Andoniadou, C.L.; (2007) Intrinsic factors controlling stem cell proliferation and differentiation in the central nervous system. Doctoral thesis , University of London.

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Abstract

Populations of neural stem cells (NSCs) residing in different locations of the central nervous system (CNS) are predicted to have similar gene expression profiles, which identify them as stem cells. The best in vitro model to study NSCs is the neurosphere - a spherical colony generated from a single NSC. Currently, there is a lack of markers to characterise NSCs and distinguish them from lineage-committed progenitor cells. One promising candidate is Sox2, which is a member of the Sox family of transcription factors and is expressed within the CNS. Sox2geo/+ mice carry a p-galactosidase/ neomycin fusion gene in the Sox2 locus. Homozygous Sox2eo mutants exhibit peri-implantation lethality since Sox2 is essential for the maintenance of the pluripotent epiblast cells in the embryo. I have established that the neurosphere-generating cellular component, throughout the developing CNS, resides within the Sox2 expressing population. Through neomycin selection, neurosphere cultures derived from Sox2Pgeol+ mice have been enriched for NSCs and have been used to characterise NSCs. Differences in the gene expression profiles of cells expressing and not expressing Sox2 have been carried out through microarray experiments. To investigate the possible function of Sox2 in NSCs I have depleted SOX2 by RNA interference, followed by microarray analysis, to identify potential targets of Sox2 that may further affect the proliferation or differentiation of stem cells. The influence of the niche surrounding stem cells within the neurosphere has been addressed through a series of culture experiments comparing NSC-enriched to non-enriched neurospheres. In order to elucidate the genes responsible for the identity of stem cells, enriched NSC cultures have been compared to their tissue of origin through microarray analysis. These studies have revealed genes expressed at significantly elevated levels within the stem cell cultures compared to the tissue samples - a predominantly differentiating population. Pair-wise comparisons between neurospheres generated from the spinal cord (11.5 dpc, 14.5 dpc) and dorsal telencephalon (14.5 dpc) were used to refine candidate genes and provide an insight into the spatial and temporal properties of NSCs. Several candidate NSC markers, including transcription factors and extracellular matrix molecules common to all populations, have been identified.

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