Objectives The functional properties of pluripotent stem cell-derived retinal pigment epithelium (RPE) 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).
Methods iPS cell lines (HDF2, HDF-9 and B59) were derived from human dermal fibroblasts. All of the three iPS cell lines were differentiated into RPE using existing protocols in the lab of Sally Temple (NSCI/NY). Imaging and physiology experiments were performed on confluent monolayers of iPSC-RPE/hfRPE grown on Transwells. Transmission electron microscopy (TEM) was used to characterize the ultrastructure of iPS-derived RPE and immunofluorescence analysis were used to analyze polarized protein localization. Microarrays were used to determine RPE gene expression and polarized cytokine secretions were measured using a multiplex ELISA technique. The stem cell-specific miRNA profile of iPS cell-derived RPE was compared to hfRPE. pH- and Ca2+-sensitive fluorescence dyes (BCECF and Fura-2) were used to monitor intracellular pH and Ca2+ activity. Intracellular microelectrodes were used to measure apical and basolateral membrane potentials (VA, VB), the ratio of apical to basolateral membrane resistance (RA/RB), and total tissue resistance (RT) and potential (TEP).
Results EM showed that iPS cell-derived RPE formed a monolayer with apical processes and predominantly apical melanosomes. However, the B59-derived RPE consisted of multiple layers. Immunofluorescence analysis showed the polarized localization of RPE specific proteins. B59-derived RPE secrete higher levels of pro-inflammatory cytokines as compared to primary human fetal RPE, HDF2 and HDF-9-derived RPE when activated by a pro-inflammatory cytokine cocktail. Intracellular recordings showed that iPSC-RPE and hfRPE had similar resting VA and VB membrane potentials (≈ -55 mV, n=12). When extracellular K+ was altered from 5 to 1 mM (mimicking a transition from dark to light), ΔVA, ΔVB, and ΔTEP values in hfRPE and B59-derived RPE were significantly different than HDF2 and HDF-9-derived RPE. RPE expression analysis showed that HDF-2 and HDF-9 derived RPE cells more closely resemble hfRPE as compared to B59-derived RPE.
Conclusions The present results suggest a set of standards that could be useful for authentication of human stem cell-derived RPE in cell based therapy and disease modeling. |
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