Molecular characterization and functional analysis of phagocytosis by human embryonic stem cell-derived RPE cells using a novel human retinal assay

Abstract

Purpose: To examine the ability of retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (HESC) to phagocytose photoreceptor outer segments, and to determine whether exposure to human retina induces any morphological changes in these cells.

Methods: HESC-RPE cells were derived from a super-confluent preparation of the Shef1 HESC line. Pigmented colonies were isolated and expanded into pigmented monolayers on Matrigel matrix-coated dishes or filters. Cells were exposed to fluorescently labeled outer segments isolated from the porcine eye and assessed for phagocytic activity at regular intervals. Expression of molecules associated with RPE phagocytosis was analyzed by RT-PCR, immunocytochemistry, and western blot. The role of Mer Tyrosine Kinase (MERTK) in the phagocytosis of outer segments was investigated using antibodies directed against MERTK to block function. In a novel approach, cells were also exposed to fresh human neural retina tissue then examined by electron microscopy for evidence of phagocytosis and changes in cell morphology.

Results: HESC-derived RPE cells are capable of phagocytosing isolated porcine outer segments and express molecules associated with RPE-specific phagocytosis, including MERTK. Pre-incubation with antibodies against MERTK blocked phagocytosis of photoreceptor outer segments, but not polystyrene beads. HESC-RPE cells also phagocytosed outer segments in a novel human retinal explant system. Furthermore co-culture adjacent to human retina tissue in this preparation resulted in the appearance of features in HESC-derived RPE cells normally observed only as the RPE matures.

Conclusions: The ingestion of photoreceptor outer segments from an isolated population and an artificial ex vivo human retina system demonstrates HESC-derived RPE cells are functional. HESC-derived RPE possess the relevant molecules required for phagocytosis, including MERTK, which is essential for the phagocytosis of outer segments but not latex beads. Furthermore, some changes observed in cell morphology after co-culture with human retina may have implications for understanding the full development and differentiation of RPE cells.

Figure 1

HESC-derived RPE cells internalize fluorescently labeled POS isolated from the porcine eye. A: The pigmented monolayer of HESC-derived RPE binds labeled POS (green). B: A Nomarski image showing pigmented HESC-derived RPE overlaid with Alexa Fluor® 488-labeled porcine POS. Evidence of internalization of outer segments is clear by 4 h. C-D: Alexa Fluor® 488-labeled porcine POS are associated with the apical surface of HESC-derived RPE (delineated by TRITC-labeled Na⁺/K⁺ ATPase immunostaining). D-E: POS are ingested by the cells and are located close to the HESC-derived RPE nuclei (DAPI, stained blue). Photomicrographs in C-E are single (y-axis) confocal optical slices (<0.8 μm) from 3 separate HESC-RPE samples. In A-B, scale bars equal 150 μm; in C-E, scale bars equal 10 μm.

Figure 2

Increasing numbers of fluorescently labeled porcine POS are phagocytosed by HESC-derived RPE over time. A monolayer of HESC-derived RPE cells was exposed to Alexa Fluor® 488-labeled porcine POS, treated with trypan blue to remove fluorescent non-internalized POS, washed, fixed, and processed for immunocytochemistry. Representative micrographs are shown from (A) 1 h and (B) 20 h together with respective Nomarski images (C and D). Alexa Fluor® 488-labeled POS are in green. Scale bars equal 50 μm. E Increased internalization of POS was observed over time. Data shown are mean±SEM p<0.001, One-way ANOVA with Bonferroni multicomparison test, (n=5); 0.5 h versus 4 h p<0.05, 0.5 h versus 20 h p<0.001, 1 h versus 20 h p<0.001, 4 h versus 20 h p<0.05.

Figure 3

HESC-RPE cells require MERTK for the internalization of POS. HESC-RPE cells express the phagocytic proteins (A) MERTK and (B) αVβ5 integrin. To determine the importance of MERTK in the phagocytosis of POS, we treated HESC-RPE cells with control IgG (C and F) or MERTK antibody (D and G) for 1 h before exposure to Alexa Fluor® 488-labeled porcine POS (C and D) or fluorescent polystyrene beads (F and G) for 5 h. Cells were treated with trypan blue, washed, and fixed. The number of internalized POS (E) and fluorescent beads (H) was then quantified per field view (150 μm × 150 μm). Data shown are mean±SEM (n=6). Pre-incubation with MERTK antibody had a significant effect on the number of outer segments ingested by HESC-RPE cells (p<0.01, Student t-test, n=6). There was no effect of MERTK antibody on the ingestion of polystyrene beads by the cells (p>0.05, Student t-test, n=6). Photomicrographs are single confocal optical slices merged with Nomarski image (<0.8 μm). Scale bars equal 50 µm. MERTK is labeled with Cy5 and counterstained with DAPI, αVβ5 is labeled with FITC.

