FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2601589334> ?p ?o ?g. }

• W2601589334 endingPage "906" @default.
• W2601589334 startingPage "894" @default.
• W2601589334 abstract "•Establishment of a standardized human BBB co-culture model based on hiPSCs and fNSCs•Reflection of physiological BBB integrity and expression of relevant transporters/TJs•Confirmation of TJ network functionality by claudin-specific TJ modulators•Validation of physiological transcellular model tightness by permeability studies In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies. In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies. The blood-brain barrier (BBB) is the most important biological barrier between the blood circulation and the central nervous system (CNS), consisting of specialized blood endothelial cells (ECs) that line the cerebral capillaries and are connected by very dense tight junctions (TJs). Anatomically, the BBB is part of the neurovascular unit, which maintains the physiological function of the brain capillary ECs and includes cellular components such as pericytes, astrocytes, neurons, and microglia (Hawkins and Davis, 2005Hawkins B.T. Davis T.P. The blood-brain barrier/neurovascular unit in health and disease.Pharmacol. Rev. 2005; 57: 173-185Crossref PubMed Scopus (1977) Google Scholar). The main functions of the BBB are the maintenance of CNS homeostasis and the prevention of penetration of neurotoxic substances as well as pathogens, such as bacteria and viruses. Besides functioning as a physical barrier, the BBB plays a major role as a transport and metabolic barrier (Neuhaus and Noe, 2010Neuhaus W. Noe C.R. Transport at the Blood–brain Barrier Transporters as Drug Carriers. Wiley-VCH Verlag, 2010: 263-298Google Scholar). Models of the BBB serve as very strong tools in drug development and are important to elucidate further physiological and pathophysiological molecular mechanisms. Besides in silico and in vivo models, a variety of cellular in vitro BBB models are available, such as transwell models, dynamic flow-based hollow-fiber models, or microfluidic devices (Avdeef et al., 2015Avdeef A. Deli M.A. Neuhaus W. In Vitro Assays for Assessing BBB Permeability. Blood-brain Barrier in Drug Discovery. John Wiley, 2015: 188-237Google Scholar). So far, primary porcine, bovine, and rodent ECs are characterized by the best functionality, tightest barrier integrity, and lowest permeability (Vastag and Keseru, 2009Vastag M. Keseru G.M. Current in vitro and in silico models of blood-brain barrier penetration: a practical view.Curr. Opin. Drug Discov. Dev. 2009; 12: 115-124PubMed Google Scholar). Disadvantages associated with the use of primary cells are the time- and cost-intensive isolation processes, the variabilities between cells of different isolations, and the high consumption of animals for each new isolation. Access to human primary brain material is very limited and restricted to biopsy or autopsy material from patients with diseases such as epilepsy or brain tumors. The use of EC lines for BBB modeling helps to circumvent the disadvantages of primary cells. Immortalized cells of different species, such as murine EC lines (MBEC4, b.END3, b.END5, cEND, cerebEND) as well as cell lines from rat (RBE4), cow (t-BBEC-117), pig (PBMEC/C1-2), and human (hCMEC/D3, hBMEC, TY10, and BB19) exist (Eigenmann et al., 2013Eigenmann D.E. Xue G. Kim K.S. Moses A.V. Hamburger M. Oufir M. Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies.Fluids Barriers CNS. 2013; 10: 33Crossref PubMed Scopus (241) Google Scholar, Avdeef et al., 2015Avdeef A. Deli M.A. Neuhaus W. In Vitro Assays for Assessing BBB Permeability. Blood-brain Barrier in Drug Discovery. John Wiley, 2015: 188-237Google Scholar). These cell lines have the advantage of being usable over many passages with a higher reproducibility of the results compared with primary cells. Notably, almost all immortalized cell lines form barriers with a transendothelial electrical resistance (TEER) below 150 Ω cm2 (Deli et al., 2005Deli M.A. Abraham C.S. Kataoka Y. Niwa M. Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology.Cell Mol. Neurobiol. 2005; 25: 59-127Crossref PubMed Scopus (539) Google Scholar). For drug transport and barrier functionality studies, a minimal tightness of the BBB models with TEER values between 150 and 200 Ω cm2 has been defined (Gaillard and de Boer, 2000Gaillard P.J. de Boer A.G. Relationship between permeability status of the blood-brain barrier and in vitro permeability coefficient of a drug.Eur. J. Pharm. Sci. 2000; 12: 95-102Crossref PubMed Scopus (154) Google Scholar). However, compared with physiological TEER values of more than 1,500 Ω cm2, which have been measured in capillaries of rat or frog brains (Crone and Olesen, 1982Crone C. Olesen S.P. Electrical resistance of brain microvascular endothelium.Brain Res. 1982; 241: 49-55Crossref PubMed Scopus (367) Google Scholar, Butt et al., 1990Butt A.M. Jones H.C. Abbott N.J. Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study.J. Physiol. 1990; 429: 47-62Crossref PubMed Scopus (602) Google Scholar), the discrepancies with current in vitro models are significant. Another important aspect is the species differences that exist between humans and other mammalian subsets. In particular, the expression and functionality of important BBB transporters such as P-glycoprotein are described (Takeuchi et al., 2006Takeuchi T. Yoshitomi S. Higuchi T. Ikemoto K. Niwa S. Ebihara T. Katoh M. Yokoi T. Asahi S. Establishment and characterization of the transformants stably-expressing MDR1 derived from various animal species in LLC-PK1.Pharm. Res. 2006; 23: 1460-1472Crossref PubMed Scopus (112) Google Scholar, Warren et al., 2009Warren M.S. Zerangue N. Woodford K. Roberts L.M. Tate E.H. Feng B. Li C. Feuerstein T.J. Gibbs J. Smith B. et al.Comparative gene expression profiles of ABC transporters in brain microvessel endothelial cells and brain in five species including human.Pharmacol. Res. 2009; 59: 404-413Crossref PubMed Scopus (188) Google Scholar). Therefore, there is a significant need for adequate human BBB models for academic research and the pharmaceutical industry. Minimal requirements would be the reproducibility of results, characteristic permeability of reference components, expression of main BBB transporters, and physiological cell morphology (Cecchelli et al., 2007Cecchelli R. Berezowski V. Lundquist S. Culot M. Renftel M. Dehouck M.P. Fenart L. Modelling of the blood-brain barrier in drug discovery and development.Nat. Rev. Drug Discov. 2007; 6: 650-661Crossref PubMed Scopus (486) Google Scholar). In recent promising studies, various stem cell types have been used as an alternative source for BBB remodeling. Stem cells are self-renewable, can be subsequently differentiated into mature somatic cell types, and serve as a virtually unlimited independent cell source. In particular, hematopoietic stem cells from human umbilical cord blood (Cecchelli et al., 2014Cecchelli R. Aday S. Sevin E. Almeida C. Culot M. Dehouck L. Coisne C. Engelhardt B. Dehouck M.P. Ferreira L. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.PLoS One. 2014; 9: e99733Crossref PubMed Scopus (216) Google Scholar), circulating endothelial progenitor cells mobilized from bone marrow (Boyer-Di Ponio et al., 2014Boyer-Di Ponio J. El-Ayoubi F. Glacial F. Ganeshamoorthy K. Driancourt C. Godet M. Perriere N. Guillevic O. Couraud P.O. Uzan G. 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), as well as human induced pluripotent stem cells (hiPSCs) (Lippmann et al., 2012Lippmann E.S. Azarin S.M. Kay J.E. Nessler R.A. Wilson H.K. Al-Ahmad A. Palecek S.P. Shusta E.V. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells.Nat. Biotechnol. 