Parkinson's disease (PD) is the second most common neurodegenerative disorder following Alzheimer's disease. PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra region of the midbrain and the presence of intracellular inclusions, notably Lewy bodies composed of the alpha-synuclein protein (encoded by the SNCA gene), in the surviving neurons. The precise molecular and cellular mechanisms governing PD's onset, progression, and pathology remain incompletely understood. The development of advanced research models is pivotal in unravelling the mysteries surrounding PD's pathogenesis and progression.

Although traditional monolayer cell culture models have significantly enhanced our understanding of the disease, their results have not consistently translated into clinical success. These models lack the desired cellular diversity and fail to mimic in vivo physiological conditions. To address these limitations, our goal was to establish a human midbrain organoid model that closely replicates in vivo cellular diversity, tissue cytoarchitecture, and physiology. Within this thesis, we introduce a robust novel method for deriving human midbrain organoids from both healthy individuals and PD patients induced pluripotent cells (hiPSCs). We characterized the cellular diversity within these midbrain organoids using single-cell RNA sequencing (scRNA-seq), revealing the presence of dopaminergic neuron populations resembling in vivo substantia nigra neurons. We demonstrated the utility of the midbrain organoid model by capturing a comprehensive transcriptomic profile of dopaminergic neurons in response to SNCA triplication mutation and oxidative stress. The single-cell transcriptomics highlighted perturbations in oxidative phosphorylation and protein translation in response to SNCA-3X mutation. The vulnerability of dopaminergic neurons to oxidative stress was associated with the enrichment of cholesterol biosynthesis and synaptic signalling processes.

Recent studies utilizing single-cell RNA sequencing have revealed that neuronal models derived from human induced pluripotent stem cells (iPSCs) using morphogens and small molecules exhibit undesired cellular heterogeneity and noticeable variability between different batches. Our aim is to develop a monolayer neuronal model that can offer highly homogeneous substantia nigra neurons on a large scale within a short timeframe through the ectopic expression of transcription factors (TFs). In this study, we validated a significant finding: ectopic expression of ATOH1 alone is sufficient to generate Tyrosine hydroxylase (TH)-positive neurons. Additionally, we identified several TF combinations that also induced TH-positive neurons. Single-cell RNA sequencing analysis of neuronal culture generated through ectopic expression of combination of (ATOH1 or NGN2), FOXA2, LMX1A, NURR1, PITX3, SOX6, and MSX1 revealed three distinct TH-positive neuronal clusters. However, these neurons were also found to be positive for PRPH, a peripheral nervous system marker. The characterization of TH-positive neurons induced by ATOH1 alone, with or without midbrain patterning factors such as SHH and FGF8b, also showed positivity for PRPH. Further refinement of the TF combination is needed to correct the regional identity of the TH-positive neurons. Additionally, the generated neurons require characterization beyond scRNA-seq/qPCR techniques.

Human post-mortem material remains the gold standard for formulating hypotheses and subsequently testing them in both in vivo and in vitro models of PD. It also serves as an invaluable tool to validate hypotheses derived from experimental models of PD, confirming their relevance to human disease. In this study, we generated a comprehensive single-nucleus RNA sequencing dataset of a prominent PD-affected region of the midbrain, the substantia nigra pars compacta, from a large cohort of 15 sporadic PD patients and 14 control individuals. This study involved transcriptomic-level characterization of all major cell types, including their subpopulations, present in the brain. We demonstrated specificity of GWAS PD associated genes to different cell types. Importantly, we identified glial subpopulations enriched in the TH gene, which was found to be depleted in PD samples. This dataset provides a valuable resource for future hypothesis-driven experiment and represents a significant step forward in understanding PD's disease mechanisms.