Dissecting Dopaminergic Neuron Specification at Single Cell Resolution - Insights from 3D brain organoids and xenograft models

Midbrain dopaminergic neurons exhibit a wide diversity in their projection patterns, disease vulnerability, and functions, playing key roles in voluntary motor control, cognition, and reward processing. Parkinson’s Disease, one of the most common neurodegenerative disorders, is characterized by the selective degeneration of the A9 dopaminergic neuron subtype, leading to severe motor dysfunction. Despite advancements in symptomatic treatments, current approaches fail to halt disease progression, highlighting the need for novel therapeutic strategies such as cell replacement therapy. Generating authentic human dopaminergic neurons for transplantation and therapeutic purposes relies on a comprehensive understanding of the factors driving their development. However, the molecular mechanisms underlying the specification of midbrain dopaminergic neurons into distinct subtypes remain poorly understood. Due to the inaccessibility of developing and adult human brain tissue, novel methodologies are needed for investigating these processes in a human-relevant context. This thesis investigates the development, diversity, and specification of human dopaminergic neurons using advanced human stem cell models, with a particular focus on their application in cell replacement therapies for Parkinson’s Disease. In the first part, I established ventral midbrain-patterned organoids that recapitulate developmental trajectories and the molecular identities of distinct dopaminergic neuron subtypes. To overcome limitations in conventional organoid technology, silk scaffolding was also introduced, enhancing cell viability and neuronal maturation. In the second part, I linked the molecular identities of human dopaminergic neurons to their projection patterns using homotopic transplantation models. Finally, co-grafting experiments examined the influence of support cell types on dopaminergic neuron maturation and lineage commitment, identifying critical factors shaping subtype identity. These findings advance our understanding of human dopaminergic neuron development and subtype specification, offering valuable insights for refining cell replacement therapies for Parkinson’s Disease.