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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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Expression of BBB markers, stability, reproducibility and functional properties of a monolayer of human BLECs.(A) BLECs were obtained by the co-culture of CD34+-derived ECs with pericytes for 6 days in a transwell system. (B–C) Paracellular permeability to lucifer yellow of EC monolayers either cultured alone or with pericytes. (D–E) Paracellular permeability in a co-culture of CD34+-derived ECs with pericytes at day 6 obtained from (D) different donors and at (E) different laboratories (P = Portugal (LF); F = France (RC); S = Switzerland (BE)). From B to E, results are Mean ± SEM (n≥4). (F) Expression of BBB markers in BLECs as obtained by immunofluorescence. (G) Electron micrographs of ECs co-cultured with pericytes for 6 days (1) or alone (2). (1) Occlusion of the intercellular space between the ECs. WGA-HRP penetrates partially the intracellular cleft (arrow). (2) No occlusion of the intercellular space between the ECs in 84% of the cases. WGA-HRP penetrates from the luminal compartment (asterisk) through the entire intercellular cleft and is deposited in the underlying matrix (arrows). (H) Gene expression of tight junctions and influx transporters in BLECs and CD34+-derived ECs. Results are Mean ± SEM (n = 3). (I) Gene expression of efflux transporters and large molecule receptors in BLECS, i.e., CD34+-derived ECs co-cultured with pericytes for 6 days. β actin was used as housekeeping gene (J) Expression of P-gp as evaluated by immunofluorescence. In G and K, bar corresponds to 50 µm. *P<0.05, **P<0.01, ***P<0.001.
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pone-0099733-g001: Expression of BBB markers, stability, reproducibility and functional properties of a monolayer of human BLECs.(A) BLECs were obtained by the co-culture of CD34+-derived ECs with pericytes for 6 days in a transwell system. (B–C) Paracellular permeability to lucifer yellow of EC monolayers either cultured alone or with pericytes. (D–E) Paracellular permeability in a co-culture of CD34+-derived ECs with pericytes at day 6 obtained from (D) different donors and at (E) different laboratories (P = Portugal (LF); F = France (RC); S = Switzerland (BE)). From B to E, results are Mean ± SEM (n≥4). (F) Expression of BBB markers in BLECs as obtained by immunofluorescence. (G) Electron micrographs of ECs co-cultured with pericytes for 6 days (1) or alone (2). (1) Occlusion of the intercellular space between the ECs. WGA-HRP penetrates partially the intracellular cleft (arrow). (2) No occlusion of the intercellular space between the ECs in 84% of the cases. WGA-HRP penetrates from the luminal compartment (asterisk) through the entire intercellular cleft and is deposited in the underlying matrix (arrows). (H) Gene expression of tight junctions and influx transporters in BLECs and CD34+-derived ECs. Results are Mean ± SEM (n = 3). (I) Gene expression of efflux transporters and large molecule receptors in BLECS, i.e., CD34+-derived ECs co-cultured with pericytes for 6 days. β actin was used as housekeeping gene (J) Expression of P-gp as evaluated by immunofluorescence. In G and K, bar corresponds to 50 µm. *P<0.05, **P<0.01, ***P<0.001.

