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A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.

Cecchelli R, Aday S, Sevin E, Almeida C, Culot M, Dehouck L, Coisne C, Engelhardt B, Dehouck MP, Ferreira L - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Blood Brain Barrier Laboratory, University of Artois, Lens, France.

ABSTRACT
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.

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Functional properties of BLECs.(A) Effect of P-gp protein inhibition on active transport of drugs. (B) Efflux ratio of small (sucrose) and large (HSA and IgG) molecules. In A and B: Mean ± SEM (n = 3–7). (C) Transendothelial electrical resistance (TEER) of monocultures of CD34+-derived ECs or co-cultures of ECs with pericytes for 6 days. The TEER of the co-culture of ECs was compared with the gold standard of bovine brain microvascular endothelial cells co-cultured with bovine astrocytes for 12 days on insert filters 30 mm diameter. Values are Mean ± SEM, n = 4. ***P<0.001; ns means P>0.05. (D) Expression of adhesion molecules by ECs in co-culture with pericytes. The expression of the adhesion molecules was assessed by flow cytometry analysis on untreated and treated ECs by TNFα (10 ng/mL) for 24 h. (E) Correlation (r2 = 0.84; Pearson r = 0.9160) between our human in vitro data (Kp,uu,brain) and human Kp,uu,CSF data (obtained from ref. [18]). Kp,uu,CSF = (In vivo concentration of unbound drug in cerebrospinal fluid (CSF))/(In vivo concentration of unbound drug in plasma). Kp,uu,brain = (In vitro concentration of unbound drug in brain)/(In vitro concentration of unbound drug in plasma). Kp,uu,brain were calculated from flux experiments using triplicate filters as described in Text S1. Materials and Methods. Values are Mean ± SEM (n = 3).
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pone-0099733-g002: Functional properties of BLECs.(A) Effect of P-gp protein inhibition on active transport of drugs. (B) Efflux ratio of small (sucrose) and large (HSA and IgG) molecules. In A and B: Mean ± SEM (n = 3–7). (C) Transendothelial electrical resistance (TEER) of monocultures of CD34+-derived ECs or co-cultures of ECs with pericytes for 6 days. The TEER of the co-culture of ECs was compared with the gold standard of bovine brain microvascular endothelial cells co-cultured with bovine astrocytes for 12 days on insert filters 30 mm diameter. Values are Mean ± SEM, n = 4. ***P<0.001; ns means P>0.05. (D) Expression of adhesion molecules by ECs in co-culture with pericytes. The expression of the adhesion molecules was assessed by flow cytometry analysis on untreated and treated ECs by TNFα (10 ng/mL) for 24 h. (E) Correlation (r2 = 0.84; Pearson r = 0.9160) between our human in vitro data (Kp,uu,brain) and human Kp,uu,CSF data (obtained from ref. [18]). Kp,uu,CSF = (In vivo concentration of unbound drug in cerebrospinal fluid (CSF))/(In vivo concentration of unbound drug in plasma). Kp,uu,brain = (In vitro concentration of unbound drug in brain)/(In vitro concentration of unbound drug in plasma). Kp,uu,brain were calculated from flux experiments using triplicate filters as described in Text S1. Materials and Methods. Values are Mean ± SEM (n = 3).

Mentions: The inhibition of P-gp protein by verapamil or elacridar, and the concomitant blocking of the active transport of drugs to outside the cell [14], leads to a significant increase in the accumulation of the antitumor drug vincristine (Fig. 2A). This result demonstrates that P-gp is functionally active in BLECs. The higher efflux ratio of IgG as compared to human serum albumin shows receptor-mediated transport of this macromolecule across the polarized monolayer from the abluminal to the luminal side (Fig. 2B). In addition, BLECs have the ability to form a monolayer that has a TEER similar to monolayers of bovine BECs (Fig. 2C) and higher than monolayers of human hCMEC/D3 cell line (<40 Ω×cm2) [2] or monolayers of cord blood-derived ECs co-cultured with astrocytes [5]. Moreover, BLECs express constitutively the adhesion molecule PECAM-1 and ICAM-2, typically found in hBECs [15]. These molecules are up-regulated in ECs exposed to tumor necrosis factor alpha (TNF-α) mediated by the activation of the pleiotropic nuclear factor–kB (NF-kB) [16]. Accordingly, BLECs show an up-regulation in the expression of ICAM-1, ICAM-2, CD40 and VCAM-1 after stimulation with 10 ng/mL TNF-α for 24 h, as hBECs [2] (Fig. 2D). Taken together, these results show that our in vitro model is functional and can reproduce key aspects of the human BBB activity.


