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. 2005 Mar 15;19(6):756–767. doi: 10.1101/gad.1272305

Figure 3.

Figure 3.

p21 loss increases the expansion but then decreases the maintenance of self-renewal of neural stem cells in vitro. (A) Neurosphere expansion and self-renewal during bulk (10 cell/μL in 500 μL) passaging in vitro. Data presented as mean ± SEM. p21-/- neurospheres from post-natal 1 and 60 d of age show an initial increase in expansion, whereas p21-/- neurospheres from post-natal 480 d show an initial decrease in expansion compared to p21+/+ neurospheres. Moreover, p21-/- neurospheres from all ages show eventual self-renewal exhaustion. For cultures derived from mice at 1 d of age, there was a significant interaction between genotype and passage number [F (8, 80) = 4.31, p < 0.05] with increased neurosphere numbers in p21-/- cultures on passages 1, 2, and 3 (ps < 0.05), but decreased neurosphere numbers on passages 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) = 17.90, p < 0.05] with increased neurosphere numbers in p21-/- cultures on primary and passage 1 (ps < 0.05), but decreased neurosphere numbers 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) = 248.85, p < 0.05] with decreased neurosphere numbers in p21-/- cultures on primary and all passages relative to p21+/+ cultures (ps < 0.05). (B) Neurosphere expansion and self-renewal during the passaging of single neurospheres (of the same size) in vitro. Data presented as mean ± SEM. No effects are observed on primary passaging of single neurospheres from 60 or 480 d of age, but neurospheres from both ages show eventual self-renewal exhaustion. For neurospheres derived from mice at 60 d of age, there was a significant interaction between genotype and passage number [F (2, 56) = 3.66, p < 0.05] with decreased secondary neurosphere numbers in p21-/- cultures on passage 3 (p < 0.05); thereafter, too few p21-/- of appropriate size were available for single neurosphere passaging. Similarly, for neurospheres derived from mice at 480 d of age, there was a significant effect of genotype [F (1, 15) = 4.92, p < 0.05] with decreased secondary neurosphere numbers in p21-/- cultures on passages 2 and 3 (p < 0.05); there-after, too few p21-/- of appropriate size were available for single neurosphere passaging.