Figure 5. Pre-tumorigenic SVZ Neural Stem/Progenitor Cells Exhibit Growth Advantage and Altered Differentiation.
A. SVZ neurospheres from pre-symptomatic mutant mice exhibit hypertrophy. SVZ cells of 4-week-old mice were cultured as neurospheres. As shown in representative images, Nf1/p53- (Mut3:hGFAP-cre;Nf1flox/+;p53−/+) or Nf1/p53/Pten- (Mut4:hGFAP-cre;Nf1flox/+;p53−/+;Ptenflox/+) deficient neurospheres were larger than those from wild type (WT). Scale bar, 200 μm. B. Neurosphere proliferation was measured by two hours of bromodeoxyuridine (BrdU) pulsing on day three cultures and subsequent FACS analysis. Mut3 and Mut4 cells had significantly increased S phase, compared to wt cells (p<0.05). C. Neurosphere cell death was measured by Annexin V labeling and subsequent FACS analysis. Mut3 or Mut4 cells undergo significantly reduced cell death compared to wt cells. 7-amino-actinomycin (7-AAD) is a vital dye. D. SVZ neurospheres from pre-symptomatic mutant mice show increased self-renewal potential, as shown by methylcellulose assay compared to wt (**p<0.005). E. Quantification of differentiated cells reveals significant increase in Tuj1- or nestin-positive cells and significant decrease in Gfap-positive cells in Mut3 and Mut4 cultures compared to those of wt control (* p<0.05, **p< 0.005). All error bars are +/− SEM. F. Neurosphere cultures from pre-symptomatic mutant mice exhibit retention of heterozygosity of tumor suppressor genes. Genomic DNA from the ear (E) or neurospheres (N) from four-week-old Mut3 and Mut4 mice was subjected to semi-quantitative PCR genotyping. Representative data for cre, Nf1, p53 and Pten status for neurospheres indicate retention of wild type alleles in all Mut3 and Mut4 neurospheres.