Human induced pluripotent stem cells (BIONi010-C) generate tight cell monolayers with blood-brain barrier traits and functional expression of large neutral amino acid transporter 1 (SLC7A5)

Goldeman, Charlotte; Andersen, Mads Varis Nis; Al-Robai, A.; Buchholtz, T.; Svane, N.; Ozgür, Burak; Holst, Bjørn; Shusta, Eric V.; Hall, Vanessa Jane; Saaby, Lasse; Hyttel, Poul; Brodin, Birger

doi:10.1016/j.ejps.2020.105577

Cited by 15 publications

(14 citation statements)

References 56 publications

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“…However, the reproducibility of measurements with chopstick electrodes can also be a serious concern due to large variations in electrode positioning, the small electrode surfaces, and their geometry related to the membrane. To compare how measuring devices influence TEER values, three TEER measurement devices (chopstick and chamber electrodes, and a cellZscope system) were used to assess TEER values across three different cell culture models: bEnd.3 mouse brain endothelial cell line [41], Caco-2 human epithelial cell line [42], and human endothelial cells derived from Bioni010-C induced pluripotent stem cells [43]. The three types of cell culture models represent (1) low barrier tightness (TEER <25 Ω•cm 2 ), (2) medium-to-high barrier integrity (TEER ~1000-1500 Ω•cm 2 ), and (3) extremely tight barrier properties (TEER >5000 Ω•cm 2 ), respectively (Figure 5).…”

Section: Influence Of Measuring Devices: Differences Between Chopstic...mentioning

confidence: 99%

Transendothelial Electrical Resistance Measurement across the Blood–Brain Barrier: A Critical Review of Methods

et al. 2021

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The blood–brain barrier (BBB) represents the tightest endothelial barrier within the cardiovascular system characterized by very low ionic permeability. Our aim was to describe the setups, electrodes, and instruments to measure electrical resistance across brain microvessels and culture models of the BBB, as well as critically assess the influence of often neglected physical and technical parameters such as temperature, viscosity, current density generated by different electrode types, surface size, circumference, and porosity of the culture insert membrane. We demonstrate that these physical and technical parameters greatly influence the measurement of transendothelial electrical resistance/resistivity (TEER) across BBB culture models resulting in severalfold differences in TEER values of the same biological model, especially in the low-TEER range. We show that elevated culture medium viscosity significantly increases, while higher membrane porosity decreases TEER values. TEER data measured by chopstick electrodes can be threefold higher than values measured by chamber electrodes due to different electrode size and geometry, resulting in current distribution inhomogeneity. An additional shunt resistance at the circumference of culture inserts results in lower TEER values. A detailed description of setups and technical parameters is crucial for the correct interpretation and comparison of TEER values of BBB models.

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Section: Influence Of Measuring Devices: Differences Between Chopstic...mentioning

confidence: 99%

Transendothelial Electrical Resistance Measurement across the Blood–Brain Barrier: A Critical Review of Methods

et al. 2021

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“…Cells were incubated for 10 min with the respective inhibitor concentration and 10 µM [ 13 C 6 , 15 N]-L-leucine. L-leucine is the gold-standard amino acid widely used to characterize LAT-1 HD [ 5 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. IC 50 was determined with three independent experiments with at least three replicates.…”

Section: Resultsmentioning

confidence: 99%

“…Direct measurement of amino acid uptake can especially be realized in heterologous expression systems with low intracellular levels of amino acids, such as Xenopus laevis oocytes [ 28 ]. Although 13 C-isotopologes of amino acids have been used for uptake experiments [ 10 ], especially in the context of metabolism studies [ 29 ], previous investigations of L-leucine uptake in the context of transporter characterization were primarily based on the radioactive isotopologes [ 14 C]-L-leucine or [ 3 H]-L-leucine [ 5 , 10 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. Nevertheless, through comprehensive uptake studies with different amino acids and the consideration of the respective metabolism, the specific contribution of LAT-1 HD for L-leucine uptake and, hence, functional characterization can be performed [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine

