Name: Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier
Uploaded: 2023-10-20T12:30:00
Description: Watch this Scientific Journal Video about Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier at JoVE.com

constructing the 3d human-stem-cell-derived in vitro blood brain barrier

Un modèle 3D de barrière hémato-encéphalique dérivé de cellules souches pluripotentes humaines

The blood-brain barrier (BBB) is a key microvascular network separating the central nervous system (CNS) from the periphery to maintain an ideal environment for proper neuronal function. It has a critical role in regulating the influx and efflux of substances into the CNS by maintaining metabolic homeostasis1,2,3,4, clearing waste4,5,6, and protecting the brain from pathogens and toxins7,8.
The primary cell type of the BBB is the endothelial cell (EC). Endothelial cells, derived from the mesoderm lineage, form the walls of the vasculature1,9. Microvascular ECs form tight junctions with each other to greatly decrease the permeability of their membrane10,11,12,13,14 while expressing transporters to facilitate the movement of nutrients into and out of the CNS1,4,12,14. Microvascular ECs are encircled by pericytes (PCs)-mural cells that regulate microvascular function and homeostasis and are critical for regulating the permeability of the BBB to molecules and immune cells15,16,17. The astrocyte, a major glial cell type, is the final cell type comprising the BBB. Astrocyte end-feet wrap around the EC-PC vascular tubes while the cell bodies extend into the brain parenchyma, forming a connection between neurons and vasculature1. Distinct solute and substrate transporters are localized on astrocyte&#160;end-feet (e.g., aquaporin 4 [AQP-4]) that have a critical role in BBB function18,19,20,21.
The BBB is critical in maintaining proper brain health function, and dysfunction of the BBB has been reported in many neurological diseases, including Alzheimer&#39;s disease (AD)22,23,24,25, multiple sclerosis7,26,27,28, epilepsy29,30, and stroke31,32. It is increasingly recognized that cerebrovascular abnormalities play a central role in neurodegeneration, contributing to increased susceptibility to ischemic and hemorrhagic events. For example, more than 90% of AD patients have cerebral amyloid angiopathy (CAA), a condition characterized by the deposition of amyloid &#946; (A&#946;) along the cerebral vasculature. CAA increases BBB permeability and decreases BBB function, leaving the CNS vulnerable to ischemia, hemorrhagic events, and accelerated cognitive decline33.
We recently developed an in vitro model of the human BBB, derived from&#160;patient-induced pluripotent stem cells, which incorporates ECs, PCs, and astrocytes encapsulated in a 3D matrix (Figure 1A). The iBBB recapitulates physiologically relevant interactions, including vascular tube formation and localization of astrocyte end-feet with vasculature24. We applied the iBBB to model the susceptibility of CAA mediated by APOE4 (Figure 1B). This enabled us to identify the causal cellular and molecular mechanisms by which APOE4 promotes CAA, and leverage these insights to develop therapeutic strategies that reduce CAA pathology and improve learning and memory in vivo in APOE4 mice24. Here, we provide a detailed protocol and video tutorial for reconstructing the BBB from human iPSCs and modeling CAA in vitro.

Pleins feux sur l’auteur : Une approche personnalisée de l’étude de la maladie d’Alzheimer à l’aide d’un modèle in vitro de barrière hémato-encéphalique 

Reconstruction de la barrière hémato-encéphalique in vitro pour modéliser et cibler thérapeutiquement les maladies neurologiques

Our goal is to determine the molecular and cellular mechanisms that mediate Alzheimer's disease pathogenesis to uncover therapeutic and diagnostic opportunities. Currently, the molecular pathways and mechanisms involved in the onset and development of Alzheimer's disease are poorly understood. This is due to mouse models and other in vitro Alzheimer's disease models failing to recapitulate unique human biology that is crucial to the development of the disease.We have developed an in vitro model of the blood-brain barrier that can be applied to study cerebrovascular pathologies associated with Alzheimer's disease, dementia, and other forms of neurodegeneration. This model has been employed to show that APOE4, the strongest risk factor for Alzheimer's disease, acts in part through a parasite-specific pathway to increase pathogenic amyloid burden Using induced pluripotent stem cells, we can construct human brain tissue in vitro that can be used to investigate disease pathology and drug screenings. Such an in vitro blood-brain barrier model enables personalized therapeutic and diagnostic approaches.Our 3D in vitro blood-brain barrier model demonstrates physiologically relevant interactions, including vascular tube formation and localization of astrocyte endfeet with vasculature. It is also capable of modeling disease-relevant phenotypes, including amyloid pathology. Finally, the use of patient-derived induced pluripotent stem cells allows the study of any desired genetic background.Using the in vitro blood-brain barrier model, we discovered pathways that mediate pathological cerebrovascular amyloid accumulation. This led us to explore therapeutic strategies that can reverse this pathology in aged APOE4 mice, which we are currently following up on.

Watch this Scientific Journal Video about Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier at JoVE.com

Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier  

Construction de la barrière hémato-encéphalique in vitro in vitro dérivée de cellules souches humaines en 3D

Assembler la barrière hémato-encéphalique dérivée de cellules souches pluripotentes humaines, ou iBBB, à l’aide des cellules endothéliales dissociées, des parasites et des astrocytes, Combiner les trois types de cellules dans un tube conique tout en incluant 10% de plus que le nombre calculé de cellules pour tenir compte des erreurs de pipetage. Essorez le mélange cellulaire à 300 g pendant trois minutes. Aspirer le surnageant, en laissant la pastille cellulaire avec environ 50 microlitres de milieu.À l’aide d’une pipette P200, remettre délicatement en suspension la pastille cellulaire dans le milieu résiduel pour créer une suspension unicellulaire. Placez le tube contenant la boue cellulaire résultante sur de la glace et ajoutez-y une quantité suffisante de matrice de membrane basale GF réduite selon le nombre souhaité d’iBBB. Mélangez les cellules de manière homogène sans introduire de bulles d’air.Pipeter 50 microlitres du mélange de matrice cellulaire résultant dans un puits d’une boîte de culture à fond de verre à 48 puits ou 96 puits et le répartir uniformément dans le fond de la boîte. Incuber la boîte à 37 degrés Celsius pendant 30 à 40 minutes pour polymériser la matrice tout en encapsulant les cellules. Enfin, ajoutez 500 microlitres de milieu astrocytaire supplémenté dans le puits et maintenez les iBBB en culture.Pour une plaque de 96 puits, ajoutez 100 à 150 microlitres de milieu dans le puits jusqu’à ce qu’ils soient suffisamment développés pour les analyses en aval, ce qui prend généralement deux semaines. L’iBBB correctement formé est apparu sous la forme d’un disque translucide unique solidifié sous un microscope à fond clair. Après 24 heures, des cellules individuelles uniformément réparties ont été identifiées.Au bout de deux semaines, des structures plus distinctes, bien que difficiles à définir, étaient visibles.

Research

JoVE Journal

Neuroscience

Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...