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Jie Gong, Huey Cai, Lucian V Del Priore, Mark Anthony Fields; Increased Reactive Oxygen Species (ROS) production in Patient-specific iPSC-derived RPE from AMD patients. Invest. Ophthalmol. Vis. Sci. 2021;62(8):255.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress plays an important role in RPE cell injury and is also a risk factor of AMD. Tert-butyl hydrogen peroxide (TBHP) is an agent used to induce oxidative stress in RPE and serves as a reproducible model that mimics key aspects of AMD pathology. We have previously demonstrated our ability to derive iPSC-derived RPE from AMD patients and age-matched controls. These cells perform critical functions such as phagocytosis of photoreceptor outer segments, the ability to form tight junctions, and retinol metabolism. A decrease in metabolic capacity was also observed in iPSC derived RPE from AMD patients. Here we seek to detect the cellular ROS production of iPSC-derived RPE from AMD patients and age-matched controls.
IPSCs were generated from AMD (n=3) and age-matched patients with no history of AMD (n = 2) using mRNA. IPSCs were differentiated into RPE using an established protocol. RPE lines were verified by morphology, immunohistochemistry and confocal microscopy. Cells were labeled with 20µM 2’, 7’ –dichlorofluorescein diacetate (DCFDA), and then cultured for another 2 hours with or without 150 µM TBHP, and then quantified using a BioTek FLx800 plate reader.
Human iPSC-derived RPE expressed specific RPE cell markers RPE65, CRALBP, and ZO-1. In the presence of TBHP, both groups of iPSC-derived RPE cells expressed higher level of ROS. In normal control group, ROS were 42.10% (p<0.001) higher in TBHP than untreated group. In AMD group, ROS were 23.70% (p<0.001) higher in TBHP than untreated group. A comparison of iPSC- derived RPE cells with PBS treatment from normal controls (n = 2 ) vs. AMD patients (n = 3) revealed no significant difference in ROS production in all 5 lines. However, iPSC-derived RPE from normal controls with TBHP treatment revealed higher ROS production 18.47% (p<0.05) vs AMD patients with TBHP treatment.
In this study, we generated RPE from AMD patients and individuals with no history of AMD. Higher levels of ROS production was observed in iPSC-derived RPE from both AMD patients and normal controls. However, no differences were observed when comparing TBHP-treated AMD and control lines. Further, exploration of various inflammation related growth factors and metabolic pathways among normal and AMD-derived RPE should reveal mechanisms of disease-related phenotypes and pave the way for novel therapeutic strategies.
This is a 2021 ARVO Annual Meeting abstract.
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