Contribution of brain pericytes in blood-brain barrier formation and maintenance: A transcriptomic study of cocultured human endothelial cells derived from hematopoietic stem cells

The formation, maintenance, and repair of the blood-brain barrier (BBB) are critical for central nervous system homeostasis. The interaction of endothelial cells with brain pericytes is known to induce BBB characteristics in brain endothelial cells during embryogenesis and could be used to differentiate human endothelial cells from stem cell source in in vitro BBB models. However, the molecular events involved in BBB maturation are not fully understood. To this end, human endothelial cells derived from hematopoietic stem cells were cultivated with either primary bovine or cell line-derived human brain pericytes to induce BBB formation. Subsequently, the transcriptomic profiles of solocultured vs. cocultured endothelial cells were analyzed over time by the Massive Analysis of cDNA Ends (MACE) technology. This RNA sequencing method is a 3'-end targeted, tag-based, reduced representation transcriptome profiling technique that can reliably quantify all polyadenylated transcripts including those with low expression. By analyzing the generated transcriptomic profiles, we can explore the molecular processes responsible for the functional changes observed in endothelial cells in coculture with brain pericytes (e.g. barrier tightening, changes in the expression of transporters and receptors). Our results identified several up- and downregulated genes and signaling pathways that provide a valuable data source to further delineate complex molecular processes that are involved in BBB formation and BBB maintenance. In addition, this data provides a source to identify novel targets for central nervous system drug delivery strategies. Overall design: 27 samples were analysed by genome-wide gene expression profiling of solo- and cocultured ECs at 0, 24, 48 and 96 hours after putting ECs in coculture (3 biological replicates each consisting of 3 technical replicates) using the MACE method to identify differentially expressed genes upon pericyte introduction.