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FNU RUCHI, Barbara Corneo, Janine Davis, Becky King, Qin Wan, Kiyoharu Miyagishima, Sally Temple, Sheldon Miller, Marc Ferrer, Kapil Bharti; A Multiplex High Content High Throughput Screening Platform Using iPS Cells Derived RPE. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2230.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) derived from pluripotent stem cells has provided hope for a cell-based therapy for degenerative eye diseases. But, the currently available RPE cells are not fully differentiated and continue to express fetal RPE markers. The aim of this study is to develop a multiplex high through put screening platform to identify compounds that can improve RPE maturation when differentiated from stem cells.
Induced pluripotent stem cells (iPSC) derived RPE monolayers were authenticated using molecular, imaging, and physiological assays. Cells were grown in 96-well and 384-well plates at two differentiation stages. Fetal human RPE was used as a positive control and iPS cells were used as a negative control. The endogenous expression of eight different genes was simultaneously quantified using the multiplex QuantiGene Plex technology.
iPSC- derived RPE monolayers showed characteristic RPE features like pigmentation, polygonal morphology, apical processes, and the expression of several RPE signature genes. The monolayers showed physiological and electrophysiological responses similar to the primary human RPE: (1) a transepithelial resistance (RT) of > 150 ohms per cm2; (2) a resting membrane potential (VA and VB) close to -55mV resulting in a transepithelial potential (TEP) of ≈2.5 mV; (3) ability to depolarize in response to a reduced apical K conc.; (4) changes in cytoplasmic calcium levels in response to an apical ATP stimulus. These cells maintained their characteristic RPE morphology in 96-well and 384-well plates. Simultaneous quantification of eight genes using the QuantiGene Plex technology confirmed the fetal like state of these RPE cells. The expression of fetal specific genes SOX2 and PAX6 was higher and the expression of mature RPE specific genes TYROSINASE, RPE65, CSPG5, BEST1, and RDH5 was lower as compared to primary fetal RPE.
Expression, imaging, and physiological analyses confirm the RPE nature of iPSC- derived RPE-like cells. These cells maintain their RPE characteristics when grown in 96-well and 384-well plates. QuantiGene Plex successfully measures the endogenous expression of eight genes in a single well of a 96-well or a 384-well plate. This high content assay will be used to screen for compounds that will improve the iPSC to RPE maturation process.
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