Vascularization of cerebral organoids: Improving in vitro modelling of the neurovascularity

Brain organoids (BOs) are multicellular, self-organized, stem cell-derived in vitro 3D systems aim to recapitulate morphological and physiological features of the human brain. These emerging models are attractive for developmental biology, disease modelling and drug screening, and represent an alternative to animal experimentation in neuroscience. However, low oxygen and nutrients diffusion due to a missing vascularity limits their growth and survival, hence their translatability to clinical applications. Vascularization of BOs poses several technical challenges, including the simultaneous differentiation of two tissues of different origin, uneven distribution and limited penetration of the vascular networks within the microtissue, and the absence of luminal perfusion. To mitigate these issues, we devised an encapsulation approach in which human brain microvascular endothelial cells (HBMVECs) were delivered to developing cerebral organoids (COs), a self-patterned type of BOs, from a progressively degrading surrounding biomaterial. The composition of the media and the concentration of the hydrogel were tuned to promote both neurodevelopment and endothelial network formation. Using this strategy, we identified a higher density of vascular-like networks that expanded towards the organoid centre. By using pathway inhibitors and fluorescent endothelial cells, the origin of these endothelial networks was revealed to arise from both endogenous differentiation and the exogenously introduced HBMVECs. Endogenous endothelial contributions were enhanced in unguided BO protocols and varied across cell lines. Vascularized COs displayed typical blood–brain barrier (BBB) characteristics, including claudin 5 expression, astrocytes and pericyte-like cells interactions, and laminin and collagen IV depositions. They also showed smaller necrotic cores and increased permeability to an external dye, although intraluminal flow was not demonstrated. Fluorescence-activated cell sorting (FACS), RNA sequencing and immunohistochemistry (IHC) revealed enhanced cortical regionalization with PAX6 expression and expanded radial glia layers, while later neuroglial differentiation remained unaltered upon vascularization, as confirmed by calcium imaging and IHC. Vascularized COs have also been integrated into microfluidic devices to incorporate directional flow within the vessels, but further optimization is required. Overall, these findings establish an accessible model of the human neurovascularity as a platform for disease investigation and drug discovery.

Funder

Research Ireland
Engineering and Physical Sciences Research Council
European Regional Development Fund

Publisher

University of Galway

Rights

CC BY-NC-ND

cbn

Collections

University of Galway Theses (PhD Theses)