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Laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity
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 gelatino...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106710/ https://www.ncbi.nlm.nih.gov/pubmed/32228708 http://dx.doi.org/10.1186/s12987-020-00186-4 |
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author | Aoki, Hiromasa Yamashita, Misaki Hashita, Tadahiro Iwao, Takahiro Matsunaga, Tamihide |
author_facet | Aoki, Hiromasa Yamashita, Misaki Hashita, Tadahiro Iwao, Takahiro Matsunaga, Tamihide |
author_sort | Aoki, Hiromasa |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7106710 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-71067102020-04-01 Laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity Aoki, Hiromasa Yamashita, Misaki Hashita, Tadahiro Iwao, Takahiro Matsunaga, Tamihide Fluids Barriers CNS Research 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. BioMed Central 2020-03-30 /pmc/articles/PMC7106710/ /pubmed/32228708 http://dx.doi.org/10.1186/s12987-020-00186-4 Text en © The Author(s) 2020 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. |
spellingShingle | Research Aoki, Hiromasa Yamashita, Misaki Hashita, Tadahiro Iwao, Takahiro Matsunaga, Tamihide Laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity |
title | Laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity |
title_full | Laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity |
title_fullStr | Laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity |
title_full_unstemmed | Laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity |
title_short | Laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity |
title_sort | laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106710/ https://www.ncbi.nlm.nih.gov/pubmed/32228708 http://dx.doi.org/10.1186/s12987-020-00186-4 |
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