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Justin R Chang, Balendu Shekhar Jha, Madhu Lal, Ruchi Sharma, Marc Ferrer, Kapil Bharti; Discovering Novel Genes and Pathways Involved in Maintenance of Epithelial Phenotype in Retinal Pigment Epithelium Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):238. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Proliferative vitreoretinopathy (PVR) causes retinal pigment epithelium (RPE) dedifferentiation where RPE loses its epithelial characteristics. The goal of this study is to perform a series of high throughput screening assays and bioinformatics analyses to identify novel genes and pathways that are involved in maintenance of RPE epithelial phenotype to facilitate vision recovery after PVR surgery.
RPE cells derived from a reporter line of human induced pluripotent stem cells (iPSCs) expressing RPE-specific GFP and constitutive housekeeping RFP were optimized to be cultured in 384-well plates. A library of 875 miRNA mimics was tested, and top 5% GFP up- and down-regulators (GFP, RFP signals measured by MetaXpress) were identified. Multiple miRNA databases were used to analyze these selected mimics for their target genes. Targets that had >90% scores and common occurrences between multiple databases per miRNA were chosen for subsequent siRNA screening. siRNA screening results and Ingenuity Pathway Analysis (IPA) software were used to compile a list of genes for further assays.
The miRNA assay had a high reproducibility with correlation coefficients of 0.87, 0.94, 0.91 in GFP, RFP, Hoechst signals, respectively, between two identical screenings. The top 5% of miRNA mimics (21 mimics) that decreased GFP by 50% compared to control and the top 5% miRNA mimics (10 mimics) that increased GFP by 250% compared to control were selected for target analysis. miRNA databases and IPA predicted novel genes and pathways that are involved in regulating the RPE epithelial phenotype. The results were confirmed by a subsequent siRNA screening comprising of 122 siRNAs. Small molecule inhibitors and activators of these pathways are currently being tested to modulate in vitro RPE phenotype.
HTS on iPSC-RPE identified novel genes and pathways involved in RPE epithelial phenotype maintenance, and potential therapeutic targets for RPE-associated conditions like PVR. Additionally, these pathways can provide potential targets to enhance the protocol of RPE differentiation from pluripotent stem cells.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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