Ketone bodies supplementation restores the barrier function, induces a metabolic switch, and elicits beta-hydroxybutyrate diffusion across a monolayer of iPSC-derived brain microvascular endothelial cells.

Glucose constitutes the main source of energy for the central nervous system (CNS), its entry occurring at the blood-brain barrier (BBB) via the presence of glucose transporter 1 (GLUT1). However, under food intake restrictions, the CNS can utilize ketone bodies (KB) as an alternative source of energy. Notably, the relationship between the BBB and KBs and its effect on their glucose metabolism remains poorly understood. In this study, we investigated the effect of glucose deprivation on the brain endothelium in vitro, and supplementation with KBs using induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cell-like cells (iBMECs). Glucose-free environment significantly decreased cell metabolic activity and negatively impacted the barrier function. In addition, glucose deprivation did not increase GLUT1 expression but also resulted in a decrease in glucose uptake and glycolysis. Supplementation of glucose-deprived iBMECs monolayers with KB showed no improvement and even worsened upon treatment with acetoacetate. However, under a hypoglycemic condition in the presence of KBs, we noted a slight improvement of the barrier function, with no changes in glucose uptake. Notably, hypoglycemia and/or KB pre-treatment elicited a saturable beta-hydroxybutyrate diffusion across iBMECs monolayers, such diffusion occurred partially via an MCT1-dependent mechanism. Taken together, our study highlights the importance of glucose metabolism and the reliance of the brain endothelium on glucose and glycolysis for its function, such dependence is unlikely to be covered by KBs supplementation. In addition, KB diffusion at the BBB appeared induced by KB pre-treatment and appears to involve an MCT1-dependent mechanism.

Human-Induced Pluripotent Stem Cell-Based Model of the Blood-Brain at 10 Years: A Retrospective on Past and Current Disease Models.

The initial discovery and derivation of induced pluripotent stem cells (iPSCs) by Yamanaka and colleagues in 2006 revolutionized the field of personalized medicine, as it opened the possibility to model diseases using patient-derived stem cells. A decade of adoption of iPSCs within the community of the blood-brain barrier (BBB) significantly opened the door for modeling diseases at the BBB, a task until then considered challenging, if not impossible.In this book chapter, we provided an extensive review of the literature on the use of iPSC-based models of the human BBB to model neurological diseases including infectious diseases (COVID-19, Streptococcus, Neisseria) neurodevelopmental diseases (adrenoleukodystrophy, Allan-Herndon-Dudley Syndrome, Batten's disease, GLUT1 deficiency syndrome), and neurodegenerative diseases (Alzheimer's disease, the current findings and observations, but also the challenges and limitations inherent to the use of iPSC-based models in reproducing the human BBB during health and diseases in a Petri dish.

In Vitro Models of the Human Blood-Brain Barrier Utilising Human Induced Pluripotent Stem Cells: Opportunities and Challenges.

The blood-brain barrier (BBB) is a component of the neurovascular unit formed by specialized brain microvascular endothelial cells surrounded by astrocytes end-feet processes, pericytes, and a basement membrane. The BBB plays an important role in the maintenance of brain homeostasis and has seen a growing involvement in the pathophysiology of various neurological diseases. On the other hand, the presence of such a barrier remains an important challenge for drug delivery to treat such illnesses.Since the pioneering work describing the isolation and cultivation of primary brain microvascular cells about 50 years ago until now, the development of an in vitro model of the BBB that is scalable, capable to form tight monolayers, and predictive of drug permeability in vivo remained extremely challenging.The recent description of the use of induced pluripotent stem cells (iPSCs) as a modeling tool for neurological diseases raised momentum into the use of such cells to develop new in vitro models of the BBB. This chapter will provide an exhaustive description of the use of iPSCs as a source of cells for modeling the BBB in vitro, describe the advantages and limitations of such model, as well as describe their prospective use for disease modeling and drug permeability screening platforms.

Abeta peptides disrupt the barrier integrity and glucose metabolism of human induced pluripotent stem cell-derived brain microvascular endothelial cells.

Amyloid ß (Aß) peptides are key components of Alzheimer's disease and cerebral amyloid angiopathy and have been associated with detrimental effects at the blood-brain barrier (BBB) in vivo. Yet, the cellular and molecular mechanisms by which such peptides exert their effect on the brain vasculature remain unclear. This study aimed to assess the cellular response of induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMECs) to Aß peptides. Changes in the barrier function, efflux transporters activity, glucose uptake, and metabolism were assessed in such model. Although iPSC-derived BMECs sustained prolonged exposure (<72 h) to a high level of Aß peptides including Aß42, such cells also suffered from a loss of barrier integrity, coupled with reduced glucose uptake and impaired bioenergetic activity. Taken together, this study shows the ability of iPSC-derived BMECs to reproduce features observed in other models and suggests that Aß peptides may compromise the BBB via different targets.

