The derivation of human brain capillary endothelial cells is of utmost importance for drug discovery programs focusing on diseases of the central nervous system. Here, we describe a two-step differentiation protocol to derive brain capillary-like endothelial cells from human pluripotent stem cells. The cells were initially differentiated into endothelial progenitor cells followed by specification into a brain capillary-like endothelial cell phenotype using a protocol that combined the induction, in a time-dependent manner, of VEGF, Wnt3a, and retinoic acid signaling pathways and the use of fibronectin as the extracellular matrix. The brain capillary-like endothelial cells displayed a permeability to lucifer yellow of 1 x 10(-3) cm/min, a transendothelial electrical resistance value of 60 Omega cm(2) and were able to generate a continuous monolayer of cells expressing ZO-1 and CLAUDIN-5 but moderate expression of P-glycoprotein. Further maturation of these cells required coculture with pericytes. The study presented here opens a new approach for the study of soluble and non-soluble factors in the specification of endothelial progenitor cells into brain capillary-like endothelial cells.

Stimulation of adult neurogenesis by targeting the endogenous neural stem cells (NSCs), located in hippocampus and subventricular zone (SVZ), with nanoformulations has been proposed for brain repair in cases of neurodegenerative diseases. Unfortunately, it is relatively unknown the nanoformulation properties to facilitate their accumulation in the neurogenic niches after intravenous injection. Here, we have screened different gold-based formulations having variable morphology, surface chemistry and responsiveness to light for their capacity to cross the blood brain barrier (BBB) and accumulate preferentially in the neurogenic niches. Results obtained in a human in vitro BBB model showed that gold nanoparticles (Au NPs) and gold nanorods (Au NRs) conjugated with medium density of transferrin (Tf) peptides (i.e. between 169 and 230 peptides per NP) crossed more efficiently the BBB than the remaining formulations. This is due to a relatively lower avidity of these formulations to Tf receptor (TfR) and lower accumulation in the lysosomes, as compared to the other formulations. We further show that the near infrared light (NIR) irradiation of Au NRs, under a certain concentration and at specific cell culture time, lead to the opening of the BBB. Finally, we demonstrate that Au NRs conjugated with Tf administered intravenously in mice and activated by NIR had the highest accumulation in the neurogenic niches. Our results open the possibility of targeting more effectively the neurogenic niches by controlling the properties of the nanoformulations.

The alkaloid piperine from black pepper (Piper nigrum L.) and several synthetic piperine analogs were recently identified as positive allosteric modulators of γ-aminobutyric acid type A (GABAA) receptors. In order to reach their target sites of action, these compounds need to enter the brain by crossing the blood–brain barrier (BBB). We here evaluated piperine and five selected analogs (SCT-66, SCT-64, SCT-29, LAU397, and LAU399) regarding their BBB permeability. Data were obtained in three in vitro BBB models, namely a recently established human model with immortalized hBMEC cells, a human brain-like endothelial cells (BLEC) model, and a primary animal (bovine endothelial/rat astrocytes co-culture) model. For each compound, quantitative UHPLC-MS/MS methods in the range of 5.00–500 ng/mL in the corresponding matrix were developed, and permeability coefficients in the three BBB models were determined. In vitro predictions from the two human BBB models were in good agreement, while permeability data from the animal model differed to some extent, possibly due to protein binding of the screened compounds. In all three BBB models, piperine and SCT-64 displayed the highest BBB permeation potential. This was corroborated by data from in silico prediction. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human BBB models, and moderate to high in the animal BBB model. Efflux ratios (ER) calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux transporters.

Around 7-17% of metastatic breast cancer patients will develop brain metastases, associated with a poor prognosis. To reach the brain parenchyma, cancer cells need to cross the highly restrictive endothelium of the Blood-Brain Barrier (BBB). As treatments for brain metastases are mostly inefficient, preventing cancer cells to reach the brain could provide a relevant and important strategy. For that purpose an in vitro approach is required to identify cellular and molecular interaction mechanisms between breast cancer cells and BBB endothelium, notably at the early steps of the interaction. However, while numerous studies are performed with in vitro models, the heterogeneity and the quality of BBB models used is a limitation to the extrapolation of the obtained results to in vivo context, showing that the choice of a model that fulfills the biological BBB characteristics is essential. Therefore, we compared pre-established and currently used in vitro models from different origins (bovine, mice, human) in order to define the most appropriate tool to study interactions between breast cancer cells and the BBB. On each model, the BBB properties and the adhesion capacities of breast cancer cell lines were evaluated. As endothelial cells represent the physical restriction site of the BBB, all the models consisted of endothelial cells from animal or human origins. Among these models, only the in vitro BBB model derived from human stem cells both displayed BBB properties and allowed measurement of meaningful different interaction capacities of the cancer cell lines. Importantly, the measured adhesion and transmigration were found to be in accordance with the cancer cell lines molecular subtypes. In addition, at a molecular level, the inhibition of ganglioside biosynthesis highlights the potential role of glycosylation in breast cancer cells adhesion capacities.

