Understanding photoreceptor outer segment phagocytosis: Use and utility of RPE cells in culture

Mazzoni, Francesca; Safa, Hussein; Finnemann, Silvia C.

doi:10.1016/j.exer.2014.01.010

Cited by 178 publications

(189 citation statements)

References 86 publications

(152 reference statements)

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“…Lastly, we showed that most (if not all) of the phagocytosis observed was mediated through the aVb5 receptor, which is a key player involved in the initial specific recognition of ROS in RPE cells. 34,35 Additional studies are needed to determine whether primary RPE or RPE derived from either human embryonic stem cells or induced pluripotent stem cells (iPSC) would behave similarly on spontaneously aged or nitrite-modified ECM surfaces. Ongoing experiments in our laboratory are aimed at understanding the effects of BM-aging on iPSC-derived RPE cells derived from AMD patients and unaffected controls.…”

Section: Discussionmentioning

confidence: 99%

“…In vivo, approximately 3% to 5% of the distal tips of photoreceptor outer segments are shed on a daily basis and proper RPE phagocytosis is necessary to maintain the health and integrity of the neural retina and the choriocapillaris. 33,34 RPE phagocytosis uses aVb5 integrin receptors on the RPE surface for initial outer segment cell surface binding, the receptor MerTK for internalization, and the scavenger receptor CD36. 31,35,36 Disruption of any step in this process can lead to pathology.…”

Section: Discussionmentioning

confidence: 99%

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Reengineering Human Bruch's Membrane Increases Rod Outer Segment Phagocytosis by Human Retinal Pigment Epithelium

Moreira

Cai

Tezel

et al. 2015

Trans. Vis. Sci. Tech.

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Purpose: We have shown previously that Bruch's membrane (BM) aging decreases retinal pigment epithelium (RPE) phagocytosis. Herein, we determine the effects of BM reengineering on RPE phagocytosis.Methods: BM explants were dissected from young and old donor eyes. Some old BM explants were reengineered by cleaning with Triton X-100 and/or coating with extracellular matrix (ECM) ligands. ARPE-19 cell-derived ECM (ARPE-ECM) modified (''aged'') by sodium nitrite was subjected to similar treatments. ARPE-19 cells were then cultured to confluence onto the different surfaces. Fluorescently-labeled bovine rod outer segments (ROS) were fed to cells with or without aVb5 integrin antibody. Image acquisition and phagocytosis quantification was performed by fluorescence microscopy and ImageJ analysis.Results: Cleaning old donor-derived BM with detergent does not increase the uptake of ROS, but a combination of cleaning and coating with ECM ligands significantly increases RPE phagocytosis (54.9 6 6.2 vs. 83.5 6 6.5 arbitrary units; P , 0.05) to levels closer to young donor BM (123.6 6 9.9 arbitrary units). Similar effects were observed on nitrite-modified ARPE-ECM subjected to the same treatments. Incubation of aVb5 blocking antibody with ROS significantly decreased RPE phagocytosis. Conclusions:The detrimental effects of aging BM on RPE phagocytosis can be reversed by reengineering the BM surface with detergent cleaning and recoating with ECM ligands.Translation Relevance: These results demonstrate that the therapeutic success of transplanted RPE cells may require, at least in part, reengineering of diseased BM to make it a more suitable environment for attachment, survival and proper functioning of the RPE.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Reengineering Human Bruch's Membrane Increases Rod Outer Segment Phagocytosis by Human Retinal Pigment Epithelium

Moreira

Cai

Tezel

et al. 2015

Trans. Vis. Sci. Tech.

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“…Photoreceptors (PRs) and the retinal pigment epithelium (RPE) are commonly considered the most important targets of stem cell therapies [1][2][3]. PRs and RPE interact functionally and structurally [2][3][4][5][6], appear to polarize each other [7], and are reported to show improved engraftment when transplanted together [8,9]. After engraftment, stem cell-derived PRs must survive and form functional synaptic connections.…”

mentioning

confidence: 99%

Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures

Singh

Mallela

Cornuet

et al. 2015

Stem Cells and Development

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Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltagegated Na + and K + currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.

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“…The different phases of uptake can also distinguished in vitro by using appropriate experimental procedures 39 . Pulse-chase experiments can be performed using temperature switch: POS binding occurs at 20 °C while internalization needs a temperature of 37 °C to proceed 32,40,7 .…”

Section: Representative Resultsmentioning

confidence: 99%

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Parinot

Rieu

Chatagnon

et al. 2014

JoVE

Self Cite

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Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

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Understanding photoreceptor outer segment phagocytosis: Use and utility of RPE cells in culture

Cited by 178 publications

References 86 publications

Reengineering Human Bruch's Membrane Increases Rod Outer Segment Phagocytosis by Human Retinal Pigment Epithelium

Reengineering Human Bruch's Membrane Increases Rod Outer Segment Phagocytosis by Human Retinal Pigment Epithelium

Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

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