2012; 30: 783-791Crossref PubMed Scopus (481) Google Scholar) have been used for BBB modeling with promising in vivo-like characteristics, e.g. TEER values up to 5,000 Ω cm2 (Lippmann et al., 2014Lippmann E.S. Al-Ahmad A. Azarin S.M. Palecek S.P. Shusta E.V. A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources.Sci. Rep. 2014; 4: 4160Crossref PubMed Scopus (307) Google Scholar). The addition of stimulating compounds such as retinoic acid (RA) during differentiation (Lippmann et al., 2014Lippmann E.S. Al-Ahmad A. Azarin S.M. Palecek S.P. Shusta E.V. A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources.Sci. Rep. 2014; 4: 4160Crossref PubMed Scopus (307) Google Scholar) and co-culturing with individual niche cell types, such as pericytes, astrocytes, and neural cells, have further improved BBB properties (Lippmann et al., 2013Lippmann E.S. Al-Ahmad A. Palecek S.P. Shusta E.V. Modeling the blood-brain barrier using stem cell sources.Fluids Barriers CNS. 2013; 10: 2Crossref PubMed Scopus (96) Google Scholar, Cecchelli et al., 2014Cecchelli R. Aday S. Sevin E. Almeida C. Culot M. Dehouck L. Coisne C. Engelhardt B. Dehouck M.P. Ferreira L. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.PLoS One. 2014; 9: e99733Crossref PubMed Scopus (216) Google Scholar). The aim of this study was to systematically investigate the individual impact of the different cell types on hiPSC-derived BBB endothelial cell (hiPS-EC) function as well as gene expression and therefore establish the most predictive BBB model. Furthermore, we standardized the methods to differentiate the corresponding BBB cell types from hiPSCs as well as human multipotent stem cells. Thus, it will be technically feasible to generate large quantities of human cell types from a single cell source and, in combination with new detection methods, to develop standardized higher-throughput in vitro assays in drug discovery and toxicity testing. hiPSCs as well as multipotent neural stem cell (NSCs) provide an effective cell source to generate functional brain cells and have the advantage of being independent of postnatal brain tissue biopsy samples. For our studies, we used the recently published hiPSC lines IMR90-4 and ARiPS (Kadari et al., 2014Kadari A. Lu M. Li M. Sekaran T. Thummer R.P. Guyette N. Chu V. Edenhofer F. Excision of viral reprogramming cassettes by Cre protein transduction enables rapid, robust and efficient derivation of transgene-free human induced pluripotent stem cells.Stem Cell Res. Ther. 2014; 5: 47Crossref PubMed Scopus (26) Google Scholar) to differentiate them into BBB ECs (Lippmann et al., 2012Lippmann E.S. Azarin S.M. Kay J.E. Nessler R.A. Wilson H.K. Al-Ahmad A. Palecek S.P. Shusta E.V. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells.Nat. Biotechnol. 2012; 30: 783-791Crossref PubMed Scopus (481) Google Scholar) and hiPS-NSCs. As a physiological control, NSCs were additionally isolated from fetal brain tissue (fNSCs). The growth characteristics of hiPSCs are similar to embryonic stem cells, forming compact colonies with defined borders, which typically appear in phase-contrast microscopy (Figure 1D). Colonies were characterized by immunofluorescence staining for pluripotency-associated markers, including OCT3/4 (Figure 1A), SOX2 (Figure 1B), and TRA1-81 (Figure 1C); flow cytometry analyses demonstrated at least 90% positive staining (data not shown). For differentiation of hiPSCs into NSCs, a recently published protocol was used employing neurogenic media in adherent culture (Yan et al., 2013Yan Y. Shin S. Jha B.S. Liu Q. Sheng J. Li F. Zhan M. Davis J. Bharti K. Zeng X. et al.Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells.Stem Cells Transl. Med. 2013; 2: 862-870Crossref PubMed Scopus (120) Google Scholar) with slight modifications described by Günther et al., 2016Günther K. Appelt-Menzel A. Keong Kwok C. Walles H. Metzger M. Edenhofer F. Rapid monolayer neural induction of induced pluripotent stem cells yields stably proliferating neural stem cells.J. Stem Cell Res. Ther. 2016; 6: 6Crossref Google Scholar. Morphology of hiPS-NSCs (Figure 1H) appeared as typical rosette-like structures, whereas fNSCs (Figure 1L) were more heterogeneous, some of them with elongated processes. After culturing both NSC types in NSC medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), NSCs could be further expanded in vitro. NSC identity was confirmed by staining for early NSC markers such as SOX1 (Figures 1E and 1I), SOX2 (Figures 1F and 1J), and NESTIN (Figures 1G and 1K). In contrast, the expression of pluripotency and astroglial markers could not be detected (data not shown). For differentiation of hiPSCs and NSCs to BBB ECs and astrocytes, the in vivo neurodevelopmental process has to be mimicked in vitro. For BBB capillary ECs, a co-differentiation of neural and ECs was initiated by treatment with a so-called unconditioned medium (Lippmann et al., 2014Lippmann E.S. Al-Ahmad A. Azarin S.M. Palecek S.P. Shusta E.V. A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources.Sci. Rep. 2014; 4: 4160Crossref PubMed Scopus (307) Google Scholar). The purification of hiPS-ECs was performed using an EC medium with RA and sub-cultivation on a collagen IV-/fibronectin-coated matrix. After differentiation for 10 days, hiPS-ECs showed a typical elongated spindle-shaped morphology, and the cell size was increased compared with the original hiPSCs (Figure 2A). The hiPS-ECs were characterized by immunofluorescence staining for the typical EC marker von Willebrand factor (vWF, Figure 2B). The TJ-associated protein ZO1 as well as the BBB-relevant glucose transporter 1 (GLUT1) were homogeneously expressed at the cell-cell borders (Figures 2C and 2D). The expression of the adherence junction protein vascular endothelial cadherin (CDH5) was also detectable, the endothelial proteins angiopoietin receptor 2 (TIE2) and PECAM1 (CD31) appeared weaker and less distinct (Figures S1A–S1C). The functionality of the hiPS-ECs was tested by an uptake assay with fluorescein isothiocyanate (FITC)-labeled acetylated low-density lipoprotein. Substance was taken up by 92.4% of the BBB hiPS-ECs (Figure S1D), and the fluorescence intensity was lower compared with the control cell line, human umbilical vein endothelial cells (Figures S1D–S1F). Astrocytes differentiated from hiPSCs (hiPS-As, Figures 2E–2H) as well as human primary fetal brain astrocytes from the cerebral cortex (astrocytes, Figures 2I–2L) were characterized by immunofluorescence staining for intermediate filament protein glial fibrillary acidic protein (GFAP, Figures 2F and 2J) as well as for glial-specific calcium-binding protein B (S100β, Figures 2G and 2K). The differentiation efficiency of hiPSCs, analyzed by flow cytometry, was relatively high with 53.8% GFAP-positive astrocytes (H), although human primary fetal brain astrocytes were nearly 100% positive for GFAP (L). As for astrocytes, human primary pericytes were heterogenic in their morphology and marker expression. Cells showed elongated fibroblast- or MSC-like morphology and were positively stained for alpha smooth muscle actin (αSMA, Figure 2N) and platelet-derived growth factor receptor-beta (PDGFRβ, Figure 2O). Flow cytometry demonstrated 85.2% PDGFRβ-positive cells (Figure 2P). To investigate the influence of different cell types on BBB hiPS-EC integrity, diverse sets of BBB co-cultures were established. As shown in Figure 3A, the differentiated hiPS-ECs were cultured on a collagen IV-/fibronectin-coated transwell membrane. The different types of co-culture cells were seeded in coated wells in the bottom compartment without direct contact with the hiPS-ECs. We studied the influence of primary fetal brain astrocytes, hiPS-As, primary fetal brain pericytes, and NSCs derived from hiPSCs (hiPS-NSCs) or isolated from fetal brain (fNSCs) on BBB hiPS-EC integrity. Moreover, we established several combinations of the above-mentioned cell types to study synergistic effects. After 2 days of co-culture, the TEER was measured as the first important readout characterizing the paracellular tightness of hiPS-ECs. The cell density of co-culture cells was kept constant independent from the co-culture system. A significant increase in TEER compared with the hiPS-EC mono-cultures (TEER = 1,198 ± 265) could