Multi‐omic study of a cellular model of the retinal pigment epithelium producing extracellular deposits

Abstract

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.