Engineering the human blood-brain barrier at the capillary scale using a double-templating technique.

In vitro blood-brain barrier (BBB) models have played an important role in studying processes such as immune cell trafficking and drug delivery, as well as contributing to the understanding of mechanisms of disease progression. Many biological and pathological processes in the cerebrovasculature occur in capillaries and hence the lack of robust hierarchical models at the capillary scale is a major roadblock in BBB research. Here we report on a double-templating technique for engineering hierarchical BBB models with physiological barrier function at the capillary scale. We first demonstrate the formation of hierarchical vascular networks using human umbilical vein endothelial cells. We then characterize barrier function in a BBB model using brain microvascular endothelial-like cells (iBMECs) differentiated from induced pluripotent stem cells (iPSCs). Finally, we characterize immune cell adhesion and transmigration in response to perfusion with the inflammatory cytokine tumor necrosis factor-alpha, and show that we can recapitulate capillary-scale effects, such as leukocyte plugging, observed in mouse models. Our double-templated hierarchical model enables the study of a wide range of biological and pathological processes related to the human BBB.

Long-Term Cryopreservation Preserves Blood-Brain Barrier Phenotype of iPSC-Derived Brain Microvascular Endothelial Cells and Three-Dimensional Microvessels.

Brain microvascular endothelial cells derived from induced pluripotent stem cells (dhBMECs) are a scalable and reproducible resource for studies of the human blood-brain barrier, including mechanisms and strategies for drug delivery. Confluent monolayers of dhBMECs recapitulate key in vivo functions including tight junctions to limit paracellular permeability and efflux and nutrient transport to regulate transcellular permeability. Techniques for cryopreservation of dhBMECs have been reported; however, functional validation studies after long-term cryopreservation have not been extensively performed. Here, we characterize dhBMECs after 1 year of cryopreservation using selective purification on extracellular matrix-treated surfaces and ROCK inhibition. One-year cryopreserved dhBMECs maintain functionality of tight junctions, efflux pumps, and nutrient transporters with stable protein localization and gene expression. Cryopreservation is associated with a decrease in the yield of adherent cells and unique responses to cell stress, resulting in altered paracellular permeability of Lucifer yellow. Additionally, cryopreserved dhBMECs reliably form functional three-dimensional microvessels independent of cryopreservation length, with permeabilities lower than non-cryopreserved two-dimensional models. Long-term cryopreservation of dhBMECs offers key advantages including increased scalability, reduced batch-to-batch effects, the ability to conduct well-controlled follow up studies, and support of multisite collaboration from the same cell stock, all while maintaining phenotype for screening pharmaceutical agents.

Brain microvascular endothelial cell dysfunction in an isogenic juvenile iPSC model of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of cytosine-adenine-guanine (CAG) repeats in the huntingtin gene, which leads to neuronal loss and decline in cognitive and motor function. Increasing evidence suggests that blood-brain barrier (BBB) dysfunction may contribute to progression of the disease. Studies in animal models, in vitro models, and post-mortem tissue find that disease progression is associated with increased microvascular density, altered cerebral blood flow, and loss of paracellular and transcellular barrier function. Here, we report on changes in BBB phenotype due to expansion of CAG repeats using an isogenic pair of induced pluripotent stem cells (iPSCs) differentiated into brain microvascular endothelial-like cells (iBMECs). We show that CAG expansion associated with juvenile HD alters the trajectory of iBMEC differentiation, producing cells with ~ two-fold lower percentage of adherent endothelial cells. CAG expansion is associated with diminished transendothelial electrical resistance and reduced tight junction protein expression, but no significant changes in paracellular permeability. While mutant huntingtin protein (mHTT) aggregates were not observed in HD iBMECs, widespread transcriptional dysregulation was observed in iBMECs compared to iPSCs. In addition, CAG expansion in iBMECs results in distinct responses to pathological and therapeutic perturbations including angiogenic factors, oxidative stress, and osmotic stress. In a tissue-engineered BBB model, iBMECs show subtle changes in phenotype, including differences in cell turnover and immune cell adhesion. Our results further support that CAG expansion in BMECs contributes to BBB dysfunction during HD.

Three-dimensional microenvironment regulates gene expression, function, and tight junction dynamics of iPSC-derived blood-brain barrier microvessels.

The blood-brain barrier (BBB) plays a pivotal role in brain health and disease. In the BBB, brain microvascular endothelial cells (BMECs) are connected by tight junctions which regulate paracellular transport, and express specialized transporter systems which regulate transcellular transport. However, existing in vitro models of the BBB display variable accuracy across a wide range of characteristics including gene/protein expression and barrier function. Here, we use an isogenic family of fluorescently-labeled iPSC-derived BMEC-like cells (iBMECs) and brain pericyte-like cells (iPCs) within two-dimensional confluent monolayers (2D) and three-dimensional (3D) tissue-engineered microvessels to explore how 3D microenvironment regulates gene expression and function of the in vitro BBB. We show that 3D microenvironment (shear stress, cell-ECM interactions, and cylindrical geometry) increases BBB phenotype and endothelial identity, and alters angiogenic and cytokine responses in synergy with pericyte co-culture. Tissue-engineered microvessels incorporating junction-labeled iBMECs enable study of the real-time dynamics of tight junctions during homeostasis and in response to physical and chemical perturbations.