Appropriately differentiated ARPE-19 cells regain phenotype and gene expression profiles similar to those of native RPE cells

Abstract

Purpose: The RPE cell line ARPE-19 provides a dependable and widely used alternative to native RPE. However, replication of the native RPE phenotype becomes more difficult because these cells lose their specialized phenotype after multiple passages. Compounding this problem is the widespread use of ARPE-19 cells in an undifferentiated state to attempt to model RPE functions. We wished to determine whether suitable culture conditions and differentiation could restore the RPE-appropriate expression of genes and proteins to ARPE-19, along with a functional and morphological phenotype resembling native RPE. We compared the transcriptome of ARPE-19 cells kept in long-term culture with those of primary and other human RPE cells to assess the former's inherent plasticity relative to the latter.

Methods: ARPE-19 cells at passages 9 to 12 grown in DMEM containing high glucose and pyruvate with 1% fetal bovine serum were differentiated for up to 4 months. Immunocytochemistry was performed on ARPE-19 cells grown on filters. Total RNA extracted from ARPE-19 cells cultured for either 4 days or 4 months was used for RNA sequencing (RNA-Seq) analysis using a 2 × 50 bp paired end protocol. The RNA-Seq data were analyzed to identify the affected pathways and recognize shared ontological classification among differentially expressed genes. RPE-specific mRNAs and miRNAs were assessed with quantitative real-time (RT)-PCR, and proteins with western blotting.

Results: ARPE-19 cells grown for 4 months developed the classic native RPE phenotype with heavy pigmentation. RPE-expressed genes, including RPE65, RDH5, and RDH10, as well as miR-204/211, were greatly increased in the ARPE-19 cells maintained at confluence for 4 months. The RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of the genes in the differentiated ARPE-19 cells. Of the 16,757 genes with detectable signals, nearly 1,681 genes were upregulated, and 1,629 genes were downregulated with a fold change of 2.5 or more differences between 4 months and 4 days of culture. Gene Ontology analysis showed that the upregulated genes were associated with visual cycle, phagocytosis, pigment synthesis, cell differentiation, and RPE-related transcription factors. The majority of the downregulated genes play a role in cell cycle and proliferation.

Conclusions: The ARPE-19 cells cultured for 4 months developed a phenotype characteristic of native RPE and expressed proteins, mRNAs, and miRNAs characteristic of the RPE. Comparison of the ARPE-19 RNA-Seq data set with that of primary human fetal RPE, embryonic stem cell-derived RPE, and native RPE revealed an important overall similar expression ratio among all the models and native tissue. However, none of the cultured models reached the absolute values in the native tissue. The results of this study demonstrate that low-passage ARPE-19 cells can express genes specific to native human RPE cells when appropriately cultured and differentiated.

Figure 1

ARPE-19 cells develop RPE phenotype characteristics during differentiation. ARPE-19 cells were grown for various time periods as outlined in the Methods section, and the morphology of the cells was examined with phase-contrast microscopy. A: Subconfluent cells 1 day after plating. B: Cells grown for 4 days. C: Cells grown for 4 weeks. D: Cells grown for 4 months. Magnification, 100X.

Figure 2

Confocal micrographs of ARPE-19 cells cultured for 4 months. The ARPE-19 cells were plated on laminin-coated Transwell^® filter membrane at a density of 3 × 10⁵ cells/cm² and grown for 4 months as outlined in the Methods section. Rabbit anti- zonula occludens-1 (ZO-1), mouse anti-premelanosome protein (PMEL), mouse anti-claudin-2 primary antibodies, and Alexa Fluor secondary antibodies were used to immunostain the ARPE-19 cells at 4 months post-confluency; rhodamine-phalloidin was used to stain actin. A: PMEL immunostaining correlates with the presence of melanosome pigmentation. B: ZO-1 immunostaining shows good junctional development and localization at the cell borders containing a mixture of elongated and polygonal cells. C: Actin immunostaining reveals circumferential actin distribution throughout the height of the cells. D: Claudin-2 immunostaining shows tight junction localization uniformly across the monolayer. E: ZO-1 immunostaining shows good junctional development and localization at the cell borders regardless of the secondary antibody used. F: Claudin-2 immunofluorescent labeling shows colocalization with ZO-1 in the tight junction complexes (merged image of D and E). Images were taken using an Andor Revolution XD spinning disk confocal microscope using the 40X Plan Fluor oil immersion objective. Scale bar = 15 μm.

Figure 3

Volcano plot showing differentially expressed genes in the differentiated RPE cells. ARPE-19 cells grown for either 4 days or 4 months in DMEM were used for total RNA extractions and were used for RNA-Seq analysis, as described in the Methods section. Volcano plot for the 27,939 genes of the RPE cells cultured for 4 days and for 4 months. Representative highly up- or downregulated genes at high statistical significance are labeled.

Figure 4

Expression of visual cycle genes increases in the differentiated RPE cells. ARPE-19 cells grown for either 4 days or 4 months were used for total RNA and protein extractions and were used for real-time quantitative PCR and western blotting, respectively, as described in the Methods section. A: Real-time PCR analysis of RPE-specific mRNA expression. The values are mean ± standard deviation (SD), n = 4. *p<0.001 compared with control. B: Western blot analysis of the expression of the RPE-specific proteins. α-Tubulin expression shows that the amount of protein used in the different samples is similar.

