Nestin-EGFP transgenic mice: Visualization of the self-renewal and multipotency of CNS stem cells

We generated transgenic mice carrying enhanced green fluorescent protein (E GFP) under the control of the nestin second-intronic enhancer (E/nestin:EGF P). Flow cytometry followed by in vitro assays revealed that in situ EGFP e xpression in the embryonic brain correlated with the mitotic index, the cog eneration of both neurons and glia, and the frequency of neurosphere format ion in vitro. High-level EGFP expressors derived from embryos included a di stinct subpopulation of cells that were self-renewable and multipotent, cri teria that define neural stem cells (NSCs). Such cells were largely absent among lower-level or non-EGFP expressors, thereby permitting us to enrich f or NSCs using EGFP expression level. In adults, although E/nestin:EGFP-posi tive cells included the NSC population, the frequency of neurosphere format ion did not correlate directly with the level of EGFP expression. However, moderately EGFP-expressing cells in adults gained EGFP intensity when they formed neurospheres, suggesting embryonic and adult NSCs exist in different microenvironments in vivo.