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

Summary

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.

Abstract

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'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 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.

Introduction

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 homeostasis^1,2,3,4, clearing waste^4,5,6, and protecting the brain from pathogens and toxins^7,8.

The primary cell type of the BBB is the endothelial cell (EC). Endothelial cells, derived from the mesoderm lineage, form the walls of the vasculature^1,9. Microvascular ECs form tight junctions with each other to greatly decrease the permeability of their membrane^{10,11,12,13,14} while expressing transporters to facilitate the movement of nutrients into and out of the CNS^1,4,12,14. 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 cells^15,16,17. 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 end-feet (e.g., aquaporin 4 [AQP-4]) that have a critical role in BBB function^18,19,20,21.

The BBB is critical in maintaining proper brain health function, and dysfunction of the BBB has been reported in many neurological diseases, including Alzheimer's disease (AD)^22,23,24,25, multiple sclerosis^7,26,27,28, epilepsy^29,30, and stroke^31,32. 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 β (Aβ) 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 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.

Protocol

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

Representative Results

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,…

Discussion

BBB dysfunction is a co-morbidity, and potentially, a cause or exacerbating factor in numerous neurological diseases^7,40,41. 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…

Materials

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