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Human iPSC-derived blood-brain barrier microvessels: validation of barrier function and endothelial cell behavior.

ABSTRACT: Microvessels of the blood-brain barrier (BBB) regulate transport into the brain. The highly specialized brain microvascular endothelial cells, a major component of the BBB, express tight junctions and efflux transporters which regulate paracellular and transcellular permeability. However, most existing models of BBB microvessels fail to exhibit physiological barrier function. Here, using (iPSC)-derived human brain microvascular endothelial cells (dhBMECs) within templated type I collagen channels we mimic the cylindrical geometry, cell-extracellular matrix interactions, and shear flow typical of human brain post-capillary venules. We characterize the structure and barrier function in comparison to non-brain-specific microvessels, and show that dhBMEC microvessels recapitulate physiologically low solute permeability and quiescent endothelial cell behavior. Transcellular permeability is increased two-fold using a clinically relevant dose of a p-glycoprotein inhibitor tariquidar, while paracellular permeability is increased using a bolus dose of hyperosmolar agent mannitol. Lastly, we show that our human BBB microvessels are responsive to inflammatory cytokines via upregulation of surface adhesion molecules and increased leukocyte adhesion, but no changes in permeability. Human iPSC-derived blood-brain barrier microvessels support quantitative analysis of barrier function and endothelial cell dynamics in quiescence and in response to biologically- and clinically-relevant perturbations.

SUBMITTER: Linville RM

PROVIDER: S-EPMC6289621 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Human iPSC-derived blood-brain barrier microvessels: validation of barrier function and endothelial cell behavior.

Linville Raleigh M RM DeStefano Jackson G JG Sklar Matt B MB Xu Zinnia Z Farrell Alanna M AM Bogorad Max I MI Chu Chengyan C Walczak Piotr P Cheng Linzhao L Mahairaki Vasiliki V Whartenby Katharine A KA Calabresi Peter A PA Searson Peter C PC

Biomaterials 20181025

Microvessels of the blood-brain barrier (BBB) regulate transport into the brain. The highly specialized brain microvascular endothelial cells, a major component of the BBB, express tight junctions and efflux transporters which regulate paracellular and transcellular permeability. However, most existing models of BBB microvessels fail to exhibit physiological barrier function. Here, using (iPSC)-derived human brain microvascular endothelial cells (dhBMECs) within templated type I collagen channel ...[more]

PMID: 30391800

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Project description:BACKGROUND:Pericytes of the blood-brain barrier (BBB) are embedded within basement membrane between brain microvascular endothelial cells (BMECs) and astrocyte end-feet. Despite the direct cell-cell contact observed in vivo, most in vitro BBB models introduce an artificial membrane that separates pericytes from BMECs. In this study, we investigated the effects of pericytes on BMEC barrier function across a range of in vitro platforms with varied spatial orientations and levels of cell-cell contact. METHODS:We differentiated RFP-pericytes and GFP-BMECs from hiPSCs and monitored transendothelial electrical resistance (TEER) across BMECs on transwell inserts while pericytes were either directly co-cultured on the membrane, indirectly co-cultured in the basolateral chamber, or embedded in a collagen I gel formed on the transwell membrane. We then incorporated pericytes into a tissue-engineered microvessel model of the BBB and measured pericyte motility and microvessel permeability. RESULTS:We found that BMEC monolayers did not require co-culture with pericytes to achieve physiological TEER values (> 1500 Ω cm2). However, under stressed conditions where TEER values for BMEC monolayers were reduced, indirectly co-cultured hiPSC-derived pericytes restored optimal TEER. Conversely, directly co-cultured pericytes resulted in a decrease in TEER by interfering with BMEC monolayer continuity. In the microvessel model, we observed direct pericyte-BMEC contact, abluminal pericyte localization, and physiologically-low Lucifer yellow permeability comparable to that of BMEC microvessels. In addition, pericyte motility decreased during the first 48 h of co-culture, suggesting progression towards pericyte stabilization. CONCLUSIONS:We demonstrated that monocultured BMECs do not require co-culture to achieve physiological TEER, but that suboptimal TEER in stressed monolayers can be increased through co-culture with hiPSC-derived pericytes or conditioned media. We also developed the first BBB microvessel model using exclusively hiPSC-derived BMECs and pericytes, which could be used to examine vascular dysfunction in the human CNS.

