Polarized Human Retinal Pigment Epithelium Exhibits Distinct Surface Proteome on Apical and Basal Plasma Membranes

Abstract

Surface proteins localized on the apical and basal plasma membranes are required for a cell to sense its environment and relay changes in ionic, cytokine, chemokine, and hormone levels to the inside of the cell. In a polarized cell, surface proteins are differentially localized on the apical or the basolateral sides of the cell. The retinal pigment epithelium (RPE) is an example of a polarized cell that performs a variety of functions that are dependent on its polarized state including trafficking of ions, fluid, and metabolites across the RPE monolayer. These functions are absolutely crucial for maintaining the health and integrity of adjacent photoreceptors, the photosensitive cells of the retina. Here we present a series of approaches to identify and validate the polarization state of cultured primary human RPE cells using immunostaining for RPE apical/basolateral markers, polarized cytokine secretion, electrophysiology, fluid transport, phagocytosis, and identification of plasma membrane proteins through cell surface capturing technology. These approaches are currently being used to validate the polarized state and the epithelial phenotype of human induced pluripotent stem (iPS) cell derived RPE cells. This work provides the basis for developing an autologous cell therapy for age-related macular degeneration using patient specific iPS cell derived RPE.

Keywords: Cell surface capturing; Cytokine secretion; Electrophysiology; Fluid transport; Immunocytochemistry; Monolayer; Phagocytosis; Polarization; Retinal pigment epithelium.

Fig. 1

TEM micrograph of hfRPE monolayer grown on transwell membrane. A polarized monolayer of hfRPE cells is observed as indicated by apical processes, apically localized melanosomes, and nuclei located close to the basolateral membrane

Fig. 2

Polarized distribution of known RPE markers in the monolayer. Immunofluorescence localization of RPE basolateral marker Collagen IV (a) and apical marker Ezrin (b) in primary cultures hfRPE grown on transwell inserts. DAPI (blue) labels the nuclei located close to the basolateral membrane

Fig. 3

Differential electrical responses evoked by apical or basal application of ATP. The continuous trace represents transepithelial potential (TEP) and open squares represent total tissue resistance (R_T). The apical or basal bath perfusion of 100 μM ATP is indicated by black bars above the graph. A time scale bar is located at the bottom of the graph. In the native tissue, ATP acts on purinergic P2Y₂ receptors on RPE apical membrane, increasing intracellular IP₃ and thus stimulating Ca²⁺ release from the endoplasmic reticulum, to cause activation of basolateral membrane Ca²⁺-activated Cl⁻ channels followed by a decrease in apical membrane K⁺ conductance, thus leading to electrical responses across the RPE monolayer [14]. Consistent with this, upon the apical application in the primary cultures RPE, ATP produced a dramatic biphasic response in TEP and a markedly decrease in R_T; but induced much smaller TEP responses with little R_T changes after basal perfusion, suggesting the majority of P2Y₂ receptors are located on RPE apical membrane

Fig. 4

Differential electrical responses evoked by apical or basal application of the ClC₂ channel activator. The ClC₂ activator (10 μM) produced a similar increase in TEP and a decrease in R_T upon either the apical or basal bath application, suggesting the basal localization of ClC₂ channels in RPE. The activation of ClC₂ on the RPE basal membrane leads to the efflux of intracellular Cl⁻ and depolarization of RPE basolateral membrane, thus causing TEP increase and R_T drop. The larger effect induced by apical application may be due to the much larger RPE apical surface area

Fig. 5

Polarized cytokine secretion to determine the polarity of iPSC-derived RPE monolayer. RPE from three different RPE samples, namely Line A, B, and C, were tested for VEGF secretion on apical and basal side. RPE monolayers were fed with fresh media 24 h before the media collection. The collected media was spun down and used for the analysis. The results demonstrate the RPE monolayers grown in vitro secrete the VEGF cytokine in a manner replicating in vivo function

Fig. 6

Fluid transport response induced by apical application of ATP. Top panel: Transepithelial fluid transport (J_V). Bottom panel: Black trace represents transepithelial potential (TEP) and red trace represents transepithelial resistance (R_T). Application of ATP is indicated by the black bar above the top graph. The apical (AP) application of 100 μM ATP activated the P2Y₂ receptors on RPE apical membrane, which resulted in a reversible increase in apical-to-basal fluid adsorption. Time scale bar indicated 50 min

Fig. 7

Phagocytic capability of polarized primary hfRPE monolayer. Fluorescently labeled photoreceptor outer segments (POS) were observed in hfRPE cells indicating its normal phagocytic capability

Fig. 8

Immunostaining of a G-protein coupled receptor RPE marker. Immunofluorescence localization of a known RPE marker, OA1 (red) in cultures hfRPE (a) and fibroblasts (b). In each figure, the Central panel is an en face view of the cell culture insert, shown as the maximum-intensity projection through the z-axis. Images of the cross section through the Z-plane are shown in the top and the right panels. F-actin (green) was used to visualize the cytoskeleton and cell boundary in the cell; DAPI (blue) labels the nuclei located close to the basolateral membrane

Fig. 9

Immunostaining of a transmembrane protein RPE marker. Immunofluorescence localization of a known RPE marker, GPNMB (red), F-actin (green) in cultures hfRPE (a) and fibroblasts (b)

Fig. 10

Immunostaining of a novel RPE cell maker. Immunofluorescence localization of a novel RPE marker, SLC5A12 (red) and F-actin (green) in cultured hfRPE (a) and fibroblasts (b)

Fig. 11

Western blot of a novel RPE marker GRIK1 is enriched in the membrane and cytosolic fractions of hfRPE cells. (A) Nuclear wash buffer, (B) nuclear fraction, (C) mitochondrial fraction, (D) cytosolic fraction, (E) membrane fraction

Fig. 12

Western blot of known RPE marker GPR143 is enriched in the membrane fraction of hfRPE cells. (A) Nuclear wash buffer, (B) nuclear fraction, (C) mitochondrial fraction, (D) cytosolic fraction, (E) membrane fraction