Event Abstract Back to Event Derivation of a human in vitro BBB model from hiPSCs Catarina Praça1, 2, 3, Susana C. Rosa4, Romeo Cecchelli3, Marie-Pierre Dehouck3 and Lino S. Ferreira2, 4 1 CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Doctoral Programme in Experimental Biology and Biomedicine (PDBEB), University of Coimbra, Portugal 2 Institute for Interdisciplinary Research (IIIUC), University of Coimbra, Portugal 3 Faculté des Sciences Jean Perrin, Université d´Artois, France 4 CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal Introduction: The barriers of the central nervous system are essential protectors of the brain from the rest of the body. The blood-brain barrier (BBB) is the most selective one, being the major responsible for keeping the neuronal microenvironment homeostasis. Using BBB models is possible to make reliable predictions of drug-BBB interactions and also to study biological and pathological aspects of the barrier. Recently we have generated a stable and reproducible human in vitro BBB model derived from cord blood hematopoietic stem cells[1]. The cells were initially differentiated into endothelial cells (ECs) followed by the induction of BBB properties by co-culture with bovine pericytes. The brain-like endothelial cells (BLECs) expressed tight junctions and transporters typically observed in brain endothelium and maintained the expression of most BBB properties for at least 20 days [1]. However, so far, the derivation of disease models of human BBB has not been reported. Induced pluripotent stem cells (iPSCs) represent a promising source of BLECs with specific disease phenotypes such as Alzheimer, Parkinson and other neurodegenerative diseases. This is not currently possible using the human BBB system developed by us from human cord blood hematopoietic stem cells. Here, we describe a procedure to derive BLECs from iPSCs using well-characterized progenitor cells. We further document the effect of extracellular matrix (ECM) and soluble factors in this inductive process, and we report their functional activity. Results and Discussion: Vascular progenitor cells isolated from iPSCs express high levels of CD31 (~ 91%) and GLUT-1 (~ 91%), moderate levels of claudin-5 (~ 26%), occludin (~ 11%) and ZO-1 (~ 24%), and low levels of P-gp (~ 1%). Immunocytochemistry results showed that the expression of occludin and claudin-5 is not entirely at cell junctions, disclosing an immature BBB phenotype. These cells were matured for 4 passages in specific cell media conditions (not disclosed) and specific native decellularized matrices. Along the maturation process, there is an increase in the co-localization of the CD31 marker with some of the BBB markers (Claudin-5, ZO-1 and PgP), suggesting a specification for the BBB phenotype. Functionally, these cells when cultured in the transwell systems, present relatively low permeability to Lucifer yellow (1.24±0.14 ×10-3 cm/min), TEER values of 55±0.58Ωcm2 and are able to generate a continuous monolayer presenting ZO-1 and claudin-5 in the cell membrane. Conclusion: In this work we report a methodology to differentiate hiPSCs into ECs with BBB properties (BLECs). Our results further highlight the importance of spcefic soluble factors and neurovascular unit ECM in the differentiation of vascular progenitor cells. FCT Fundação para a Ciência e Tecnologia (SFRH/BD/51678/2011); FCT Fundação para a Ciência e Tecnologia (PTDC/SAU-TOX/121); COMPETE funding (Project “Stem cell based platforms for Regenerative and Therapeutic Medicine”, Centro-07-ST24-FEDER-002008)References:[1] Cecchelli, R., et al. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells. PLoS One 9, e99733 (2014). Keywords: Cell Differentiation, Extracellular Matrix, stem cell, modeling Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Synthetic scaffolds as extracellular matrices Citation: Praça C, Rosa SC, Cecchelli R, Dehouck M and Ferreira LS (2016). Derivation of a human in vitro BBB model from hiPSCs. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02905 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Catarina Praça Susana C Rosa Romeo Cecchelli Marie-Pierre Dehouck Lino S Ferreira Google Catarina Praça Susana C Rosa Romeo Cecchelli Marie-Pierre Dehouck Lino S Ferreira Google Scholar Catarina Praça Susana C Rosa Romeo Cecchelli Marie-Pierre Dehouck Lino S Ferreira PubMed Catarina Praça Susana C Rosa Romeo Cecchelli Marie-Pierre Dehouck Lino S Ferreira Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This "blood-brain barrier" function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood-brain barrier models with a focus on their validation regarding a set of well-established blood-brain barrier characteristics. As an ideal cell culture model of the blood-brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described.

