Engineering developmentally-relevant Wnt signaling events to improve iPSC-derived blood-brain barrier models

Project summary Mounting evidence indicates that vascular damage and blood-brain barrier (BBB) dysfunction contribute to the progression of Alzheimer’s disease and related dementias (ADRDs). However, it remains challenging to study and understand connections between BBB dysfunction and ADRDs. In vitro BBB models represent valuable tools for investigating vascular contributions to ADRDs due to higher throughput and ease of manipulation. Historically, in vitro BBB models were constructed from primary brain microvascular endothelial cells (BMECs)— the principal functional component of the BBB—but after removal from the brain, BMECs rapidly de-differentiate and lose BBB-specific properties that are crucial for studying ADRDs. Strategies have been developed to differentiate human induced pluripotent stem cells (iPSCs) into endothelial cells with BBB attributes, but such approaches remain imperfect even after a decade of refinement. For example, iPSC-derived cells with a strong BBB phenotype lack robust endothelial character, but artificial reinforcement of endothelial identity via transcription factor overexpression completely ablates BBB properties. Conversely, efforts to imbue endothelial progenitors or mature endothelial cells with a BBB phenotype, through transgene overexpression or small molecule and growth factor treatments, have been largely ineffective. In vivo, BBB development is driven specifically by Wnt7a, and this Wnt ligand can only be transduced into active β-catenin signaling when membrane receptors GPR124 and RECK are present on the endothelium—for example, deletion of Wnt7a, GPR124, or RECK activity yields defects in BBB and neurovascular development. Given the importance of this pathway for BBB development, we hypothesize that specific activation of Wnt7 signaling in naïve iPSC-derived endothelial progenitors will impart representative BBB identity more effectively than prior efforts using generic activation of Wnt/β-catenin signaling. We will investigate this hypothesis using CRISPR and synthetic biology approaches to activate Wnt7 signaling in naïve iPSC-derived endothelial progenitors in simple two-dimensional models (Aim 1) and more complex three-dimensional neurovascular assemblies (Aim 2). Outcomes from this project are expected to yield improved in vitro human BBB models that are better suited for studying causes and impacts of vascular disturbance in ADRDs.