PhD The University of Melbourne Melbourne, Victoria, Australia
Abstract: Human pluripotent stem cell-derived neural organoids/assembloids have provided invaluable models in which to study human neural development, disease and advance drug therapies. Yet lacking in these models is the critical importance of the neurovascular niche and blood brain barrier (BBB). Efforts to date have shown primitive incorporation of endothelial cells into assembloids or cultured organoids onto rudimentary blood vessel networks. Here we present a high-throughput platform for generating healthy and patient specific neurovascular assembloids by integrating independently differentiated human pluripotent stem cell-derived endothelial cells, pericytes, astrocytes, and neurons. These assembloids exhibit in vivo-like features, including tight junction integrity, selective permeability and elaborate vasculature and neuronal maturation over time. Functional stress assays, with mitochondrial and calcium imaging readout in defined neural or endothelial populations, confirm the spatially organised neurovascular crosstalk and coordinated cell-cell (neural-vasculature) communication. Extensive transcriptomic sequencing and developmental profiling validate the reproducibility and temporal precision of these assembloids, uncovering mechanisms of cell-autonomous stress responses and dynamic intercellular signalling during maturation. We utilised this novel throughput model to demonstrate a previously uncharacterised role of pericytes in initiating neurovascular dysfunction in amyotrophic lateral sclerosis patients carrying C9orf72 mutations. By enabling the study of cell-specific contributions to BBB dysfunction and neurovascular disease pathophysiology, these assembloids provide a scalable and robust platform for advancing personalised medicine and therapeutic discovery.
