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

Ein 3D-Blut-Hirn-Schranken-Modell, das aus menschlichen pluripotenten Stammzellen gewonnen wurde

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.

Autor im Rampenlicht: Ein personalisierter Ansatz zur Erforschung der Alzheimer-Krankheit unter Verwendung eines In-vitro-Blut-Hirn-Schrankenmodells 

Rekonstruktion der Blut-Hirn-Schranke in vitro zur Modellierung und therapeutischen Behandlung neurologischer Erkrankungen

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  

Konstruktion der aus menschlichen Stammzellen gewonnenen 3D-In-vitro-Blut-Hirn-Schranke

Zusammenbau der aus humanen pluripotenten Stammzellen gewonnenen Blut-Hirn-Schranke (iBBB) unter Verwendung der dissoziierten Endothelzellen, Parasiten und Astrozyten. Kombinieren Sie die drei Zelltypen in einem konischen Röhrchen und fügen Sie dabei 10 % mehr als die berechnete Anzahl von Zellen hinzu, um Pipettierfehler zu berücksichtigen. Schleudern Sie die Zellmischung bei 300 g für drei Minuten herunter. Saugen Sie den Überstand an und lassen Sie das Zellpellet mit ca. 50 Mikrolitern des Mediums zurück.Mit einer P200-Pipette wird das Zellpellet vorsichtig in das Restmedium resuspendiert, um eine Einzelzellaufschlämmung zu erzeugen. Legen Sie das Röhrchen mit der resultierenden Zellaufschlämmung auf Eis und geben Sie eine ausreichende Menge reduzierter GF Basement Membrane Matrix entsprechend der gewünschten Anzahl von iBBBs hinzu. Mischen Sie die Zellen homogen und führen Sie keine Luftblasen ein.Pipettieren Sie 50 Mikroliter der resultierenden Zellmatrixmischung in eine Vertiefung einer 48-Well- oder 96-Well-Glasboden-Kulturschale und verteilen Sie sie gleichmäßig auf dem Boden der Schale. Inkubieren Sie die Schale bei 37 Grad Celsius für 30 bis 40 Minuten, um die Matrix zu polymerisieren und gleichzeitig die Zellen einzukapseln. Zum Schluss geben Sie 500 Mikroliter ergänztes Astrozytenmedium in die Vertiefung und halten die iBBBs in Kultur.Für eine 96-Well-Platte werden 100 bis 150 Mikroliter Medium in die Bohrung gegeben, bis sie für nachgelagerte Assays ausreichend entwickelt sind, was in der Regel zwei Wochen dauert. Die richtig geformte iBBB erschien unter einem Hellfeldmikroskop als erstarrte, einzelne durchscheinende Scheibe. Nach 24 Stunden wurden gleichmäßig verteilte Einzelzellen identifiziert.Nach zwei Wochen waren deutlichere Strukturen sichtbar, wenn auch schwer zu definieren.

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 ...