Presence of a mutation in PSEN1 or PSEN2 gene is associated with an impaired brain endothelial cell phenotype in vitro.

BACKGROUND: Alzheimer's disease (AD) is the most common form of neurodegenerative disease. It is an irreversible condition marked by irreversible cognitive loss, commonly attributed to the loss of hippocampal neurons due to the formation of senile plaques and neurofibrillary tangles. Although the sporadic form is the most prevalent, the presence of familial form (involving several genes such as APP, PSEN1, and PSEN2) of the disease is commonly used as a model for understanding the pathophysiology of the disease. The aim of this study is to investigate the effect of a mutation on PSEN1 and PSEN2 genes on the BBB function using induced pluripotent stem cells (iPSCs). METHODS: iPSC lines from patients suffering from a familial form of Alzheimer's disease and harboring mutations in PSEN1 or PSEN2 were used in this study and compared to a control iPSC line. Cells were differentiated into brain microvascular endothelial cells (BMECs) following established differentiation protocols. Barrier function was assessed by measuring TEER and fluorescein permeability, drug transporter activity was assessed by uptake assay, glucose uptake and metabolism assessed by cell flux analyzer, mitochondrial potential by JC-1, and lysosomal acidification by acridine orange. RESULTS: iPSC-derived BMECs from the FAD patient presenting a mutation in the PSEN1 gene showed impaired barrier function compared to the FAD patient harboring a mutation in PSEN2 and to the control group. Such impaired barrier function correlated with poor tight junction complexes and reduced drug efflux pump activity. In addition, both PSEN1 and PSEN2-BMECs displayed reduced bioenergetics, lysosomal acidification, autophagy, while showing an increase in radical oxygen species (ROS) production. Finally, PSEN1- and PSEN2-BMECs showed an elevated secretion of Aß1-40 peptides compared to control-BMECs. CONCLUSIONS: Our study reports that iPSC-derived BMECs obtained from FAD patients showed impaired barrier properties and BMEC metabolism. In particular, mutation in the PSEN1 gene was associated with a more detrimental phenotype than mutation in PSEN2, as noted by a reduced barrier function, reduced drug efflux pump activity, and diminished glucose metabolism. Therefore, assessing the contribution of genetic mutations associated with Alzheimer's disease will allow us to better understand the contribution of the BBB in dementia, but also other neurodegenerative diseases.

Neurolysin substrates bradykinin, neurotensin and substance P enhance brain microvascular permeability in a human in vitro model.

Increased brain microvascular permeability and disruption of blood-brain barrier (BBB) function are among hallmarks of several acute neurodegenerative disorders, including stroke. Numerous studies suggest the involvement of bradykinin (BK), neurotensin (NT) and substance P (SP) in BBB impairment and oedema formation after stroke; however, there is paucity of data in regard to the direct effects of these peptides on the brain microvascular endothelial cells (BMECs) and BBB. The present study aimed to evaluate the direct effects of BK, NT and SP on the permeability of BBB in an in vitro model based on human induced pluripotent stem cell (iPSC)-derived BMECs. Our data indicate that all three peptides increase BBB permeability in a concentration-dependent manner in an in vitro model formed from two different iPSC lines (CTR90F and CTR65M) and widely used hCMEC/D3 human BMECs. The combination of BK, NT and SP at a sub-effective concentration also resulted in increased BBB permeability in the iPSC-derived model indicating potentiation of their action. Furthermore, we observed abrogation of BK, NT and SP effects with pretreatment of pharmacological blockers targeting their specific receptors. Additional mechanistic studies indicate that the short-term effects of these peptides are not mediated through alteration of tight-junction proteins claudin-5 and occludin, but likely involve redistribution of F-actin and secretion of vascular endothelial growth factor. This is the first experimental study to document the increased permeability of the BBB in response to direct action of NT in an in vitro model. In addition, our study confirms the expected but not well-documented, direct effect of SP on BBB permeability and adds to the well-recognised actions of BK on BBB. Lastly, we demonstrate that peptidase neurolysin can neutralise the effects of these peptides on BBB, suggesting potential therapeutic implications.

Hyaluronan impairs the barrier integrity of brain microvascular endothelial cells through a CD44-dependent pathway.

