Limits...
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.

Show MeSH

Related in: MedlinePlus

Evaluation of Wnt signaling in the induction of BBB properties in ECs.(A) Schematic representation of the methodology used to assess the modulation of Wnt signaling. CD34+-derived ECs were seeded in a transwell insert coated with Matrigel at a density of 80,000 cells. Wnt ligands were added in the culture medium at the basolateral side. (B) qRT-PCR results showing differences in expression of claudin-1 and Lef1 genes on CD34+-derived ECs cultured with or without Wnt3a. Values are Mean ± SEM, n = 4. (C) Schematic representation of the methodology used to assess the effect of abrogation of Wnt signaling. CD34+-derived ECs were seeded in a transwell insert coated with Matrigel at a density of 80,000 cells and cultured in medium supplemented with XAV 939 (0.1 and 1 µM). In the bottom of the transwell was seeded 45,000 bovine pericytes. After 4 days of coculture, the paracellular permeability and cell organization were evaluated. (D) Fluorescence microscopy images showing the expression of ZO-1 in untreated ECs or ECs treated with XAV 939 (1 µM) for 4 days. Scale bar corresponds to 50 µm. (E) Paracellular permeability of untreated ECs or ECs treated with 0.1 or 1 µM XAV939 for 4 days. Results are Mean ± SEM (n = 4).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4061029&req=5

pone-0099733-g006: Evaluation of Wnt signaling in the induction of BBB properties in ECs.(A) Schematic representation of the methodology used to assess the modulation of Wnt signaling. CD34+-derived ECs were seeded in a transwell insert coated with Matrigel at a density of 80,000 cells. Wnt ligands were added in the culture medium at the basolateral side. (B) qRT-PCR results showing differences in expression of claudin-1 and Lef1 genes on CD34+-derived ECs cultured with or without Wnt3a. Values are Mean ± SEM, n = 4. (C) Schematic representation of the methodology used to assess the effect of abrogation of Wnt signaling. CD34+-derived ECs were seeded in a transwell insert coated with Matrigel at a density of 80,000 cells and cultured in medium supplemented with XAV 939 (0.1 and 1 µM). In the bottom of the transwell was seeded 45,000 bovine pericytes. After 4 days of coculture, the paracellular permeability and cell organization were evaluated. (D) Fluorescence microscopy images showing the expression of ZO-1 in untreated ECs or ECs treated with XAV 939 (1 µM) for 4 days. Scale bar corresponds to 50 µm. (E) Paracellular permeability of untreated ECs or ECs treated with 0.1 or 1 µM XAV939 for 4 days. Results are Mean ± SEM (n = 4).

Mentions: To determine whether the activation of Wnt is required for the induction of barrier properties in CD34+-derived ECs, we cultured these cells alone up to 5 days and then exposed them to Wnt ligands/agonists. ECs respond rapidly to BIO, a specific pharmacological inhibitor of glycogen synthase kinase-3 (GSK-3) and thus an activator of Wnt signaling, or Wnt3a by increasing the expression of active β-catenin (Fig. 5A). The paracellular permeability of Wnt3a-treated ECs to Lucifer Yellow was statistical lower (P<0.01, n = 4) for short-term (5 days of monoculture + 1 day of Wnt3a treatment) and long-term (1 day of monoculture + 5 day of Wnt3a treatment) as compared to untreated cells (Figs. 5A and 5B). The effect of Wnt7a and BIO was only observed after 5-day treatment (Figs. 5A and 5C). During the induction process by Wnt3a or BIO, there is an increase in the expression and nuclear localization of total β-catenin (Fig. 5D), the up-regulation of β-catenin-associated transcription factor LEF1 gene (Figs. 6A and 6B), the up-regulation of claudin-1 gene expression (Figs. 6A and 6B) and the localization of claudin-1 at the cell-cell contacts (Fig. 5D). The localization of claudin-1 at the periphery of the cells might explain the restrictive permeability of ECs in co-culture with pericytes. To further confirm the role of Wnt pathway in the induction of BBB properties, we abrogated the Wnt signaling in ECs co-cultured with pericytes. ECs were seeded in a transwell insert coated with Matrigel while pericytes were seeded in the bottom of the transwell (Figs. 6C–6E). ECs were treated with the Wnt antagonist XAV-939 for 4 days by adding the inhibitor in the luminal side of the insert. The abrogation of Wnt pathway, in conditions that did not affect cell viability, increased the paracellular permeability of the EC monolayer to lucifer yellow. Overall, our gain and loss function experiments indicate that Wnt signaling is required for the BBB properties in CD34+-derived ECs co-cultured with pericytes.


