INCREASING THE COMPLEXITY OF MIDBRAIN ORGANOID SYSTEMS FOR DEVELOPMENTAL STUDIES AND DISEASE MODELLING

The discovery of iPSC technology revolutionized the biomedical field, allowing the
development of translatable and complex 2D and 3D cell culture systems. Organoids are 3D
models containing multiple cell types that mimic complex microenvironments. This is highly
advantageous to understand human development, physiology and disease, especially in
inaccessible areas such as the brain. Human midbrain-specific organoids have been
developed to study the midbrain (abundant in dopaminergic neurons). In Parkinson’s Disease
(PD), dopaminergic neurons in the substantia nigra of the midbrain degenerate, causing a
broad spectrum of clinical features. Midbrain organoids (MO) are rich in dopaminergic neurons,
and contain spatially organized groups of neural cells and progenitors. MO generated from PD
patients’ cells recapitulate dopaminergic neuron degeneration. In this thesis, we first
demonstrated that dopaminergic neuron PD phenotypes and drug rescue effects were similar
between MO and mice. After, we identified different neuronal clusters, progenitor cells, radial
glia and mesenchymal cells in MO by scRNA-Seq. As expected, due to the neuro-ectodermal
patterning of the MO’ starting cell population, we confirmed the absence of mesoderm-derived
cell types, such as microglia and endothelial cells. This represents a limitation for the system
in terms of cellular and molecular complexity. Microglia in the human brain perform
surveillance, defence and homeostasis functions; they phagocytose metabolic waste products
and cell debris. We successfully developed a novel protocol to integrate functional microglia
into our MO model. SnRNA-Seq analysis and electrophysiological results suggested a
reduction of stress levels and higher maturation of neurons in the presence of microglia,
respectively. We then aimed to vascularise MO, which would better recapitulate the brain
environment and improve oxygen and nutrient supply into the organoid core (a common 3D
culture limitation). We integrated an endothelial network into MO by fusion with vascular
organoids, and observed the presence of blood vessel components like pericytes and basal
lamina. Furthermore, vascularized assembloids showed decreased levels of cell death and
hypoxia. Finally, by co-culturing microglia with vascularized assembloids, we modelled the
neurovascular unit in 3D. Altogether, this work contributes to the development of advanced 3D
region-specific organoids, which better recapitulate the complexity of the human brain. These
novel MO systems represent one step further into modelling neuroinflammation and blood
brain barrier disruption, typical from neurodegenerative disorders such as PD, which might
lead to more reliable and personalized medical approaches.