New neurons are born in the subgranular zone (SGZ) of the hippocampus throughout life. Research on adult SGZ neurogenesis is extremely exciting, as it may help us understand hippocampal functions like learning and memory and even to utilize endogenous neurogenesis for regenerative medicine. Nestin-expressing (nestin+) stem cells are thought to be the source of a continuous supply of progenitor cells, some of which eventually mature into neurons. Technical limitations have prevented comprehensive study of the nature and regulation of SGZ stem-like cells in vivo. To address this major knowledge gap, we developed a nestin-CreERT2/R26R-YFP transgenic mouse to track adult stem-like cells and their progeny in the adult brain. I will test if some nestin+ SGZ cells are stem cells in vivo. I present pilot data that demonstrate I can phenotype and quantify stem-like cells, and I provide evidence that the labeled cells are stable over time. To expand on these results and to clarify the markers that can identify stem-like cells in the SGZ, for Aim 1 I will use multiple sets of stem cell markers to characterize labeled cells in our transgenic mouse. To clarify the contribution of nestin+ vs. GFAP+/GLAST+ stem-like cells to adult neurogenesis, I will also count and phenotype YFP+ cells and their progeny in GLAST-CreERT2/R26R-YFP transgenic mice. In order to understand regulation of stem-like cells, in Aim 2 I assess stem cell properties in vitro using the neurosphere assay and flow cytometry. By comparing recombined YFP+ and non-recombined stem-like cells from the nestin-CreERT2/R26R-YFP mouse, I explore whether nestin+ cells maintain characteristics of self-renewal and multipotency. Finally, in Aim 3, I use our transgenic mouse to determine the functional contribution of nestin+ stem-like cells and their progeny to neurogenesis after stimulating proliferation by voluntary exercise or after chemical ablation of cell proliferation by an anti-mitotic compound, Ara-C. This proposal will allow, for the first time, clarification of the nature and regulation of adult SGZ stem cells in vivo. In advancing our knowledge of this key aspect of adult neurogenesis, the basic and functional studies proposed have tremendous potential to positively impact stem cell biology and biomedical research.