be obtained by triple culture of hiPS-ECs, hiPS-NSCs, and pericytes (1,723 ± 90 Ω cm2) as well as by quadruple culture of hiPS-ECs, hiPS-NSCs, astrocytes, and pericytes (1,757 ± 320 Ω cm2) (Figure 3B). Maximal absolute TEER values ranged between 2,000 Ω cm2 (mono-culture) and 2,500 Ω cm2 (triple and quadruple culture), approximately (Figure 3C). In addition to TEER measurements, we analyzed BBB models according to a characteristic gene expression profile via qRT-PCR. Genes included the efflux transporter ABCB1, the glutamate transporter SLC1A1, the glucose transporter SLC2A1, and TJ component OCLN. The hiPS-EC mono-culture (100%) was compared with the different co-culture settings (Figure 3D), and a 1.5-fold regulation in gene expression was set as an arbitrary biological threshold. In concordance with the TEER experiments, the most robust upregulation was observed for the quadruple culture, indicating effective modeling of the BBB phenotype. Under these conditions, the expression of ABCB1 was on average upregulated by 1.5-fold, SLC1A1 by 1.3-fold, SLC2A1 by 1.7-fold, and OCLN by 1.6-fold compared with hiPS-ECs from mono-cultures, however statistical significance was reached only for SLC2A1. The triple culture of hiPS-ECs, hiPS-NSCs, and pericytes revealed only moderate upregulation of 1.3-fold for ABCB1, 1.5-fold for SLC1A1, 1.2-fold for SLC2A1, and 1.4-fold for OCLN. Other co-culture systems also yielded moderate upregulation in expression of these genes, however, with higher variances as in the quadruple culture. Noteworthy, no significant effects were observed on gene expression and TEER of hiPS-ECs using the control colon carcinoma cell line Caco-2 (data not shown). As the quadruple culture showed the strongest enhancement of the phenotypical development of BBB properties, further analyses focused on the comparison between the mono-culture and the quadruple setup. To characterize the functionality of the efflux transporter P-glycoprotein, we performed transport studies with the substrate rhodamine 123 with and without inhibiting the transporter by use of verapamil (Figure 3E). The permeability coefficients (PCcell) for rhodamine 123 could be significantly increased in the mono-culture setup as well as in the quadruple culture by adding verapamil, indicating the correct transporter functionality and polarization in the cell membrane. To characterize the paracellular permeability of the cell layers in a molecular size-dependent manner, transport studies with several paracellular marker molecules such as lucifer yellow (∼0.44 kDa), fluorescein (∼0.33 kDa), and FITC-labeled dextrans (4 and 40 kDa) were accomplished (Table 1). As expected, lucifer yellow and fluorescein permeated very similarly (PCcell ∼1.5 μm/min). FITC-labeled dextran (4 kDa) migrated about 100-fold slower than these two small paracellular markers, and the PCs for 40 kDa FITC-labeled dextran were even smaller (PCcell 0.003–0.0054 μm/min). Corresponding to the TEER values, comparison with the mono-culture revealed a lower, statistically significant different permeability of 40 kDa FITC-labeled dextran across the quadruple cultures.Table 1Overview of the Results of the Permeability Studies with Paracellular Marker MoleculesPCall (μm/min)PCcell (μm/min)Transport RankingMono-cultureLucifer yellow1.41 ± 0.251.52 ± 0.291Fluorescein1.39 ± 0.591.53 ± 0.671FITC-labeled dextran 4 kDa0.0166 ± 0.00370.0166 ± 0.00373FITC-labeled dextran 40 kDa0.0054 ± 0.00070.0054 ± 0.00074Quadruple CultureLucifer yellow1.44 ± 0.341.58 ± 0.401Fluorescein1.26 ± 0.271.33 ± 0.292FITC-labeled dextran 4 kDa0.0106 ± 0.00160.0106 ± 0.00163FITC-labeled dextran 40 kDa0.0030 ± 0.0004∗0.0030 ± 0.0004∗4Data are presented as means ± SEM, n = 6–8 from three to four independent experiments. Statistically significant difference, ∗p < 0.05, in comparison with the mono-culture setup. PC, permeability coefficient.Permeability coefficients and transport rankings are compared between mono- and quadruple culture models. Open table in