Title

In vitro analysis of stem cell generated dopaminergic neurons

Date of Completion

January 2008

Keywords

Biology, Neuroscience

Degree

Ph.D.

Abstract

Santiago Ramon y Cajal, one of the most outstanding neuroscientists of the last century, suggested, that "neuroepithelial cells, which share embryonic stem cell properties, are the support elements of the early brain". Arturo Alvarez-Buylla proposed that astrocytes, the "support" elements of the adult brain, might be a reminiscent of early neuroepithelial cells, that causes a fraction of them to behave as stem cells. To gain more insight in the strengths and limitations of both populations embryonic- and adult stem cells, I performed a side-by-side in vitro analysis. This thesis summarizes the results of a comparative study focused on the differentiation potential of adult mouse neural stem cells (aNSCs) isolated from the largest brain germinal region, the subventricular zone (SVZ), and mouse embryonic stem cells (ESC) to form midbrain Dopaminergic (mDA) neurons. My data indicate that when presented with in vitro conditions that promote mDA neuron specification for embryonic cultures, aNSCs instead generate forebrain-like DA neurons, demonstrating their restricted and prescribed nature. In addition, to explore the regulation of the DA phenotype and provide a way to remodel Parkinson's disease (PD), I took advantage of the aphakia (ak) mouse, which due to a spontaneous mutation in the homeobox gene Pitx3, results in selective loss of the substantia nigra pars compacta (SNpc) DA neurons, the same population lost in patients with PD disease. To investigate the connection between Pitx3 and SNpc DA neurodegeneration, both an ak- and a nuclear transfer ak ESC line were generated for the present study. This 'loss of function' in vitro system allowed us to examine characteristic features in mature DA neuron development and the role Pitx3 plays in the differentiation process. I found that ak cultures generated a smaller pool of tyrosine hydroxyalase + (TH) neurons with predominantly immature characteristics, like the higher Nestin (NES) levels and the lower percentage of TH neurons co-expressing markers, which facilitate neurotransmission. Those results raise therefore the possibility that absence of Pitx3 might profoundly change the molecular composition of even surviving mDA neuronal populations in the ak mouse. ^