Hyaluronan (HA) constitutes the most abundant extracellular matrix component during brain development, only to become a minor component rapidly after birth and in adulthood to remain in specified regions. HA signaling has been associated with several neurological disorders, yet the impact of HA signaling at the blood-brain barrier (BBB) function remains undocumented. In this study, we investigated the impact of HA on BBB properties using human-induced pluripotent stem cell (iPSC) -derived and primary human and rat BMECs. The impact of HA signaling on developmental and mature BMECs was assessed by measuring changes in TEER, permeability, BMECs markers (GLUT1, tight junction proteins, P-gp) expression and localization, CD44 expression and hyaluronan levels. In general, HA treatment decreased barrier function and reduced P-gp activity with effects being more prominent upon treatment with oligomeric forms of HA (oHA). Such effects were exacerbated when applied during BMEC differentiation phase (considered as developmental BBB). We noted a hyaluronidase activity as well as an increase in CD44 expression during prolonged oxygen-glucose deprivation stress. Inhibition of HA signaling by antibody blockade of CD44 abrogated the detrimental effects of HA treatment. These results suggest the importance of HA signaling through CD44 on BBB properties.

Comparative study of expression and activity of glucose transporters between stem cell-derived brain microvascular endothelial cells and hCMEC/D3 cells.

Glucose constitutes a major source of energy of mammalian brains. Glucose uptake at the blood-brain barrier (BBB) occurs through a facilitated glucose transport, through glucose transporter 1 (GLUT1), although other isoforms have been described at the BBB. Mutations in GLUT1 are associated with the GLUT1 deficiency syndrome, yet none of the current in vitro models of the human BBB maybe suited for modeling such a disorder. In this study, we investigated the expression of glucose transporters and glucose diffusion across brain microvascular endothelial cells (BMECs) derived from healthy patient-derived induced pluripotent stem cells (iPSCs). We investigated the expression of different glucose transporters at the BBB using immunocytochemistry and flow cytometry and measured glucose uptake and diffusion across BMEC monolayers obtained from two iPSC lines and from hCMEC/D3 cells. BMEC monolayers showed expression of several glucose transporters, in particular GLUT1, GLUT3, and GLUT4. Diffusion of glucose across the monolayers was mediated via a saturable transcellular mechanism and partially inhibited by pharmacological inhibitors. Taken together, our study suggests the presence of several glucose transporters isoforms at the human BBB and demonstrates the feasibility of modeling glucose across the BBB using patient-derived stem cells.

Cerebral hypoxia/ischemia selectively disrupts tight junctions complexes in stem cell-derived human brain microvascular endothelial cells.

BACKGROUND: Cerebral hypoxia/ischemia (H/I) is an important stress factor involved in the disruption of the blood-brain barrier (BBB) following stroke injury, yet the cellular and molecular mechanisms on how the human BBB responds to such injury remains unclear. In this study, we investigated the cellular response of the human BBB to chemical and environmental H/I in vitro. METHODS: In this study, we used immortalized hCMEC/D3 and IMR90 stem-cell derived human brain microvascular endothelial cell lines (IMR90-derived BMECs). Hypoxic stress was achieved by exposure to cobalt chloride (CoCl2) or by exposure to 1 % hypoxia and oxygen/glucose deprivation (OGD) was used to model ischemic injury. We assessed barrier function using both transendothelial electrical resistance (TEER) and sodium fluorescein permeability. Changes in cell junction integrity were assessed by immunocytochemistry and cell viability was assessed by trypan-blue exclusion and by MTS assays. Statistical analysis was performed using one-way analysis of variance (ANOVA). RESULTS: CoCl2 selectively disrupted the barrier function in IMR90-derived BMECs but not in hCMEC/D3 monolayers and cytotoxic effects did not drive such disruption. In addition, hypoxia/OGD stress significantly disrupted the barrier function by selectively disrupting tight junctions (TJs) complexes. In addition, we noted an uncoupling between cell metabolic activity and barrier integrity. CONCLUSIONS: In this study, we demonstrated the ability of IMR90-derived BMECs to respond to hypoxic/ischemic injury triggered by both chemical and environmental stress by showing a disruption of the barrier function. Such disruption was selectively targeting TJ complexes and was not driven by cellular apoptosis. In conclusion, this study suggests the suitability of stem cell-derived human BMECs monolayers as a model of cerebral hypoxia/ischemia in vitro.

Analysis of Cancer-Targeting Alkylphosphocholine Analogue Permeability Characteristics Using a Human Induced Pluripotent Stem Cell Blood-Brain Barrier Model.

