Stem Cell Migration

Volume 750 of the series Methods in Molecular Biology pp 61-77


Isolation and Characterization of Adult Neural Stem Cells

  • Florian A. Siebzehnrubl
  • , Vinata Vedam-Mai
  • , Hassan Azari
  • , Brent A. Reynolds
  • , Loic P. DeleyrolleAffiliated withMcKnight Brain Institute, University of Florida Email author 

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It has been thought for a long time that the adult brain is incapable of generating new neurons, or that neurons cannot be added to its complex circuitry. However, recent technology has resulted in an explosion of research demonstrating that neurogenesis, or the birth of new neurons from adult stem cells constitutively occurs in two specific regions of the mammalian brain; namely the subventricular zone and hippocampal dentate gyrus. Adult CNS stem cells exhibit three main characteristics: (1) they are “self-renewing,” i.e., they possess a theoretically unlimited ability to produce progeny indistinguishable from themselves, (2) they are proliferative (undergoing mitosis) and (3) they are multipotent for the different neuroectodermal lineages of the CNS, including the different neuronal, and glial subtypes. CNS stem cells and all progenitor cell types are broadly termed “precursors.” In this chapter, we describe methods to identify, isolate and experimentally manipulate stem cells of the adult brain. We outline how to prepare a precursor cell culture from naive brain tissue and how to test the “stemness” potential of different cell types present in that culture, which is achieved in a three-step paradigm. Following their isolation, stem/progenitor cells are expanded in neurosphere culture. Single cells obtained from these neurospheres are sorted for the expression of surface markers by flow cytometry. Finally, putative stem cells from cell sorting will be subjected to the so-called neural colony-forming cell assay, which allows discrimination between stem and progenitor cells. At the end of this chapter we will also describe how to identify neural stem cells in vivo.

Key words

Neural stem cell Neurosphere assay Flow cytometry Neural colony-forming cell assay Immunohistochemistry