Figure 5

Retinyl ester synthesis in the differentiated ARPE-19 cells. Cells cultured for 4 months on Transwell^® permeable membranes were incubated for 16 h with or without 4 μM all-trans retinol provided via the outside (basal) chamber. The cells were harvested, and the extracted retinoids were then analyzed with normal-phase high performance liquid chromatography (HPLC). A: Chromatogram overlay of retinoid extracts from ARPE-19 cells treated with (black) or without (red) all-trans retinol. The inset shows the absorbance spectrum from the peak observed in the treated ARPE-19 cells (black). B: Chromatogram of the authentic all-trans retinyl palmitate standard and its corresponding absorbance spectrum (inset).

Figure 6

Differentiation of RPE cells increases non-visual cycle gene expression. ARPE-19 cells grown for either 4 days or 4 months were used for total RNA and protein extractions and were used for real-time quantitative PCR and western blotting, respectively, as described in the Methods section. A: Real-time PCR analysis of RPE-specific non-visual cycle mRNA expression. The values are mean ± standard deviation (SD), n = 4. *p<0.001 compared with control. B: Western blot analysis of the expression of the RPE-specific non-visual cycle proteins. α-Tubulin expression shows that the amount of protein used in different samples is similar.

Figure 7

Epithelial-specific gene expression is increased in the differentiated ARPE-19 cells. ARPE-19 cells grown for either 4 days or 4 months were used for total RNA and protein extractions. The samples were then used for real-time quantitative PCR and western blotting, respectively, as described in the Methods section. A: Real-time PCR analysis of epithelial- and mesenchymal-specific mRNA expression. The values are mean ± standard deviation (SD), n = 4. *p<0.001 compared with control. B: Western blot analysis of the expression of the epithelial- and mesenchymal-specific proteins. α-Tubulin expression shows that the amount of protein used in different samples is similar.

Figure 8

Expression of genes and miRNAs involved in regulating RPE differentiation is increased. ARPE-19 cells grown for either 4 days or 4 months were used for total RNA extractions. The samples were then used for real-time quantitative PCR, as described in the Methods section. A: Real-time PCR analysis of RPE-specific miRNAs, miR-204 and miR-211, expression. B: Real-time PCR analysis of MITF and its target genes TRPM1 and TRPM3 mRNA expression. The values are mean ± standard deviation (SD), n = 4. *p<0.001 compared with control.

Figure 9

Protein network visualization of genes differentially expressed in the differentiated RPE cells. Significantly altered up- and downregulated genes were selected from the RNA sequencing (RNA-Seq) analysis of the RPE cells cultured for 4 days and for 4 months and were classified by their involvement in specific pathways. The STRING 10 database was used to visualize the known and predicted protein–protein interactions. Upregulated core set genes are indicated with red arrows and the downregulated genes with green arrows. The lines and arrows in different colors indicate the data sources: experimental (red), database (blue), and text-mining (green).

Figure 10

Comparative gene expression analysis between current RNA-Seq experiments and previous transcriptome studies of human primary and stem cell–derived RPE cells with RNAs sequencing (RNA-Seq). A: The Venn diagram shows the number of common and unique gene expression profiles between the 1,000 most highly expressed mRNA of the ARPE-19 cells cultured for 4 months (ARPE-19 4M) and RPE derived from human embryonic stem cells (H1 and H9) and fetal eyes (HF). The union of the four data sets gives rise to 1,511 genes, taken as the total for calculating the percentage shown. B: The Venn diagram shows the shared and unique genes among the 1,000 most highly expressed genes out of > 20,000 mRNA between the current RNA-Seq data (ARPE-19 4M), human embryonic stem cell-derived RPE cells (H1), and macular region of the RPE/choroid (RPE/CHOR_MAC). The union of these three data sets is 1,735 genes, taken as the total for calculating the percentages shown. C–H: Scatter plots with regression lines show the relations between each comparison. The regression line is in red. R = Pearson correlation coefficient; S = Spearman correlation coefficient. The x-axis corresponds to the gene expression observed in the ARPE-19 cells cultured for 4 months. The y-axis displays the RNA-Seq data set derived from the source.

Figure 11

Comparison of visual cycle and RPE signature gene expression. Fragments per thousand bases of gene per million bases mapped (FPKM) values obtained from RNA sequencing (RNA-Seq) of the ARPE-19 cells cultured for 4 months (ARPE-19 4M), RPE derived from human embryonic stem cells (H1 and H9) and fetal eyes (HF), and native RPE from the nasal (RPE/CHOR_NAS), temporal (RPE/CHOR_TMP), and macular (RPE/CHOR_MAC) regions of the RPE/choroid were used for the analysis. A: Comparison of the expression of the visual cycle genes. B: Comparison of the expression of RPE-specific genes. The x-axis indicates the expression levels of the genes analyzed. The y-axis indicates the transcript expression levels quantified as FPKM on a log₁₀ scale.