a new tab Data are presented as means ± SEM, n = 6–8 from three to four independent experiments. Statistically significant difference, ∗p < 0.05, in comparison with the mono-culture setup. PC, permeability coefficient. Permeability coefficients and transport rankings are compared between mono- and quadruple culture models. Paracellular permeability is functionally linked to the expression of junctional molecules, especially of claudins (CLDN). Therefore, the expression of major TJ and TJ-associated molecules was analyzed. In addition to occludin (OCLN, Figure 3D), we determined mRNA expression of CLDN3, CLDN4, CDH5, and TJP1 (ZO-1) of quadruple cultures in direct comparison with mono-cultures, however upregulation was mostly below the threshold of 1.5-fold, and no statistical significant effects were detected (data not shown). The expression of all analyzed genes could be qualitatively confirmed representatively in mono-cultures by gel electrophoresis of PCR products (Figure S2). At the protein level, the presence of the TJ proteins CLDN1, CLDN4, and CLDN5 was also confirmed, again without any statistically significant change in expression as shown by western blot analysis (Figures 4A and 4B ). In order to confirm the role of claudins for paracellular tightness from BBB hiPS-EC layers, the effects of claudin-specific TJ modulators on TEER were investigated (Figure 4C). These TJ modulators were based on the claudin-binding domain of the Clostridium perfringens enterotoxin (Protze et al., 2015Protze J. Eichner M. Piontek A. Dinter S. Rossa J. Blecharz K.G. Vajkoczy P. Piontek J. Krause G. Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5.Cell Mol. Life Sci. 2015; 72: 1417-1432Crossref PubMed Scopus (41) Google Scholar). Data revealed a significant time- and concentration-dependent decrease of TEER after addition of cCPEwt, which binds with high affinity to CLDN3/4 and interacts with CLDN1. Furthermore, incubation with CLDN5-binding cCPEY306W/S313H decreased TEER. On the contrary, application of the non-binding control cCPEY306A/L315A showed no effects on TEER progression. Interestingly, 1 μg/mL cCPEwt reduced TEER to 32% ± 3% after 4 hr, whereas 1 μg/mL cCPEY306W/S313H (76% ± 10%) did not significantly disrupt the barrier. Since cCPE_Y306W/S313H has a higher affinity for CLDN5 than cCPE_wt (Kd ∼30 nM versus Kd ≫ 1 μM; Protze et al., 2015Protze J. Eichner M. Piontek A. Dinter S. Rossa J. Blecharz K.G. Vajkoczy P. Piontek J. Krause G. Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5.Cell Mol. Life Sci. 2015; 72: 1417-1432Crossref PubMed Scopus (41) Google Scholar), the results indicated that, in our model, other claudins next to claudin-5 contribute strongly to the high TEER values and formation of the paracellular barrier. To characterize the TJs on the ultrastructural level, cells were fixed, and freeze-fracture electron microscopy (EM) was performed. Intramembranous TJ particles were found on the protoplasmic face (P face, PF) and exoplasmic face (E face, EF) of the plasma membrane (Figure 5). On the E face, TJ strands were detected as particles and particle-free grooves. On the P face, TJ strands were detected partly as continuous strands and partly as beaded particles (Figure 5). Quadruple cultures and mono-cultures showed variable although similar complex networks of meshes formed by branched strands with mixed P/E face association. A tendency to higher complexity was found for the quadruple cultures (mean number of meshes in the strand network, 33.0 ± 5.0 versus 26.1 ± 2.8; rectangular area with strands, 1.1 ± 0.1 μm2 versus 0.9 ± 0.1 μm2; mesh density, 33.4 ± 2.7 μm−2 versus 30.6 ± 2.8 μm−2; n > 20). However, no significant differences were obtained for any of these morphometric parameters. In sum, on the ultrastructural level, for BBB hiPS-ECs, TJs similar to those of brain capillary ECs of the BBB were found (Wolburg et al., 1994Wolburg H. Neuhaus J. Kniesel U. Krauss B. Schmid E.M. Ocalan M. Farrell C. Risau W. Modulation of tight junction