This work is supported by NIH 3-UG3-NS115064-01, R01NS14239, Cure Alzheimer&#39;s Fund, NASA 80ARCO22CA004, Chan-Zuckerberg Initiative, MJFF/ASAP Foundation, and Brain Injury Association of America. C.G. is supported by NIH F31NS130909. Figure 1A was created with BioRender.com.

1. Differentiating iPSCs into iBBB cells
NOTE: These differentiation protocols have been previously described in Mesentier-Louro et al.34.
<ol>
	<li>Coating cell culture plates
	<ol>
		<li>Thaw reduced growth factor (GF) membrane matrix overnight at 4 &#176;C. Dilute 500 &#181;L of basement membrane matrix in 49.5 mL of DMEM. Keep this solution cold to prevent premature polymerization of the coating solution.</li>
		<li>Add 1-2 mL per well of a 6-well...

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

BBB dysfunction is a co-morbidity, and potentially, a cause or exacerbating factor in numerous neurological diseases7,40,41. However, it is nearly impossible to study the molecular and cell biology underlying the dysfunction and breakdown of the BBB in humans with neurovascular disease. The inducible-BBB (iBBB) presented in this protocol provides an in vitro system that recapitulates important cell interactions of the B...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6e10 amyloid-&#946; antibody</td>
			<td>Biolegend</td>
			<td>SIG-39320</td>
			<td>Used at 1:1000</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>Peprotech</td>
			<td>20-14E</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488, 555, 647 secondary antibodies</td>
			<td>Invitrogen</td>
			<td>Various</td>
			<td>Used at 1:1000</td>
		</tr>
		<tr>
			<td>Amyloid-beta 40 fibril</td>
			<td>AnaSpec</td>
			<td>AS-24235</td>
			<td></td>
		</tr>
		<tr>
			<td>Amyloid-beta 42 fibril</td>
			<td>AnaSpec</td>
			<td>AS-20276</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquaporin-4 antibody</td>
			<td>Invitrogen</td>
			<td>PA5-53234</td>
			<td>Used at 1:300</td>
		</tr>
		<tr>
			<td>Astrocyte basal media and supplements</td>
			<td>ScienCell</td>
			<td>1801</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 serum-free supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>Peprotech</td>
			<td>120-05ET</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Cyamn Chemical</td>
			<td>13112</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with GlutaMAX medium</td>
			<td>Gibco</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Millipore-Sigma</td>
			<td>D3072-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-basic</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G slide mounting medium</td>
			<td>VWR</td>
			<td>100502-406</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>R&#38;D Systems</td>
			<td>1099/10</td>
			<td></td>
		</tr>
		<tr>
			<td>GeltrexTM LDEV-Free hESC-qualified Reduced Growth Factor Basement&#160;</td>
			<td>Gibco</td>
			<td>A1413302</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom 48-well Culture Dishes</td>
			<td>Mattek Corporation</td>
			<td>P48G-1.5-6-F</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342&#160;</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Endothelial Serum-free medium</td>
			<td>Gibco</td>
			<td>11111044</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Tocris</td>
			<td>6053</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Non-essential Amino Acid Solution (MEM-NEAA)&#160;</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Millipore-Sigma</td>
			<td>S30-100mL</td>
			<td>Use serum to match secondary antibody host</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)&#160;</td>
			<td>ThermoFisher</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-BB</td>
			<td>Peprotech</td>
			<td>100-14B</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGFRB (Platelet-derived growth factor receptor beta) antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF385</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS), pH 7.4</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>Pecam1 (Platelet endothelial cell adhesion molecule 1) antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF806</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PiggyBac plasmid (PB_iETV2_P2A_GFP_Puro)</td>
			<td>AddGene</td>
			<td>&#160;Catalog #168805</td>
			<td></td>
		</tr>
		<tr>
			<td>S100B antibody</td>
			<td>Sigma-Aldrich</td>
			<td>S2532-100uL</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>SB43152</td>
			<td>Reprocell</td>
			<td>04-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioflavin T</td>
			<td>Chem Impex</td>
			<td>22870</td>
			<td>Used at 25uM</td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-250mL</td>
			<td></td>
		</tr>
		<tr>
			<td>VE-cadherin (CD144) antibody</td>
			<td>R&#38;D systems</td>
			<td>AF938</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>VEGF-A</td>
			<td>Peprotech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>R&#38;D Systems</td>
			<td>1254/10</td>
			<td></td>
		</tr>
		<tr>
			<td>ZO-1</td>
			<td>Invitrogen</td>
			<td>MA3-39100-A488</td>
			<td>Dilution = 1:500</td>
		</tr>
	</tbody>
</table>

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 the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

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.

A properly formed iBBB solidifies into a single translucent disc (Figure 3A). It is normal for the iBBB to detach from the surface onto which it was first pipetted after a few days. This cannot be avoided, but is not a major concern to the proper formation of the iBBB if care is taken when changing media to not accidentally aspirate the iBBB. After 24 h, evenly distributed, single cells can be identified under a brightfield microscope (Figure 3B). After 2 weeks,...

Watch this Scientific Journal Video about A Personalized Approach Towards Investigating Alzheimer's Disease Using an In Vitro Blood-Brain Barrier Model at JoVE.com

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

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

reconstruction-blood-brain-barrier-vitro-to-model-therapeutically