In vitro models of the blood brain-barrier using iPSC-derived cells

Abstract

The blood-brain barrier (BBB) constitutes the interface between the blood and the brain tissue. Its primary function is to maintain the tightly controlled microenvironment of the brain. Models of the BBB are useful for studying the development and maintenance of the BBB as well as diseases affecting it. Furthermore, BBB models are important tools in drug development and support the evaluation of the brain-penetrating properties of novel drug molecules. Currently used in vitro models of the BBB include immortalized brain endothelial cell lines and primary brain endothelial cells of human and animal origin. Unfortunately, these cell lines and primary cells have failed to recreate physiologically relevant control of transport in vitro. Human-induced pluripotent stem cell (iPSC)-derived brain endothelial cells have proven a promising alternative source of brain endothelial-like cells that replicate tight cell layers with low para-cellular permeability. Given the possibility to generate large amounts of iPSC-derived brain endothelial cells they are a feasible alternative when modelling the BBB in vitro. This thesis aimed to develop iPSC-derived models of the BBB that display a barrier like phenotype and characterize these models in terms of specific properties. The BBB model development was based on investigations into mechanisms important for barrier formation in iPSC-derived endothelial cells and development of high-quality supporting cells. The possibilities to use the model in drug discovery, and in determination of brain penetrating capacity of drug substances were specifically considered. These studies have increased knowledge of molecular mechanisms behind the restricted permeability across iPSC-derived endothelial cells and identified transcriptional changes that occur in iPSC-derived endothelial cells upon coculture with relevant cell types of the neurovascular unit. Furthermore, high quality iPSC-derived astrocytic cells were developed, and the biological relevance and model diversity between astrocytic models were evaluated. Both astrocytes and brain endothelial cells have been adapted to xeno-free culture conditions and used in the BBB models, demonstrating a xeno-free BBB model. Finally, a more biologically relevant microphysiological dynamic BBB model was generated. This model demonstrated improved permeability modelling and compatibility with high-throughput substance permeability screening. Taken together these results show that iPSC-derived BBB models are useful for studying BBB-specific properties in vitro and that both marker expression and functional evaluation of iPSC-derived cells are important in assessing cell identity and cell quality. In addition, these results show that iPSC derived BBB models are feasible for high-throughput permeability studies.