The human blood brain barrier (BBB) is a selective barrier formed by human brain endothelial cells (hBECs), which is important to ensure adequate neuronal function and protect the central nervous ...system (CNS) from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.
The market for neuropharmaceuticals is potentially one of the largest sectors of the global pharmaceutical market owing to the increase in average life expectancy and the fact that many neurological ...disorders have been largely refractory to pharmacotherapy. The brain is a delicate organ that can efficiently protect itself from harmful compounds and precisely regulate its microenvironment. Unfortunately, the same mechanisms can also prove to be formidable hurdles in drug development. An improved understanding of the regulatory interfaces that exist between blood and brain may provide novel and more effective strategies to treat neurological disorders.
Novel 6‐alkyl‐ and 6‐alkenyl‐3‐fluoro‐2‐pyridinaldoximes have been synthesised by using a mild and efficient chemoselective hydrogenation of 6‐alkynyl‐3‐fluoro‐2‐pyridinaldoxime scaffolds, without ...altering the reducible, unprotected, sensitive oxime functionality and the C−F bond. These novel 6‐alkyl‐3‐fluoro‐2‐pyridinaldoximes may find medicinal application as antidotes to organophosphate poisoning. Indeed, one low‐molecular‐weight compound exhibited increased affinity for sarin‐inhibited acetylcholinesterase (hAChE) and greater reactivation efficiency or resurrection for sarin‐inhibited hAChE, compared with those of 2‐pyridinaldoxime (2‐PAM) and 1‐({4‐(aminocarbonyl)pyridiniomethoxy}methyl)‐2‐(hydroxyimino)methylpyridinium chloride (HI‐6), two pyridinium salts currently used as antidote by several countries. In addition, the uncharged 3‐fluorinated bifunctional hybrid showed increased in vitro blood–brain barrier permeability compared with those of 2‐PAM, HI‐6 and obidoxime. These promising features of novel low‐molecular‐weight alkylfluoropyridinaldoxime open up a new era for the design, synthesis and discovery of central non‐quaternary broad spectrum reactivators for organophosphate‐inhibited cholinesterases.
Permeable antidotes: The chemoselective hydrogenation of 6‐alkynyl‐3‐fluoro‐2‐pyridinaldoximes to afford 6‐alkyl‐3‐fluoro‐2‐pyridinaldoximes under mild hydrogenation conditions, without altering the C−F and oxime functionalities, is described. One of the resulting products acts as a low‐molecular‐weight non‐quaternary fluorinated hybrid reactivator of sarin‐inhibited human‐acetylcholinesterase, with unprecedented blood–brain barrier permeability efficacy.
Recent events demonstrated that organophosphorus nerve agents are a serious threat for civilian and military populations. The current therapy includes a pyridinium aldoxime reactivator to restore the ...enzymatic activity of acetylcholinesterase located in the central nervous system and neuro-muscular junctions. One major drawback of these charged acetylcholinesterase reactivators is their poor ability to cross the blood-brain barrier. In this study, we propose to evaluate glucoconjugated oximes devoid of permanent charge as potential central nervous system reactivators. We determined their
reactivation efficacy on inhibited human acetylcholinesterase, the crystal structure of two compounds in complex with the enzyme, their protective index on intoxicated mice, and their pharmacokinetics. We then evaluated their endothelial permeability coefficients with a human
model. This study shed light on the structural restrains of new sugar oximes designed to reach the central nervous system through the glucose transporter located at the blood-brain barrier.
Although brain metastases are the most common brain tumors in adults, there are few treatment options in this setting. To colonize the brain, circulating tumor cells must cross the blood–brain ...barrier (BBB), which is situated within specialized, restrictive microvascular endothelium. Understanding how cancer cells manage to transmigrate through the BBB might enable this process to be prevented. In vitro models are dedicated tools for characterizing the cellular and molecular mechanisms that underlie transendothelial migration process, as long as they accurately mimic the brain endothelium's in vivo characteristics. The objective of the present study was to adapt an existing in vitro model of the human BBB for use in studying cancer cell transmigration. The model is based on the coculture of endothelial cells (ECs, derived from cord blood hematopoietic stem cells) and brain pericytes. To allow the migration of cancer cells into the lower compartment, our model had to be transposed onto inserts with a larger pore size. However, we encountered a problem when culturing ECs on large (3-μm)-pore inserts: the cells crossed the membrane and formed a non-physiological second layer on the lower face of the insert. Using 3-μm-pore inserts (in a 12-well plate format), we report here on a method that enables the maintenance of a single monolayer of ECs on the insert's upper face only. Under these chosen conditions, the ECs exhibited typical BBB properties found in the original model (including restricted paracellular permeability and the expression of continuous tight junctions). This modified in vitro model of the human BBB enabled us to investigate the migratory potential of the MDA-MB-231 cell line (derived from highly metastatic human breast cancer cells). Last, the results obtained were compared with the rate of transmigration through endothelia with no BBB features.
