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Aims/Purpose: To study the changes on the metallome and transcriptome of a cellular model of the retinal pigment epithelium (RPE) during its in vitro degeneration after 4 months in culture. Methods: Primary cultures of human fetal RPE cells were established in P12 transwell inserts and cultured for 4 months. Cell pigmentation and barrier function was followed up using optical microscopy and measurement of the transepithelial electrical resistance (TEER). Samples were collected at 21, 91 and 133 days in culture and analysed for gene expression, metal homeostasis of Ca, Na, P, Mg and P, and extracellular deposit formation through RNA sequencing (RNA‐Seq), elemental mass spectrometry (ICP‐MS) and immunocytochemistry assays, respectively. Results: RPE cells formed a differentiated monolayer at 21 days in culture, expressing specific genes and proteins of RPE and giving increasing TEER values. However, after 133 days in culture, loss of tight junction proteins (i.e. CLDN19 and BEST1) and electrical resistance was seen, together with loss of pigmentation and increase of apolipoprotein E deposition in the extracellular space. Gene expression analysis showed an enrichment on apoptotic processes, biomolecule phosphorylation, and signs of epithelial‐to‐mesenchymal transition (EMT). Degeneration of RPE cells concurred with an increase of Na and P content in the cytosol and decrease of Mg, while a decrease of Mg, P and Cu was determined in the insoluble fraction of cells. Time‐dependent changes of metals were accompanied with the alteration of metal transporters and antioxidant enzymes using metals as cofactors. Conclusions: Degeneration of RPE cells in culture was characterized by depigmentation of cells, improper barrier function, downregulation of antioxidant protection, loss of epithelial phenotype, and changes on the levels of Na, P, Mg and Cu, as well as of the expression of their homeostatic proteins. [ABSTRACT FROM AUTHOR]

Aims/Purpose: To study transcriptomic and metabolomic changes during a 6‐month follow‐up of an in vitro model of the retinal pigment epithelium (RPE) producing extracellular deposits. Methods: Primary cultures of human fetal RPE cells were established in P12 transwell inserts for 6 months and harvested at 4, 12, 17 and 25 weeks. Progressive accumulation of extracellular material was determined by apolipoprotein E (APOE) and hydroxyapatite (HAP) localization. Transcriptome was analysed by RNA sequencing (RNAseq) and differential expression analysis was conducted using the platform Dr. Tom. Untargeted metabolomics was carried out by high‐resolution mass spectrometry (LC‐IM‐QTOF) and bioinformatic analysis was performed using Mass Professional Profiler software. Results: RPE cells formed a fully differentiated monolayer at 4 weeks in culture, but tight junctions and electrical resistance decreased from 12 weeks onwards, coinciding with the formation of extracellular deposits containing APOE and HAP. RNAseq showed alterations of cell migration, adhesion and differentiation, and oxidation–reduction, lipid metabolic and apoptotic processes during RPE ageing (4 vs 25 weeks, Q‐value < 0.001). Untargeted metabolomics provided 350 non‐redundant compounds, and principal component analysis showed a clear separation between time points and good reliability within each group (55% of cumulative variance). Moderated t‐test (Benjamin‐Hochberg correction) provided 103 compounds differentially altered during RPE follow‐up (p‐value < 0.001, fold‐change > 2), belonging to trans‐sulfuration, one carbon metabolism, glutathione metabolism, Kennedy pathway for sphingolipids and cysteine and methionine catabolism. Conclusions: Time‐dependent changes were observed in the cell culture model, including progressive sub‐RPE accumulation of APOE and HAP, and alterations in cell adhesion and migration, lipid metabolism and sphingolipids pathway contributing to photoreceptor protection and calcium mobilization. [ABSTRACT FROM AUTHOR]

Purpose: During ageing, the retinal pigment epithelium (RPE) undergoes several morphological and physiological alterations such as loss of melanin, formation of extracellular deposits and metal dyshomeostasis. We examined the age‐related changes in the transcriptome and metallome (metal content in cellular cytosol, membrane and culture media) of the ageing RPE to give an insight into the origin of risk factors linked with neurodegenerative eye diseases like AMD. Methods: We established a primary human RPE cells culture model to study the maturation (10, 21 and 59 days in culture) and ageing (91 and 133 days in culture) of cells in vitro. We analyzed the transcriptome by RNA sequencing (RNAseq) and determined the metallome by inductively coupled plasma mass spectrometry (ICP‐MS). Localization of specific proteins was carried out by immunocytochemistry. Results: RPE differentiation markers increased significantly from 10 to 59 days in culture, followed by a decrease from 91 days onwards, as determined by RNAseq and immunocytochemistry assays. After 59 days in culture, there was an alteration in the expression of genes associated with essential biological processes, some of them being phosphorylation, oxidation‐reduction and ion transport. Metal content (i.e., Na, P, Mg, Ca, Cu and Zn) in the cellular cytosol and membranes showed mixed tendencies with time. Interestingly, cytosolic zinc levels decreased from 10 to 59 days (maturing RPE) but increased at 91 and 133 days (ageing RPE). This biphasic change was mirrored by the gene expression changes of specific isoforms of the zinc‐binding metallothioneins including MT1G and MT2A. Conclusions: Gene expression and metal content showed time‐dependent changes in the in vitro RPE cell culture model. We observed a specific association between zinc levels and metallothionein expressions during maturation and ageing that, if confirmed by further evidences, may shed light for the metal dyshomeostasis observed during AMD. [ABSTRACT FROM AUTHOR]