Neural stem cells have been identified in both the cerebellum and forebrain of fetal and adult mice. These cells can form neurospheres in culture and differentiate into both glia and neurons either in vitro or in vivo. In embryonic day 14 forebrain, neural stem cells are found to exist exclusively in a subpopulation with the Side Population (SP) phenotype, and express Abcg2, a member of the ABC transporter family that is responsible for the SP phenotype in hematopoietic stem cells (HSCs). The expression of Abcg2 in stem cells in the cerebellum has not been characterized. We have generated an Abcg2/GFP knock-in mouse model in which expression of GFP is under control of the endogenous Abcg2 locus and used this model to demonstrate that Abcg2 expression can be used for HSC enrichment. Here we report the use of this mouse model to explore the relationship between Abcg2 expression and neural stem cell function in neonatal cerebellum. Single cells were prepared from cerebellum of 4–9 day old mice by digesting with papain. We then stained the cells with anti-CD45 and anti-Ter119 antibody to exclude the resident hematopoietic cells in subsequent flow cytometry analysis and cell sorting. We found that a small but consistent subpopulation of cells, comprising 0.7±0.12% of total CD45−Ter119- single cell preparations, expressed the Abcg2/GFP allele. To determine whether these GFP+ cells were enriched for neural stem cells, we sorted the CD45−Ter119- cells into GFP+ and GFP− subpopulations and analyzed for their neurosphere forming activity in the presence of epidermal growth factor and basic fibroblast growth factor. We found that the GFP+ subpopulation formed 21 fold more neurospheres compared with the GFP− subpopulation. These neurosphere forming cells can self-renew as evidenced by their capacity to form secondary neurospheres when replated. These results demonstrate that similar to what is seen with HSCs and with embryonic forebrain cells, Abcg2 is expressed in the neural stem cells in neonatal cerebellum, and Abcg2/GFP expression in this mouse model could also be used as a marker to prospectively purify neural stem cells from cerebellum. Ongoing studies are focused on defining the in vivo multilineage differentiation potential of the Abcg2/GFP+ cells and determining whether Abcg2 expression could be used as a marker for purification of medulloblastoma stem cells.

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