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Amanda-Jayne Francis Carr, Carolyn Swann, Monte J Radeke, Tim Rudd, Jennifer Man, Hector Keun, Toby Athersuch, Peter J Coffey; Using ARPE-19 cells to investigate pathways associated with retinal pigment epithelium differentiation.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2469.
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© ARVO (1962-2015); The Authors (2016-present)
Human ARPE-19 cells are widely used as a model system to study retinal pigment epithelial (RPE) cells, however, achieving a reliable RPE cell phenotype is a challenge due to epithelial-to-mesenchymal transition over repeated passage. We have shown that ARPE-19 cells can be redirected towards the RPE phenotype in defined growth medium, resulting in re-establishment of epithelial morphology, pigmentation, and RPE gene and protein expression. Here, we have performed an omics comparison of ARPE-19 cell differentiation in a novel growth medium to investigate pathways associated with RPE cell differentiation.
ARPE-19 cells were cultured in DMEM (high pyruvate + high glucose) or X-Vivo medium for 6 weeks during which morphological changes were examined by microscopy. Cells were sampled weekly over the culture period for RNA-seq transcriptome profiling, Western blot, immunocytochemistry and metabolomics studies (n=3). RNA-seq libraries were created from cells sampled each week in DMEM and X-Vivo and sequenced using the Ion PGM next generation sequencing platform. Transcriptional profiles were compared to those observed in fetal RPE using Ingenuity Pathway Analysis. For metabolomic analysis, cell metabolites were analysed using Gas/Liquid-Chromatography Mass spectrometry.
ARPE-19 cells cultured in X-Vivo developed cobblestone morphology and pigmentation over 6 weeks in culture, whilst cells cultured in DMEM remained fibroblastic in appearance. RNA-seq showed a temporal increase in RPE markers, such as CRALBP, PEDF, MerTK and Pmel17 after culture in X-Vivo medium, these remained low in cells cultured in DMEM. However, a number of key RPE genes, such as RPE65, LRAT and BEST were expressed at low levels in both conditions. Pathway Analysis of RNA-seq data identified a number of pathways altered during RPE differentiation, including TGFβ, ERK, EGF and TNF signalling. Metabolomic analysis suggests that ARPE-19 metabolite consumption is altered by culture in different medium.
Changes in the morphology, pigmentation, and transcriptome of ARPE-19 cells cultured in X-vivo suggest that this culture medium induces RPE cell differentiation. However, following 6 weeks in culture a full RPE transcription profile comparable to fetal RPE was not achieved. Nevertheless, ARPE-19 cell provide a useful model system for the investigation of RPE cell differentiation.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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