Significant BBB differences between species. Derivation of human BBB models from human pluripotent stem cells and adult stem/progenitor cells. Derivation of pathological BBB models from human induced pluripotent stem cells. BBB models are useful to study the permeability of drugs and nanoformulations. The development of novel neuropharmaceuticals requires the evaluation of blood–brain barrier (BBB) permeability and toxicity. Recent studies have highlighted differences in the BBB among different species, with the most important differences involving the expression of P-glycoprotein (P-gp), multidrug resistance-associated proteins, transporters, and claudins. In addition, functional studies have shown that brain pharmacokinetics of P-glycoprotein substrates are different in humans and rodents. Therefore, human BBB models may be an important platform for initial drug screening before in vivo studies. This strategy might help to reduce costs in drug development and failures in clinical studies. We review the differences in the BBB among species, recent advances in the generation of human BBB models, and their applications in drug discovery and delivery. The development of novel neuropharmaceuticals requires the evaluation of blood–brain barrier (BBB) permeability and toxicity. Recent studies have highlighted differences in the BBB among different species, with the most important differences involving the expression of P-glycoprotein (P-gp), multidrug resistance-associated proteins, transporters, and claudins. In addition, functional studies have shown that brain pharmacokinetics of P-glycoprotein substrates are different in humans and rodents. Therefore, human BBB models may be an important platform for initial drug screening before in vivo studies. This strategy might help to reduce costs in drug development and failures in clinical studies. We review the differences in the BBB among species, recent advances in the generation of human BBB models, and their applications in drug discovery and delivery. BBB models are important tools with which to study both the transport mechanism of neuropharmaceuticals from the blood to the brain and the barrier from fundamental and pathological points of view [1Cecchelli R. et al.Modelling of the blood–brain barrier in drug discovery and development.Nat. Rev. Drug Discov. 2007; 6: 650-661Crossref PubMed Scopus (487) Google Scholar]. In the central nervous system (CNS) drug industry, very few small molecules [typically with a molecular mass (Mr) below 400–500 Da] can cross the BBB [2Pardridge W.M. Blood–brain barrier drug targeting: the future of brain drug development.Mol. Interv. 2003; 3: 90-105Crossref PubMed Scopus (598) Google Scholar]. In a study evaluating more than 7000 drugs, only 5% could cross the BBB and affect the CNS [3Ghose A.K. et al.A knowledge-based approach in designing combinatorial or medicinal chemistry libraries for drug discovery. 1. A qualitative and quantitative characterization of known drug databases.J. Comb. Chem. 1999; 1: 55-68Crossref PubMed Scopus (1689) Google Scholar]. The development of more effective neuropharmaceuticals that can cross the BBB requires a better understanding of the expression and functionality of transporters in the human and rodent BBBs because rodents are typically used in preclinical assays. Quantitative-targeted absolute proteomics (QTAP) studies showed pronounced differences between rodents and primates in the expression of transporters, tight junctions, and receptors [4Hoshi Y. et al.Quantitative atlas of blood–brain barrier transporters, receptors, and tight junction proteins in rats and common marmoset.J. Pharm. Sci. 2013; 102: 3343-3355Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 5Ito K. et al.Quantitative membrane protein expression at the blood–brain barrier of adult and younger cynomolgus monkeys.J. Pharm. Sci. 2011; 100: 3939-3950Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 6Shawahna R. et al.Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels.Mol. Pharm. 2011; 8: 1332-1341Crossref PubMed Scopus (285) Google Scholar, 7Uchida Y. et al.Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors.J. Neurochem. 2011; 117: 333-345Crossref PubMed Scopus (610) Google Scholar, 8Uchida Y. et al.A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice.Fluids Barriers CNS. 2013; 10: 21Crossref PubMed Scopus (161) Google Scholar]. In addition, positron emission tomography (PET) showed significant differences among species (rats, guinea pigs, minipigs, monkeys, and humans) in the brain pharmacokinetics of P-gp substrate radioligands [9Syvanen S. et al.Species differences in blood–brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport.Drug Metab. Dispos. 2009; 37: 635-643Crossref PubMed Scopus (274) Google Scholar]. PET functional studies indicated that the human BBB is more permeable than the rodent BBB to P-gp substrates. The differences observed between primates and rodents were likely due to a combination of factors such as species-related differences in P-gp transport (turnover of the protein) and variations in the distribution of the molecules within the brain [9Syvanen S. et al.Species differences in blood–brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport.Drug Metab. Dispos. 2009; 37: 635-643Crossref PubMed Scopus (274) Google Scholar]. CNS-targeting drug discovery programs in the pharmaceutical industry generally start by the screening of a large library of compounds against a specific molecular target, followed by an analysis of the absorption, distribution, metabolism, and excretion (ADME) properties of the drug [10Lipinski C.A. Drug-like properties and the causes of poor solubility and poor permeability.J. Pharmacol. Toxicol. Methods. 2000; 44: 235-249Crossref PubMed Scopus (2776) Google Scholar]. Screening is typically performed in non-primate cells, and the ADME analyses are performed in rodents and then extrapolated to humans [11Hsiao P. et al.Verapamil P-glycoprotein transport across the rat blood–brain barrier: cyclosporine, a concentration inhibition analysis, and comparison with human data.J. Pharm. Exp. Ther. 2006; 317: 704-710Crossref PubMed Scopus (85) Google Scholar, 12Lee Y.J. et al.In vivo evaluation of P-glycoprotein function at the blood–brain barrier in nonhuman primates using [11C]verapamil.J. Pharmacol. Exp. Ther. 2006; 316: 647-653Crossref PubMed Scopus (94) Google Scholar]. However, these extrapolations may be affected by the significant differences among species in the expression of transporters, receptors, and tight junction proteins (see below) which influence BBB permeability. Thus, it is very important to complement the results obtained in animals with the results collected from in vitro human BBB models [9Syvanen S. et al.Species differences in blood–brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport.Drug Metab. Dispos. 2009; 37: 635-643Crossref PubMed Scopus (274) Google Scholar, 13Lippmann E.S. et al.Derivation of blood–brain barrier endothelial cells from human pluripotent stem cells.Nat. Biotechnol. 2012; 30: 783-791Crossref PubMed Scopus (483) Google Scholar]. The goal of this review is to highlight recent advances in the generation and use of stem cell-based human BBB models for drug discovery and neuropharmaceutical transport, and to summarize the differences between human and non-human BBB. A review on this topic of research is justified by recent advances (during the past 5 years) in the evaluation of the BBB differences among different species [4Hoshi Y. et al.Quantitative atlas of blood–brain barrier transporters, receptors, and tight junction proteins in rats and common marmoset.J. Pharm. Sci. 2013; 102: 3343-3355Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 5Ito K. et al.Quantitative membrane protein expression at the blood–brain barrier of adult and younger cynomolgus monkeys.J. Pharm. Sci. 2011; 100: 3939-3950Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 7Uchida Y. et al.Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors.J. Neurochem. 2011; 117: 333-345Crossref PubMed Scopus (610) Google Scholar, 8Uchida Y. et al.A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice.Fluids Barriers CNS. 2013; 10: 21Crossref PubMed Scopus (161) Google Scholar, 9Syvanen S. et al.Species differences in blood–brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport.Drug Metab. Dispos. 2009; 37: 635-643Crossref PubMed Scopus (274) Google Scholar] and in the creation of new human cell models [13Lippmann E.S. et al.Derivation of blood–brain barrier endothelial cells from human pluripotent stem cells.Nat. Biotechnol. 