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      In Vitro Modeling of Blood-Brain Barrier with Human iPSC-Derived Endothelial Cells, Pericytes, Neurons, and Astrocytes via Notch Signaling

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          Summary

          The blood-brain barrier (BBB) is composed of four cell populations, brain endothelial cells (BECs), pericytes, neurons, and astrocytes. Its role is to precisely regulate the microenvironment of the brain through selective substance crossing. Here we generated an in vitro model of the BBB by differentiating human induced pluripotent stem cells (hiPSCs) into all four populations. When the four hiPSC-derived populations were co-cultured, endothelial cells (ECs) were endowed with features consistent with BECs, including a high expression of nutrient transporters ( CAT3, MFSD2A) and efflux transporters ( ABCA1, BCRP, PGP, MRP5), and strong barrier function based on tight junctions. Neuron-derived Dll1, which activates Notch signaling in ECs, was essential for the BEC specification. We performed in vitro BBB permeability tests and assessed ten clinical drugs by nanoLC-MS/MS, finding a good correlation with the BBB permeability reported in previous cases. This technology should be useful for research on human BBB physiology, pathology, and drug development.

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          Highlights

          • •

            BBB are generated with hiPSC-derived BECs, pericytes, neurons, and astrocytes

          • •

            ciBECs highly express BBB-specific transporters and showed barrier function

          • •

            Mechanism of ciBEC specification activates Notch signaling via Dll1 in neurons

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            BBB permeability of clinical drugs by nanoLC-MS/MS correlates with previous cases

          Abstract

          Yamamizu and colleagues generated an in vitro model of the BBB by differentiating hiPSCs into brain endothelial cells (BECs), pericytes, neurons, and astrocytes. They found that neuron-derived Dll1, which activates Notch signaling in ECs, was essential for the BEC specification. They also performed in vitro BBB permeability tests and assessed clinical drugs by nanoLC-MS/MS, finding a good correlation with the clinical cases.

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          A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes.

          Shinsuke Nakagawa, Mária A Deli, Hiroko Kawaguchi … (2009)
          Blood-brain barrier (BBB) characteristics are induced and maintained by cross-talk between brain microvessel endothelial cells and neighbouring elements of the neurovascular unit. While pericytes are the cells situated closest to brain endothelial cells morphologically and share a common basement membrane, they have not been used in co-culture BBB models for testing drug permeability. We have developed and characterized a new syngeneic BBB model using primary cultures of the three main cell types of cerebral microvessels. The co-culture of endothelial cells, pericytes and astrocytes mimick the anatomical situation in vivo. In the presence of both pericytes and astrocytes rat brain endothelial cells expressed enhanced levels of tight junction (TJ) proteins occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. Further morphological evidence of the presence of interendothelial TJs was provided by electron microscopy. The transendothelial electrical resistance (TEER) of brain endothelial monolayers in triple co-culture, indicating the tightness of TJs reached 400Omegacm(2) on average, while the endothelial permeability coefficients (P(e)) for fluorescein was in the range of 3x10(-6)cm/s. Brain endothelial cells in the new model expressed glucose transporter-1, efflux transporters P-glycoprotein and multidrug resistance protein-1, and showed a polarized transport of rhodamine 123, a ligand for P-glycoprotein. To further characterize the model, drug permeability assays were performed using a set of 19 compounds with known in vivo BBB permeability. Good correlation (R(2)=0.89) was found between in vitroP(e) values obtained from measurements on the BBB model and in vivo BBB permeability data. The new BBB model, which is the first model to incorporate pericytes in a triple co-culture setting, can be a useful tool for research on BBB physiology and pathology and to test candidate compounds for centrally acting drugs.
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            Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology.