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)

Evaluation of Wnt signaling in the induction of BBB properties in ECs.(A) Schematic representation of the methodology used to assess the modulation of Wnt signaling. CD34+-derived ECs were seeded in a transwell insert coated with Matrigel at a density of 80,000 cells. Wnt ligands were added in the culture medium at the basolateral side. (B) qRT-PCR results showing differences in expression of claudin-1 and Lef1 genes on CD34+-derived ECs cultured with or without Wnt3a. Values are Mean ± SEM, n = 4. (C) Schematic representation of the methodology used to assess the effect of abrogation of Wnt signaling. CD34+-derived ECs were seeded in a transwell insert coated with Matrigel at a density of 80,000 cells and cultured in medium supplemented with XAV 939 (0.1 and 1 µM). In the bottom of the transwell was seeded 45,000 bovine pericytes. After 4 days of coculture, the paracellular permeability and cell organization were evaluated. (D) Fluorescence microscopy images showing the expression of ZO-1 in untreated ECs or ECs treated with XAV 939 (1 µM) for 4 days. Scale bar corresponds to 50 µm. (E) Paracellular permeability of untreated ECs or ECs treated with 0.1 or 1 µM XAV939 for 4 days. Results are Mean ± SEM (n = 4).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099733-g006: Evaluation of Wnt signaling in the induction of BBB properties in ECs.(A) Schematic representation of the methodology used to assess the modulation of Wnt signaling. CD34+-derived ECs were seeded in a transwell insert coated with Matrigel at a density of 80,000 cells. Wnt ligands were added in the culture medium at the basolateral side. (B) qRT-PCR results showing differences in expression of claudin-1 and Lef1 genes on CD34+-derived ECs cultured with or without Wnt3a. Values are Mean ± SEM, n = 4. (C) Schematic representation of the methodology used to assess the effect of abrogation of Wnt signaling. CD34+-derived ECs were seeded in a transwell insert coated with Matrigel at a density of 80,000 cells and cultured in medium supplemented with XAV 939 (0.1 and 1 µM). In the bottom of the transwell was seeded 45,000 bovine pericytes. After 4 days of coculture, the paracellular permeability and cell organization were evaluated. (D) Fluorescence microscopy images showing the expression of ZO-1 in untreated ECs or ECs treated with XAV 939 (1 µM) for 4 days. Scale bar corresponds to 50 µm. (E) Paracellular permeability of untreated ECs or ECs treated with 0.1 or 1 µM XAV939 for 4 days. Results are Mean ± SEM (n = 4).
Mentions: To determine whether the activation of Wnt is required for the induction of barrier properties in CD34+-derived ECs, we cultured these cells alone up to 5 days and then exposed them to Wnt ligands/agonists. ECs respond rapidly to BIO, a specific pharmacological inhibitor of glycogen synthase kinase-3 (GSK-3) and thus an activator of Wnt signaling, or Wnt3a by increasing the expression of active β-catenin (Fig. 5A). The paracellular permeability of Wnt3a-treated ECs to Lucifer Yellow was statistical lower (P<0.01, n = 4) for short-term (5 days of monoculture + 1 day of Wnt3a treatment) and long-term (1 day of monoculture + 5 day of Wnt3a treatment) as compared to untreated cells (Figs. 5A and 5B). The effect of Wnt7a and BIO was only observed after 5-day treatment (Figs. 5A and 5C). During the induction process by Wnt3a or BIO, there is an increase in the expression and nuclear localization of total β-catenin (Fig. 5D), the up-regulation of β-catenin-associated transcription factor LEF1 gene (Figs. 6A and 6B), the up-regulation of claudin-1 gene expression (Figs. 6A and 6B) and the localization of claudin-1 at the cell-cell contacts (Fig. 5D). The localization of claudin-1 at the periphery of the cells might explain the restrictive permeability of ECs in co-culture with pericytes. To further confirm the role of Wnt pathway in the induction of BBB properties, we abrogated the Wnt signaling in ECs co-cultured with pericytes. ECs were seeded in a transwell insert coated with Matrigel while pericytes were seeded in the bottom of the transwell (Figs. 6C–6E). ECs were treated with the Wnt antagonist XAV-939 for 4 days by adding the inhibitor in the luminal side of the insert. The abrogation of Wnt pathway, in conditions that did not affect cell viability, increased the paracellular permeability of the EC monolayer to lucifer yellow. Overall, our gain and loss function experiments indicate that Wnt signaling is required for the BBB properties in CD34+-derived ECs co-cultured with pericytes.

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