Lipoprotein transport across the blood-brain barrier (BBB) is of critical importance for the delivery of essential lipids to the brain cells. The occurrence of a low density lipoprotein (LDL) ...receptor on the BBB has recently been demonstrated. To examine further the function of this receptor, we have shown using an in vitro model of the BBB, that in contrast to acetylated LDL, which does not cross the BBB, LDL is specifically transcytosed across the monolayer. The C7 monoclonal antibody, known to interact with the LDL receptor-binding domain, totally blocked the transcytosis of LDL, suggesting that the transcytosis is mediated by the receptor. Furthermore, we have shown that cholesterol-depleted astrocytes upregulate the expression of the LDL receptor at the BBB. Under these conditions, we observed that the LDL transcytosis parallels the increase in the LDL receptor, indicating once more that the LDL is transcytosed by a receptor-mediated mechanism. The nondegradation of the LDL during the transcytosis indicates that the transcytotic pathway in brain capillary endothelial cells is different from the LDL receptor classical pathway. The switch between a recycling receptor to a transcytotic receptor cannot be explained by a modification of the internalization signals of the cytoplasmic domain of the receptor, since we have shown that LDL receptor messengers in growing brain capillary ECs (recycling LDL receptor) or differentiated cells (transcytotic receptor) are 100% identical, but we cannot exclude posttranslational modifications of the cytoplasmic domain, as demonstrated for the polymeric immunoglobulin receptor. Preliminary studies suggest that caveolae are likely to be involved in the potential transport of LDL from the blood to the brain.
We previously developed an in vitro model of the human blood-brain barrier (BBB) based on the use of endothelial cells derived from CD34
-hematopoietic stem cells and cultured with brain pericytes. ...The purpose of the present study was to provide information on the protein expression levels of the transporters, receptors, tight junction/adherence junction molecules, and transporter-associated molecules of human brain-like endothelial cells (hBLECs). The absolute protein expression levels were determined by liquid chromatography-mass spectrometry-based quantitative targeted absolute proteomics and compared with those from human brain microvessels (hBMVs). The protein levels of CD144, CD147, MRP4, Annexin A6 and caveolin-1 showed more than 3-fold abundance in hBLECs, those of MCT1, Connexin 43, TfR1, and claudin-5 showed less than 3-fold differences, and the protein levels of other drug efflux transporters and nutrient transporters were less represented in hBLECs than in hBMVs. It is noteworthy that BCRP was more expressed than MDR1 in hBLECs, as this was the case for hBMVs. These results suggest that transports mediated by MCT1, TfR1, and claudin-5-related tight junction function reflect the in vivo BBB situation. The present study provided a better characterization of hBLECs and clarified the equivalence of the transport characteristics between in vitro BBB models and in vivo BBB models using LC-MS/MS-based protein quantification.
Pediatric diffuse intrinsic pontine glioma (DIPG) represents one of the most devastating and lethal brain tumors in children with a median survival of 12 months. The high mortality rate can be ...explained by the ineligibility of patients to surgical resection due to the diffuse growth pattern and midline localization of the tumor. While the therapeutic strategies are unfortunately palliative, the blood-brain barrier (BBB) is suspected to be responsible for the treatment inefficiency. Located at the brain capillary endothelial cells (ECs), the BBB has specific properties to tightly control and restrict the access of molecules to the brain parenchyma including chemotherapeutic compounds. However, these BBB specific properties can be modified in a pathological environment, thus modulating brain exposure to therapeutic drugs. Hence, this study aimed at developing a syngeneic human blood-brain tumor barrier model to understand how the presence of DIPG impacts the structure and function of brain capillary ECs.
