Laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity

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Abstract

Background

In vitro blood–brain barrier (BBB) models using human induced pluripotent stem (iPS) cell-derived brain microvascular endothelial-like cells (iBMELCs) have been developed to predict the BBB permeability of drug candidates. For the differentiation of iBMELCs, Matrigel, which is a gelatinous protein mixture, is often used as a coating substrate. However, the components of Matrigel can vary among lots, as it is obtained from mouse sarcoma cells with the use of special technics and also contains various basement membranes. Therefore, fully defined substrates as substitutes for Matrigel are needed for a stable supply of iBMELCs with less variation among lots.

Methods

iBMELCs were differentiated from human iPS cells on several matrices. The barrier integrity of iBMELCs was evaluated based on transendothelial electrical resistance (TEER) values and permeability of fluorescein isothiocyanate-dextran 4 kDa (FD4) and Lucifer yellow (LY). Characterization of iBMELCs was conducted by RT-qPCR and immunofluorescence analysis. Functions of efflux transporters were defined by intracellular accumulation of the substrates in the wells of multiwell plates.

Results

iBMELCs differentiated on laminin 221 fragment (LN221F-iBMELCs) had higher TEER values and lower permeability of LY and FD4 as compared with iBMELCs differentiated on Matrigel (Matrigel-iBMELCs). Besides, the gene and protein expression levels of brain microvascular endothelial cells (BMEC)-related markers were similar between LN221F-iBMELCs and Matrigel-iBMELCs. Moreover, both Matrigel- and LN221F-iBMELCs had functions of P-glycoprotein and breast cancer resistance protein, which are essential efflux transporters for barrier functions of the BBB.

Conclusion

The fully defined substrate LN221F presents as an optimal coating matrix for differentiation of iBMELCs. The LN221F-iBMELCs had more robust barrier function for a longer period than Matrigel-iBMELCs with characteristics of BMECs. This finding will contribute the establishment of an iBMELC supply system for pharmacokinetic and pathological models of the BBB.

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Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells

Antje Appelt-Menzel, Alevtina Cubukova, Katharina Günther … (2017)

Summary 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.

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Species differences in blood-brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport.

Örjan Lindhe, Jakob Knudsen, Margareta Hammarlund-Udenaes … (2009)

Species differences occur in the brain concentrations of drugs, but the reasons for these differences are not yet apparent. This study was designed to compare brain uptake of three radiolabeled P-glycoprotein (P-gp) substrates across species using positron emission tomography. Brain concentrations and brain-to-plasma ratios were compared; [(11)C]verapamil in rats, guinea pigs, and monkeys; [(11)C](S)-(2-methoxy-5-(5-trifluoromethyltetrazol-1-yl)-phenylmethylamino)-2(S)-phenylpiperidine (GR205171) in rats, guinea pigs, monkeys, and humans; and [(18)F]altanserin in rats, minipigs, and humans. The fraction of the unbound radioligand in plasma was studied along with its metabolism. The effect of P-gp inhibition was investigated by administering cyclosporin A (CsA). Pronounced species differences were found in the brain and brain-to-plasma concentrations of [(11)C]verapamil, [(11)C]GR205171, and [(18)F]altanserin with higher brain distribution in humans, monkeys, and minipigs than in rats and guinea pigs. For example, the brain-to-plasma ratio of [(11)C]GR205171 was almost 9-fold higher in humans compared with rats. The species differences were still present after P-gp inhibition, although the increase in brain concentrations after P-gp inhibition was somewhat greater in rats than in the other species. Differences in plasma protein binding and metabolism did not explain the species-related differences. The findings are important for interpretation of brain drug delivery when extrapolating preclinical data to humans. Compounds found to be P-gp substrates in rodents are likely to also be substrates in higher species, but sufficient blood-brain barrier permeability may be retained in humans to allow the compound to act at intracerebral targets.

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The extracellular matrix protein laminin α2 regulates the maturation and function of the blood-brain barrier.

Michael J Menezes, Freyja K McClenahan, Cindy V Leiton … (2014)

Laminins are major constituents of the gliovascular basal lamina of the blood-brain barrier (BBB); however, the role of laminins in BBB development remains unclear. Here we report that Lama2(-/-) mice, lacking expression of the laminin α2 subunit of the laminin-211 heterotrimer expressed by astrocytes and pericytes, have a defective BBB in which systemically circulated tracer leaks into the brain parenchyma. The Lama2(-/-) vascular endothelium had significant abnormalities, including altered integrity and composition of the endothelial basal lamina, inappropriate expression of embryonic vascular endothelial protein MECA32, substantially reduced pericyte coverage, and tight junction abnormalities. Additionally, astrocytic endfeet were hypertrophic and lacked appropriately polarized aquaporin4 channels. Laminin-211 appears to mediate these effects at least in part by dystroglycan receptor interactions, as preventing dystroglycan expression in neural cells led to a similar set of BBB abnormalities and gliovascular disturbances, which additionally included perturbed vascular endothelial glucose transporter-1 localization. These findings provide insight into the cell and molecular changes that occur in congenital muscular dystrophies caused by Lama2 mutations or inappropriate dystroglycan post-translational modifications, which have accompanying brain abnormalities, including seizures. Our results indicate a novel role for laminin-dystroglycan interactions in the cooperative integration of astrocytes, endothelial cells, and pericytes in regulating the BBB.

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Author and article information

Contributors

Hiromasa Aoki: aokihiromasa812@gmail.com

Misaki Yamashita: y.misaki47@gmail.com

Tadahiro Hashita: thashita@phar.nagoya-cu.ac.jp

Takahiro Iwao: tiwao@phar.nagoya-cu.ac.jp

Tamihide Matsunaga: tmatsu@phar.nagoya-cu.ac.jp

Journal

Journal ID (nlm-ta): Fluids Barriers CNS

Journal ID (iso-abbrev): Fluids Barriers CNS

Title: Fluids and Barriers of the CNS

Publisher: BioMed Central (London )

ISSN (Electronic): 2045-8118

Publication date (Electronic): 30 March 2020

Publication date PMC-release: 30 March 2020

Publication date Collection: 2020

Volume: 17

Electronic Location Identifier: 25

Affiliations

GRID grid.260433.0, ISNI 0000 0001 0728 1069, Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, , Nagoya City University, ; 3-1 Tanabe-dori, Mizuho-ku, Nagoya, 467-8603 Japan

Article

Publisher ID: 186

DOI: 10.1186/s12987-020-00186-4

PMC ID: 7106710

PubMed ID: 32228708

SO-VID: f7fed087-da6c-4f93-b35c-e8e9d432b50d

License:

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 7 November 2019

Date accepted : 17 March 2020

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100009619, Japan Agency for Medical Research and Development;

Award ID: JP19be0304322

Award Recipient : Tadahiro Hashita

Funded by: FundRef http://dx.doi.org/10.13039/501100001691, Japan Society for the Promotion of Science;

Award ID: JP17K08422

Award Recipient : Tadahiro Hashita

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ScienceOpen disciplines: Neurology

Keywords: human induced pluripotent stem cell,brain microvascular endothelial cell,laminin 221,differentiation,blood–brain barrier

Data availability:

ScienceOpen disciplines: Neurology

Keywords: human induced pluripotent stem cell, brain microvascular endothelial cell, laminin 221, differentiation, blood–brain barrier

Laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity

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Nanoparticles to cross the blood-brain barrier (BBB)

Most cited references 14

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells

Species differences in blood-brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport.

The extracellular matrix protein laminin α2 regulates the maturation and function of the blood-brain barrier.

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