A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.

Cecchelli R, Aday S, Sevin E, Almeida C, Culot M, Dehouck L, Coisne C, Engelhardt B, Dehouck MP, Ferreira L - PLoS ONE (2014)

Functional properties of BLECs.(A) Effect of P-gp protein inhibition on active transport of drugs. (B) Efflux ratio of small (sucrose) and large (HSA and IgG) molecules. In A and B: Mean ± SEM (n = 3–7). (C) Transendothelial electrical resistance (TEER) of monocultures of CD34+-derived ECs or co-cultures of ECs with pericytes for 6 days. The TEER of the co-culture of ECs was compared with the gold standard of bovine brain microvascular endothelial cells co-cultured with bovine astrocytes for 12 days on insert filters 30 mm diameter. Values are Mean ± SEM, n = 4. ***P<0.001; ns means P>0.05. (D) Expression of adhesion molecules by ECs in co-culture with pericytes. The expression of the adhesion molecules was assessed by flow cytometry analysis on untreated and treated ECs by TNFα (10 ng/mL) for 24 h. (E) Correlation (r2 = 0.84; Pearson r = 0.9160) between our human in vitro data (Kp,uu,brain) and human Kp,uu,CSF data (obtained from ref. [18]). Kp,uu,CSF = (In vivo concentration of unbound drug in cerebrospinal fluid (CSF))/(In vivo concentration of unbound drug in plasma). Kp,uu,brain = (In vitro concentration of unbound drug in brain)/(In vitro concentration of unbound drug in plasma). Kp,uu,brain were calculated from flux experiments using triplicate filters as described in Text S1. Materials and Methods. Values are Mean ± SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061029&req=5

pone-0099733-g002: Functional properties of BLECs.(A) Effect of P-gp protein inhibition on active transport of drugs. (B) Efflux ratio of small (sucrose) and large (HSA and IgG) molecules. In A and B: Mean ± SEM (n = 3–7). (C) Transendothelial electrical resistance (TEER) of monocultures of CD34+-derived ECs or co-cultures of ECs with pericytes for 6 days. The TEER of the co-culture of ECs was compared with the gold standard of bovine brain microvascular endothelial cells co-cultured with bovine astrocytes for 12 days on insert filters 30 mm diameter. Values are Mean ± SEM, n = 4. ***P<0.001; ns means P>0.05. (D) Expression of adhesion molecules by ECs in co-culture with pericytes. The expression of the adhesion molecules was assessed by flow cytometry analysis on untreated and treated ECs by TNFα (10 ng/mL) for 24 h. (E) Correlation (r2 = 0.84; Pearson r = 0.9160) between our human in vitro data (Kp,uu,brain) and human Kp,uu,CSF data (obtained from ref. [18]). Kp,uu,CSF = (In vivo concentration of unbound drug in cerebrospinal fluid (CSF))/(In vivo concentration of unbound drug in plasma). Kp,uu,brain = (In vitro concentration of unbound drug in brain)/(In vitro concentration of unbound drug in plasma). Kp,uu,brain were calculated from flux experiments using triplicate filters as described in Text S1. Materials and Methods. Values are Mean ± SEM (n = 3).
Mentions: The inhibition of P-gp protein by verapamil or elacridar, and the concomitant blocking of the active transport of drugs to outside the cell [14], leads to a significant increase in the accumulation of the antitumor drug vincristine (Fig. 2A). This result demonstrates that P-gp is functionally active in BLECs. The higher efflux ratio of IgG as compared to human serum albumin shows receptor-mediated transport of this macromolecule across the polarized monolayer from the abluminal to the luminal side (Fig. 2B). In addition, BLECs have the ability to form a monolayer that has a TEER similar to monolayers of bovine BECs (Fig. 2C) and higher than monolayers of human hCMEC/D3 cell line (<40 Ω×cm2) [2] or monolayers of cord blood-derived ECs co-cultured with astrocytes [5]. Moreover, BLECs express constitutively the adhesion molecule PECAM-1 and ICAM-2, typically found in hBECs [15]. These molecules are up-regulated in ECs exposed to tumor necrosis factor alpha (TNF-α) mediated by the activation of the pleiotropic nuclear factor–kB (NF-kB) [16]. Accordingly, BLECs show an up-regulation in the expression of ICAM-1, ICAM-2, CD40 and VCAM-1 after stimulation with 10 ng/mL TNF-α for 24 h, as hBECs [2] (Fig. 2D). Taken together, these results show that our in vitro model is functional and can reproduce key aspects of the human BBB activity.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Blood Brain Barrier Laboratory, University of Artois, Lens, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus