Heymans et al. Fluids Barriers CNS (2020) 17:48 https://doi.org/10.1186/s12987-020-00208-1 Fluids and Barriers of the CNS RESEARCH Open Access Contribution of brain pericytes in blood– brain barrier formation and maintenance: a transcriptomic study of cocultured human endothelial cells derived from hematopoietic stem cells Marjolein Heymans1, Ricardo Figueiredo2,3, Lucie Dehouck1, David Francisco4, Yasuteru Sano5, Fumitaka Shimizu5, Takashi Kanda5, Rémy Bruggmann4, Britta Engelhardt6, Peter Winter2, Fabien Gosselet1 and Maxime Culot1* Abstract Formation, maintenance, and repair of the blood–brain barrier (BBB) are critical for central nervous system homeo- stasis. The interaction of endothelial cells (ECs) with brain pericytes is known to induce BBB characteristics in brain ECs during embryogenesis and can be used to diferentiate human ECs 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 ECs 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 profles of solocultured vs. cocultured ECs were analysed over time by Massive Analysis of cDNA Ends (MACE) technology. This RNA sequencing method is a 3′-end targeted, tag-based, reduced representation transcriptome profling technique, that can reliably quantify all polyadenylated transcripts including those with low expression. By analysing the generated transcriptomic profles, we can explore the molecular processes responsible for the functional changes observed in ECs in coculture with brain pericytes (e.g. barrier tightening, changes in the expression of transporters and receptors). Our results identifed 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. Keywords: Blood–brain barrier1, Transcriptome2, BBB formation3, In vitro4, Central nervous system5, Brain endothelial cells6, Human hematopoietic stem cells7, Brain pericytes8 Background Brain capillary endothelial cells (ECs) display unique characteristics when compared to ECs from peripheral vasculature, e.g. tight junctions, low pinocytic activity, expression of metabolic enzymes, transporters, receptors *Correspondence: [email protected] 1 Laboratoire de la Barrière Hémato-Encéphalique (LBHE), Univ. Artois, UR and efux pumps [24]. Tese characteristics are known to 2465, 62300 Lens, France be the blood-brain barrier (BBB) phenotype which con- Full list of author information is available at the end of the article stitute the BBB [2, 11]. Te BBB is the interface between © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Heymans et al. Fluids Barriers CNS (2020) 17:48 Page 2 of 28 the systemic circulation and the central nervous system system using the Massive Analysis of cDNA Ends and is essential to maintain brain homeostasis, thereby (MACE) technology. Tis RNA sequencing method is a restricting the entry of many pathogens, toxins and com- 3′-end targeted, tag-based, reduced representation tran- pounds into the brain [1]. Several of these brain capillary scriptome profling technique, that can reliably quantify EC characteristics mentioned above are demonstrated all polyadenylated transcripts including those with low not to be intrinsic to brain ECs, however, they result from expression. By analysing the generated transcriptomic the regulation of cellular and non-cellular factors pro- profles, we can explore the molecular processes respon- duced by diferent cell types of the neurovascular unit sible for the functional changes observed in ECs in cocul- (NVU), e.g. astrocytes, pericytes, neurons, neuroglia and ture with brain pericytes. peripheral immune cells [9, 11, 30]. Te specifc cross- To specifcally focus on the pericyte-EC interaction, talk between brain ECs and brain pericytes is known to we decomposed the model in either solo- or cocultured induce BBB characteristics (e.g. expression and func- ECs. Human ECs were cultivated (in a non-contact set- tionality of tight junction proteins, decreasing leukocyte up) with either human pericytes (CHP) or with bovine adhesion molecule expression, decreasing transcytosis brain pericytes (CBP). In both coculture conditions, the and induction of the basement membrane) in ECs dur- ECs display BBB characteristics like restrictive tight junc- ing embryogenesis in vivo [11, 30]. Pericytes are a type tions, low paracellular permeability to integrity mark- of vascular cells embedded in the basement membrane, ers and functional expression of polarized uptake and thereby they wrap the cerebral capillary walls, with a efux transporters [7]. We subsequently compared the pericyte coverage being the highest in neural tissue [30]. transcriptomic profle of cocultured ECs to the tran- Te latter implicates the importance of pericytes for scriptomic profle of solocultured ECs to delineate the BBB functioning, which is as well indicated by studies transcriptional changes occurring in the ECs during bar- that relate pericytes to barrier function and regulation rier establishment. Besides the transcriptomic data, BBB of infammatory responses [9, 22]. Te pericyte-brain EC functions were assessed by drug accumulation and per- interaction is also used to diferentiate ECs from stem cell meability studies to preliminary validate the physiological source to human brain-like ECs which are used in in vitro relevance of the used in vitro model. BBB models [7, 21, 23, 36]. Tese in vitro models should Transcriptomic profling was done using high-through- display barrier tightening, i.e. induced by coculture, in put mRNA sequencing in combination with the digital order to be of use for pharmaceutical screening. How- gene expression profling technique of GenXPro (Frank- ever, the underlying molecular events involved in devel- furt am Main, Germany), the MACE technology. MACE opment, maturation and maintenance of BBB features, performs gene expression profling by sequencing part of are not fully understood and difcult to study in vivo, the 3′-end of mRNA transcripts. Since each sequenced especially in humans. In particular, the BBB regulation read represents one single mRNA molecule, the MACE related to the communication between pericytes and technique can accurately quantify polyadenylated tran- brain ECs remains largely unknown [5, 15, 22]. scripts using a considerably lower sequencing depth than In the present study, we make use of a human in vitro that of standard RNA-sequencing protocols, for which BBB model developed by Cecchelli et al. [7] consisting in the number of fragments per transcript depends on the ECs derived from hematopoietic stem cells which are co- length of the transcript. cultivated with brain pericytes. After 5 days of coculture Our results provide a transcriptomic landscape of with brain pericytes, the ECs were shown to display fea- human brain-like ECs in solo- or coculture with brain tures of the BBB which were absent when the cells were pericytes that was used to identify interesting gene pro- cultivated alone: the co-cultivated ECs display a continu- fles over time, soloculture enriched transcripts, cocul- ous expression of ZO-1, occludin, JAM-A, claudin-1 and ture enriched transcripts, etc., which might prove to be claudin-5 at cell–cell contacts resulting in a lower perme- valuable in the further delineation of complex molecular ability to non-permeant marker than when the cells were processes involved in BBB formation and regulation. Te solocultivated. Tese cocultivated ECs also express sev- transcriptomic profle could also be used as a source for eral transporters typically observed in brain endothelium novel targets for central nervous system drug delivery in vivo (e.g. ABCB1 and ABCG2) [7]. strategies. To study the molecular processes responsible for the observed changes in ECs (i.e. barrier tightening, changes Materials and methods in the expression of transporters and receptors) in cocul- Compounds −1 ture with brain pericytes of ECs derived from hematopoi- Te compounds lucifer yellow (LY; Mw = 457.25 g mol ), −1 etic stem cells cultivated with brain pericytes from either rhodamine 123 (R123; Mw = 380.82 g mol ) and −1 primary bovine or cell line human origin in a Transwell