Project description:Autoreactive T lymphocytes crossing the blood-brain barrier (BBB) into the central nervous system (CNS) play a crucial role in the initiation of demyelination and neurodegeneration in multiple sclerosis (MS). Recently, extracellular vesicles (EV) secreted by BBB endothelial cells (BBB-EC) have emerged as a unique form of cell-to-cell communication that contributes to cerebrovascular dysfunction. However, the precise impact of different size-based subpopulations of BBB-EC-derived EV (BBB-EV) on the early stages of MS remains unclear. Therefore, our objective was to investigate the content and function of distinct BBB-EV subpopulations in regulating BBB integrity and their role in T cell transendothelial migration, both in vitro and in vivo. Our study reveals that BBB-ECs release two distinct size based EV populations, namely small EV (sEV; 30-150 nm) and large EV (lEV; 150-300 nm), with a significantly higher secretion of sEV during inflammation. Notably, the expression patterns of cytokines and adhesion markers differ significantly between these BBB-EV subsets, indicating specific functional differences in the regulation of T cell migration. Through in vitro experiments, we demonstrate that lEV, which predominantly reflect their cellular source, play a major role in BBB integrity loss and the enhanced migration of pro-inflammatory Th1 and Th17.1 cells. Conversely, sEV appear to protect BBB function by inducing an anti-inflammatory phenotype in BBB-EC. These findings align with our in vivo data, where the administration of sEV to mice with experimental autoimmune encephalomyelitis (EAE) results in lower disease severity compared to the administration of lEV, which exacerbates disease symptoms. In conclusion, our study highlights the distinct and opposing effects of BBB-EV subpopulations on the BBB, both in vitro and in vivo. These findings underscore the need for further investigation into the diagnostic and therapeutic potential of BBB-EV in the context of MS.

Project description:Transwell-based models of the blood-brain barrier (BBB) incorporating monolayers of human brain microvascular endothelial cells (dhBMECs) derived from induced pluripotent stem cells show many of the key features of the BBB, including expression of transporters and efflux pumps, expression of tight junction proteins, and physiological values of transendothelial electrical resistance. The fabrication of 3D BBB models using dhBMECs has so far been unsuccessful due to the poor adhesion and survival of these cells on matrix materials commonly used in tissue engineering.To address this issue, we systematically screened a wide range of matrix materials (collagen I, hyaluronic acid, and fibrin), compositions (laminin/entactin), protein coatings (fibronectin, laminin, collagen IV, perlecan, and agrin), and soluble factors (ROCK inhibitor and cyclic adenosine monophosphate) in 2D culture to assess cell adhesion, spreading, and barrier function.Cell coverage increased with stiffness of collagen I gels coated with collagen IV and fibronectin. On 7 mg mL-1 collagen I gels coated with basement membrane proteins (fibronectin, collagen IV, and laminin), cell coverage was high but did not reliably reach confluence. The transendothelial electrical resistance (TEER) on collagen I gels coated with basement membrane proteins was lower than on coated transwell membranes. Agrin, a heparin sulfate proteoglycan found in basement membranes of the brain, promoted monolayer formation but resulted in a significant decrease in transendothelial electrical resistance (TEER). However, the addition of ROCK inhibitor, cAMP, or cross-linking the gels to increase stiffness, resulted in a significant improvement of TEER values and enabled the formation of confluent monolayers.Having identified matrix compositions that promote monolayer formation and barrier function, we successfully fabricated dhBMEC microvessels in cross-linked collagen I gels coated with fibronectin and collagen IV, and treated with ROCK inhibitor and cAMP. We measured apparent permeability values for Lucifer yellow, comparable to values obtained in the transwell assay. During these experiments we observed no focal leaks, suggesting the formation of tight junctions that effectively block paracellular transport.

Dataset Information

Human iPSC-derived blood-brain barrier microvessels: validation of barrier function and endothelial cell behavior.

Publications

Human iPSC-derived blood-brain barrier microvessels: validation of barrier function and endothelial cell behavior.

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