Quantification and Clonal Culture of Neural Stem Cells from the Hippocampus of Adult Mouse.

Abstract

Visualizing markers for neural stem cells (NSCs) and morphological analysis are frequently used for identification of NSCs in tissues. However, NSCs are defined as cells with the ability to both self-renew and produce descendants that can differentiate into neurons, astrocytes and oligodendrocytes. The neural colony forming cell (NCFC) assay is a single-step semisolid based assay for the identification of NSCs. In this assay, NSCs generate clonally derived colonies due to their high proliferative potential. The relative comparison of NSC populations between tissues is possible by counting the colonies obtained from the NCSC assay. Furthermore, the colonies can be isolated to establish monolayer cultures of clonal NSCs. Using clonal cultures of NSCs, it is possible to assess differentiation stage and differentiation potential of each NSC. Here, we describe a semi quantitative method for the enumeration of NSCs using the NCFC assay, with slight modification from the original protocol (Louis et al., Stem Cells 26:988-996, 2008). A method to establish monolayer culture of NSCs from a colony derived from NCFC assay is also described.