Blood–brain barrier (BBB) breakdown and immune cell infiltration into the CNS are early hallmarks of multiple sclerosis (MS). The mechanisms leading to BBB dysfunction are incompletely understood and generally thought to be a consequence of neuroinflammation. Here, we have challenged this view and asked if intrinsic alterations in the BBB of MS patients contribute to MS pathogenesis. To this end, we made use of human induced pluripotent stem cells derived from healthy controls and MS patients and differentiated them into brain microvascular endothelial cell (BMEC)-like cells as in vitro model of the BBB. MS-derived BMEC-like cells showed impaired junctional integrity, barrier properties and efflux pump activity when compared to healthy controls. Also, MS-derived BMEC-like cells displayed an inflammatory phenotype with increased adhesion molecule expression and immune cell interactions. Activation of Wnt/β-catenin signalling in MS-derived endothelial progenitor cells enhanced barrier characteristics and reduced the inflammatory phenotype. Our study provides evidence for an intrinsic impairment of BBB function in MS patients that can be modelled in vitro. Human iPSC-derived BMEC-like cells are thus suitable to explore the molecular underpinnings of BBB dysfunction in MS and will assist in the identification of potential novel therapeutic targets for BBB stabilization.

Endothelial cells (ECs) in the central nervous system (CNS) acquire their specialized blood–brain barrier (BBB) properties in response to extrinsic signals, with Wnt/β-catenin signaling coordinating multiple aspects of this process. Our knowledge of CNS EC development has been advanced largely by animal models, and human pluripotent stem cells (hPSCs) offer the opportunity to examine BBB development in an in vitro human system. Here, we show that activation of Wnt signaling in hPSC-derived naïve endothelial progenitors, but not in matured ECs, leads to robust acquisition of canonical BBB phenotypes including expression of GLUT-1, increased claudin-5, decreased PLVAP, and decreased permeability. RNA-seq revealed a transcriptome profile resembling ECs with CNS-like characteristics, including Wnt-upregulated expression of LEF1, APCDD1, and ZIC3. Together, our work defines effects of Wnt activation in naïve ECs and establishes an improved hPSC-based model for interrogation of CNS barriergenesis.

The blood-brain barrier (BBB), formed by specialized endothelial cells (ECs), regulates the extracellular composition of the central nervous system (CNS). Little is known about whether there are regional specializations of the BBB that may control the function of specific neural circuits. We use single cell RNA-seq to characterize ECs from nine CNS regions in male mice: cortex, hippocampus, cerebellum, spinal cord, striatum, thalamus, hypothalamus, midbrain, and medulla/pons. Although there is a core BBB transcriptional profile, there are significant regional specializations. Stra6, a retinoid transporter, is highly enriched in the BBB of the nucleus accumbens shell (ShNAc) and ventral cochlear nucleus, and is controlled by dietary vitamin A, through endothelial RARƔ. EC Stra6 regulates the deposition of retinoids specifically in the ShNAc and cochlear nucleus, and is required for the function of the ShNAc, in a retinoid-dependent manner. Thus regional specializations of the BBB can regulate the function of local brain regions.

Brain mural cells regulate development and function of the blood-brain barrier and control blood flow. Existing in vitro models of human brain mural cells have low expression of key mural cell genes, including NOTCH3 . Thus, we asked whether activation of Notch3 signaling in hPSC-derived neural crest could direct the differentiation of brain mural cells with an improved transcriptional profile. Overexpression of the Notch3 intracellular domain (N3ICD) induced expression of mural cell markers PDGFRβ, TBX2, FOXS1 , KCNJ8 , SLC6A12 , and endogenous Notch3. The resulting N3ICD-derived brain mural cells produced extracellular matrix, self-assembled with endothelial cells, and had functional K _ATP channels. ChIP-seq revealed that Notch3 serves as a direct input to relatively few genes in the context of this differentiation process. Our work demonstrates that activation of Notch3 signaling is sufficient to direct the differentiation of neural crest to mural cells and establishes a developmentally relevant differentiation protocol.

The blood–brain barrier (BBB) is a term used to describe the specialized properties of vasculature in the central nervous system (CNS) that regulate the exchange of ions, molecules and cells, thereby maintaining CNS homeostasis. These BBB properties are largely possessed by vascular endothelial cells, which exhibit molecular specializations that distinguish them from endothelial cells in other organs. BBB development – orchestrated by complex interactions between endothelial cells, neural progenitor cells, pericytes, astrocytes, neurons and other cell types that form the neurovascular unit – is initiated concurrently with CNS angiogenesis and continues during expansion and maturation of the vasculature. Importantly, the Wnt/β-catenin signaling pathway plays a central role in barriergenesis, the initial acquisition of BBB phenotype in endothelial cells. In this Review, we summarize current understanding of BBB development, including timing and underlying cellular and molecular mechanisms. We also discuss established and emerging techniques for studying BBB development and highlight important unanswered questions in the field.