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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.

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.

SUBMITTER: Aoki H

PROVIDER: S-EPMC7106710 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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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.

Aoki Hiromasa H Yamashita Misaki M Hashita Tadahiro T Iwao Takahiro T Matsunaga Tamihide T

Fluids and barriers of the CNS 20200330 1

<h4>Background</h4>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 an ...[more]

PMID: 32228708

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Project description:Multiple echo-time arterial spin labelling (multi-TE ASL) offers estimation of blood-tissue exchange dynamics by probing the T2 relaxation of the labelled spins. In this study, we provide a recipe for robust assessment of exchange time (Texch) as a proxy measure of blood-brain barrier (BBB) integrity based on a test-retest analysis. This includes a novel scan protocol and an extension of the two-compartment model with an "intra-voxel transit time" (ITT) to address tissue transit effects. With the extended model, we intend to separate the underlying two distinct mechanisms of tissue transit and exchange. The performance of the extended model in comparison with the two-compartment model was evaluated in simulations. Multi-TE ASL sequence with two different bolus durations was used to acquire in vivo data (n = 10). Cerebral blood flow (CBF), arterial transit time (ATT) and Texch were fitted with the two models, and mean grey matter values were compared. Additionally, the extended model also extracted ITT parameter. The test-retest reliability of Texch was assessed for intra-session, inter-session and inter-visit pairs of measurements. Intra-class correlation coefficient (ICC) and within-subject coefficient of variance (CoV) for grey matter were computed to assess the precision of the method. Mean grey matter Texch and ITT values were found to be 227.9 ± 37.9 ms and 310.3 ± 52.9 ms, respectively. Texch estimated by the extended model was 32.6 ± 5.9% lower than the two-compartment model. A significant ICC was observed for all three measures of Texch reliability (P < 0.05). Texch intra-session CoV, inter-session CoV and inter-visit CoV were found to be 6.6%, 7.9%, and 8.4%, respectively. With the described improvements addressing intra-voxel transit effects, multi-TE ASL shows good reproducibility as a non-invasive measure of BBB permeability. These findings offer an encouraging step forward to apply this potential BBB permeability biomarker in clinical research.

Project description:Simple Summary Numerous studies over the past few decades have revealed that the interactions of gastric cancer cells with laminins through integrins play important roles in tumor cell proliferation, infiltration, and metastasis. However, the association between gastric cancer cells and the laminin E8 fragment, which is the smallest integrin-binding component, has not been investigated. In this study, we revealed that the laminin 511-E8 fragment had a greater impact on the adhesion, morphology, and proliferation of gastric cancer cells than full-length laminin 511. Thus, the laminin 511-E8 fragment is considered to be suitable for investigating the interaction between gastric cancer cells and extracellular matrices in tumor invasion and metastasis. Further, the involvement of Cdc42 in the laminin 511-E8 fragment-induced enhanced adhesion of gastric cancer cells was suggested. Abstract Background: The interaction between cancer cells and laminin (Ln) is a key event in tumor invasion and metastasis. Previously, we determined the effect of full-length Ln511 on gastric cancer cells. However, the interactions between the Ln511-E8 fragment, a truncated protein of Ln511, and gastric cancer cells have not been investigated. Methods: We investigated the adhesion properties of gastric cancer cells to full-length Ln511 and Ln511-E8 fragments. Results: The proliferation of four gastric cancer cell lines (SH-10-TC, MKN74, SC-6-JCK, and MKN45) was highest on the Ln511-E8 fragment. Further, a larger cytoplasm was observed in SH-10-TC and MKN74 cells cultured on full-length Ln511 or Ln511-E8 fragments. The percentage of adhesive cells was highest on the Ln511-E8 fragment in all four cell lines. Moreover, adhesion of the gastric cancer cells to Ln511-E8 fragment-coated plates was reduced by the Cdc42 GTPase inhibitor in a dose-dependent manner, suggesting the involvement of Cdc42 in the Ln511-E8 fragment-induced enhanced adhesion of gastric cancer cells. Conclusions: The Ln511-E8 fragment had a greater impact on the adhesion, morphology, and proliferation of gastric cancer cells than full-length laminin. Thus, the Ln511-E8 fragment is suitable for investigating the interaction between gastric cancer cells and extracellular matrices in tumor invasion and metastasis.

Dataset Information

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

Publications

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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