| Abstract No.: | C-A3007 |
| Country: | Canada |
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| Title: | CHARACTERIZATION AND ISOLATION OF QUIESCENT NEURAL STEM CELLS |
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| Authors/Affiliations: | 4 Renee Phillips*; 1 Sandy Vascotto; 2 David Hess; 3 Susan Meakin;
1 Robarts Research Institute, London, ON, Canada; 2 Robarts Research Institute and the University of Western Ontario, London, ON, Canada; 3 University of Western Ontario, London, ON, Canada
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| Content: | Objectives: Accurate study of neural stem cell (NSC) properties and potential clinical applications require reliable NSC isolation techniques. The most widely used method of culturing NSC, the neurosphere method, enriches for lineage-specific precursor cells as opposed to NSCs. In our research, a population of cells that demonstrates enrichment for NSC characteristics has been isolated using a novel culture technique.
Materials and Methods: Early postnatal murine subventricular tissues are dissociated into single cell suspensions and cultured under defined conditions in which the vast majority of cells die. The remaining cells form neurospheres, symmetrical heterogeneous aggregates of differentiated and partially differentiated cells, in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF). The efficiency of this culture technique is determined by analyzing neurosphere formation frequency and size as compared to traditional culture techniques. Expression of stem cell marker proteins such as nestin, prominin 1 (mouse homology of CD133), aldehyde dehydrogenase (ALDH) activity and sox2 was determined using Western blotting and flow cytometry techniques.
Results: Applying our novel culture technique resulted in the survival of approximately 5.2% of the initial population. These remaining quiescent (Q) cells form neurospheres in the presence of EGF and FGF at a frequency of approximately 29.8% (SD = 15.6) relative to that obtained through traditional neurosphere culturing methods in which constant EGF and FGF exposure is applied. While Q cells do not express nestin or sox2, neurospheres arising from Q cells do express these NSC markers, as determined by Western blotting. Flow cytometry experiments have revealed expression of both prominin 1 and ALDH active cells in tissues harvested from the subventricular zone. The subpopulation of prominin 1 expressing cells is distinct from that of the subpopulation showing high ALDH activity. Similar experiments are ongoing for both Q cells and neurospheres.
Conclusion: In contrast to results obtained using the traditional neurosphere assay, our novel culture technique allowed us to purify a population of quiescent cells lacking nestin or sox 2, traditional stem cell markers. These quiescent cells do give rise to neurospheres when stimulated and the neurospheres contain nesting and sox 2 positive cells. Thus, this novel culture technique in combination with fluorescence activated cell sorting (FACS) may provide a more precise method for isolating a true population of NSC than neurosphere assays or FACS alone.
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