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Ernesto F Moreira, Jie Gong, Sibylle Rosendahl, Amanda Barrett, Mark Anthony Fields, Zsolt Ablonczy, Baerbel Rohrer, Lucian V Del Priore; Altered Transepithelial Resistance of Induced Pluripotent Stem Cell-derived Retinal Pigment Epithelium Obtained from Age-related Macular Degeneration (AMD) Patients. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6034. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously reported the generation of induced pluripotent stem cells (iPSC) derived from fibroblasts of patients with advanced macular degeneration, as well as the differentiation of iPSCs into retinal pigment epithelium (RPE). Here, we report the transepithelial resistance (TER) analysis of iPSC-derived RPE cells from AMD patients and unaffected controls.
Fibroblasts were grown and expanded from punch biopsies obtained from patients with advanced (dry or wet) AMD and unaffected controls. Sendai virus technology (ThermoFisher Scientific) was used to deliver Yamanaka factors to reprogram fibroblasts into iPSCs. Subsequently, iPSCs were differentiated into RPE using our recently published protocol (Gong et al, 2015). Pure iPSC-RPE cells, grown in transwell plates for 1-2 months, were characterized morphologically (honeycomb monolayer appearance and pigmentation) and by immunocytochemistry for RPE specific molecules (i.e., OTX2, MITF, Bestrophin). RPE trans-epithelial resistance (TER) was assessed with a Voltohmmeter averaging three measurements in each (duplicate) transwell.
iPSC-RPE cell lines derived from AMD patients (N=4) and controls (N=2) formed characteristic monolayers showing typical honeycomb organization and pigmentation. These cells also expressed specific RPE cell markers important for their differentiation and function, including MITF, RPE65. Baseline TER analysis showed that dry AMD samples had a significantly reduced TER (29 +/-10 (#1) and 37 +/-16 (#2) and 117 +/-21 (#3)) as compared to the wet AMD and controls (411 +/-16 (wet AMD); 629 +/-26 (control #1); 330 +/- 48 (control #2)).
These studies suggest that intrinsic differences in RPE cells between AMD patients and unaffected controls may exist in the ability to make tight junctions necessary for RPE barrier function. In addition, these results further indicate the potential use of these cells to investigate features of AMD based on the genotype and/or phenotype of the patients. Ongoing experiments are aimed at analyzing complement activation and VEGF levels in the supernatant of iPSC-RPE before and after challenging them with specific AMD-relevant stressors, in addition to determining expression of tight-junction markers for these 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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