Since the initial report ( 1), the authors sought to improve the quality and the yield of these iBMECs resulting in three major iterations of the original protocol. ( E) Volcano plots depicting gene ontology of biological processes using the top 100 negative loading genes of PC1, demonstrating expression of genes involved in endothelial cell processes. ( D) Volcano plots depicting gene ontology of biological processes using the top 100 positive loading genes of PC1 demonstrating expression of genes involved in epithelial cell processes. ( C) Principal component analysis plot approximating relative relationship of 109 distinct cell samples across 22 library preparations from previously published and newly generated bulk RNA-sequencing data. Epi-iBMECs generated by our group are molecularly equivalent to those reported in the literature. ( B) Heatmap showing Pearson correlation coefficients between previously reported Epi-iBMEC transcriptomes and Epi-iBMECs generated in the current study. ( A) Schematic diagrams for differentiation of hPSCs to Epi-iBMECs highlighting changes (marked in red) implemented since its initial description as well as differentiation of hPSCs into generic endothelial cells (iECs). Metaanalysis of global high-throughput gene expression profiles reveal Epi-iBMECs possess an epithelial transcriptomic signature. This addition of supportive extracellular physical cues is meant to bolster endothelial cell (EC) maturation and result in a pure population of iBMECs.įig. A human serum-free endothelial base medium is then used to stimulate EC expansion and finally, the heterogenous population of cells is subcultured from Matrigel onto a CollagenIV/Fibronectin matrix. This benchmark protocol relies on performing a sequence of defined steps starting with the differentiation of PSCs into both neural and endothelial lineages. These cells have been reported to meet the need for a reliable and reproducible in vitro model of the human BBB that can be used to screen drugs and understand mechanisms of neurological diseases ( 25, 26). ( 1) which has been both used and modified in various subsequent studies ( 2– 6, 9, 11, 13– 24) ( Fig. Here, we assess the cellular identity of human pluripotent stem cell (hPSC)-derived brain microvascular endothelial cells (iBMECs) possessing blood–brain barrier (BBB) attributes developed by Lippmann et al. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery. Overexpression of key endothelial ETS transcription factors ( ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood–brain barrier (BBB)-like properties has been reported. Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics.
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