Optimization of iPSC-RPE oxidative stress model to be used as screening tool for novel AMD drug discovery

Abstract

Purpose : The purpose of this study was to optimize a method of oxidative stress induction in inducible pluripotent stem cell-derived retinal pigment epithelial cells (iPSC-RPE) and test the cytoprotective effect of the antioxidant, Mn(III)TMPyP. This model mimics early AMD changes in the RPE and will serve as a screening tool to identify lead candidates to treat AMD.

Methods : iPSC-RPE (Phenocell, France) were exposed to either tert-butyl hydroperoxide (tBHP; 0.2 – 1.2 mM) or sodium iodate (NaIO₃; 4 – 20 mM) for 24h. The tBHP experiments were performed on cells 17, 21, and 25 days post start of differentiation (DIV17, DIV21, DIV25), to evaluate if susceptibility to oxidative stress changes throughout differentiation. The NaIO₃ experiments were performed on DIV22 and cells were left untreated as control or treated with antioxidant (0.005% w/v of Mn(III)TMPyP) 24h prior to NaIO₃ exposure. Resazurin and lactate dehydrogenase release assays for cell viability were performed immediately afterwards. Data was analyzed as 4 parameter logistic curves, and half-maximal inhibitory concentration (IC₅₀) for resazurin assay and half-maximal effective concentration (EC₅₀) for LDH assay were calculated in Prism 10 (GraphPad, La Jolla, CA).

Results : For the tBHP induced experiments, mean IC₅₀ of tBHP was 0.75 (DIV17), 0.59 (DIV21), and 0.69 (DIV25) mM; mean EC₅₀ was 0.66 (DIV17), 0.55 (DIV21), and 0.62 (DIV25) mM. The difference between DIV17 and DIV21 IC₅₀ was significant (n = 3; **P=0.008), however, the EC₅₀ data did not reach statistical significance.
While mean IC₅₀ for NaIO₃ induced experiments was 8.84 mM for control and 11.76 mM for antioxidant; mean EC₅₀ was 5.12 for control and 10.47 for antioxidant. The difference between antioxidant treatment and control was significant in both assays (*** P<0.001; control n = 8, antioxidant n = 7).

Conclusions : There is a need for efficacious AMD therapies that target early disease manifestations such as oxidative stress to prevent disease progression. The method described herein serves as an in vitro platform to test efficacy of drug candidates in preventing oxidative stress-induced cellular death of RPE. Future studies will investigate if, in addition to antioxidants, other therapeutics can reduce cell death in this model, to serve as a high throughput screen of potential therapeutics and dose ranging studies for drug discovery.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.