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Characterization of human dopaminergic neurons in the developing mesencephalon and upon differentiation of stem cells- for replacement therapy in Parkinson´s disease

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Author

Summary, in English

Cell-replacement therapy is a promising approach for treating patients with Parkinson ́s disease (PD). For this purpose, there is a need for developing a protocol that can generate high numbers of human transplantable mesencephalic dopaminer- gic (mesDA) neurons. For decades, studies have therefore been made in mouse and other model organisms in order to elucidate key-factors that can be used for proper characterization and patterning of cells to become mesDA neurons. However, limited numbers of studies have been performed in human to confirm the expression and role of these factors. In this thesis, I have analysed the developing human mesencephalon for expression of key-fate determining proteins, which are known to be important in mesDA neuron development in the mouse. These key-factors were shown to exhibit a similar spatiotemporal expression pattern in the human brain, suggesting a conserved role for these proteins in mesDA neuron development across species. We were also able to confirm that human mesDA neurons are derived from radial glial cells in the floor plate (FP), positioned in the most ventral part of the mesencephalon. With the help of human specific mesDA markers we could optimize and develop a protocol that successfully patterns human embryonic stem cells (hESCs) into functional mesDA neuron progenitors. This protocol is one of the first to allow the generation of authen- tic mesDA neuron progenitors through a FP stage, mimicking early human mesDA neuron development. Furthermore, these cells survive transplantation and can restore motor deficits in a rat Parkinson ́s disease (PD) model. Thus, these cells show a prom- ising potential to be further developed for clinical use, treating patients with PD.

In addition to patterning cells to a mesencephalic (midbrain) fate, we were also able to regionalize hESCs to neural progenitors resembling those of the human em- bryonic forebrain and hindbrain. This protocol, in combination with the generation of a SOX1-GFP hESC reporter cell line and the expression of the cell-surface marker CORIN by human FP cells, allowed us to isolate pure populations of regionalized neu- roepithelial and FP cells for deep sequencing. From this study, we identified several microRNAs with potential roles in the specification and development of human neural progenitors populations, including mesDA neuron progenitors. This opens up the possibility to further explore the mechanisms behind the specification of cells within the human central nervous system and can potentially be used for further development and optimization of protocols specifying human neural progenitor subtypes.

Publishing year

2013

Language

English

Publication/Series

Lund University Faculty of Medicine Doctoral Dissertation Series

Volume

2013:135

Document type

Dissertation

Publisher

Developmental Neurobiology

Topic

  • Neurosciences

Keywords

  • Parkinson´s disease
  • stem cells
  • ventral mesencephalon
  • dopaminergic neurons
  • cell replacement therapy
  • human fetal tissue
  • radial glia
  • microRNA
  • BAC recombineering
  • reporter cell lines

Status

Published

Research group

  • Developmental and Regenerative Neurobiology

ISBN/ISSN/Other

  • ISSN: 1652-8220
  • ISBN: 978-91-87651-10-6

Defence date

12 December 2013

Defence time

09:15

Defence place

Segerfalk lecture hall, BMC A10, Sölvegatan 17, Lund

Opponent

  • Elena Cattaneo (professor)