1. Stem Cell-Based Human Blood–Brain Barrier Models for Drug Discovery and Delivery 
S. Aday, R. Cecchelli, D. Hallier-Vanuxeem, M.P. Dehouck, L. Ferreira 
Trends in Biotechnology 
Volume 34, Issue 5, Pages (May 2016)
DOI: /j.tibtech
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2. Figure 1
Structure and Functionality of the Blood–Brain Barrier (BBB). (A) Brain barriers. The brain has several barriers, including (i) the BBB, (ii) the outer blood–cerebrospinal fluid (CSF)–brain barrier, and (iii) the blood–CSF barrier. (B) BBB structure. The BBB is formed by endothelial cells (ECs) that are in close association with astrocyte end feet and pericytes, forming a physical barrier. (C) BBB transport. Routes for molecular traffic across the BBB are shown. Some transporters are energy-dependent (e.g., P-glycoprotein, P-gp) and act as efflux transporters. AZT, azidothymidine. (D) Tight junctions. Tight junctions are typically located on the apical region of ECs. The tight junctions form complex networks that result in multiple barriers that restrict the penetration of polar drugs into the brain.
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3. Figure 2
In Vitro Human Blood–Brain Barrier (BBB) Models Derived From Stem Cells. (A) Cord blood-derived mononuclear cells (MNCs) or CD34+ cells as a source of brain-like endothelial cells (ECs). Recent studies have shown that MNCs or CD34+ cells may be an interesting source of ECs that are particularly responsive to instructive induction by the cells of the neurovascular unit (astrocytes or pericytes). These ECs were co-cultured with astrocytes for 14 days [14] or pericytes for 6 days [15]. In both cases the ECs showed markers of a BBB phenotype, including increased numbers of tight junctions and upregulation of GLUT-1 and P-gp; however, significant differences were observed in transendothelial electrical resistance (TEER) and permeability to small molecules (see main text). (B) Induced pluripotent stem cells (iPSCs) as a source of brain-like ECs. Undifferentiated iPSCs were differentiated simultaneously into ECs and neural cells, and then brain-like ECs were purified on a selective matrix [13]. When the brain-like ECs were co-cultured with astrocytes, the ECs exhibited a high TEER (between 700 and 1450Ω.cm2) and formed networks of tight junctions. (C) In vitro transwell assay is commonly used to evaluate the permeability and electrical resistance (TEER) of EC monolayers formed on a porous membrane and co-cultured with NVU cells such as astrocytes and pericytes. In permeability measurements, radio- or fluorescent-labeled solute or nanoparticles are introduced to the apical (luminal) side of the transwell, and the amount in the basolateral (abluminal) side is measured as a function of time. Voltohmmeters composed of two sets of electrodes are used for in vitro TEER measurements. In vivo, radiolabeled solutes are injected into the blood flow, and the penetration into the brain measured using imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT).
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4. Figure 3
Human Blood–Brain Barrier (BBB) Models for Drug Screening. Human BBB models can be used to predict the in vivo transport of drugs with different properties. (A) Some studies have shown a good correlation (R2=0.98) between the human BBB model and in vivo rodent brain uptake measured by in situ brain perfusion [13]. There was a 40-fold dynamic range of permeability coefficient (Pe) values. (B) We have shown recently that an in vitro human BBB model generated from hematopoietic stem/progenitor cells correlates well with human pharmacokinetic parameters [15]. In this case, the unbound brain-to-plasma concentration ratio (Kp,uu,brain) was calculated by the in vitro BBB model and correlated with the ratio of unbound cerebrospinal fluid (CSF)-to-plasma concentration (Kp,uu,CSF) in humans. For the nine compounds tested, a good correlation (R2=0.89) was found. Kp,uu,CSF = (in vivo concentration of unbound drug in the CSF)/(in vivo concentration of unbound drug in the plasma). Kp,uu,brain = (in vitro concentration of unbound drug in the brain)/(in vitro concentration of unbound drug in the plasma).
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5. Figure 4
Human Blood–Brain Barrier (BBB) Models To Study the Transport of Nanoparticles Containing Neuropharmaceuticals. (A) Uptake of nanoparticles. Uptake of 500nm silica nanoparticles functionalized with hydroxyl groups (NB nanoparticles), polyethylene imine (PEI) (PEIB nanoparticles), or human prion protein (PrPB nanoparticles) (data from [69]). NB nanoparticles interact with the electron-dense plasma membrane (arrowheads) or (b) within cellular extensions. (c) PrPB nanoparticles show less binding to the electron-dense regions at the plasma membrane. (d) Cellular protrusions embrace PEIB nanoparticles. Structures resembling clathrin-coated pits are indicated with white arrowheads. (B) Mechanisms of nanoparticle penetration though the BBB. Nanoparticles can pass through the BBB by receptor-mediated transcytosis, paracellular diffusion, transcellular diffusion, and adsorption-mediated transcytosis.
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