An in vitro model of glucose transporter 1 deficiency syndrome at the blood-brain barrier using induced pluripotent stem cells.

Glucose is an important source of energy for the central nervous system. Its uptake at the blood-brain barrier (BBB) is mostly mediated via glucose transporter 1 (GLUT1), a facilitated transporter encoded by the SLC2A1 gene. GLUT1 Deficiency Syndrome (GLUT1DS) is a haploinsufficiency characterized by mutations in the SLC2A1 gene, resulting in impaired glucose uptake at the BBB and clinically characterized by epileptic seizures and movement disorder. A major limitation is an absence of in vitro models of the BBB reproducing the disease. This study aimed to characterize an in vitro model of GLUT1DS using human pluripotent stem cells (iPSCs). Two GLUT1DS clones were generated (GLUT1-iPSC) from their original parental clone iPS(IMR90)-c4 by CRISPR/Cas9 and differentiated into brain microvascular endothelial cells (iBMECs). Cells were characterized in terms of SLC2A1 expression, changes in the barrier function, glucose uptake and metabolism, and angiogenesis. GLUT1DS iPSCs and iBMECs showed comparable phenotype to their parental control, with exception of reduced GLUT1 expression at the protein level. Although no major disruption in the barrier function was reported in the two clones, a significant reduction in glucose uptake accompanied by an increase in glycolysis and mitochondrial respiration was reported in both GLUT1DS-iBMECs. Finally, impaired angiogenic features were reported in such clones compared to the parental clone. Our study provides the first documented characterization of GLUT1DS-iBMECs generated by CRISPR-Cas9, suggesting that GLUT1 truncation appears detrimental to brain angiogenesis and brain endothelial bioenergetics, but maybe not be detrimental to iBMECs differentiation and barriergenesis. Our future direction is to further characterize the functional outcome of such truncated product, as well as its impact on other cells of the neurovascular unit.

Effects of glyphosate and aminomethylphosphonic acid on an isogeneic model of the human blood-brain barrier.

Glyphosate is a pesticide used for occupational and non-occupational purposes. Because glyphosate targets a metabolic pathway absent in animals, it is considered safe for humans. Yet, case reports of accidental exposure to concentrated solutions following self-inflicted poisoning documented neurological lesions suggesting a neurotoxicity. In this study, we investigated the effect of acute exposure to glyphosate (GPH) on the blood-brain barrier in vitro based on induced pluripotent stem cells (iPSCs) and compared to two chemical analogs: aminomethylphosphonic acid (AMPA) and glycine (GLY), for concentrations ranging from 0.1 µM to 1000 µM. GPH treatment (1 and 10 µM) for 24 h showed an increase BBB permeability to fluorescein, with similar outcomes for AMPA. In addition to its ability to disrupt the barrier function, GPH show evidence of permeability across the BBB. Although no detrimental effects were observed on neuron differentiation at high doses, we noted changes in neuronal cell metabolic activity and glucose uptake in brain microvascular endothelial cells (BMECs) following treatment with 100 µM GPH or AMPA. Taken together, our data indicates that accidental exposure to high level of GPH may result in neurological damage via an opening of the blood-brain barrier and an alteration of glucose metabolism.

Gliotoxin penetrates and impairs the integrity of the human blood-brain barrier in vitro.

Cerebral fungal infections represent an important public health concern, where a key element of pathophysiology is the ability of the fungi to cross the blood-brain barrier (BBB). Yet the mechanism used by micro-organisms to cross such a barrier and invade the brain parenchyma remains unclear. This study investigated the effects of gliotoxin (GTX), a mycotoxin secreted by Aspergillus fumigatus, on the BBB using brain microvascular endothelial cells (BMECs) derived from induced pluripotent stem cells (iPSCs). We observed that both acute (2 h) and prolonged (24 h) exposure to GTX at the level of 1 µM or higher compromised BMECs monolayer integrity. Notably, acute exposure was sufficient to disrupt the barrier function in iPSC-derived BMECs, resulting in decreased transendothelial electrical resistance (TEER) and increased fluorescein permeability. Further, our data suggest that such disruption occurred without affecting tight junction complexes, via alteration of cell-matrix interactions, alterations in F-actin distribution, through a protein kinase C-independent signaling. In addition to its effect on the barrier function, we have observed a low permeability of GTX across the BBB. This fact can be partially explained by possible interactions of GTX with membrane proteins. Taken together, this study suggests that GTX may contribute in cerebral invasion processes of Aspergillus fumigatus by altering the blood-brain barrier integrity without disrupting tight junction complexes.

Isogenic blood-brain barrier models based on patient-derived stem cells display inter-individual differences in cell maturation and functionality.

The blood-brain barrier (BBB) constitutes an important component of the neurovascular unit formed by specialized brain microvascular endothelial cells (BMECs) surrounded by astrocytes, pericytes, and neurons. Recently, isogenic in vitro models of the BBB based on human pluripotent stem cells have been documented, yet the impact of inter-individual variability on the yield and phenotype of such models remains to be documented. In this study, we investigated the impact of inter-individual variability on the yield and phenotype of isogenic models of the BBB, using patient-derived induced pluripotent stem cells (iPSCs). Astrocytes, BMECs, and neurons were differentiated from four asymptomatic patient-derived iPSCs (two males, two females). We differentiated such cells using existing differentiation protocols and quantified expression of cell lineage markers, as well as BBB phenotype, barrier induction, and formation of neurite processes. iPSC-derived BMECs showed barrier properties better than hCMEC/D3 monolayers; however, we noted differences in the expression and activity among iPSC lines. In addition, we noted differences in the differentiation efficiency of these cells into neural stem cells and progenitor cells (as noted by differences in expression of cell lineage markers). Such differences were reflected later in the terminal differentiation, as seen as ability to induce barrier function and to form neurite processes. Although we demonstrated our ability to obtain an isogenic model of the BBB with different patients' iPSCs, we also noted subtle differences in the expression of cell lineage markers and cell maturation processes, suggesting the presence of inter-individual polymorphisms.