Figure 2.

Pten deletion in clonal neurosphere cultures enhances stem cell self-renewal and neurogenesis over long-term serial clonal passages compared with the gradual senescence and decreased neurogenesis in wild-type cultures. a, Neurosphere cultures generated from the adult SVZ of Pten knock-out (Mutant) and wild-type (Control) mice were cultured at clonal density (1000 cells/ml), passaged, and reseeded at clonal density every 2 weeks. Clonal neurosphere numbers, representative of the number of stem cells present in the culture, were significantly higher in mutant cultures at all passages (p < 0.001). b, The clonal neurospheres were then dissociated and the total cell number was counted at each passage followed by reculturing at clonal density. After the first passage, mutant cultures consistently produced higher total cell numbers even after long-term serial clonal passaging while control cultures produced decreased cell numbers with time, eventually senescing by passage 24. Data are means ± SEM; n = 4. c, Immunocytochemistry for TUJ1⁺ cells (green) and the counterstain Hoescht (blue) are shown at passage 4 and 16 indicating the clonal neurospheres from mutant mice maintained their robust neurogenesis even at late clonal passages while the ability to generate neurons was dramatically attenuated in control cultures over time. Scale bar, 60 μm. n = 4.