Advancing human brain organoids: Modeling glia and the blood-brain barrier in vitro

In this dissertation, we aimed to develop new human brain organoid (hBO)-based models to study the human brain in development and disease, with a focus on astrocytes, microglia, and the blood–brain barrier (BBB). We also set out to create a more complex in vitro model of the BBB and neurovascular unit (NVU) by connecting a vessel-like structure to an hBO on a microfluidic chip. A central motivation for this work is the need for better in vitro BBB models to test how therapeutic compounds affect and cross the barrier. HBOs have the potential to model the human brain and BBB with higher complexity, but they still face limitations such as variability, dependence on animal-derived components, and a lack of (perfused) vasculature. We therefore investigated whether hBOs can be cultured without Matrigel. We found that while Matrigel strongly affects early morphogenesis and regional identity, it is not strictly necessary for correct cytoarchitecture and growth. A hyaluronic acid-based hydrogel supported neuronal identity and reduced non-ectodermal differentiation, particularly after slicing and culturing on an air–liquid interface. In a literature review we then explored how hBOs can be used to model early neurovascular development. Current approaches to vascularize hBOs partly recapitulate processes such as angiogenesis, NVU formation, and early barriergenesis. However, these models do not yet represent a functional BBB due to the lack of mature barrier properties and flow. Next, we studied how endothelial cell-derived factors influence hBO development, focusing on astrocytes. Exposure to endothelial-conditioned medium led radial glia to adopt a more mature transcriptomic profile. Proteomic analysis identified potential interactions between endothelial-secreted factors and early radial glia markers that may underlie this effect. To further model BBB interactions, we developed a microfluidic chip that connects an hBO to a vessel-like structure. This system allows direct physical contact between endothelial cells and hBO astrocytes, which form endfeet-like structures. It can be used to study how compounds or blood-borne cells cross the BBB and affect brain tissue. We also addressed the modeling of human microglia in vitro, highlighting the importance of understanding microglial development and reviewing current approaches and challenges. Finally, we established an in vitro model for ischemic stroke by exposing microglia-containing hBOs to oxygen-glucose deprivation and reoxygenation. This led to stabilization of HIF1α and induced changes in microglial gene expression and morphology, although responses varied between iPSC lines. Overall, this work shows that changes in culture conditions and treatments (such as extracellular matrices, cell-derived signals, co-culture systems, and hypoxic stimuli) can influence the development, function, and cellular composition of hBOs. Together, we provide new methods and systems for modeling the human brain, with a focus on astrocytes, microglia, and the BBB.