A human syngeneic in vitro BBB model consisting of a triple culture of human (ECs) (differentiated from CD34
-stem cells), pericytes and astrocytes was developed. Once validated in terms of BBB phenotype, this model was adapted to develop a blood-brain tumor barrier (BBTB) model specific to pediatric DIPG by replacing the astrocytes by DIPG-007, -013 and -014 cells. The physical and metabolic properties of the BBTB ECs were analyzed and compared to the BBB ECs. The permeability of both models to chemotherapeutic compounds was evaluated.
In line with clinical observation, the integrity of the BBTB ECs remained intact until 7 days of incubation. Both transcriptional expression and activity of efflux transporters were not strongly modified by the presence of DIPG. The permeability of ECs to the chemotherapeutic drugs temozolomide and panobinostat was not affected by the DIPG environment.
This original human BBTB model allows a better understanding of the influence of DIPG on the BBTB ECs phenotype. Our data reveal that the chemoresistance described for DIPG does not come from the development of a "super BBB". These results, validated by the absence of modification of drug transport through the BBTB ECs, point out the importance of understanding the implication of the different protagonists in the pathology to have a chance to significantly improve treatment efficiency.
The derivation of human brain capillary endothelial cells is of utmost importance for drug discovery programs focusing on diseases of the central nervous system. Here, we describe a two-step ...differentiation protocol to derive brain capillary-like endothelial cells from human pluripotent stem cells. The cells were initially differentiated into endothelial progenitor cells followed by specification into a brain capillary-like endothelial cell phenotype using a protocol that combined the induction, in a time-dependent manner, of VEGF, Wnt3a, and retinoic acid signaling pathways and the use of fibronectin as the extracellular matrix. The brain capillary-like endothelial cells displayed a permeability to lucifer yellow of 1 × 10−3 cm/min, a transendothelial electrical resistance value of 60 Ω cm2 and were able to generate a continuous monolayer of cells expressing ZO-1 and CLAUDIN-5 but moderate expression of P-glycoprotein. Further maturation of these cells required coculture with pericytes. The study presented here opens a new approach for the study of soluble and non-soluble factors in the specification of endothelial progenitor cells into brain capillary-like endothelial cells.
•Derivation of BCLECs from iPSCs in chemically defined medium in two steps•Specification of EPCs into BCLECs requires the activation of VEGF, Wnt3a, and RA•Fibronectin seems a sufficient substrate for the specification of EPCs into BCLECs
In this article, Ferreira and colleagues describe a protocol for the derivation of brain capillary-like endothelial cells from iPSCs under fully chemically defined medium and extracellular matrix in two steps. The protocol requires the modulation of three signaling pathways (VEGF, Wnt3a, and retinoic acid) and the use of fibronectin as a single extracellular matrix component.
The blood-brain barrier (BBB) is a dynamic cellular complex that is responsible for the maintenance of brain homeostasis. To understand the BBB's key cellular and molecular mechanisms, in vitro ...models combining endothelial cells and astrocytes can be used to reproduce most of the barrier's in vivo features (low paracellular permeability and the expression of specific transporters). However, these models lack pericytes - a poorly characterized cell type which appears to be of crucial importance to understand BBB's function in healthy and diseased states. The present study sought to identify and characterize this cell population - which lacks a specific marker - by comparing its phenotype with that of vascular smooth muscle cells. Even if pericytes and smooth muscle cells shared many markers in vitro, our results showed that they could be distinguished by their different P-glycoprotein expression and γ-glutamyltranspeptidase activity. Two different three-cell-type culture models were described, including pericytes to mimic the neurovascular unit. In the first model, endothelial cells were cultured alone on a filter, away from glial cells and pericytes, allowing endothelial cell phenotype characterization. In the second model, glial cells were at the bottom of the well while pericytes and endothelial cells were cultured together in the filter: close interactions were observed in peg-and-socket contacts. In both models low paracellular permeability and P-glycoprotein functionality were demonstrated. These models are likely to be useful tools for understanding the pericytes' role in BBB physiology and could be of value in investigating the pericytes' influence on BBB in diseased states.
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