structure in blood-brain barrier endothelial cells Effects of tissue culture, second messengers and cocultured astrocytes.J. Cell Sci. 1994; 107: 1347-1357PubMed Google Scholar). Transmission EM also revealed the presence of complex TJs, constricting the paracellular gap and connecting two neighboring hiPS-ECs (Figures 5E–5G). However, no significant differences of TJs between mono- and quadruple culture models were found. In addition to the restriction of paracellular permeability, the BBB is also a barrier for transcellular transport. To describe these properties and to perform a first assessment about the qualification for drug transport studies, permeation of several reference drugs was studied across the mono-culture and quadruple culture setup. Calculated PCs across the total barrier comprising the cell layer and the membrane support (PCall) revealed mean permeabilities from 3.44 to 26.94 μm/min (Table 2). Especially in the case of transport studies of compounds migrating via the transcellular route, correction of the PC for the barrier formed by the membrane support itself is essential to obtain the permeability only across the cell layer (PCcell). This correction procedure revealed significantly increased PCs. In addition to accounting for cell layer variabilities, diazepam was used as an internal standard for each compound, and the permeability rankings were calculated with the PCcell data normalized to the PCcell data of diazepam. These rankings showed that diazepam permeated fastest followed by caffeine, ibuprofen, celecoxib, diclofenac, loratadine, and rhodamine 123 across the mono-culture model. This ranking was according to the classification based on literature data for diazepam and caffeine as fast, ibuprofen, celecoxib, and diclofenac as medium, and loratadine and rhodamine 123 as slow permeating compounds (Nakazono et al., 1992Nakazono T. Murakami T. Sakai S. Higashi Y. Yata N. Application of microdialysis for study of caffeine distribution into brain and cerebrospinal fluid in rats.Chem. Pharm. Bull. (Tokyo). 1992; 40: 2510-2515Crossref PubMed Scopus (17) Google Scholar, Neuhaus et al., 2012Neuhaus W. Mandikova J. Pawlowitsch R. Linz B. Bennani-Baiti B. Lauer R. Lachmann B. Noe C.R. Blood-brain barrier in vitro models as tools in drug discovery: assessment of the transport ranking of antihistaminic drugs.Pharmazie. 2012; 67: 432-439PubMed Google Scholar, Novakova et al., 2014Novakova I. Subileau E.A. Toegel S. Gruber D. Lachmann B. Urban E. Chesne C. Noe C.R. Neuhaus W. Transport rankings of non-steroidal antiinflammatory drugs across blood-brain barrier in vitro models.PLoS One. 2014; 9: e86806Crossref PubMed Scopus (64) Google Scholar). In the case of the quadruple culture, the mean ratio to diazepam was significantly decreased for caffeine, from 0.499 to 0.251, leading to a switch in the ranking position from second to fourth place in comparison with the mono-culture setup.Table 2Overview of the Results of the Transport StudiesSubstanceDiazepamRatio to DiazepamTransport RankingPCall (μ" @default.
• W2601589334 created "2017-04-07" @default.
• W2601589334 creator A5003008756 @default.
• W2601589334 creator A5004893893 @default.
• W2601589334 creator A5023308750 @default.
• W2601589334 creator A5024765436 @default.
• W2601589334 creator A5045190178 @default.
• W2601589334 creator A5063860814 @default.
• W2601589334 creator A5064000953 @default.
• W2601589334 creator A5074701019 @default.
• W2601589334 creator A5079423786 @default.
• W2601589334 creator A5089794891 @default.
• W2601589334 date "2017-04-01" @default.
• W2601589334 modified "2023-10-11" @default.
• W2601589334 title "Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells" @default.
• W2601589334 cites W1185979967 @default.
• W2601589334 cites W1517640635 @default.
• W2601589334 cites W1924595537 @default.
• W2601589334 cites W1969012119 @default.
• W2601589334 cites W1973626590 @default.
• W2601589334 cites W1976936511 @default.
• W2601589334 cites W1992866322 @default.