2012; 30: 783-791Crossref PubMed Scopus (483) Google Scholar, 14Boyer-Di Ponio J. et al.Instruction of circulating endothelial progenitors in vitro towards specialized blood–brain barrier and arterial phenotypes.PLoS ONE. 2014; 9: e84179Crossref PubMed Scopus (79) Google Scholar, 15Cecchelli R. et al.A stable and reproducible human blood–brain barrier model derived from hematopoietic stem cells.PLoS ONE. 2014; 9: e99733Crossref PubMed Scopus (216) Google Scholar, 16Lippmann E.S. et al.A retinoic acid-enhanced, multicellular human blood–brain barrier model derived from stem cell sources.Sci. Rep. 2014; 4: 4160Crossref PubMed Scopus (308) Google Scholar], particularly models derived from stem cells. Among the barriers of the brain (Figure 1), the BBB is the most closely scrutinized [17Ballabh P. et al.The blood–brain barrier: an overview: structure, regulation, and clinical implications.Neurobiol. Dis. 2004; 16: 1-13Crossref PubMed Scopus (1627) Google Scholar, 18Cardoso F.L. et al.Looking at the blood–brain barrier: molecular anatomy and possible investigation approaches.Brain Res. Rev. 2010; 64: 328-363Crossref PubMed Scopus (443) Google Scholar, 19Gabathuler R. Approaches to transport therapeutic drugs across the blood–brain barrier to treat brain diseases.Neurobiol. Dis. 2010; 37: 48-57Crossref PubMed Scopus (651) Google Scholar, 20Liddelow S.A. Fluids and barriers of the CNS: a historical viewpoint.Fluids Barriers CNS. 2011; 8: 2Crossref PubMed Scopus (58) Google Scholar, 21Wilhelm I. et al.In vitro models of the blood–brain barrier.Acta Neurobiol. Exp. 2011; 71: 113-128PubMed Google Scholar]. The BBB is an active and complex diffusion barrier between the blood and the CNS, transporting nutrients essential for the normal metabolism of brain cells [19Gabathuler R. Approaches to transport therapeutic drugs across the blood–brain barrier to treat brain diseases.Neurobiol. Dis. 2010; 37: 48-57Crossref PubMed Scopus (651) Google Scholar, 21Wilhelm I. et al.In vitro models of the blood–brain barrier.Acta Neurobiol. Exp. 2011; 71: 113-128PubMed Google Scholar] while providing protection against many toxic compounds and pathogens [18Cardoso F.L. et al.Looking at the blood–brain barrier: molecular anatomy and possible investigation approaches.Brain Res. Rev. 2010; 64: 328-363Crossref PubMed Scopus (443) Google Scholar]. The BBB is maintained by a complex crosstalk between brain endothelial cells (BECs) and cells from the neurovascular unit (pericytes, astrocytes, microglia, neurons) [22Abbott N.J. et al.Astrocyte-endothelial interactions at the blood–brain barrier.Nat. Rev. Neurosci. 2006; 7: 41-53Crossref PubMed Scopus (3769) Google Scholar, 23Hawkins B.T. Davis T.P. The blood–brain barrier/neurovascular unit in health and disease.Pharmacol. Rev. 2005; 57: 173-185Crossref PubMed Scopus (1979) Google Scholar] (Figure 1). The morphological and functional characteristics of BECs in the BBB differ from endothelial cells (ECs) in peripheral vessels. Peripheral capillaries have pores between the cells that are normally 6–7 nm in size [24Deeken J.F. Loscher W. The blood–brain barrier and cancer: transporters, treatment, and Trojan horses.Clin. Cancer Res. 2007; 13: 1663-1674Crossref PubMed Scopus (552) Google Scholar]. Brain capillaries are 50–100-fold tighter than peripheral capillaries and thus have lower paracellular permeability to hydrophilic solutes [25Abbott N.J. Astrocyte–endothelial interactions and blood–brain barrier permeability.J. Anat. 2002; 200: 629-638Crossref PubMed Scopus (920) Google Scholar]. Intercellular junctions (tight junctions and adherens junctions) between adjacent cells contribute to the tightness of the BBB. BECs also differ from ECs of peripheral vessels in the morphology of the cytoplasm, the intracellular trafficking and the expression of specific transporters. The BEC has a cytoplasm with uniform thickness, no fenestrations, very low pinocytotic activity, and contains specific transporters and enzymes such as γ-glutamyl transpeptidase and alkaline phosphatase [26Joo F. Endothelial cells of the brain and other organ systems: some similarities and differences.Prog. Neurobiol. 1996; 48: 255-273Crossref PubMed Scopus (113) Google Scholar, 27Wolburg H. Lippoldt A. Tight junctions of the blood–brain barrier: development, composition and regulation.Vascul. Pharmacol. 2002; 38: 323-337Crossref PubMed Scopus (905) Google Scholar]. Tight junctions between BECs form a diffusion barrier, which strongly restricts penetration of water-soluble compounds into the brain (Figure 1) [24Deeken J.F. Loscher W. The blood–brain barrier and cancer: transporters, treatment, and Trojan horses.Clin. Cancer Res. 2007; 13: 1663-1674Crossref PubMed Scopus (552) Google Scholar]. Occludin and claudins make important contributions to tight junction structure, whereas junctional adhesion molecules (JAM-1, -2, -3; IgG superfamily proteins) and cytoplasmic tight junction accessory proteins [zonula occludens-1, -2, and -3 (ZO-1, -2, -3), AF-6, 7H6, and cingulin] are involved in the formation and maintenance of the tight junctions [18Cardoso F.L. et al.Looking at the blood–brain barrier: molecular anatomy and possible investigation approaches.Brain Res. Rev. 2010; 64: 328-363Crossref PubMed Scopus (443) Google Scholar, 22Abbott N.J. et al.Astrocyte-endothelial interactions at the blood–brain barrier.Nat. Rev. Neurosci. 2006; 7: 41-53Crossref PubMed Scopus (3769) Google Scholar]. As a consequence of the tight junctions between BECs, brain capillaries have a high transendothelial electrical resistance (TEER) (1000–2000 Ω.cm2) compared with the TEER of peripheral capillaries (10 Ω.cm2) [24Deeken J.F. Loscher W. The blood–brain barrier and cancer: transporters, treatment, and Trojan horses.Clin. Cancer Res. 2007; 13: 1663-1674Crossref PubMed Scopus (552) Google Scholar, 28Butt A.M. et al.Electrical resistance across the blood–brain barrier in anaesthetized rats: a developmental study.J. Physiol. 1990; 429: 47-62Crossref PubMed Scopus (603) Google Scholar, 29Hirase T. et al.Occludin as a possible determinant of tight junction permeability in endothelial cells.J. Cell Sci. 1997; 110: 1603-1613Crossref PubMed Google Scholar]. This high TEER prevents polar and ionic substances from entering the brain [24Deeken J.F. Loscher W. The blood–brain barrier and cancer: transporters, treatment, and Trojan horses.Clin. Cancer Res. 2007; 13: 1663-1674Crossref PubMed Scopus (552) Google Scholar, 28Butt A.M. et al.Electrical resistance across the blood–brain barrier in anaesthetized rats: a developmental study.J. Physiol. 1990; 429: 47-62Crossref PubMed Scopus (603) Google Scholar]. In addition, BECs in brain capillaries have higher volumes of mitochondria owing to the enhanced energy requirements of the active transport systems and additional enzymatic aspects, all of which serve to protect the brain [19Gabathuler R. Approaches to transport therapeutic drugs across the blood–brain barrier to treat brain diseases.Neurobiol. Dis. 2010; 37: 48-57Crossref PubMed Scopus (651) Google Scholar, 24Deeken J.F. Loscher W. The blood–brain barrier and cancer: transporters, treatment, and Trojan horses.Clin. Cancer Res. 2007; 13: 1663-1674Crossref PubMed Scopus (552) Google Scholar]. BEC polarity is a key feature of the BBB (Figure 1). This polarity is necessary for the transcellular transport of solutes from one side of the cell to the other. Transporters for small hydrophilic molecules necessary for the survival of brain cells (amino acids, glucose, etc.) are located both in the luminal (apical) and abluminal (basolateral) sides of BECs [22Abbott N.J. et al.Astrocyte-endothelial interactions at the blood–brain barrier.Nat. Rev. Neurosci. 2006; 7: 41-53Crossref PubMed Scopus (3769) Google Scholar]. However, some transporters and enzymes are located preferentially in one side of the cell. For example, K+-dependent p-nitrophenyl phosphatase is located in the luminal but not abluminal side of the BEC, whereas Na+/K+-ATPase is located in the abluminal but not the luminal side [30Betz A.L. et al.Polarity of the blood–brain barrier: distribution of enzymes between the luminal and antiluminal membranes of brain capillary endothelial cells.Brain Res. 1980; 192: 17-28Crossref PubMed Scopus (272) Google Scholar]. Moreover, relevant differences have been found in the lipid composition of the luminal and abluminal cell membranes of the BECs. For example, the main phospholipid in the luminal membrane is phosphatidylcholine, whereas the main phospholipid in the abluminal membrane is sphingomyelin [31Tewes B.J. Galla H-J. Membrane fractionation of brain capillary endothelial cells and analysis of lipid polarity.in: Couraud P. Scherman D. Biology and Physiology of the Blood–Brain Barrier. Plenum Press, 1996: 97-101Crossref Google Scholar]. Only recently, the differences in the BBBs of rodents and primates have been highlighted (Table 1). BBB permeability-related transporters, receptors, and tight junction proteins were analyzed using liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based QTAP to study inter-species (between rats and marmoset) and intra-species (between Sprague–Dawley and Wistar rats) differences [4Hoshi Y. et al.Quantitative atlas of blood–brain barrier transporters, receptors, and tight junction proteins in rats and common marmoset.J. Pharm. Sci. 2013; 102: 3343-3355Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar]. In this method, a peptide probe specific for each target molecule was used to quantify its level of expression with high sensitivity and accuracy [7Uchida Y. et al.Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors.J. Neurochem. 