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Karolina Ploessl, Annie Lauer, Christina Kiel, Caroline Brandl, Felix Grassmann, Bernhard HF Weber; An iPSC-derived RPE cell repository with high and low genetic AMD risk as model systems to study AMD pathology in vitro. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4916.
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© ARVO (1962-2015); The Authors (2016-present)
Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries. Currently, the molecular processes underlying AMD pathogenesis are not yet understood, although environmental and genetic factors have been recognized as key determinants to a person’s individual risk to develop the disease. To gain further insights into the molecular pathology of AMD, we generated an iPSC-derived RPE cell repository with cell lines derived from well defined genetic AMD backgrounds. Since oxidative stress plays an important role in AMD pathogenesis, we sought to initially determine the effect of oxidative stress induced by Paraquat (PQ) treatment on the different cell lines.
AMD patients and controls were genotyped for 13 AMD-associated SNPs at 8 different loci known to be highly correlated with AMD risk. Genetic risk scores for AMD were calculated using the model published by Grassmann et al., 2012. Fibroblast or PBMC cultures were established from patients with very low (category 1) and very high (category 5) AMD risk scores, reprogrammed to iPSCs and differentiated into RPE cells according to Brandl et al., 2014. RPE cell properties were assessed by TEER measurement and immunocytochemistry. Oxidative stress was induced by treatment with PQ and cellular responses were measured by MTT assay, qRT-PCR, and ELISA.
Four RPE cell lines corresponding to AMD risk score category 1, and four cell lines corresponding to category 5 have successfully been established and characterized. All cell lines showed high TEER values above 800 Ω*cm2 indicating a tight monolayer structure. Staining patterns for ZO1 as a tight junction marker and BEST1 as a RPE specific ion channel were distinct in all cell lines. Oxidative stress induced with 1 mM PQ for 24h significantly decreased cell viability of all cell lines whereas VEGF mRNA expression was upregulated as shown by qRT PCR. Secreted VEGF protein levels in basal supernatants of PQ treated cells were also significantly elevated.
Having established RPE lines with known genetic AMD background and a reproducible protocol to induce oxidative stress in these cells, we now aim to gain deeper insights into molecular processes responsible for regulation of VEGF expression following oxidative stress and to determine the influence of the genetic AMD risk on molecular disease mechanisms.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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