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Lucian V Del Priore, Jie Gong, Ernesto F Moreira, Hannah E Bowrey, Zsolt Ablonczy, Mark Fields; Characterization of Human Stem Cells Generated from AMD Patients . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3173.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigmented epithelium (RPE) derived from human induced pluripotent stem (iPS) cells is a promising source of cells for the replacement of degenerated RPE in patients with age-related macular degeneration (AMD), as use of patient-specific iPS will avoid graft rejection. Here we demonstrate that iPS can be derived from fibroblasts obtained from older patients with AMD, and that these cells can be used to generate autologous RPE despite advanced patient age and the presence of severe AMD in the donors.
Patients with advanced AMD exhibiting either geographic atrophy or exudative AMD were recruited. Fibroblasts obtained from skin biopsies were grown to confluence (dry AMD n=8, wet AMD n=7, age-matched control n=5) in DMEM containing 10% Fetal Bovine Serum (FBS). Supernatant was collected 24 hours later and the VEGF levels were measured by ELISA. iPS were generated from these fibroblasts using Sendai virus reprogramming and functional RPE tests were performed.
Fibroblasts were obtained successfully from over 20 patients with advanced AMD and 5 age-matched controls. VEGF secretion from these fibroblasts was as follows: dry AMD 185 + 60 pg/ml, wet AMD 118 + 38 pg/ml, aged control 230 + 191 pg/ml. VEGF secretion by both dry and wet AMD patients were significantly lower than aged-matched controls (dry AMD vs aged control P < 0.0001, wet AMD vs aged control p=0.02). VEGF secretion in wet AMD patients was significantly lower than dry AMD patient (P=0.0002). Patient-specific iPS have been generated from 6 of these samples, and 5 RPE cell lines have been generated. iPS cells were all positive for stem cell markers including SSEA4, TRA-1-60, OCT4, and TRA-1-8. Cells differentiated towards an RPE fate formed a hexagonal monolayer within 30 days with a transepithelial resistance of 154.8 + 0.23 Ω*cm2. Monolayers were uniformly positive for Bestrophin, MITF, RPE65 and ZO-1. iPS-derived RPE were able to ingest outer segments, and this phagocytic ability was blocked by specific addition of avB5 integrin antibodies.
Patient-specific fibroblasts have been reprogrammed into iPS from AMD patients up to age 80 with no decline is this ability with advancing patient age. Interestingly, supernatant VEGF levels were lower in fibroblasts from AMD patients compared to age-matched controls. iPS can be differentiated towards RPE with cells expressing RPE markers and performing phagocytic functions.
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