Modelling Huntington’s and Parkinson’s disease using human neural progenitor cells and brain organoids

Neurodegenerative diseases like Huntington’s disease (HD) and Parkinson’s disease (PD) are still not fully understood, mainly because of the lack of reliable human-based experimental models that can truly capture the complexity and progressive nature of these conditions. While animal models have shed some light on the mechanisms behind these diseases, they often miss the mark when it comes to replicating crucial aspects of human neuropathology, which limits their usefulness in predicting outcomes and translating findings to human treatments. In this light, human stem cell-based in vitro models offer a promising alternative for exploring disease-specific traits in a context that is more relevant to humans. The main goal of this PhD thesis is to explore and validate various in vitro human models for studying neurodegenerative diseases, particularly focusing on HD and PD. By merging two-dimensional neural cultures with three-dimensional brain organoid systems derived from pluripotent stem cells, this research aims to assess the strengths and weaknesses of each model in capturing region-specific and disease-relevant characteristics. The first part of the thesis focuses on the generation and characterization of human neural progenitor cells (NPCs) as a two-dimensional in vitro model for HD. These NPCs were used to investigate disease-associated phenotypes and to establish a platform suitable for gene therapy-based screening approaches, highlighting their potential utility for preclinical therapeutic testing. In the second part, the work addresses the development of human striatal organoids (hStrOs) as a three-dimensional model of the brain region primarily affected in HD. Medium spiny neurons (MSNs), which constitute approximately 95% of the striatal neuronal population, are selectively vulnerable in HD. Here, hStrOs were generated from human embryonic stem cells (hESCs) with the aim of optimizing and validating the differentiation protocol. Although patient-derived cells were not used at this stage, this approach allowed the assessment of striatal identity and cellular composition, providing a robust foundation for future disease modelling applications. The final part of the thesis focused on midbrain organoids (hMOs) derived from induced pluripotent stem cells of PD patients, modelling the brain region most affected in PD. In this context, the progressive degeneration of dopaminergic neurons and their terminals is a hallmark of the disease. Patient-specific hMOs were characterized using advanced imaging techniques and transcriptomic profiling through RNA sequencing, enabling the identification of cellular and molecular alterations associated with PD pathology.