2011; 117: 333-345Crossref PubMed Scopus (610) Google Scholar]. For many functional membrane proteins, the levels of expression correlate with the levels of activity.Table 1Protein Expression Amounts (fmol/μg Protein) in Different SpeciesaProtein expression levels are shown as means±SEM. Abbreviations: ABC, ATP-binding cassette; BCRP, breast cancer resistance protein; GLUT1, glucose transporter 1; INSR, insulin receptor; LAT1, L-type amino-acid transporter 1; LRP1, low-density lipoprotein receptor-related protein 1; MCT1, monocarboxylate transporter 1; ND, not determined; OAT3, organic anion transporter 3; MDR1, multidrug resistance protein 1; MRP4, multidrug resistance-associated protein 4; SLC, solute carrier; TfR1, transferrin receptor 1; ULQ, under limit of quantification – the quantification limit is indicated in parentheses (fmol/μg protein).,bMouse data, Uchida et al. [8]; rat and marmoset data, Hoshi et al. [4]; monkey data, Ito et al. [5]; Human data, Uchida et al. [7].Protein Symbol/AliasddY MouseFVB MouseC57BL/6J MouseSprague–Dawley (SD) RatWistar RatMarmosetIndonesian Adult MonkeyChinese Adult MonkeyHumanABC TransportersABCB1/MDR1/P-gp16.40 ± 1.3014.20 ± 1.6017.80 ± 1.2019.00 ± 2.0019.20 ± 1.106.48 ± 1.31 (peptide 1)6.31 ± 1.08 (peptide 2)2.65 ± 0.12 (peptide 1)ND (peptide 2)5.12 ± 0.91 (peptide 1)6.24 ± 1.01 (peptide 2)6.06 ± 1.69ABCC4/MRP41.33 ± 0.141.27 ± 0.211.51 ± 0.271.60 ± 0.291.46 ± 0.080.32 ± 0.060.20 ± 0.040.30 ± 0.010.19 ± 0.07ABCG2/BCRP3.74 ± 0.323.21 ± 0.495.48 ± 0.374.15 ± 0.295.74 ± 0.5016.50 ± 1.4014.10 ± 0.30 (peptide 1)ND (peptide 2)14.20 ± 1.40 (peptide 1)14.40 ± 1.20 (peptide 2)8.14 ± 2.26SLC TransportersSLC2A1/GLUT182.10 ± 3.0090.90 ± 3.90101.00 ± 4.0084.00 ± 4.1098.20 ± 7.00145.00 ± 20.00118.00 ± 7.00131.00 ± 22.00139.00 ± 46.00SLC7A5/LAT12.54 ± 1.552.11 ± 0.821.17 ± 0.363.41 ± 0.742.58 ± 0.84NDULQ (0.33)ULQ (0.33)0.43 ± 0.09SLC16A1/MCT117.30 ± 1.3019.90 ± 1.0013.70 ± 0.5011.60 ± 0.6013.50 ± 0.803.04 ± 0.351.15 ± 0.220.75 ± 0.372.27 ± 0.85SLC22A8/OAT31.78 ± 0.151.65 ± 0.522.29 ± 0.402.13 ± 0.491.37 ± 0.18NDULQ (0.40)ULQ (0.40)ULQ (0.35)Tight JunctionsClaudin-56.16 ± 0.205.50 ± 0.498.07 ± 1.47NDND8.03 ± 0.984.01 ± 0.227.17 ± 0.773.62 ± 0.96cShawahna et al. [6].ReceptorsINSR0.74 ± 0.210.64 ± 0.011.13 ± 0.180.78 ± 0.111.15 ± 0.340.66 ± 0.161.84 ± 0.101.46 ± 0.221.09 ± 0.21LRP11.36 ± 0.420.98 ± 0.071.37 ± 0.331.09 ± 0.141.16 ± 0.21ND1.30 ± 0.081.29 ± 0.051.51 ± 0.26TfR14.34 ± 0.813.89 ± 0.665.22 ± 0.476.74 ± 0.398.93 ± 1.16NDNDND2.34 ± 0.76a Protein expression levels are shown as means ± SEM. Abbreviations: ABC, ATP-binding cassette; BCRP, breast cancer resistance protein; GLUT1, glucose transporter 1; INSR, insulin receptor; LAT1, L-type amino-acid transporter 1; LRP1, low-density lipoprotein receptor-related protein 1; MCT1, monocarboxylate transporter 1; ND, not determined; OAT3, organic anion transporter 3; MDR1, multidrug resistance protein 1; MRP4, multidrug resistance-associated protein 4; SLC, solute carrier; TfR1, transferrin receptor 1; ULQ, under limit of quantification – the quantification limit is indicated in parentheses (fmol/μg protein).b Mouse data, Uchida et al. 8Uchida Y. et al.A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice.Fluids Barriers CNS. 2013; 10: 21Crossref PubMed Scopus (161) Google Scholar; rat and marmoset data, Hoshi et al. 4Hoshi Y. et al.Quantitative atlas of blood–brain barrier transporters, receptors, and tight junction proteins in rats and common marmoset.J. Pharm. Sci. 2013; 102: 3343-3355Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar; monkey data, Ito et al. 5Ito K. et al.Quantitative membrane protein expression at the blood–brain barrier of adult and younger cynomolgus monkeys.J. Pharm. Sci. 2011; 100: 3939-3950Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar; Human data, Uchida et al. 7Uchida Y. et al.Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors.J. Neurochem. 2011; 117: 333-345Crossref PubMed Scopus (610) Google Scholar.c Shawahna et al. 6Shawahna R. et al.Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels.Mol. Pharm. 2011; 8: 1332-1341Crossref PubMed Scopus (285) Google Scholar. Open table in a new tab Proteomic studies in different species have shown that, with regard to the expression of P-gp protein, the human BBB (6.06 ± 1.69 fmol/μg total protein) is closer to cynomolgus monkey (4.71 ± 1.30 fmol/μg total protein) and marmoset BBBs (6.48 ± 1.31 fmol/μg total protein) than to mouse (14.1 ± 2.1 fmol/μg total protein) or rat (19.1 ± 1.00 fmol/μg total protein) BBBs [4Hoshi Y. et al.Quantitative atlas of blood–brain barrier transporters, receptors, and tight junction proteins in rats and common marmoset.J. Pharm. Sci. 2013; 102: 3343-3355Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 5Ito K. et al.Quantitative membrane protein expression at the blood–brain barrier of adult and younger cynomolgus monkeys.J. Pharm. Sci. 2011; 100: 3939-3950Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 7Uchida Y. et al.Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors.J. Neurochem. 2011; 117: 333-345Crossref PubMed Scopus (610) Google Scholar, 32Kamiie J. et al.Quantitative atlas of membrane transporter proteins: development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria.Pharm. Res. 2008; 25: 1469-1483Crossref PubMed Scopus (416) Google Scholar] (Table 1). For these assays, human BECs were collected from different donors and BEC purity was monitored by the levels of γ-glutamyl transpeptidase. Because transporter expression is not necessarily correlated with activity/function [33Lescale-Matys L. et al.Regulation of the ovine intestinal Na+/glucose co-transporter (SGLT1) is dissociated from mRNA abundance.Biochem. J. 1993; 291: 435-440Crossref PubMed Scopus (120) Google Scholar, 34Molina-Arcas M. et al.Equilibrative nucleoside transporter-2 (hENT2) protein expression correlates with ex vivo sensitivity to fludarabine in chronic lymphocytic leukemia (CLL) cells.Leukemia. 2005; 19: 64-68Crossref PubMed Scopus (60) Google Scholar], functional methods such as PET and single-photon emission computed tomography (SPECT) have recently been used to quantitatively evaluate BBB permeability to compounds in humans. These nuclear imaging methods evaluate the density and function of molecular targets in vivo by externally monitoring the distribution of intravenously administered, target-selective radiotracers in the tissue. The activity of P-gp, a protein that is responsible for the efflux of many compounds from BECs to the blood, has been evaluated using PET and various radioligands in multiple species [9Syvanen S. et al.Species differences in blood–brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport.Drug Metab. Dispos. 2009; 37: 635-643Crossref PubMed Scopus (274) Google Scholar, 12Lee Y.J. et al.In vivo evaluation of P-glycoprotein function at the blood–brain barrier in nonhuman primates using [11C]verapamil.J. Pharmacol. Exp. Ther. 2006; 316: 647-653Crossref PubMed Scopus (94) Google Scholar, 35Elsinga P.H. et al.PET Studies on P-glycoprotein function in the blood–brain barrier: how it affects uptake and binding of drugs within the CNS.Curr. Pharm. 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PET studies have shown that the penetration efficiencies of P-gp substrates [18F]-altanserin and [11C] -R205171 were 4.5- and 8.6-fold greater in humans than in rodents, respectively [9Syvanen S. et al.Species differences in blood–brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport.Drug Metab. Dispos. 2009; 37: 635-643Crossref PubMed Scopus (274) Google Scholar]. These results can be explained by lower P-gp function in the human BBB than in rodent BBBs. Proteomic studies in mouse, rat, cynomolgus monkey, and human [4Hoshi Y. et al.Quantitative atlas of blood–brain barrier transporters, receptors, and tight junction proteins in rats and common marmoset.J. Pharm. Sci. 2013; 102: 3343-3355Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 5Ito K. et al.Quantitative membrane protein expression at the blood–brain barrier of adult and younger cynomolgus monkeys.J. Pharm. Sci. 2011; 100: 3939-3950Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 7Uchida Y. et al.Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors.J. Neurochem. 2011; 117: 333-345Crossref PubMed Scopus (610) Google Scholar, 32Kamiie J. et al.Quantitative atlas of membrane transporter proteins: development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria.Pharm. Res. 2008; 25: 1469-1483Crossref PubMed Scopus (416) Google Scholar] have revealed significant differences between rodents and primates in several transporters. According to these studies, multidrug resistance-associated protein 4 (MRP4), monocarboxylate transporter 1 (MCT1/SLC16A1), L-type amino acid transporter (LAT1/SLC7A5), and organic anion transporter 3 (OAT3/SLC22A8) were expressed at greater than twofold higher levels in the mouse/rat BBB than in the human BBB, whereas glucose transporter 1 (SLC2A1/GLUT-1) and insulin receptor (INSR) were expressed at similar levels (less than twofold variation) in the mouse/rat and human BBBs (Table 1). In addition, the expression profiles of MRP4

