Construction and Functional Evaluation of a Three-Dimensional Blood–Brain Barrier Model Equipped With Human Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells
Abstract:Purpose
The purpose of this study was to construct and validate an in vivo three-dimensional blood–brain barrier (3D-BBB) model system equipped with brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs).
Methods
The 3D-BBB system was constructed by seeding hiPS-BMECs onto the capillary lane of a MIMETAS OrganoPlate® 3-lane coated with fibronectin/collagen IV. hiPS-BMECs were incubated under continuous switchba… Show more
“…BMECs cultured in this system expressed BBB markers such as Claudin-5, ZO-1, Glut-1 and Breast Cancer Resistance Protein (BCRP) and exhibited low permeability to LY. Notably, the authors found that ABCG2 (gene encoding BCRP) expression in BMECs was 3.3-fold higher in their 3D system compared to 2D monolayer culture measured by qRT-PCR, and that transport of BCRP substrates in their 3D system was comparable to that in previous in vivo rat studies (Kurosawa et al, 2022). It is possible that these systems could be further improved and lead to better in vitro BBB models if coatings alternative to C4/Fn were tested and optimized.…”
The blood-brain barrier (BBB) is a highly impermeable barrier separating circulating blood and brain tissue. A functional BBB is critical for brain health, and BBB dysfunction has been linked to the pathophysiology of diseases such as stroke and Alzheimer’s disease. A variety of models have been developed to study the formation and maintenance of the BBB, ranging from in vivo animal models to in vitro models consisting of primary cells or cells differentiated from human pluripotent stem cells (hPSCs). These models must consider the composition and source of the cellular components of the neurovascular unit (NVU), including brain microvascular endothelial cells (BMECs), brain pericytes, astrocytes, and neurons, and how these cell types interact. In addition, the non-cellular components of the BBB microenvironment, such as the brain vascular basement membrane (BM) that is in direct contact with the NVU, also play key roles in BBB function. Here, we review how extracellular matrix (ECM) proteins in the brain vascular BM affect the BBB, with a particular focus on studies using hPSC-derived in vitro BBB models, and discuss how future studies are needed to advance our understanding of how the ECM affects BBB models to improve model performance and expand our knowledge on the formation and maintenance of the BBB.
“…BMECs cultured in this system expressed BBB markers such as Claudin-5, ZO-1, Glut-1 and Breast Cancer Resistance Protein (BCRP) and exhibited low permeability to LY. Notably, the authors found that ABCG2 (gene encoding BCRP) expression in BMECs was 3.3-fold higher in their 3D system compared to 2D monolayer culture measured by qRT-PCR, and that transport of BCRP substrates in their 3D system was comparable to that in previous in vivo rat studies (Kurosawa et al, 2022). It is possible that these systems could be further improved and lead to better in vitro BBB models if coatings alternative to C4/Fn were tested and optimized.…”
The blood-brain barrier (BBB) is a highly impermeable barrier separating circulating blood and brain tissue. A functional BBB is critical for brain health, and BBB dysfunction has been linked to the pathophysiology of diseases such as stroke and Alzheimer’s disease. A variety of models have been developed to study the formation and maintenance of the BBB, ranging from in vivo animal models to in vitro models consisting of primary cells or cells differentiated from human pluripotent stem cells (hPSCs). These models must consider the composition and source of the cellular components of the neurovascular unit (NVU), including brain microvascular endothelial cells (BMECs), brain pericytes, astrocytes, and neurons, and how these cell types interact. In addition, the non-cellular components of the BBB microenvironment, such as the brain vascular basement membrane (BM) that is in direct contact with the NVU, also play key roles in BBB function. Here, we review how extracellular matrix (ECM) proteins in the brain vascular BM affect the BBB, with a particular focus on studies using hPSC-derived in vitro BBB models, and discuss how future studies are needed to advance our understanding of how the ECM affects BBB models to improve model performance and expand our knowledge on the formation and maintenance of the BBB.
