The isolation, purification and analysis of stem cells

Neural stem cells generate a wide spectrum of cell types in developing and adult nervous systems. These cells are marked by expression of the intermediate filament nestin. The regulatory elements of the nestin gene were used to generate transgenic mice in which neural stem cells of the embryonic and adult brain are marked by the expression of the green fluorescent protein (GFP). These animals were used as a reporter line for studying neural stem and progenitor cells in the developing and adult nervous systems as well as in various non-neural tissues. The data demonstrates that in these nestin-GFP animals, GFP-positive cells faithfully reflect the distribution of nestin positive cells and accurately mark the neurogenic areas of the adult brain and mark stem cells from other non-neural tissue. Furthermore, unusual features of nestin-GFP-positive cells in the subgranular cell layer of the dentate gyrus were identified. Nestin-GFP cells can be isolated with high purity using fluorescent activated cell sorting and can generate multipotential neurospheres. In the adult brain, nestin-GFP cells are >1400 fold more efficient in generating neurospheres than GFP-negative cells, and despite their small number, give rise to 70 times more neurospheres than the GFP-negative population. Further characterization demonstrates the expression of a panel of differentiation markers in GFP-positive cells in the nestin-GFP transgenics, and show that these cells can be divided into two groups based on the strength of their GFP signal: GFP-bright cells express GFAP but not βIII-tubulin, whereas GFP-dim cells express βIII-tubulin, but not GFAP. These two classes of cells represent distinct classes of neuronal precursors in the adult mammalian brain and may reflect different stages of neuronal differentiation. Using a recently established method for determining stem cells based on there ability to efflux certain DNA intercolating dyes “SP” method, nestin-GFP was further demonstrated to enrich stem cell populations from the eye, heart, muscle, kidney, liver, muscle, and pancreas. Furthermore, using cell culture methods, nestin-GFP cells from the hair follicle are able to form both neuronal and epidermal cell types and are the hair follicle stem cells of the bulge region. Together, the results indicate that GFP-positive cells in the transgenic animals accurately represent neural stem and progenitor cells in multiple tissues and suggest that these nestin-GFP-expressing cells encompass the majority of the neural stem cells in the adult brain.