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Tarun Bansal, Qin Wan, Rong Li, Patricia Lederman, Barbara Corneo, Sally Temple, Sunita D'Souza, Kapil Bharti, Sheldon S. Miller; Physiological Differences Between iPSC-RPE and Human Fetal RPE. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2697.
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© ARVO (1962-2015); The Authors (2016-present)
Pluripotent stem cell-derived retinal pigment epithelium (RPE) has been used as a potential therapeutic intervention for people suffering from degenerative eye diseases. However, the functional properties of these RPE cells have not been fully authenticated. In this study, we compared the physiology of induced pluripotent stem (iPS) cell-derived RPE with previously well-characterized cultured human fetal RPE (hfRPE).
Human dermal fibroblasts derived iPS cell lines (HDF2/9) were differentiated using existing protocols in the lab of Sally Temple (NSCI/NY). Confluent monolayers of hfRPE/iPSC-RPE grown on Transwells were used for imaging and physiology experiments. We used pH- and Ca2+-sensitive fluorescence dyes (BCECF and Fura-2) to monitor intracellular pH and Ca2+ activity, while simultaneously recording transepithelial potential (TEP) and resistance (RT). Intracellular microelectrodes were used to measure apical/basolateral membrane potentials (VA/VB), the ratio of apical to basolateral membrane resistance (RA/RB), and RT.
Intracellular recordings showed that iPSC-RPE had a similar resting VA and VB as the hfRPE (≈ -55 mV, n=12); however, when extracellular K+ was altered from 5 to 1 mM (mimicking the transition from dark to light), ΔVA, ΔVB, and ΔTEP values in hfRPE were significantly different than iPSC-RPE (ΔVA = 23.2 ± 7.5 mV for hfRPE and 10.9 ± 1.6 mV for HDF9-RPE; n=6, p < 0.005). Intracellular Ca2+ imaging revealed that HDF2-RPE showed an impaired function of SERC ATPase, as compared to hfRPE or HDF9-RPE. All three cell lines showed the presence of apical membrane Na/H exchangers, apical P2Y2 receptors, differential apical/basolateral CO2 permeability, and basolateral membrane Cl/HCO3 exchangers. Using immunofluorescence we identified several proteins whose polarization is identical in these cell types (EZRIN, ALDH1A3, and BEST1). In contrast, several other proteins showed differences in localization (COLLAGEN IV, CFTR, DCT, SLC16A1, and TYRP1).
Our results suggest that a thorough functional authentication of iPSC-RPE would be prudent before such cells are used for transplantation. We seek to provide a set of standards that will be useful for comparison of native human RPE and stem cell-derived RPE.
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