“…However, SLC7A3 has low expression levels in iPSC-derived BMEC-like cells, mouse brain endothelial cells [ 66 ], and human endothelial cells [ 67 ] compared to the relatively higher expression of SLC7A1 . Further, SLC7A1 is highly and selectively expressed in brain endothelial cells with respect to other neurovascular cell types in single-cell RNA-sequencing datasets [ 66 ], and CAT-1 has previously been shown to be functionally active in iPSC-derived BBB models [ 68 , 69 ], marking it as an interesting candidate for further evaluation. Fig.…”
Background
The hormone leptin exerts its function in the brain to reduce food intake and increase energy expenditure to prevent obesity. However, most obese subjects reflect the resistance to leptin even with elevated serum leptin. Considering that leptin must cross the blood–brain barrier (BBB) in several regions to enter the brain parenchyma, altered leptin transport through the BBB might play an important role in leptin resistance and other biological conditions. Here, we report the use of a human induced pluripotent stem cell (iPSC)-derived BBB model to explore mechanisms that influence leptin transport.
Methods
iPSCs were differentiated into brain microvascular endothelial cell (BMEC)-like cells using standard methods. BMEC-like cells were cultured in Transwell filters, treated with ligands from a nuclear receptor agonist library, and assayed for leptin transport using an enzyme-linked immune sorbent assay. RNA sequencing was further used to identify differentially regulated genes and pathways. The role of a select hit in leptin transport was tested with the competitive substrate assay and after gene knockdown using CRISPR techniques.
Results
Following a screen of 73 compounds, 17β-estradiol was identified as a compound that could significantly increase leptin transport. RNA sequencing revealed many differentially expressed transmembrane transporters after 17β-estradiol treatment. Of these, cationic amino acid transporter-1 (CAT-1, encoded by SLC7A1) was selected for follow-up analyses due to its high and selective expression in BMECs in vivo. Treatment of BMEC-like cells with CAT-1 substrates, as well as knockdown of CAT-1 expression via CRISPR-mediated epigenome editing, yielded significant increases in leptin transport.
Conclusions
A major female sex hormone, as well as an amino acid transporter, were revealed as regulators of leptin BBB transport in the iPSC-derived BBB model. Outcomes from this work provide insights into regulation of hormone transport across the BBB.
“…As discussed in previous sections, such new technology has not yet yielded clinically relevant systems and results regarding brain penetration of molecules. However, recent advancements in brain organ on chip or 3D platforms (Piantino et al., 2022) using human‐induced pluripotent stem cell‐derived BMEC provided promising results (Kurosawa et al., 2022). However, more research needs to be conducted in this area to have a complete model that will be fit for disease pathology understanding as well as the function of BBB transporters.…”
One challenge in central nervous system (CNS) drug discovery has been ensuring the blood-brain barrier (BBB) penetration of compounds at an efficacious concentration that provides suitable safety margins for clinical investigation. Research providing for the accurate prediction of brain penetration of compounds during preclinical discovery is important to a CNS program. In the BBB, P-glycoprotein (Pgp) (ABCB1) and breast cancer resistance protein (BCRP) (ABCG2) transporters have been demonstrated to play a major role in the active efflux of endogenous compounds and xenobiotics out of the brain microvessel cells and back to the systemic circulation. In the past 10 years, there has been significant technological improvement in the sensitivity of quantitative proteomics methods, in vivo imaging, in vitro methods of organoid and microphysiological systems, as well as in silico quantitative physiological based pharmacokinetic and systems pharmacology models. Scientists continually leverage these advancements to interrogate the distribution of compounds in the CNS which may also show signals of substrate specificity of P-gp and/ or BCRP. These methods have shown promise toward predicting and quantifying the unbound concentration(s) within the brain relevant for efficacy or safety. In this review, the authors have summarized the in vivo, in vitro, and proteomics advancements toward understanding the contribution of P-gp and/or BCRP in restricting the entry of compounds to the CNS of either healthy or special populations. Special emphasis has been provided on recent investigations on the application of a proteomics-informed approach to predict steady-state drug concentrations in the brain. Moreover, future perspectives regarding the role of these transporters in newer modalities are discussed.
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