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Stacey K. Moreno, Peter D. Westenskow, Tim U. Krohne, Mandy Lehmann, Alison L. Dorsey, Saiyong Zhu, Sheng Ding, Martin Friedlander; Characterization Of Phagocytic Activity In OCT4-only Derived iPS-RPE Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):859.
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Retinal pigment epithelial (RPE) cells derived from induced pluripotent (iPS) cells have the potential to be used as treatment for diseases in which the native RPE are degenerated or defective such as age-related macular degeneration (AMD). In order for iPS-RPE to function as normal RPE they must have the ability to phagocytose photoreceptor outer segments (POS) in the same manner as normal RPE cells. We have developed a flow cytometry based assay to characterize phagocytosis in iPS-RPE.
We generated iPS using retroviral delivered OCT4 and small molecules and derived RPE using directed differentiation techniques. The iPS-RPE cells strongly resemble fetal hRPE as confirmed using multiple methods. The iPS-RPE are treated with fluorescently labeled porcine outer segments over different time-points and are analyzed by flow cytometry to determine rate and efficiency of binding and internalization. Trypsin is used to release the cells from the surface and to cleave bound outer segments in order to distinguish between outer segments that are bound to the cell surface and those that are internalized. To determine receptor expression patterns during and after exposure to POS cells are labeled with antibodies to cell surface receptors thought to be involved in POS phagocytosis (such as integrin alpha v beta 5).
iPS-RPE cells phagocytose POS with slightly slower rates of binding and internalization as compared to ARPE-19 cells. Cell surface receptors involved in phagocytosis are expressed on both iPS-RPE and ARPE-19 before exposure to POS. After treating with outer segments both cell types decrease expression of integrin alpha v beta 5 on their surface and increase intracellular expression of the integrin. Other phagocytosis receptors are currently being studied.
OCT4-only derived iPS-RPE express cell surface receptors known to be involved in photoreceptor phagocytosis and have the ability to phagocytose POS. Although slightly less effective as compared to cultured ARPE-19 cells iPS-RPE cells are a promising replacement for damaged RPE cells in the diseased retina.
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