ORGANOIDS AS TOOLS TO STUDY BRAIN DEVELOPMENT AND DISEASE

Human brain organoids (ORG) are a widely used in vitro model that contribute to our knowledge about the biology and pathophysiology of the nervous central system (CNS). They are 3D multicellular clusters that mimic the cytoarchitecture and the developmental pathways that occur in vivo. The most debated limitation of ORG is the lack of the endothelial component. Thus, at first we have developed a co-culture in vitro system composed by the Blood Brain Barrier (BBB) and ORG. The in vitro BBB is composed by human brain microvascular endothelial cells (HBMEC) and human astrocytes (HA). ORG are generated from induced pluripotent stem cells (iPSCs) cultured with different differentiation media for 36 days, and then co-cultured with the in vitro generated BBB. To evaluate the strength of our model, organoids in the absence (ORG) or in the presence (BBB-ORG) of the BBB were compared for their maturation stage and BDNF levels. In addition, the effects of magnesium (Mg) were investigated with a focus on excitotoxicity and neurodevelopment. Indeed, Mg is known to play crucial role in the CNS. It protects the CNS from inflammation, from excitotoxicity driven by NMDA-R over-activation, it has positive effects on the BBB integrity and it is involved in brain development. However, the mechanisms of action are still poorly understood and studied. To shed light on the Magnesium (Mg)-driven mechanisms involved in brain development and protection, an inorganic and an organic Mg salt (Mg sulphate, MgSO4, and Mg pidolate, MgPid, respectively) were added to the culture media to reach the extracellular concentrations of 1, the physiological extracellular Mg concentration, or 5 mM, to mimic Mg supplementation. After the treatment, the possible modulation of molecules involved in excitotoxicity and neurodevelopment was investigated by qRT-PCR, western blot or ELISA. In particular, we analysed the modulation of NMDA-R, GABA-R and their neurotransmitters glutamate and GABA, neurotrophic factors, dopamine and its receptors and the Mg channels TRPM6 and TRPM7. The maturation stage and the cortical layer organization were assessed by immunofluorescence using CTIP2, TBR2 and SOX2 antibodies to detect the cortical, the subcortical neurons and the neural progenitor cells, respectively. Toluidine blue staining and ultrastructural analysis using transmission electron microscopy were performed to further confirm the results. 6 Our data demonstrate that BDNF levels are higher in the BBB-ORG model because of the presence of the endothelial component, which is the main responsible for its secretion. Moreover, the cortical layer is more organized in the presence of the BBB than in its absence, underlying its importance. High concentrations of Mg salts lowers the levels of NMDA-R and increases GABA-Rs levels only in the presence of the BBB, thus suggesting a role in Mg in counteracting excitotoxicity. These results suggest that Mg supplementation might ameliorates the outcome of patients when administered as adjunctive therapy. Particularly, MgPid performs better than MgSO4. Interestingly, high concentration of Mg salts concentration results in a higher maturation stage of the BBB-ORG. The increased levels of released and intra-org BDNF detected in the presence of 5 mM MgPid could explain the faster maturation of the ORG. This study confirms that the BBB-ORG is a useful tool to study the crosstalk between BBB and brain in both physiological and pathological conditions, and emphasises the importance of the continuous search for a model that closely resembles the in vivo context. Moreover, although further analyses are needed, these studies unveil the role of Mg in brain development and in modulating the release of BDNF from the BBB. In addition, preliminary studies on the neuroinvasiveness of SARS-Cov-2 were conducted on ORG compared to BBB-ORG, and also on HBMEC and HA alone. Our results reveal once again that the presence of the BBB is fundamental for in vitro studies. Indeed, the in vitro generated BBB is crucial in protecting the organoids from viral infection, with HA being more susceptible than HBMEC to viral infection. In conclusion, the BBB-ORG model might be useful to disclose physiological and pathophysiological events in the CNS.