Mentions: To induce BBB properties in CD34+-derived ECs, cells were seeded in a transwell system and co-cultured with pericytes (Fig. 1A). Pericytes were selected after a screening of different cell types from the neurovascular unit (Figs. S2A and S2B) and because of their role in the stabilization/maturation of BBB [10], [11]. Under these conditions, the permeability of ECs decreases during the first 3 days until it reaches a stationary phase at day 4, maintaining its stability up to 20 days (Fig. 1B). At day 6, the cells had low permeability to Lucifer yellow values (0.61±0.15×10−3 cm/min, n = 60) similarly to the values found in other BBB models [12] (Fig. 1C), they showed a continuous expression of ZO-1, occludin, JAM-A, claudin-1 and claudin-5 at cell-cell contacts (Fig. 1F) and they were able to block the passage of wheat germ agglutinin (WGA)- horseradish peroxidase (HRP) in contrast with monolayers of CD34+-derived ECs where WGA-HRP reached the underlying matrix (Fig. 1G). Importantly, the induction of BBB properties in CD34+-derived ECs is highly reproducible since similar permeability results were obtained for cells derived from multiple human donors (Fig. 1D) and in 3 different laboratories (Fig. 1E). Furthermore, the BBB properties of CD34+-derived ECs require the presence of pericytes, since pericyte-conditioned medium does not have the same BBB-inductive properties, and are lost if the pericytes are removed from the co-culture system (Figs. S2C and S3B). These results show that the crosstalk between the two cells is important to maintain the BBB properties. Cells co-cultured with pericytes for 6 days express transcripts encoding tight junctions such as ZO-1 and claudin-1 higher than in ECs in monoculture, express claudin-3 and occludin at similar levels as ECs in monoculture, and express claudin-5 at lower levels as EC in monoculture (Fig. 1H). Importantly, the expression of influx transporters, specifically the expression of aminoacid (SLC7A5, SLC16A1) and glucose (SLC2A1) transporters and receptors (e.g. transferrin receptor; TFRC) was increased when the cells were co-cultured with pericytes relatively to cells cultured alone. The results are consistent with previous results showing that the induction of BBB properties in ECs correlate with an up-regulation of specific transporter systems, most prominently SLC2A1 (Glut-1) [4], [13]. In addition, ECs co-cultured with pericytes for 6 days express transcripts of key efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance protein (MRP; subfamily of the ATP-binding cassette (ABC) transporters) family, and they express large molecule receptors such as low-density lipoprotein receptor-related protein 1 (LRP1), the receptor for advanced glycation end products (RAGE) and transferrin receptor (hTrf) (Fig. 1I). The expression of RAGE, organic cation/carnitine transporter (OCTN2; also known as SLC22A5) (Fig. S3A) and P-gp protein (Fig. 1J) was further confirmed by immunofluorescence. As in hBECs, RAGE is mainly located at the luminal side of cells while OCTN2 is located at the abluminal side. Overall, ECs co-cultured with pericytes for 6 days have BBB properties at gene, protein and permeability levels, and from now on are named as brain-like endothelial cells (BLECs).


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)

Expression of BBB markers, stability, reproducibility and functional properties of a monolayer of human BLECs.(A) BLECs were obtained by the co-culture of CD34+-derived ECs with pericytes for 6 days in a transwell system. (B–C) Paracellular permeability to lucifer yellow of EC monolayers either cultured alone or with pericytes. (D–E) Paracellular permeability in a co-culture of CD34+-derived ECs with pericytes at day 6 obtained from (D) different donors and at (E) different laboratories (P = Portugal (LF); F = France (RC); S = Switzerland (BE)). From B to E, results are Mean ± SEM (n≥4). (F) Expression of BBB markers in BLECs as obtained by immunofluorescence. (G) Electron micrographs of ECs co-cultured with pericytes for 6 days (1) or alone (2). (1) Occlusion of the intercellular space between the ECs. WGA-HRP penetrates partially the intracellular cleft (arrow). (2) No occlusion of the intercellular space between the ECs in 84% of the cases. WGA-HRP penetrates from the luminal compartment (asterisk) through the entire intercellular cleft and is deposited in the underlying matrix (arrows). (H) Gene expression of tight junctions and influx transporters in BLECs and CD34+-derived ECs. Results are Mean ± SEM (n = 3). (I) Gene expression of efflux transporters and large molecule receptors in BLECS, i.e., CD34+-derived ECs co-cultured with pericytes for 6 days. β actin was used as housekeeping gene (J) Expression of P-gp as evaluated by immunofluorescence. In G and K, bar corresponds to 50 µm. *P<0.05, **P<0.01, ***P<0.001.
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Related In: Results  -  Collection

