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Jie Gong, Mark Fields, Ernesto F Moreira, Hannah Bowrey, Zsolt Ablonczy, Baerbel Rohrer, Craig C Beeson, Lucian V Del Priore; Mitochondrial metabolism and structural analysis of Induced Pluripotent Stem Cell (iPSC) derived RPE from Age-Related Macular Degeneration (AMD) Patients.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3580.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) derived from human iPSC may provide a promising source of cells for transplantation. Physiological tests are required to confirm that these cells can function as RPE cells prior to cell transplantation. Herein we analyzed critical RPE functions such as the development of Transepithelial Resistance (TER), polarized secretion of vascular endothelium growth factor (VEGF) and pigment epithelium-derived factor (PEDF) as well as energy metabolism.
Fibroblasts cultured from axillary skin biopsies obtained from a dry AMD patient and an age-matched control were reprogramed into iPSC by using Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) delivered using non-integrating Sendai viruses. iPSC cells were subsequently differentiated into RPE cells. iPS (IMR90 Wi-cell Wisconsin USA) was also used as a young control. Pigmented iPSC derived RPE were grown into monolayers on permeable transwells and fed with medium containing 1% serum. Media collected from each chamber were examined for VEGF and PEDF levels using commercially available ELISA kits. TER was measured using an epithelial voltohmmeter. Mitochondrial respiratory function and glycolysis were measured by determining the oxygen consumption rate (OCAR) and extracellular acidification rate (ECAR) of iPS derived RPE using a Seahorse Bioscience XF96 instrument.
iPS derived RPE cells from dry AMD and aged control patients formed a hexagonal monolayer exhibiting an RPE phenotype based on pigmentation, the expression of RPE (MITF) and tight junction markers (ZO-1). They form a monolayer with TER of an average of 335+36 Ω*cm2. Polarized VEGF and PEDF secretion was observed [VEGF: basal>apical, (apical 1059+93 pg/ml; basal: 1299+29 pg/ml, p=0.03), PEDF secretion: apical>basal (apical 2185+196 ng/ml basal: 1220+460 ng/ml, p=0.009)]. Mitochondrial respiration and glycolysis revealed an apparent age-dependent decline in metabolic function. However no significance differences between cells from the AMD and the age-matched control sample could be identified.
iPS derived RPE cells exhibited physiological properties typically obtained from adult RPE cells. Additional samples are required to confirm this observation and expand it to determine whether there are differences between dry AMD and age-matched control samples.
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