, with the potential to reactivate at a later time. 2 Reactivation of latent TB usually occurs when the host's immune system is compromised. 3,4 Extrapulmonary TB is believed to result from reactivation of latent mycobacteria residing within resident reticuloendothelial cells. Ocular TB is a form of extrapulmonary TB manifesting primarily as posterior uveitis. 5 However, the location of latent TB organisms in the eye is not clear. In a study of TB-related posterior uveitis, Rao et al 6 proposed that M tuberculosis may reside in the retinal pigment epithelium (RPE). However, the mechanisms underlying phagocytosis and growth containment of the bacilli by RPE remain unknown. The normal clinical appearance of RPE in individuals before reactivation of M tuberculosis suggests that latent M tuberculosis infection does not induce necrosis or apoptosis. IMPORTANCE Mycobacterium tuberculosis is an important cause of posterior uveitis in tuberculosis-endemic regions. Clinical and histopathologic evidence suggests that retinal pigment epithelium (RPE) can harbor M tuberculosis. However, the mechanism of M tuberculosis phagocytosis and its growth in RPE is not clear.OBJECTIVE To investigate M tuberculosis phagocytosis, replication, and cytopathic effects in RPE cells compared with macrophages. DESIGN, SETTING, AND PARTICIPANTSHuman fetal RPE and monocytic leukemia macrophage (THP-1) cell lines were cultured, and RPE and THP-1 cells were exposed to avirulent M tuberculosis H37Ra. Mycobacteria were added to RPE and THP-1 cells with a 5:1 multiplicity of infection. Nonphagocytized M tuberculosis was removed after 12 hours of exposure (day 0). Cells were harvested at days 0, 1, and 5 to count live and dead cells and intracellular mycobacteria. Toll-like receptor 2 (TLR2) and TLR4 expression was determined by immunohistochemistry; intracellular bacillary load, following TLR2 and TLR4 blockade. MAIN OUTCOMES AND MEASURES Number of intracellular M tuberculosis, cell survival, and TLR2 and TLR4 expression in RPE and THP-1 cells following exposure to M tuberculosis. RESULTS At day 0, an equal number of intracellular M tuberculosis was observed per THP-1 and RPE cells (0.45 and 0.35 M tuberculosis per RPE and THP-1 cells, respectively). Mean (SD)number of intracellular M tuberculosis at day 5 was 1.9 (0.03) and 3.3 (0.01) per RPE and THP-1 cells, respectively (P < .001). Viability of infected RPE was significantly greater than that of THP-1 cells at day 5 (viable cells: 17 [8%] THP-1 vs 73% [4%] RPE; P < .05). Expression of TLR2 and TLR4 was detected in ...

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A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells

Cited by 237 publications

References 22 publications

αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition

αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition

Acetylated bacterial cellulose coated with urinary bladder matrix as a substrate for retinal pigment epithelium

Replication ofMycobacterium Tuberculosisin Retinal Pigment Epithelium

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