Cancer-targeting alkylphosphocholine (APC) analogues are being clinically developed for diagnostic imaging, intraoperative visualization, and therapeutic applications. These APC analogues derived from chemically synthesized phospholipid ethers were identified and optimized for cancer-targeting specificity using extensive structure-activity studies. While they strongly label human brain cancers associated with disrupted blood-brain barriers (BBB), APC permeability across intact BBB remains unknown. Three of our APC analogues, CLR1404 (PET radiotracer), CLR1501 (green fluorescence), and CLR1502 (near-infrared fluorescence), were tested for permeability across a BBB model composed of human induced pluripotent stem cell-derived brain microvascular endothelial cells (iPSC-derived BMECs). This in vitro BBB system has reproducibly consistent high barrier integrity marked by high transendothelial electrical resistance (TEER > 1500 Ω-cm(2)) and functional expression of drug efflux transporters. The radioiodinated and fluorescent APC analogues demonstrated fairly low permeability across the iPSC-BMEC (35 ± 5.7 (CLR1404), 54 ± 3.2 (CLR1501), and 26 ± 4.9 (CLR1502) × 10(-5) cm/min) compared with BBB-impermeable sucrose (13 ± 2.5) and BBB-permeable diazepam (170 ± 29). Only the fluorescent APC analogues (CLR1501, CLR1502) underwent BCRP and MRP polarized drug efflux transport in the brain-to-blood direction of the BBB model, and this efflux can be specifically blocked with pharmacological inhibition. None of the tested APC analogues appeared to undergo substantial P-gp transport. Limited permeability of the APC analogues across an intact BBB into normal brain likely contributes to the high tumor to background ratios observed in initial human trials. Moreover, addition of fluorescent moieties to APCs resulted in greater BMEC efflux via MRP and BCRP, and may affect fluorescence-guided applications. Overall, the characterization of APC analogue permeability across human BBB is significant for advancing future brain tumor-targeted applications of these agents.

Hypoxia selectively disrupts brain microvascular endothelial tight junction complexes through a hypoxia-inducible factor-1 (HIF-1) dependent mechanism.

The blood-brain barrier (BBB) constitutes a critical barrier for the maintenance of central nervous system homeostasis. Brain microvascular endothelial cells line the vessel walls and express tight junction (TJ) complexes that restrict paracellular passage across the BBB, thereby fulfilling a crucial role in ensuring brain function. Hypoxia, an impaired O(2) delivery, is known to cause BBB dysfunction but the mechanisms that drive this disruption remain unclear. This study discloses the relevance of the master regulator of the hypoxic response, hypoxia-inducible factor-1 (HIF-1), in hypoxia-induced barrier disruption using the rat brain endothelial cell line RBE4. Hypoxic exposure rapidly induced stabilization of the HIF-1 oxygen-dependent alpha subunit (HIF-1α) concomitantly with BBB impairment and TJ disruption mainly through delocalization and increased tyrosine phosphorylation of TJ proteins. Similar observations were obtained by normoxic stabilization of HIF-1α using CoCl(2), deferoxamine, and dimethyloxalylglycine underlining the involvement of HIF-1 in barrier dysfunction particularly via TJ alterations. In agreement inhibition of HIF-1 stabilization by 2-methoxyestradiol and YC-1 improved barrier function in hypoxic cells. Overall our data suggests that activation of HIF-1-mediated signaling disrupts TJ resulting in increased BBB permeability.

Perlecan domain V modulates astrogliosis in vitro and after focal cerebral ischemia through multiple receptors and increased nerve growth factor release.

Astrogliosis constitutes part of the central nervous system's physiological response to injury. Considered for decades to be a major challenge for brain repair, recent studies have highlighted it as a promoter of such repair mechanisms. Recently, our group demonstrated the ability of perlecan domain V (DV) to be a novel potential stroke therapy by its neuroprotective effects. However, the potential for DV to modulate astrogliosis has not been investigated. The aim of this study is to better understand the relevance of DV to astrogliosis using both in vitro and in vivo rodent models. Notably, under basal conditions, astrocytes express all three DV receptors described in the literature: integrin α2ß1, α5ß1, and α-dystroglycan (αDG). DV promoted astrocyte cell adhesion, cell migration as well as astrocyte stellation. Moreover, DV induced nerve growth factor (NGF) secretion through a αDG- and ERK-dependent pathway. In contrast, α2ß1 or α5ß1 mediated DV antiproliferative effects in astrocytes. NGF production after DV treatment acted as a strong anti-proliferative agent. Another remarkable effect of DV was that it decreased several markers of astrogliosis such as glial fibrillary acidic protein (GFAP), neurocan and phosphacan both in vitro and in vivo, suggesting the role of DV as a potential modulator of postinjury during late astrogliosis, and eventually the onset of glial scarring. Taken together, our study demonstrates the ability of DV to modulate key events of astrogliosis by promoting early astrogliosis and inhibiting glial scar formation, suggesting an additional therapeutic benefit of DV for recovery from stroke. © 2011 Wiley-Liss, Inc.