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

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

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.

Author Spotlight: A Personalized Approach Towards Investigating Alzheimer&#039;s Disease Using an In Vitro Blood-Brain Barrier Model 

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

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  

Assemble the human pluripotent stem cell derived blood-brain barrier, or iBBB, using the dissociated endothelial cells, parasites, and astrocytes, Combine the three types of cells in a conical tube while including 10%more than the calculated number of cells to account for pipetting errors. Spin down the cell mixture at 300 g for three minutes. Aspirate the supernatant, leaving the cell pellet with approximately 50 microliters of the medium.Using a P200 pipette, gently resuspend the cell pellet in the residual medium to create a single cell slurry. Place the tube containing the resultant cell slurry on ice and add a sufficient amount of reduced GF Basement Membrane Matrix to it per the desired number of iBBBs. Mix the cells homogenously while not introducing any air bubbles.Pipette 50 microliters of the resultant cell matrix mixture into a well of a 48-well or 96-well glass-bottom culture dish and distribute it evenly throughout the bottom of the dish. Incubate the dish at 37 degrees Celsius for 30 to 40 minutes to polymerize the matrix while encapsulating the cells. Finally, add 500 microliters of supplemented astrocyte medium to the well and maintain the iBBBs in culture.For a 96-well plate, add 100 to 150 microliters of medium to the well until they are sufficiently developed for downstream assays, which typically takes two weeks. The properly formed iBBB appeared as a solidified, single translucent disk under a brightfield microscope. After 24 hours, evenly distributed single cells were identified.After two weeks, more distinct structures, although difficult to define, were visible.

constructing-3d-human-stem-cell-derived-vitro-blood-brain

Research

JoVE Journal

Neuroscience

105 Views.  Icahn School of Medicine at Mount Sinai. Assemble the human pluripotent stem cell derived blood-brain barrier, or iBBB, using the dissociated endothelial cells, parasites, and astrocytes, Combine the three types of cells in a conical tube while including 10%more than the calculated number of cells to account for pipetting errors. Spin down the cell mixture at 300 g for three minutes. Aspirate the supernatant, leaving the cell pellet with approximately 50 microliters of the medium.Using a P200 pipette, gently resuspend the cel...