            Máiria Deli, Csongor Abrahám, Yasufumi Kataoka … (2005)
            (1) The specifically regulated restrictive permeability barrier to cells and molecules is the most important feature of the blood-brain barrier (BBB). The aim of this review was to summarize permeability data obtained on in vitro BBB models by measurement of transendothelial electrical resistance and by calculation of permeability coefficients for paracellular or transendothelial tracers. (2) Results from primary cultures of cerebral microvascular endothelial cells or immortalized cell lines from bovine, human, porcine, and rodent origin are presented. Effects of coculture with astroglia, neurons, mesenchymal cells, blood cells, and conditioned media, as well as physiological influence of serum components, hormones, growth factors, lipids, and lipoproteins on the barrier function are discussed. (3) BBB permeability results gained on in vitro models of pathological conditions including hypoxia and reoxygenation, neurodegenerative diseases, or bacterial and viral infections have been reviewed. Effects of cytokines, vasoactive mediators, and other pathogenic factors on barrier integrity are also detailed. (4) Pharmacological treatments modulating intracellular cyclic nucleotide or calcium levels, and activity of protein kinases, protein tyrosine phosphatases, phospholipases, cyclooxygenases, or lipoxygenases able to change BBB integrity are outlined. Barrier regulation by drugs involved in the metabolism of nitric oxide and reactive oxygen species, as well as influence of miscellaneous treatments are also listed and evaluated. (5) Though recent advances resulted in development of improved in vitro BBB model systems to investigate disease modeling, drug screening, and testing vectors targeting the brain, there is a need for checking validity of permeability models and cautious interpretation of data.
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              SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier.

              Woo Jean Kim, H. Y. Song, Yung Kim … (2003)
              The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. BBB maintenance is important in the central nervous system (CNS) because disruption of the BBB may contribute to many brain disorders, including Alzheimer disease and ischemic stroke. The molecular mechanisms of BBB development remain ill-defined, however. Here we report that src-suppressed C-kinase substrate (SSeCKS) decreases the expression of vascular endothelial growth factor (VEGF) through AP-1 reduction and stimulates expression of angiopoietin-1 (Ang-1), an antipermeability factor in astrocytes. Conditioned media from SSeCKS-overexpressing astrocytes (SSeCKS-CM) blocked angiogenesis in vivo and in vitro. Moreover, SSeCKS-CM increased tight junction proteins in endothelial cells, consequently decreasing [3H]sucrose permeability. Furthermore, immunoreactivity to SSeCKS gradually increased during the BBB maturation period, and SSeCKS-expressing astrocytes closely interacted with zonula occludens (ZO)-1-expressing blood vessels in vivo. Collectively, our results suggest that SSeCKS regulates BBB differentiation by modulating both brain angiogenesis and tight junction formation.
              Article 0 comments Cited 133 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Kohei Yamamizu
                Journal
                Journal ID (nlm-ta): Stem Cell Reports
                Journal ID (iso-abbrev): Stem Cell Reports
                Title: Stem Cell Reports
                Publisher: Elsevier
                ISSN (Electronic): 2213-6711
                Publication date PMC-release: 23 February 2017
                Publication date Collection: 14 March 2017
                Publication date (Electronic): 23 February 2017
                Volume: 8
                Issue: 3
                Pages: 634-647
                Affiliations
                [1 ]Laboratory of Stem Cell Differentiation, Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
                [2 ]Department of Life Science Frontier, CiRA, Kyoto University, Kyoto 606-8507, Japan
                [3 ]Department of Chemistry and Biochemistry, Waseda University, Tokyo 169-8555, Japan
                [4 ]Laboratory of Stem Cell Medicine, Department of Cell Growth and Differentiation, CiRA, Kyoto University, Kyoto 606-8507, Japan
                Author notes
                [∗ ]Corresponding author kohei@ 123456cira.kyoto-u.ac.jp
                Article
                Publisher ID: S2213-6711(17)30039-5
                DOI: 10.1016/j.stemcr.2017.01.023
                PMC ID: 5355679
                PubMed ID: 28238797
                SO-VID: dee1d06d-7555-4023-b03b-2fe4d0cc23aa
                Copyright © © 2017 The Author(s)
                License:

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                Date received : 4 July 2016
                Date revision received : 18 January 2017
                Date accepted : 21 January 2017
                Categories
                Subject: Article

                Keywords: induced pluripotent stem cells,blood-brain barrier,notch signaling,endothelial cells,pericytes,neurons,astrocytes,vasculature,permeability,drug kinetics
                Data availability:
                Keywords: induced pluripotent stem cells, blood-brain barrier, notch signaling, endothelial cells, pericytes, neurons, astrocytes, vasculature, permeability, drug kinetics

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