AbstractDrug delivery across the blood–brain barrier (BBB) is a formidable challenge for therapies targeting the central nervous system. Although BBB shuttle peptides enhance transport into the brain non‐invasively, their application is partly limited by lability to proteases. The present study proposes the use of cyclic peptides derived from venoms as an affordable way to circumvent this drawback. Apamin, a neurotoxin from bee venom, was minimized by reducing its complexity, toxicity, and immunogenicity, while preserving brain targeting, active transport, and protease resistance. Among the analogues designed, the monocyclic lactam‐bridged peptidomimetic MiniAp‐4 was the most permeable. This molecule is capable of translocating proteins and nanoparticles in a human‐cell‐based BBB model. Furthermore, MiniAp‐4 can efficiently deliver a cargo across the BBB into the brain parenchyma of mice.

The ST6GALNAC5 gene that encodes an α2,6-sialyltransferase involved in the biosynthesis of α-series gangliosides, was previously identified as one of the genes that mediate breast cancer metastasis to the brain. We have shown that the expression of ST6GALNAC5 in MDA-MB-231 breast cancer cells resulted in the expression of GD1α ganglioside at the cell surface. By using a human blood-brain barrier in vitro model recently developed, consisting in CD34+ derived endothelial cells co-cultivated with pericytes, we show that ST6GALNAC5 expression decreased the interactions between the breast cancer cells and the human blood-brain barrier.

