Abstract
Purpose :
Age-related macular degeneration (AMD) is partially characterized by retinal pigment epithelial (RPE) cell dysfunction, leading to secondary photoreceptor degeneration. Lack of an appropriate in vitro model that could mimic RPE dysfunction is a major limitation to understanding the mechanisms leading to development of AMD. This study aimed to compare human induced pluripotent stem RPE cells (hiPSC-RPE) derived from dry AMD patients with hiPSC-RPE derived from elderly healthy individuals in both normal conditions and under oxidative stress.
Methods :
hiPSCs were derived from erythroblasts or fibroblasts of healthy individuals (N=6, 62 ±17 y/o) and from dry AMD patients (N=6, 77 y/o ± 7). They were differentiated into hRPE cells and characterized by morphology, immunofluorescence, FACS and electronic microscopy. Senescent cells were quantified by β-galactosidase activity in basal condition. Fe-NTA added to the culture media was used to mimic oxidative environment. In vitro concentrations of Fe-NTA were determined to evaluate the toxicity level by MTT test (n=3 per cell lines). Oxidative stress was evaluated by detection of oxidized DCFH-DA (n=3 per cell lines). Lysosomal activity was measured by quantification of Cathepsine D level concentration in both normal and oxidative stress conditions. One-way ANOVA was used for statistical analysis.
Results :
Both hiPSC-RPE control and AMD cells expressed β-galactosidase activity in basal condition after 3 weeks at passage 2. Under oxidative stress, cell death was observed from 5 mM in both cell populations but in a higher proportion in hiPSC-RPE AMD (100.6 ± 3.7% versus 89.4 ± 6%, p<0.001). ROS production induced by Fe-NTA treatment was higher in hiPSC-RPE AMD cells (p<0.05). For lysosomal activity, Fe-NTA induced a dose-dependent increase of immature Cathepsine D, which was lower in hiPSC-RPE control cells (p<0.05
Conclusions :
Both hiPSC-RPE control and AMD cells have a senescent profile. FeNTA induced a higher reactive oxygen species production and increased cell death in hiPSC-RPE AMD as compared to hiPSC-RPE controls. Functional analysis showed differences in lysosomal process between hiPSC-RPE AMD and hiPSC-RPE control. These findings demonstrate that hiPSC-RPE AMD cells have a typical disease phenotype compared to hiPSC-RPE control cells in a pro-oxidant environment.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.