Bramwell, Thomas William (2009) Investigations into the use of human embryonal carcinoma stem cells as a model to study dopaminergic neurogenesis. Doctoral thesis, Durham University.
Parkinson's disease in reality arises as a result of a complex series of events, however it is strongly linked to the loss of a specific cellular population of midbrain dopaminergic neurons making it a candidate for stem cell based research. Stem cells can be cultured in vitro and via asymmetric cell division possess the capacity for both self renewal and the production of differentiated derivatives. The use of specific molecules and culture conditions can be applied to promote the differentiation of them towards particular cellular fates, in turn facilitating the possibility of producing enriched populations of cells displaying characteristics of a certain phenotype of interest. There has been much research focussed on the in vitro generation of dopaminergic neurons from various stem cell types. In this work the Tera2.cl.SP12 embryonal carcinoma stem cell line was the primary vehicle investigated for its ability to produce cells that were reflective of a dopaminergic phenotype. Retinoic acid was found to be able to up regulate the expression of a range of dopaminergic markers in the Tera2.cl.SP12 cell line over time. However it was clear that lowered oxygen culture, a method known to promote the production of neurons reflective of a dopaminergic phenotype in mesencephalic cultures, had no effect on the dopaminergic differentiation capacity of the embryonal carcinoma stem cells. The glycoprotein Wnt1 when applied to Tera2.cl.SP12 cultures in concert with retinoic acid was shown to increase the number and percentage of cells positive for the neuronal marker Beta III tubulin approximately 1.5 fold. This was accompanied by a concomitant rise in the mRNA expression of this marker, thus suggesting that the use of Wnt1 may be a means to produce cultures derived from embryonal carcinoma cells that are more neuronal, based on marker expression data. Other established methods to achieve dopaminergic differentiation such as suspension culture, stromal cell co-culture and the application of Sonic hedgehog and Fibroblast growth factor 8 are also able to induce a degree of neuronal and dopaminergic marker expression in Tera2.cl.SPI2 cultures. Overall these results suggest that the Tera2.cl.SP12 cell line might be one vehicle for the study of dopaminergic neurogenesis in vitro, in particular when Wnt1 and retinoic acid are used as a means to favourably enrich the population of cells displaying neuronal characteristics.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||08 Sep 2011 18:24|