Bay

Bajraktari-Sylejmani

Haefeli

et al. 2022

IJMS

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The solute carrier L-type amino acid transporter 1 (LAT-1/SLC7A5) is a viable target for drug delivery to the central nervous system (CNS) and tumors due to its high abundance at the blood–brain barrier and in tumor tissue. LAT-1 is only localized on the cell surface as a heterodimer with CD98, which is not required for transporter function. To support future CNS drug-delivery development based on LAT-1 targeting, we established an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay for stable isotopically labeled leucine ([13C6, 15N]-L-leucine), with a dynamic range of 0.1–1000 ng/mL that can be applied for the functional testing of LAT-1 activity when combined with specific inhibitors and, consequently, the LAT-1 inhibition capacity of new compounds. The assay was established in a 96-well format, facilitating high-throughput experiments, and, hence, can support the screening for novel inhibitors. Applicable recommendations of the US Food and Drug Administration and European Medicines Agency for bioanalytical method validation were followed to validate the assay. The assay was applied to investigate the IC50 of two well-known LAT-1 inhibitors on hCMEC/D3 cells: the highly specific LAT-1 inhibitor JPH203, which was also used to demonstrate LAT-1 specific uptake, and the general system L inhibitor BCH. In addition, the [13C6, 15N]-L-leucine uptake was determined on two human brain capillary endothelial cell lines (NKIM-6 and hCMEC/D3), which were characterized for their expressional differences of LAT-1 at the protein and mRNA level and the surface amount of CD98. The IC50 values of the inhibitors were in concordance with previously reported values. Furthermore, the [13C6, 15N]-L-leucine uptake was significantly higher in hCMEC/D3 cells compared to NKIM-6 cells, which correlated with higher expression of LAT-1 and a higher surface amount of CD98. Therefore, the UPLC-MS/MS quantification of ([13C6, 15N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.

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“…Bovine, mouse, and human brain endothelial cell cultures were applied to study GDH expression, capacity and function in brain endothelial cells across species. We have previously established, characterized and applied bovine brain endothelial cell cultures for studying glutamate transport across the blood‐brain barrier (Helms, et al., 2017; Helms et al., 2012), and in a parallel study, we characterized a human iPSC‐derived endothelial cell based blood‐brain barrier model (Goldeman et al., 2021). Mouse brain endothelial cells were established and characterized for common brain endothelial phenotypic markers (Figure 2).…”

Section: Resultsmentioning

confidence: 99%

“…The human iPSC derived model was thoroughly characterized in a parallel study showing a range of blood–brain barrier properties, although their morphology and lack of functional p‐glycoprotein also demonstrated that they do not resemble brain endothelial cells regarding all aspects (Goldeman et al. 2021). Thus, the models applied in the present study have all been thoroughly characterized concerning a range of properties, but this does not change the fact the in vitro models are simplifications of the in vivo situation, which has to be taken into account when drawing conclusions.…”

Section: Discussionmentioning

confidence: 99%

Brain endothelial cells metabolize glutamate via glutamate dehydrogenase to replenish TCA‐intermediates and produce ATP under hypoglycemic conditions

Hinca

Salcedo

Wagner

et al. 2020

Journal of Neurochemistry

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The endothelial cells of the blood–brain barrier participate in the regulation of glutamate concentrations in the brain interstitial fluid by taking up brain glutamate. However, endothelial glutamate metabolism has not been characterized, nor is its role in brain glutamate homeostasis and endothelial energy production known. The aim of this study was to investigate endothelial glutamate dehydrogenase (GDH) expression and glutamate metabolism and probe its functional significance. The primary brain endothelial cells were isolated from bovine and mouse brains, and human brain endothelial cells were derived from induced pluripotent stem cells. GDH expression on the protein level and GDH function were investigated in the model systems using western blotting, confocal microscopy, 13C‐glutamate metabolism, and Seahorse assay. In this study, it was shown that GDH was expressed in murine and bovine brain capillaries and in cultured primary mouse and bovine brain endothelial cells as well as in human‐induced pluripotent stem cell‐derived endothelial cells. The endothelial GDH expression was confirmed in brain capillaries from mice carrying a central nervous system‐specific GDH knockout. Endothelial cells from all tested species metabolized 13C‐glutamate to α‐ketoglutarate, which subsequently entered the tricarboxylic acid (TCA)‐cycle. Brain endothelial cells maintained mitochondrial oxygen consumption rates, when supplied with glutamate alone, whereas glutamate supplied in addition to glucose did not lead to additional oxygen consumption. In conclusion, brain endothelial cells directly take up and metabolize glutamate and utilize the resulting α‐ketoglutarate in the tricarboxylic acid cycle to ultimately yield ATP if glucose is unavailable.

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Human induced pluripotent stem cells (BIONi010-C) generate tight cell monolayers with blood-brain barrier traits and functional expression of large neutral amino acid transporter 1 (SLC7A5)

Cited by 15 publications

References 56 publications

Transendothelial Electrical Resistance Measurement across the Blood–Brain Barrier: A Critical Review of Methods

Transendothelial Electrical Resistance Measurement across the Blood–Brain Barrier: A Critical Review of Methods

Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine

Brain endothelial cells metabolize glutamate via glutamate dehydrogenase to replenish TCA‐intermediates and produce ATP under hypoglycemic conditions

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