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 showed iPSC-derived RPE express 11-cis-retinal indicating functional retinoid metabolism. Here, we compare human iPSC-derived RPE phagocytic function to human fetal RPE cell line.
Methods :
Human fetal RPE were commercially sourced (Lonza, Walkersville, MD). iPSCs were differentiated into RPE using an established protocol and analyzed by morphology, immunohistochemistry and confocal microscopy (2 dry AMD patients, 2 wet AMD patients and 2 age-matched controls). iPSC-derived RPE, fetal RPE were plated onto 96 well plates for 2 weeks and fed fluorescein isothiocyanate (FITC) labeled ROS for 24 hours. After washing 3 times to eliminate unbound ROS, cells containing phagocytized FITC-labeled ROS were imaged randomly by confocal microscope (Zeiss LSM 800, Carl Zeiss Microscopy, Oberkochen, Germany) and statistically analyzed by software Graphpad prism 7 (GraphPad Software, Inc., La Jolla, CA).
Results :
Human iPSC-derived RPE expressed specific RPE cell markers Bestrophin, retinal pigment epithelium-specific 65 kDa protein (RPE65) and zonula occludens-1 (ZO-1). At 24 hours FITC intensity of iPSC-derived RPE from non-AMD individuals was 0.9943±0.3336; dry AMD patients was 0.9547±0.2693; wet AMD patients was 1.263±0.2548 (the data is a combination of 2 patients in each group); fetal RPE was 1.954±0.4179. No significant differences were seen in iPS-derived RPE from dry AMD, wet AMD patients and non-AMD individuals when compared among each other. All iPS-derived RPE revealed a significant decrease in ROS-FITC intensity compared to fetal RPE. Dry AMD revealed a 2.05-time (p <0.01) decrease, wet AMD reveal a 1.55-time decrease (p <0.01) and non-AMD revealed a 1.97 time decrease in ROS-FITC intensity.
Conclusions :
Phagocytosis of ROS in human iPSC-derived RPE did not show significant differences between dry, wet AMD or non-AMD patients, although there was a decrease in phagocytized ROS when compared to human fetal RPE which may suggest a decreased ability in aged or diseased cells. Further study will be performed using more cells from AMD patients to make further comparisons.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.