Figure 4

HESC-derived RPE cells express molecules required for POS specific phagocytosis. A: RT–PCR analysis of gene expression in HESC-derived RPE cells (HESC-RPE), control nonpigmented HESC (HESC-Control) and the human RPE cell line ARPE-19. cDNA was synthesized from 3 μg of RNA in a 20 μl reaction, from which 1 μl was used for PCR amplification. Lanes to the right of each sample are genomic DNA control reactions, which lacked the reverse transcriptase during cDNA synthesis. NTC is a no RNA template sample to control for non-specific amplification. All PCR reaction products were resolved by agarose gel electrophoresis. PCR primer sequences, amplicon size, and melting temperatures are described in Table 1. Tbp was amplified in all samples as a housekeeping control gene. B: western blot analysis of protein expression in HESC-P, HESC-NP and ARPE-19 cells. Equal amounts of protein were resolved on an SDS–PAGE gel, transferred onto membranes, and hybridized with antibodies for MERTK (180 kDa), focal adhesion kinase (FAK, 125 kDa), αV integrin (125–135 kDa), and β5 integrin (100 kDa). Membranes were stripped and re-probed with GAPDH (36 kDa) as a loading control.

Figure 5

A novel in vitro assay to assess phagocytic activity in HESC-derived RPE cells. A: A schematic illustration of the novel in vitro retinal explant model system used to analyze phagocytosis. HESC-derived RPE cells were cultured on a Matrigel™-coated filter and exposed to the photoreceptor side of human or pig retina placed on a filter. The RPE-retina “sandwich” was cultured in HESC medium and held together by a plastic insert weighted down by the 6-well plate lid. B: A Nomarski image of HESC-derived RPE exposed to a porcine retina explant for 48 h in vitro. The nuclei of cells are stained with DAPI (blue). The outer nuclear layer (ONL), indicating the rod and cone nuclei, and inner nuclear layer (INL), specifying the amacrine, bipolar, and horizontal cell layers are labeled. C-E: Immunohistochemical staining for rhodopsin in HESC-derived RPE cells exposed to porcine retina for 48 h in the novel retinal explant system. C is a confocal projection and D-E are single confocal slices (<0.8 μm) merged with the Nomarski image. The rhodopsin staining (TRITC labeled) observed within pigmented HESC-derived RPE cells is indicative of POS phagocytosis. The nucleus is stained with DAPI (blue). Scale bar in B equals 50 µm; scale bar in C, D, and E equals 20 µm.

Figure 6

Electron microscopy of HESC-derived RPE cells after exposure to human retinal explant in vitro. A: Control HESC-derived RPE cells, which were not exposed to human retina, have apical microvilli (AMv) and contain melanin granules (white arrows). B: HESC-derived RPE exposed to the photoreceptor surface of human retina for 48 h appear more mature. Note the close association of the RPE apical microvilli with the photoreceptor outer segment (OS), the abundance of pigmented melanin granules within the apical region of the cell, the nucleus (N), and the numerous lipid deposits (L) located toward the basal portion of the cell. C: Apical microvilli surround a human photoreceptor outer segment. D: An outer segment is engulfed by the HESC-derived RPE cell. E: A high magnification image of lipid deposits (L) observed in the basal portion of a HESC-derived RPE after exposure to human retina. The formation of lipid deposits is indicative of the end stages of phagocytosis. Several features associated with RPE cell function are present in the cells including (F) tight junctions (black arrow) and coated pits (red box, indicated with arrows at higher magnification in G). Cells also contain a high number of coated vesicles within the apical portion of the cell (H) and develop basal end feet and infolding of the basal membrane (I). Scale bars equal 2 μm in A-C, 1 μm in D-F,I, and 500 nm in G,H.