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

pone-0099733-g001: Expression of BBB markers, stability, reproducibility and functional properties of a monolayer of human BLECs.(A) BLECs were obtained by the co-culture of CD34+-derived ECs with pericytes for 6 days in a transwell system. (B–C) Paracellular permeability to lucifer yellow of EC monolayers either cultured alone or with pericytes. (D–E) Paracellular permeability in a co-culture of CD34+-derived ECs with pericytes at day 6 obtained from (D) different donors and at (E) different laboratories (P = Portugal (LF); F = France (RC); S = Switzerland (BE)). From B to E, results are Mean ± SEM (n≥4). (F) Expression of BBB markers in BLECs as obtained by immunofluorescence. (G) Electron micrographs of ECs co-cultured with pericytes for 6 days (1) or alone (2). (1) Occlusion of the intercellular space between the ECs. WGA-HRP penetrates partially the intracellular cleft (arrow). (2) No occlusion of the intercellular space between the ECs in 84% of the cases. WGA-HRP penetrates from the luminal compartment (asterisk) through the entire intercellular cleft and is deposited in the underlying matrix (arrows). (H) Gene expression of tight junctions and influx transporters in BLECs and CD34+-derived ECs. Results are Mean ± SEM (n = 3). (I) Gene expression of efflux transporters and large molecule receptors in BLECS, i.e., CD34+-derived ECs co-cultured with pericytes for 6 days. β actin was used as housekeeping gene (J) Expression of P-gp as evaluated by immunofluorescence. In G and K, bar corresponds to 50 µm. *P<0.05, **P<0.01, ***P<0.001.
Mentions: To induce BBB properties in CD34+-derived ECs, cells were seeded in a transwell system and co-cultured with pericytes (Fig. 1A). Pericytes were selected after a screening of different cell types from the neurovascular unit (Figs. S2A and S2B) and because of their role in the stabilization/maturation of BBB [10], [11]. Under these conditions, the permeability of ECs decreases during the first 3 days until it reaches a stationary phase at day 4, maintaining its stability up to 20 days (Fig. 1B). At day 6, the cells had low permeability to Lucifer yellow values (0.61±0.15×10−3 cm/min, n = 60) similarly to the values found in other BBB models [12] (Fig. 1C), they showed a continuous expression of ZO-1, occludin, JAM-A, claudin-1 and claudin-5 at cell-cell contacts (Fig. 1F) and they were able to block the passage of wheat germ agglutinin (WGA)- horseradish peroxidase (HRP) in contrast with monolayers of CD34+-derived ECs where WGA-HRP reached the underlying matrix (Fig. 1G). Importantly, the induction of BBB properties in CD34+-derived ECs is highly reproducible since similar permeability results were obtained for cells derived from multiple human donors (Fig. 1D) and in 3 different laboratories (Fig. 1E). Furthermore, the BBB properties of CD34+-derived ECs require the presence of pericytes, since pericyte-conditioned medium does not have the same BBB-inductive properties, and are lost if the pericytes are removed from the co-culture system (Figs. S2C and S3B). These results show that the crosstalk between the two cells is important to maintain the BBB properties. Cells co-cultured with pericytes for 6 days express transcripts encoding tight junctions such as ZO-1 and claudin-1 higher than in ECs in monoculture, express claudin-3 and occludin at similar levels as ECs in monoculture, and express claudin-5 at lower levels as EC in monoculture (Fig. 1H). Importantly, the expression of influx transporters, specifically the expression of aminoacid (SLC7A5, SLC16A1) and glucose (SLC2A1) transporters and receptors (e.g. transferrin receptor; TFRC) was increased when the cells were co-cultured with pericytes relatively to cells cultured alone. The results are consistent with previous results showing that the induction of BBB properties in ECs correlate with an up-regulation of specific transporter systems, most prominently SLC2A1 (Glut-1) [4], [13]. In addition, ECs co-cultured with pericytes for 6 days express transcripts of key efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance protein (MRP; subfamily of the ATP-binding cassette (ABC) transporters) family, and they express large molecule receptors such as low-density lipoprotein receptor-related protein 1 (LRP1), the receptor for advanced glycation end products (RAGE) and transferrin receptor (hTrf) (Fig. 1I). The expression of RAGE, organic cation/carnitine transporter (OCTN2; also known as SLC22A5) (Fig. S3A) and P-gp protein (Fig. 1J) was further confirmed by immunofluorescence. As in hBECs, RAGE is mainly located at the luminal side of cells while OCTN2 is located at the abluminal side. Overall, ECs co-cultured with pericytes for 6 days have BBB properties at gene, protein and permeability levels, and from now on are named as brain-like endothelial cells (BLECs).

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