Abstract The indolo[2,1-b]quinazoline alkaloid tryptanthrin was previously identified as a potent anti-inflammatory compound with a unique pharmacological profile. It is a potent inhibitor of cyclooxygenase-2, 5-lipooxygenase-catalyzed leukotriene synthesis, and nitric oxide production catalyzed by the inducible nitric oxide synthase. To characterize the pharmacokinetic properties of tryptanthrin, we performed a pilot in vivo study in male Sprague-Dawley rats (2 mg/kg bw i. v.). Moreover, the ability of tryptanthrin to cross the blood-brain barrier was evaluated in three in vitro human and animal blood-brain barrier models. Bioanalytical UPLC-MS/MS methods used were validated according to current international guidelines. A half-life of 40.63 ± 6.66 min and a clearance of 1.00 ± 0.36 L/h/kg were found in the in vivo pharmacokinetic study. In vitro data obtained with the two primary animal blood-brain barrier models showed a good correlation with an immortalized human monoculture blood-brain barrier model (hBMEC cell line), and were indicative of a high blood-brain barrier permeation potential of tryptanthrin. These findings were corroborated by the in silico prediction of blood-brain barrier penetration. P-glycoprotein interaction of tryptanthrin was assessed by calculation of the efflux ratio in bidirectional permeability assays. An efflux ratio below 2 indicated that tryptanthrin is not subjected to active efflux.

The development of novel neuropharmaceuticals requires the evaluation of blood-brain barrier (BBB) permeability and toxicity. Recent studies have highlighted differences in the BBB among different species, with the most important differences involving the expression of P-glycoprotein (P-gp), multidrug resistance-associated proteins, transporters, and claudins. In addition, functional studies have shown that brain pharmacokinetics of P-glycoprotein substrates are different in humans and rodents. Therefore, human BBB models may be an important platform for initial drug screening before in vivo studies. This strategy might help to reduce costs in drug development and failures in clinical studies. We review the differences in the BBB among species, recent advances in the generation of human BBB models, and their applications in drug discovery and delivery.

We previously identified the alkaloids tryptanthrin (1), indirubin (2) and indolinone (3) as pharmacologically active compounds in woad (Isatis tinctoria L.). They inhibit COX-2, 5-LOX catalyzed leukotriene synthesis, and mast cell degranulation at low µM to nM concentrations, and they possess drug-like physico-chemical properties. A pilot pharmacokinetic study in rats (2 mg/kg i.v. b.w.) showed that 1 and 2 have half-lives of 30 – 40 min, whereas 3 was rapidly eliminated. In silico predictions for 1 – 3 indicated high oral absorption and favourable blood-brain transport. In animal and human in vitro blood-brain-barrier (BBB) models 1 and 3 displayed high BBB permeation. In the Caco-2 intestinal absorption model, 1 showed high permeation, while the recovery of 3 was low.

Graphical Abstract Der Wirkstofftransport in das Gehirn kann mithilfe von Peptiden, die die Blut-Hirn-Schranke passieren, verbessert werden; ihre Wirksamkeit leidet jedoch oft unter ihrer Labilität gegenüber Proteasen. In der Zuschrift auf S. 582 ff. beschreiben M. Teixidó, E. Giralt et al. ein cyclisches Peptidmimetikum (der Schlüssel), das aus einem Bienengift abgeleitet wurde. Dieser proteaseresistente Vektor transportiert Fracht effizient in das Gehirnparenchym von Mäusen und durch humane Endothelzellen.

The present study was performed in an attempt to develop an in vitro integrated testing strategy (ITS) to evaluate drug-induced neurotoxicity. A number of endpoints were analyzed using two complementary brain cell culture models and an in vitro blood-brain barrier (BBB) model after single and repeated exposure treatments with selected drugs that covered the major biological, pharmacological and neuro-toxicological responses. Furthermore, four drugs (diazepam, cyclosporine A, chlorpromazine and amiodarone) were tested more in depth as representatives of different classes of neurotoxicants, inducing toxicity through different pathways of toxicity. The developed in vitro BBB model allowed detection of toxic effects at the level of BBB and evaluation of drug transport through the barrier for predicting free brain concentrations of the studied drugs. The measurement of neuronal electrical activity was found to be a sensitive tool to predict the neuroactivity and neurotoxicity of drugs after acute exposure. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found to be well suited for OMICs analyses after both acute and long term treatment. The obtained data suggest that an in vitro ITS based on the information obtained from BBB studies and combined with metabolomics, proteomics and neuronal electrical activity measurements performed in stable in vitro neuronal cell culture systems, has high potential to improve current in vitro drug-induced neurotoxicity evaluation.

Carnitine (3-hydroxy-4-trimethylammoniobutyrate) is necessary for transfer of fatty acids through the inner mitochondrial membrane. Carnitine, not synthesized in the brain, is delivered there through the strongly polarized blood-brain barrier (BBB). Expression and presence of two carnitine transporters - organic cation/carnitine transporter (OCTN2) and amino acid transporter B(0,+) (ATB(0,+)) have been demonstrated previously in an in vitro model of the BBB. Due to potential protein kinase C (PKC) phosphorylation sites within ATB(0,+) sequence, the present study verified effects of this kinase on transporter function and localization in the BBB. ATB(0,+) can be regulated by estrogen receptor α and up-regulated in vitro, therefore its presence in vivo was verified with the transmission electron microscopy. The analyses of brain slices demonstrated ATB(0,+) luminal localization in brain capillaries, confirmed by biotinylation experiments in an in vitro model of the BBB. Brain capillary endothelial cells were shown to control carnitine gradient. ATB(0,+) was phosphorylated by PKC, what correlated with inhibition of carnitine transport. PKC activation did not change the amount of ATB(0,+) present in the apical membrane of brain endothelial cells, but resulted in transporter exclusion from raft microdomains. ATB(0,+) inactivation by a lateral movement in plasma membrane after transporter phosphorylation has been postulated.

The blood brain barrier (BBB) regulates the passage of endogenous and exogenous compounds and thus contributes to the brain homeostasis with the help of well-known proteins such as tight junction proteins, plasma membrane transporters and metabolic barrier proteins. In the last decade, proteomics have emerged as supplementary tools for BBB research. The development of proteomic technologies has provided several means to extend knowledge on the BBB and to investigate additional routes for the bypass of this barrier. Proteomics approaches have been used in vivo and also using in vitro BBB models to decipher the physiological characteristics and, under stress conditions, to understand the molecular mechanisms of brain diseases. This work has demonstrated that both quantitative global and targeted proteomics approaches are powerful and provide significant information on the brain microvessel endothelium. However, current knowledge is only partial and it is necessary to increase the studies using proteomics tools that will provide additional information concerning brain pathologies or BBB metabolism. Highly sensitive, accurate and specific protein quantification by quantitative targeted proteomics appears as an essential methodology for human BBB studies. (C) 2014 IMSS. Published by Elsevier Inc.

Located at the level of brain capillaries, the blood-brain barrier (BBB) is a crucial component of the neurovascular unit, since its highly regulated properties are needed to maintain optimal conditions for proper neuronal and glial functions. By modelling the BBB it is possible to make predictions about whether a compound's interaction with the BBB is likely to compromise its functionality. A dysfunctional BBB may either affect brain entry of an agent or indirectly generate unwanted effects on neurons and glial cells by disturbing the brain homeostasis.Since the BBB controls the exchanges between the blood and brain compartments modelling the BBB in vitro can also help to investigate the ability of compounds to cross the BBB. In this chapter, the plethora of in vitro BBB models that exist today is discussed and several methods needed to set up and use of these in vitro models in the framework of in vitro toxicity study is detailed.

The human blood brain barrier (BBB) is a selective barrier formed by human brain endothelial cells (hBECs), which is important to ensure adequate neuronal function and protect the central nervous system (CNS) from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.