Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium-Role in Dead Cell Clearance and Inflammation

Abstract

Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional phagocytes can result in debris formation and development of age-related macular degeneration (AMD). Chronic oxidative stress and inflammation play an important role in AMD pathogenesis. Only a few well-established in vitro phagocytosis assay models exist. We propose human embryonic stem cell-derived-RPE cells as a new model for studying RPE cell removal by professional phagocytes. The characteristics of human embryonic stem cells-derived RPE (hESC-RPE) are similar to native RPEs based on their gene and protein expression profile, integrity, and barrier properties or regarding drug transport. However, no data exist about RPE death modalities and how efficiently dying hESC-RPEs are taken upby macrophages, and whether this process triggers an inflammatory responses. This study demonstrates hESC-RPEs can be induced to undergo anoikis or autophagy-associated cell death due to extracellular matrix detachment or serum deprivation and hydrogen-peroxide co-treatment, respectively, similar to primary human RPEs. Dying hESC-RPEs are efficiently engulfed by macrophages which results in high amounts of IL-6 and IL-8 cytokine release. These findings suggest that the clearance of anoikic and autophagy-associated dying hESC-RPEs can be used as a new model for investigating AMD pathogenesis or for testing the in vivo potential of these cells in stem cell therapy.

Keywords: age-related macular degeneration; anoikis; autophagy; hESC-RPE; inflammation; macrophages; phagocytosis; triamcinolone.

Figure 1

Morphological and cell death analysis after blocking the attachment of human embryonic stem cells-derived retinal pigment epithelium (hESC-RPE) cells to extracellular matrix (ECM). (A) Phase contrast images (10×) of untreated control hESC-RPE cells and anoikic hESC-RPE cells which were cultured on poly-2-hydroxyethylmethacrylate (poly-HEMA) coated culture dishes for 24 h to induce cell death by detachment from the extracellular matrix. Images were captured with a Nikon Eclipse TE2000-S phase contrast microscope. Scale bar indicates 20 µm. (B) The induction of cell death by anoikis was determined by Annexin (Anx)V-FITC/PI double staining assay. Representative dot plots of AnxV/PI measurements of anoikic dying hESC-RPE cells are shown. Top: dot plots represent the measurements of forward light scattering (FSC; X axis) vs. side light scattering (SSC; Y axis). Bottom: the horizontal axis represents the intensity of staining for Annexin V (log scale) and the vertical axis shows the intensity of staining for PI (log scale). The numbers in the quadrants indicate the percentage of different cell populations. Cells in the lower left quadrant (AnxV⁻/PI⁻) are viable, those in the lower right quadrant (AnxV⁺/PI⁻) are early apoptotic, those in the upper left (AnxV⁻/PI⁺) are necrotic and those in the upper right (AnxV⁺/PI⁺) are late apoptotic cells. Data are representative of 3 independent experiments. (C) The bar charts indicate the average percentage of AnxV⁻/PI⁻ (black bars), AnxV⁺/PI⁻ (grey bars), AnxV⁻/PI⁺ (white bars) and AnxV⁺/PI⁺ (striped bars) cells from 3 independent experiments.

Figure 2

The effect of serum deprivation and H₂O₂ co-treatment on the morphology and cell viability of hESC-RPE cells. (A) Phase contrast images (10×) of untreated control, serum-deprived (2 h) and H₂O₂-treated (2 h, 1 mM) hESC-RPE cells in the presence or absence of serum. Images were captured with a Nikon Eclipse TE2000-S phase contrast microscope. Scale bar indicates 20 µm. (B) The induction of cell death by anoikis and H₂O₂-treatment (2 h, 1 mM) in the presence or absence of serum in hESC-RPE cells was determined by Annexin (Anx)V-FITC/PI double staining assay. Representative dot plots of AnxV/PI measurements of anoikic and H₂O₂-treated (2 h, 1 mM) dying hESC-RPE cells are shown. Top: dot plots represent the measurements of forward light scattering (FSC; X axis) vs. side light scattering (SSC; Y axis). Bottom: the horizontal axis represents the intensity of staining for Annexin V (log scale) and the vertical axis shows the intensity of staining for PI (log scale). The numbers in the quadrants indicate the percentage of different cell populations. Cells in the lower left quadrant (AnxV⁻/PI⁻) are viable, those in the lower right quadrant (AnxV⁺/PI⁻) are early apoptotic, those in the upper left (AnxV⁻/PI⁺) are necrotic and those in the upper right (AnxV⁺/PI⁺) are late apoptotic cells. Data are representative of 3 independent experiments. (C) The bar charts indicate the average percentage of AnxV⁻/PI⁻ (black bars), AnxV⁺/PI⁻(grey bars), AnxV⁻/PI⁺ (white bars) and AnxV⁺/PI⁺ (striped bars) cells from 3 independent experiments.

Figure 3

Autophagy induction as a result of serum deprivation and H₂O₂ co-treatment in hESC-RPE cells. Representative western blot image for the expression of LC3 in hESC-RPE cells treated with 1 mM H₂O₂ for 2 h in the presence or absence of serum. Integrated optical density was determined by densitometry for quantification of the LC3-II/LC3-I ratio using the ImageJ software. GAPDH was used as a loading control. Data are representative of three independent experiments.

Figure 4

The clearance of anoikic and autophagy-associated dying hESC-RPE cells by macrophages. (A) Representative flow cytometry dot plots demonstrating phagocytosis of anoikic and autophagy-associated dying hESC-RPE cells by macrophages after 4 h and 8 h co-incubation, respectively. Macrophages were pre-treated with 1 µM triamcinolone (TC) for 48 h. The horizontal axis represents the intensity of staining for CFDA (log scale) and the vertical axis shows the intensity of staining for CMTMR (log scale). Cells in the upper right quadrant indicate the engulfed hESC-RPE (CFDA-labeled) cells by macrophages (CMTMR-labeled). Data are representative of 3 independent experiments. (B) The phagocytosis rate of anoikic and autophagy-associated dying hESC-RPE cells by untreated and TC-pre-treated (48 h, 1 μM) macrophages after 4 h and 8 h co-incubation, respectively, is shown as determined by flow cytometry analysis. Bars represent the mean ± SD of 3 independent experiments, * p < 0.05.

Figure 5

Determination of IL-6 and IL-8 release during the engulfment of anoikic and autophagy-associated dying hESC-RPE cells by macrophages. Anoikic dying hESC-RPE cells (left panels) and autophagy-associated dying hESC-RPE cells (right panels) were co-incubated with untreated and triamcinolone (TC)-treated (48 h, 1 μM) macrophages for 4 h and 8 h, respectively, then the supernatants were collected, and the level of secreted IL-6 (A) and IL-8 (B) cytokines were measured by ELISA. Bars represent the mean ± SD of 3 independent experiments, * p < 0.05.