DEVELOPMENT OF MURINE BRAIN ORGANOIDS AND THEIR APPLICATON FOR DISEASE MODELING

Brain organoids are in vitro three-dimensional (3D) self-organized neural structures, generated from pluripotent stem cells (PSCs). All the published protocols rely on the cerebral organoids’ growth starting from human embryonic stem cells (hESCs) or induced PSCs (hiPSCs), whereas no methods are reported for the generation of rodent cerebral 3D structures. Although the human cells hold the greatest translational potential, rodent-derived brain organoids are useful for protocols’ optimization, given the shorter amount of time requested for their set-up. Organoids produced from rodent sources are time- and cost-efficient, given the possibility to study dysfunctional brain mechanisms and to screen several compounds before their validation on human-derived models. Altogether, rodent derived 3D systems are to be considered complementary to the human derived organoids. The overall aim of my thesis was the setting up of a reliable murine brain organoid model, its neurodevelopmental characterization and its application for modeling disease and drug testing. In the first part of the work, I focused on the fine-tuned generation of a murine 3D culture protocol, analyzing the timing and choice of efficient neuronal differentiating factors. We set up a three-phase protocol using SGZ-derived NSCs, isolated from E14.5 embryos as primary cell source. The established protocol allowed to obtain, in 1 month, a standardized method for the growth of murine SGZ-derived organoids, showing dorsal forebrain identity including different cell phenotypes. We extensively characterized the generated model, spacing from protein expression evaluation, whole transcriptome signature, metabolic features and functionality studies with the goal to evaluate the 3D brain murine model’s neural development and maturation, reproducibility, cellular heterogeneity, brain region identity and functionality. In the second part of the work, I exploited the murine cerebral organoid model to study in vitro the neurodevelopment defects of Allan-Herndon Dudley Syndrome (AHDS). AHDS is caused by loss-of-function mutations of the gene encoding for the transporter of the thyroid hormone triiodothyronine T3. This determines a lack of T3 into the CNS, leading to a severe neurodevelopmental delay in infants. By using murine organoids, I investigated the neurological impairment established during the pathology and provided a proof-of-concept of therapeutical approaches, gaining additional insight on the use of murine organoids for drug testing. To obtain the AHDS-like organoids, we slightly modified the three-step previously established protocol: by removing the T3 from the culture media we aimed to mimic the AHDS pathological scenario. Specifically, we investigated the morphology, cellular composition and phenotype, metabolic profile and gene expression of such model, revealing developmental defects in AHDS-like organoids. They display a higher proliferation rate (Ki67+ cells) compared to the controls, a prolonged stem phenotype (vimentin expressing cells) and the lack of the radial organization of the neuronal cells (DCX+ and TUBB3+ cells), typical of physiological neuronal development. Remarkably, the lack of T3 had a strong impact on the NSCs differentiation, determining a major commitment toward the glial lineage rather than the neuronal one. Thus, AHDS-like structures showed an abnormal balance between the neuronal and glial component, with astrocytes emerging as the most representative cell population into AHDS-like organoids, at the expense of neurons. The metabolic investigation highlighted a decreased expression of OXPHOS complexes in AHDS-like organoids and impaired fatty acid metabolism, suggesting that they also showed defect in metabolic development. Given the metabolic impairment found during AHDS-like organoid development, we decided to target mitochondrial metabolism to revert AHDS organoid neurodevelopmental defects. Mitochondrial metabolism it is well known to be essential for neuronal differentiation and maturation and the Nicotinamide Riboside (NR), a NAD+ precursor, is an already available drug to target oxidative phosphorylation. Therefore, we administered NR to the AHDS-like organoids in order to increase the oxidative metabolism and correct the AHDS neurodevelopmental defects. NR treatment reversed the abnormal phenotype, resulting in a reduced cellular proliferation, as well as in a minor stem phenotype of the AHDS-like organoids. Furthermore, NR-supplied 3D systems acquired the ability to perform a radial migration towards the organoids’ external layer during maturation both for neuronal progenitors and immature neurons. Strikingly, the imbalance between astrocytes and neuronal cells was ameliorated after NR supplementation. Those results suggested that NR treatment could represent a new strategy to promote full neuronal maturation in our 3D system, possibly targeting mitochondrial metabolism. In conclusion, the results produced during my PhD project showed the possibility to obtain functional mouse cerebral organoids, that could be exploited as modeling disease tool as well as high-throughput screening platform for novel candidate drugs. These findings illustrate murine organoids play an important role in the research community as tools, complementary to the human-derived 3D systems.