Transcriptome comparison of primary and immortalized endothelial cells of the human choroid plexus under consideration of the Wnt signaling pathway

Endothelial cells act as important regulators of vascular permeability and angiogenesis, and supply surrounding tissues with required nutrients. They exist in a highly distinct heterogeneity, ensuring optimal adaptation to the different needs of these tissues. The vessels of the human choroid plexus (CP), a cellular convolute located in the brain ventricles whose epithelial cells mainly contribute to the formation of the blood-cerebrospinal fluid (CSF) barrier (BCSFB), have fenestrated endothelial cells. By ectopic expression of human telomerase reverse transcriptase (hTERT) in primary human CP endothelial cells (HCPEnC) we recently generated immortalized HCPEnC (iHCPEnC). To determine the similarity of iHCPEnC to the primary cells in more detail, we compared primary cells of the sixth passage (HCPEnC p6) with a lower (p20) and a higher passage (p50) of iHCPEnC by transcriptome analysis via massive analysis of cDNA ends (MACE). A very high concordance of HCPEnC and iHCPEnC was observed, as only small proportions of the transcripts examined were significantly altered, supporting the suitability of iHCPEnC as an in vitro model of the CP endothelium. Differentially expressed genes (DEG) were identified and assigned to potentially affected biological processes by gene set enrichment analysis (GSEA). Several of the identified DEG were endothelium-relevant, including representatives of the Wnt signaling pathway. Although various components of the Wnt signaling pathway were detected in HCPEnC as well as iHCPEnC, activation of Wnt signaling was only marginal, supporting down-regulation of ß-catenin (CTNNB) signaling in CP endothelial cells. Our results support the suitability of iHCPEnC for use in in vitro models of BCSFB investigations. Overall design: The vasculature of the human choroid plexus is built by human choroid plexus endothelial cells (HCPEnC). To determine whether immortalized HCPEnC (iHCPEnC) exhibit stable gene expression across advanced passages, two different passages were analyzed by MACE. A lower passage of primary HCPEnC (p6; "HCPEnC p6") as well as a low passage of immortalized iHCPEnC (p20; "iHCPEnC p20") and a higher passage of iHCPEnC (p50; "iHCPEnC p50") were examined. Three biological replicates of the primary cells and of each of the two different passages of iHCPEnC were included in the analysis.