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Jie Gong, Mark Anthony Fields, Ernesto F Moreira, Yiannis Koutalos, Zsolt Ablonczy, Lucian V Del Priore; Functional Analysis of Human Protein Induced Pluripotent Stem Cell-derived Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3997. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may provide a promising source of cells for transplantation. Physiological tests are required to establish the ability of iPS to function as RPE prior to using these cells as a source of replacement RPE. Critical RPE functions include phagocytosis of shed photoreceptor rod outer segments (ROS) and the barrier function of the tight junction. Herein we analyze the function of these ips-derived RPE as measured by phagocytosis and TER (trans epithelial resistance).
Protein-induced iPS (piPS) from System Biosciences (SBI, Mountain View, CA) were seeded directly onto Matrigel coated plates in differentiation medium. From day 0-6, a combination of Noggin, DKK1, IGF-1 and bFGF were added to the base medium. From day 6-14, Activin A, SU5402 and VIP were added. Subsequently, purified piPS-derived RPE were digested and expanded in RPE medium. Adult bovine rod outer segments were labeled with FITC, fed to the PiPS-RPE cells and analyzed by fluorescent microscopy and flow cytometry. These RPE monolayers were cultured on permeable transwell membranes were measured TER by using an epithelial voltohmmeter.
Undifferentiated piPS cells expressed all pluripotent embryonic cell markers SSEA4, TRA-1-60, OCT4, and TRA-1-81, and no RPE markers were detected. After 30 days, cells formed a hexagonal monolayer exhibiting an RPE phenotype and pigmentation. Monolayer cell domes were observed, suggesting apical-basal fluid transportation. Differentiated piPS-RPE cells expressed mature RPE markers Bestrophin, MITF and RPE65, and the tight junction marker ZO-1. Transepithelial resistance was tested at an average of 154.8 + 0.23 Ω*cm2. Phagocytosis function test showed that piPSC-RPE also ingested fluorescently-labeled isolated bovine rod outer segments. Flow cytometry of piPSC-RPE revealed 90% of cells contained fluorescently labeled outer segments.
PiPS-RPE exhibited similar physiological properties to those of adult RPE cells. Additional studies are required to analyze RPE cell function further, including retinoids uptake and processing; and analyzing cell survival and function in animal models of retinal degeneration.
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