Abstract
Purpose :
We have previously demonstrated our ability to derive iPSC-derived RPE from AMD patients and age-matched controls. These cells performed critical functions such as phagocytosis of rod photoreceptor outer segments (ROS), the ability to form tight junctions, and retinol metabolism. We have previously shown that RPE cells cultured on nitrite-modified extracellular matrix (ECM), a model of Bruch’s membrane aging, exhibit a reduced ability to phagocytize ROS. Here we use an in vitro aged Bruch’s membrane model to evaluate the phagocytic ability of iPSC-derived RPE from AMD patients and age-matched controls.
Methods :
iPSCs were generated from AMD (4 atrophic) and patients with no history of AMD (n = 3) using an mRNA reprogramming method. iPSCs were then differentiated into RPE using an established protocol. RPE lines were verified by morphology, immunohistochemistry and confocal microscopy. Ingested fluorescein isothiocyanate isomer I (FITC) labeled ROS were analyzed by confocal microscopy and quantified using a BioTek FLx800 plate reader (BioTek, Winooski, VT).
Results :
Human iPSC-derived RPE expressed specific RPE cell markers retinal pigment epithelium-specific 65 kDa protein (RPE65), cellular retinaldehyde-binding protein (CRALBP), MER proto-oncogene, tyrosine kinase (MERTK), and zona occludens-1 (ZO-1). Confocal microscopy demonstrates that phagocytized ROS particles localize within the cytoplasm of iPSC-derived RPE cells. Compared to unmodified ECM, iPSC-derived RPE cells from normal controls were 24.38% (p<0.001) less able to phagocytosis on nitrite-modified ECM. Compared to unmodified ECM, iPSC-derived RPE cells from AMD patients were 14.15% (p<0.05) less able to phagocytosis on nitrite-modified ECM. A comparison of iPSC- RPE cells from normal controls (n = 3 RPE lines) vs. AMD patients (n = 4 atrophic RPE lines) on nitrite-modified ECM revealed no significant differences in their ability to phagocytize ROS.
Conclusions :
In this study, we generated RPE from AMD patients and patients with no history of AMD. Decreased of phagocytosis on nitrite-modified ECM were observed in iPSC-RPE from both AMD patients and normal controls. Further, exploration of the critical differences among normal RPE and RPE from individuals with AMD using additional lines should reveal mechanisms of disease and pave the way for novel therapeutic strategies.
This is a 2020 ARVO Annual Meeting abstract.