Abstract
Purpose :
Retinal pigment epithelial cells perform critical functions such as phagocytosis of photoreceptor rod outer segments (ROS), retinoid metabolism and secretion of growth factors to maintain retinal homeostasis. We have previously observed no significant difference in the phagocytosis of rod outer segments (ROS) by AMD-derived RPE when compared to age-matched controls. Here, we investigate the phagocytic activity of oxidized ROS (oxROS) vs non-oxidized ROS ingestion by iPSC-RPE.
Methods :
iPSCs were differentiated into RPE using an established protocol and analyzed by morphology, immunohistochemistry and confocal. The ROS were oxidized under UVC irradiation for 3 hrs. iPSC-derived RPE were plated onto 96 well plates for 2 weeks and fed fluorescein isothiocyanate (FITC) labeled oxROS and FITC-ROS for 24 hours. After washing 3 times to eliminate unbound oxROS and ROS, cells containing phagocytized FITC-labeled oxROS and ROS were imaged randomly by confocal microscope (Zeiss LSM 800, Carl Zeiss Microscopy, Oberkochen, Germany) and flow cytometry (BD Biosciences, San Jose, California). The data was statistically analyzed by software Graphpad prism 9 (GraphPad Software, Inc., La Jolla, CA).
Results :
Human iPSC-derived RPE expressed normal RPE morphology and pigmentation. These RPE cells also expressed RPE cell markers bestrophin, retinal pigment epithelium-specific 65 kDa protein (RPE65) and zonula occludens-1 (ZO-1). At 24 hours FITC intensity of ROS was 595.8 ± 172.1, FITC intensity of oxROS was 2084 ± 428.4; significant differences were observed in iPSC-derived RPE phagocytose of oxROS. iPSC-derived RPE ingestion oxROS exhibited a 3.5-fold increase (p <0.01) compared to non-oxidized ROS.
Conclusions :
Human iPSC-derived RPE displayed a significant increase in the phagocytosis of oxROS compared to non-oxidized ROS. Further study will be performed comparing the phagocytic activity of oxROS by AMD-derived RPE compared to non-diseased controls.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.