HUMAN CORTICAL BRAIN ORGANOIDS DERIVED FROM NASCENT PLURIPOTENCY FOR MODELLING ALZHEIMER’S DISEASE

Alzheimer's disease (AD) is the most common form of dementia worldwide, characterised by memory loss and cognitive decline. Three biomarkers are used to diagnose AD uniquely: amyloid-beta (Aβ) plaques, hyperphosphorylated tau tangles, and widespread brain degeneration. Despite extensive research, there is still no cure for the disease due to a lack of understanding of the initial pathological processes, that occur 15-20 years before clinical symptoms appear. Even treatments administered at early stages of AD show limited effectiveness, including the recently approved Aβ-targeting drugs, which further question the role of the amyloid cascade in AD pathogenesis. The lack of knowledge regarding the early pathological mechanisms of AD can be explained by the absence of disease models that can fully recapitulate AD progression. Traditional in vivo mouse models, generated by forcing Aβ production, do not allow for the study of earlier disease mechanisms and do not show a proper neurodegeneration due to species-specific differences of the mouse brain. Advancements in in vitro neural models have led to the development of brain organoids, innovative 3D culture systems where human induced pluripotent stem cells (hiPSCs) are guided to differentiate into brain structures mimicking embryonic development. They already showed great potential for disease modelling. However, conventionally this process involves several steps, from patient biopsy, somatic cell isolation and reprogramming, expansion of hiPSCs, generation of embryoid bodies (EBs) and differentiation into organoids, resulting in an expensive and long process (4-8 months), that limits their application to large cohorts of patients. Additionally, each process step can result in batch-to-batch and intra-batch variability. In this context, my PhD project aimed to develop a model of Alzheimer’s disease using cortical brain organoids (CBOs), reducing variability, time, and costs. To achieve this, I worked on improving the patient-specific cell source by using mRNA-based human somatic reprogramming in microfluidics. This process allows the use freshly reprogrammed hiPSCs in an early pluripotent state, which we named “nascent”, to generate organoids. By avoiding low-efficiency reprogramming approaches and the need for any in vitro passaging, we reduced the accumulation of non-sense mutations and chromatin silencing markers, known to introduce variability in hiPSC differentiation potency, together with time and costs. I also replaced EBs with the generation of epiblast-like cysts, where hiPSCs self-assemble into a pluripotent epithelium with a central lumen. Epiblast-like cysts can be strictly guided toward the neuroectodermal fate unlike EBs, which tend to differentiate also into non-neuroectodermal cell types, increasing variability among CBOs. Lastly, I modified the most used CBO differentiation protocols, designed for EBs, to work with my epiblast-like cyst culture. I first tested CBO differentiation and maturation starting from nascent hiPSCs derived from fetal BJ fibroblasts, and then I moved to familial AD (fAD) patient-derived nascent hiPSCs. My nascent hiPSC-derived CBO model of fAD consistently exhibited a significant accumulation of Aβ species, a higher Aβ42/Aβ40 ratio, and a significant increase in total tau compared to controls. Functional imaging also revealed defects such as excitotoxicity, which could be ameliorated by NMDA inhibitors, as previously reported in the literature and exploited for patient treatment. In conclusion, I developed a new reprogramming-to-organoid approach that enables the rapid generation of CBOs in less than 70 days from patient cell collection. Exploiting mRNA-based somatic reprogramming in microfluidics and nascent pluripotency, we improved the potential to faithfully model in vitro patient phenotype. This platform holds promise to be applied for large-scale studies for uncovering new early AD pathogenic mechanisms.