Characterizing the roles of distinct ventral midbrain progenitor cell types in dopaminergic neuron development

Parkinson's disease is a neurodegenerative disorder characterized by the progressive loss of midbrain dopaminergic (mesDA) neurons. With the underlying cause of the disease still unknown, current treatments can only address the symptoms of the disease but not the continual loss of mesDA neurons. Cell replacement therapy aims to replace these lost mesDA neurons and is considered a promising treatment option. However, the widespread clinical application of cell replacement therapy depends on a reliable and standardized supply of transplantable neurons. Advances in stem cell technology offer a renewable source of human mesDA neurons for transplantation but generating high-quality mesDA neurons in vitro requires a detailed understanding of their normal development in the human brain. This thesis consists of three studies that all aim to improve our understanding of mesDA neuron development and apply this knowledge to enhance the in vitro differentiation of mesDA neurons.

In paper I, we develop a new protocol for deriving human mesDA neurons from stem cells and introduce a framework for using single-cell RNA sequencing to evaluate the quality and composition of these cell preparations.

In paper II, we generate and characterize a fluorescent reporter human stem cell line designed to isolate two ventral midbrain (VM) progenitor cell types from our in vitro cultures differentiated toward a mesDA fate.

In paper III, we identify key cell types involved in both human and mouse VM development and focus on two types of floor plate radial glia that contribute to mesDA neuron development in distinct ways. One radial glia was identified as neurogenic and likely giving rise to mesDA neurons, while the other radial glia resembled traditional floor plate radial glia and expressed factors known to influence mesDA neuron development. Finally, we identify a novel transcription factor expressed by the neurogenic radial glia that regulates the timing of mesDA neurogenesis, and extracellular matrix proteins expressed by the other radial glia that improve the yield of mesDA neurons differentiated from stem cells in vitro.

In sum, the work presented in this thesis characterizes the individual roles of VM progenitor cell types and their contributions to the mesDA lineage. Furthermore, we demonstrate how knowledge gained from studying VM development can improve the generation of mesDA neurons from human stem cells for potential use in cell replacement therapy for Parkinson's disease.

List of scientific papers

I. Single-cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation. Kaneyasu Nishimura, Shanzheng Yang, Ka Wai Lee, Emilia Sif Ásgrímsdóttir, Kasra Nikouei, Wojciech Paslawski, Sabine Gnodde, Guochang Lyu, Lijuan Hu, Carmen Saltó, Per Svenningsson, Jens Hjerling-Leffler, Sten Linnarsson, Ernest Arenas. Stem Cell Reports (2023) https://doi.org/10.1016/j.stemcr.2022.10.016

II. Generation of a fluorescent hESC reporter line (Kle-033-A-1) for the isolation of distinct midbrain progenitor cell types. Emilia Sif Ásgrímsdóttir, Ernest Arenas. Stem Cell Research (2024) https://doi.org/10.1016/j.scr.2024.103523

III. Midbrain dopaminergic neuron development is regulated by two molecularly distinct subtypes of radial glia cells. Emilía Sif Ásgrímsdóttir, Luca Fusar Bassini, Pia Rivetti di Val Cervo, Daniel Gyllborg, Claudia Puigsasllosas Pastor, Kawai Lee, Christopher Grigsby, Saiful Islam, Peter Lönnerberg, Carlos Villaescusa, Carmen Saltó, Sten Linnarsson, Gioele La Manno, Enrique M. Toledo, Ernest Arenas. BioRxiv (2024) https://doi.org/10.1101/2024.03.08.584031 [Manuscript]