A flourescence activated cell sorting strategy for enrichment of adult neural progenitor cells
Doctor of Philosophy
The discovery of neural stem cells (NSC) within the adult mammalian brain continues to fuel optimism regarding the ability of potential regenerative medicine applications to provide enhanced functional recovery from brain injuries. The adult NSC population is maintained within a complex microenvironment, referred to as the niche, where a unique cellular and extracellular environment maintains and regulates the NSC population and their progeny, enabling ongoing neurogenesis throughout adulthood. Characterization of how NSC interact with the extracellular environment and other cell subpopulations is an active area of research that will generate fundamental design parameters for biomaterial and tissue engineering strategies for neural tissue repair. A major obstacle to further progress is the lack of access to purified populations of primary NSC, a challenge which became the focus of this thesis. To address this obstacle, experimental methods were developed and optimized for isolating neural stem and progenitor cells (NSPC) from the adult NSC niche with fluorescence activated cell sorting (FACS). These methods were enhanced by the incorporation of a fluorescent reporter mouse driven by the gene Sox2, a neural stem cell associated transcription factor, which allowed NSPC enrichment within the Sox2+ population. The FACS based research approach was further developed to include additional surface antigens allowing isolation of NSPC at over 34% purity. The highly enriched population of NSPC was subjected to vital dye cell cycle analysis leading to the observation that an active and quiescent fraction exists within the NSPC pool that is delineated by β1-integrin expression. Access to enriched primary adult NSPC will lead to more a more accurate understanding of NSC dynamics with implications in fundamental biological research as well as biomaterials and tissue engineering.