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

En 3D-modell av blod-hjärnbarriären härledd från humana pluripotenta stamceller

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.

Författare Spotlight: Ett personligt tillvägagångssätt för att undersöka Alzheimers sjukdom med hjälp av en in vitro blod-hjärnbarriärmodell 

Rekonstruktion av blod-hjärnbarriären in vitro för att modellera och terapeutiskt rikta in sig på neurologiska sjukdomar

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 av 3D-human-stamcells-härledd in vitro-blod-hjärnbarriär

Montera den humana pluripotenta stamcellshärledda blod-hjärnbarriären, eller iBBB, med hjälp av de dissocierade endotelcellerna, parasiterna och astrocyterna, Kombinera de tre typerna av celler i ett koniskt rör samtidigt som du inkluderar 10 % mer än det beräknade antalet celler för att ta hänsyn till pipetteringsfel. Centrifugera cellblandningen med 300 g i tre minuter. Aspirera supernatanten och lämna cellpelleten med cirka 50 mikroliter av mediet.Använd en P200-pipett och återsuspendera försiktigt cellpelleten i restmediet för att skapa en encellsuppslamning. Placera röret som innehåller den resulterande celluppslamningen på is och tillsätt en tillräcklig mängd reducerad GF Basement Membrane Matrix till den enligt önskat antal iBBB. Blanda cellerna homogent utan att införa några luftbubblor.Pipettera 50 mikroliter av den resulterande cellmatrisblandningen i en brunn med en odlingsskål med 48 brunnar eller 96 brunnar med glasbotten och fördela den jämnt över skålens botten. Inkubera skålen vid 37 grader Celsius i 30 till 40 minuter för att polymerisera matrisen samtidigt som cellerna kapslas in. Tillsätt slutligen 500 mikroliter tillskottsmedium till brunnen och behåll iBBB:erna i odlingen.För en platta med 96 brunnar, tillsätt 100 till 150 mikroliter medium till brunnen tills de är tillräckligt utvecklade för nedströmsanalyser, vilket vanligtvis tar två veckor. Den korrekt formade iBBB framträdde som en stelnad, enda genomskinlig skiva under ett ljusfältsmikroskop. Efter 24 timmar identifierades jämnt fördelade enskilda celler.Efter två veckor kunde man se mer distinkta strukturer, även om de var svåra att definiera.

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 ...