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Audrey Leguen, Christelle Monville, Anaïs Balbous-Gautier, Afsaneh Gaillard, Nicolas LEVEZIEL; ARPE-19: the best cell to model retinal diseases in vitro?. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2285.
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© ARVO (1962-2015); The Authors (2016-present)
ARPE-19 is currently used as an in vitro model for retinal diseases such as age-related degeneration (AMD). Up to now few studies show morphological and genetic expression differences between ARPE-19 and foetal or adult human retinal pigment epithelial (hRPE) cells. This study aims to compare ARPE-19 to hRPE cells derived from induced pluripotent stem cells (iPS) cells in both normal and oxidative stress condition (Fe-NTA treatment), the study hypothesis being to consider that hRPE-iPS cells are more able than ARPE-19 to model AMD in vitro.
iPS cell obtained from peripheral venous blood samples of individuals aged more than 60 years were differenciated into hRPE cells (N=4). hRPE-iPS cells were characterized by morphology, immunofluorescence, FACS and RTqPCR. In basal condition, β-galactosidase activity was measured to quantify senescent cells. Cell count was performed manually with ZEN software. Fe-NTA added to the culture media was used to mimic oxidative environment. In vitro concentrations of Fe-NTA was determined to evaluate the toxicity level characterized by cells death by MTT test (n=9). We estimated the oxidative stress produced by detection of DCFH-DA oxidized by ROS (n=9). For phagocytosis activity, cells were fed with fluorescent polyesters beads added into the media during 3 hours. The level of phagocytosis was quantified by FACS during both conditions (100 000 cells count in each experiment). One-way ANOVA was used for statistical analysis.
Only hRPE-iPS cells expressed β-galactosidase activity (64,45±8,31%, cells counts: 12 022), suggesting that these cells have a senescent profile compared to ARPE-19. Under oxidative stress, Fe-NTA levels increase up to 15 mM during 24 hours did not cause a significant decrease in cell viability (while 20 mM, p<0,0001) while inducing oxidative stress (significant from 10 mM, p<0,062). For phagocytosis activity, Fe-NTA concentrations increase induced a significant inhibition of internalization of fluorescent beads in ARPE-19 (from 10 mM, p<0,05) but not significant in hRPE-iPS cells.
Our results are consistent with our hypothesis that ARPE-19 and hRPE-iPS cells differ morphologically and in terms of phagocytosis activity and aging. By consequence, the use of hRPE-iPS cells may be considered as a more reliable model to mimic hRPE cells as an in vitro model of AMD than ARPE-19. Further studies are needed to confirm these results.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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