p21 loss compromises the relative quiescence of forebrain stem cell proliferation leading to exhaustion of their proliferation capacity - PubMed (original) (raw)

Figure 4.

p21-/- neurosphere culture expansion and exhaustion in vitro are accompanied by an initial decrease and a subsequent increase in cell cycle time, respectively, as measured by BrdU lableing in vitro, cell expansion, and clonal neurosphere size, whereas there is no change in individual cell size or cell viability. (A) Cumulative BrdU labeling of primary and twice-bulk-passaged neurosphere cultures from p21+/+ (right panel) and p21-/- (left panel) mice at 60 d of age. Data are presented as mean ± SEM. There was a significant interaction between genotype and passage [F (1, 96) = 4.46, p < 0.05] with _p21-/-_ cells in primary culture incorporating BrdU at a faster rate than _p21-/-_ cells after passage 2 (_p_ < 0.05); conversely, _p21+/+_ cells did not differ in BrdU incorporation rate between primary and passage 2 (_p_ > 0.05). Regression analyses of the BrdU incorporation data produced cell cycle estimates of 17.71 h for p21+/+ primary neurospheres, 19.89 h for p21+/+ passage 2 neurospheres, 14.27 h for p21-/- primary neurospheres, and 28.89 h for p21-/- passage 2 neurospheres. (B) Cell number expansion (mean ± SEM) during bulk passaging of neurosphere cultures from p21+/+ and p21-/- mice at 1, 60, and 480 d of age. p21-/- neurospheres from postnatal 1 and 60 d of age is initially larger but subsequently smaller than p21+/+ neurospheres, demonstrating a significant correlation between neural stem cell self-renewal and total progeny number in vitro. For cultures derived from mice at 1 d of age, there was a significant interaction between genotype and passage number [F (8, 80) = 3.30, p < 0.05] with increased cell number expansion in p21-/- cultures on primary and passages 1, 2, 3, and 5 (ps < 0.05), but decreased cell number expansion in p21-/- cultures on passages 4, 6, 7, and 8 (ps < 0.05) relative to p21+/+ cultures. Similarly, for cultures derived from mice at 60 d of age, there was a significant interaction between genotype and passage number [F (5, 50) = 9.35, p < 0.05] with increased cell number expansion in p21-/- culture on primary culture and passages 1 (ps < 0.05), but decreased cell number expansion in p21-/- cultures on passages 3, 4, and 5 (ps < 0.05) relative to p21+/+ cultures; whereas for cultures derived from mice at 480 d of age, there was a significant main effect of genotype [F (1, 8) = 15.22, p < 0.05] with decreased cell number expansion in p21-/- cultures on primary and all passages relative to p21+/+ cultures (ps < 0.05). (C) Volumes (mean ± SEM) of passage 1 and passage 4 neurospheres derived from adult p21-/- and p21+/+ mice at 60 d. There was a significant genotype by passage interaction [F (1, 16) = 9.10, p < 0.05] with p21-/- neurospheres larger in passage 1 and smaller in passage 4 than p21+/+ neurospheres. (D) Diameters (mean ± SEM) of individual cells in passage 1 and passage 4 neurosphere cultures from p21+/+ and p21-/- at 60 d of age. No significant effects were observed. (E) Proportions (mean ± SEM) of nonviable cells in primary and bulk-passaged neurosphere cultures from p21+/+ and p21-/- mice at 60 d of age. No effect of p21 loss was detected. However, there was a significant effect of passage [F (5, 64) = 24.49, p < 0.05] with primary cultures containing more dead cells than from all passaged cultures (ps < 0.05).