FIGURE 3. p16^Ink4a negatively regulates the self-renewal of CNS stem cells and gut NCSCs in culture.

SVZ and gut cells from adult p16^-/- and wild-type mice were dissociated and cultured to generate neurospheres (a, b) or adherent stem cell colonies (c–f). After 7 d (a, c, e) or 10 d (b, d, f) in culture, self-renewal (a, b), the total number of cells per colony (c, d), and the percentage of BrdU-positive cells per colony (e, f) were assayed. In each case, the p16^-/- adult neural stem cell colonies self-renewed more, proliferated more and contained more cells (*P < 0.05). No differences were observed between p16^-/- and wild-type colonies in the frequency of apoptotic cells (0.59 0.61% versus 0.35 0.48% respectively). Bmi-1^+/-p16^+/- mice were also bred to generate P0 pups, from which SVZ CNS stem cells (g) and gut NCSCs (h) were cultured. For both types of cell, p16^Ink4a deficiency significantly increased the self-renewal of Bmi-1^-/- stem cells (*P < 0.01), although it did not fully restore self-renewal to wild-type levels (*P < 0.01 relative to wild type). Note that p16^Ink4a deficiency in Bmi-1^+/+ stem cells significantly increased the self-renewal of P0 NCSCs (h) but not P0 CNS stem cells (g).