• W2601589334 cites W2001726213 @default.
• W2601589334 cites W2005408062 @default.
• W2601589334 cites W2010641543 @default.
• W2601589334 cites W2013495726 @default.
• W2601589334 cites W2014712282 @default.
• W2601589334 cites W2017058852 @default.
• W2601589334 cites W2022673067 @default.
• W2601589334 cites W2025658823 @default.
• W2601589334 cites W2030775222 @default.
• W2601589334 cites W2033812787 @default.
• W2601589334 cites W2034370816 @default.
• W2601589334 cites W2042883482 @default.
• W2601589334 cites W2047075745 @default.
• W2601589334 cites W2051306620 @default.
• W2601589334 cites W2052578334 @default.
• W2601589334 cites W2063530485 @default.
• W2601589334 cites W2065561114 @default.
• W2601589334 cites W2067733328 @default.
• W2601589334 cites W2076634690 @default.
• W2601589334 cites W2077216169 @default.
• W2601589334 cites W2079406506 @default.
• W2601589334 cites W2082931608 @default.
• W2601589334 cites W2093271288 @default.
• W2601589334 cites W2100308259 @default.
• W2601589334 cites W2107798346 @default.
• W2601589334 cites W2115381659 @default.
• W2601589334 cites W2120914031 @default.
• W2601589334 cites W2122453033 @default.
• W2601589334 cites W2136000727 @default.
• W2601589334 cites W2136578375 @default.
• W2601589334 cites W2142046859 @default.
• W2601589334 cites W2148992136 @default.
• W2601589334 cites W2155436051 @default.
• W2601589334 cites W2156890482 @default.
• W2601589334 cites W2160753648 @default.
• W2601589334 cites W2164812139 @default.
• W2601589334 cites W2317212956 @default.
• W2601589334 cites W2405462101 @default.
• W2601589334 cites W2559974977 @default.
• W2601589334 cites W45411166 @default.
• W2601589334 doi "https://doi.org/10.1016/j.stemcr.2017.02.021" @default.
• W2601589334 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5390136" @default.
• W2601589334 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28344002" @default.
• W2601589334 hasPublicationYear "2017" @default.
• W2601589334 type Work @default.
• W2601589334 sameAs 2601589334 @default.
• W2601589334 citedByCount "205" @default.
• W2601589334 countsByYear W26015893342017 @default.
• W2601589334 countsByYear W26015893342018 @default.
• W2601589334 countsByYear W26015893342019 @default.
• W2601589334 countsByYear W26015893342020 @default.
• W2601589334 countsByYear W26015893342021 @default.
• W2601589334 countsByYear W26015893342022 @default.
• W2601589334 countsByYear W26015893342023 @default.
• W2601589334 crossrefType "journal-article" @default.
• W2601589334 hasAuthorship W2601589334A5003008756 @default.
• W2601589334 hasAuthorship W2601589334A5004893893 @default.
• W2601589334 hasAuthorship W2601589334A5023308750 @default.
• W2601589334 hasAuthorship W2601589334A5024765436 @default.
• W2601589334 hasAuthorship W2601589334A5045190178 @default.
• W2601589334 hasAuthorship W2601589334A5063860814 @default.
• W2601589334 hasAuthorship W2601589334A5064000953 @default.
• W2601589334 hasAuthorship W2601589334A5074701019 @default.
• W2601589334 hasAuthorship W2601589334A5079423786 @default.
• W2601589334 hasAuthorship W2601589334A5089794891 @default.
• W2601589334 hasBestOaLocation W26015893341 @default.
• W2601589334 hasConcept C105572291 @default.
• W2601589334 hasConcept C105702510 @default.
• W2601589334 hasConcept C169760540 @default.
• W2601589334 hasConcept C201750760 @default.
• W2601589334 hasConcept C2777670902 @default.
• W2601589334 hasConcept C2778402981 @default.
• W2601589334 hasConcept C28328180 @default.
• W2601589334 hasConcept C2910776680 @default.
• W2601589334 hasConcept C529278444 @default.
• W2601589334 hasConcept C86803240 @default.

• next