TY - JOUR

T1 - Characterization of an iPSC-based barrier model for blood-brain barrier investigations using the SBAD0201 stem cell line

AU - Ozgür, Burak

AU - Puris, Elena

AU - Brachner, Andreas

AU - Appelt-Menzel, Antje

AU - Oerter, Sabrina

AU - Balzer, Viktor

AU - Holst, Mikkel Roland

AU - Christiansen, Rasmus Folmann

AU - Hyldig, Kathrine

AU - Buckley, Stephen T

AU - Kristensen, Mie

AU - Auriola, Seppo

AU - Jensen, Allan

AU - Fricker, Gert

AU - Nielsen, Morten Schallburg

AU - Neuhaus, Winfried

AU - Brodin, Birger

N1 - © 2023. The Author(s).

PY - 2023

Y1 - 2023

N2 - BACKGROUND: Blood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201.METHODS: The model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1).RESULTS: The resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm 2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane. CONCLUSIONS: In conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.

AB - BACKGROUND: Blood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201.METHODS: The model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1).RESULTS: The resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm 2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane. CONCLUSIONS: In conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.

KW - Humans

KW - Animals

KW - Cattle

KW - Mice

KW - Swine

KW - Blood-Brain Barrier/metabolism

KW - Induced Pluripotent Stem Cells/physiology

KW - Cell Line

KW - Biological Transport

KW - Brain/metabolism

KW - Membrane Transport Proteins/metabolism

KW - Cells, Cultured

U2 - 10.1186/s12987-023-00501-9

DO - 10.1186/s12987-023-00501-9

M3 - Journal article

C2 - 38115090

VL - 20

JO - Fluids and Barriers of the CNS

JF - Fluids and Barriers of the CNS

SN - 2045-8118

IS - 1

M1 - 96

ER -