CNS stem cells and gut neural crest stem celcgls (NCSCs) require Bmi-1 to self-renew normally. Images show typical neurospheres that formed after 10 d in non-adherent cultures from E14.5 CNS neural stem cells (a) or PNS NCSCs (b). a, The frequency of Bmi-1 ⁻/⁻ E14 telencephalon cells, P0 SVZ cells or P30 SVZ cells that formed multipotent CNS neurospheres was significantly reduced relative to wild-type cells (*P < 0.05). The diameter and self-renewal of the Bmi-1 ⁻/⁻ neurospheres were also significantly reduced. Self-renewal capacity is expressed as the number of secondary neurospheres generated per primary neurosphere on subcloning. b, Similar results were obtained for multipotent PNS neurospheres generated by E14.5, P0 or P30 wild-type and Bmi-1 ⁻/⁻ gut cells, except that the frequency of neurosphere-forming cells was not significantly reduced until P30. Values are the mean ± s.d. for 3–6 independent experiments. Bmi-1 deficiency also consistently reduced self-renewal when the data were normalized to control for differences in the size of neurospheres: 3.5 ± 0.8% of dissociated wild-type P0 PNS neurosphere cells formed secondary neurospheres on subcloning, as compared with 0.3 ± 0.1% of Bmi-1 ⁻/⁻ cells (P < 0.001). ND, not determined.

Bmi-1 deficiency reduces proliferation but does not increase cell death in CNS stem cell colonies. P0 SVZ cells were dissociated and plated in adherent cultures, and the number of cells per colony was counted after 4, 7 and 14 d (a). All colonies were counted after 4 d, but only colonies that contained neurons and glia were counted after 7 and 14 d. At all time points, wild-type (WT) colonies contained significantly more cells than the Bmi-1 ⁻/⁻ colonies (*P < 0.01). Similar results were obtained at E14 and P30 (not shown). Only a few cells in adherent CNS stem cell colonies (b) or in the P0 SVZ (c) stained with antibodies against activated caspase-3 (a marker of cell death). Arrows and arrowheads indicate the SVZ and caspase-3-positive cells, respectively. Note that the choroid plexus in the lateral ventricle is highly autofluorescent (c). No statistically significant difference in the frequency of dying cells was observed between wild-type and Bmi-1 ⁻/⁻ CNS stem cell colonies on the basis of the frequency of condensed, fragmented nuclei identified by 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (d), or activated caspase-3 (not shown). Bmi-1 deficiency was associated with a significant decrease in the rate of BrdU incorporation into CNS stem cell colonies (e, f; *P < 0.01), indicating reduced proliferation. A similar reduction in proliferation was observed in Bmi-1 ⁻/⁻ NCSC colonies in culture (not shown).

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).

Restricted neural progenitors from the CNS and PNS proliferate normally in the absence of Bmi-1. E14 telencephalon cells or P0 or P30 SVZ cells were dissociated and cultured at clonal density under adherent conditions. a–c, The frequency of CNS cells that formed neuron-only or glia-only colonies after 12–14 d in culture did not significantly differ between wild-type (WT) and Bmi-1 ⁻/⁻ mice (a), nor did the number of cells per neuron-only colony (b) or glia-only colony (c). d, The number of cells per glia-only colony formed by dissociated P0 gut cells did not differ between wild-type and Bmi-1 ⁻/⁻ mice. e, In BMP4-treated cultures of E14.5 gut p75⁺ cells, no difference was observed in the number of cells per neuron-only colony. f, Bmi-1 ⁻/⁻ neural progenitor colonies in the absence of Nrg proliferated at a significantly lower rate than did wild-type colonies (*P < 0.01), but Bmi-1 ⁻/⁻ colonies in the presence of Nrg proliferated similarly to wild-type colonies (although the difference was still statistically significant, *P < 0.05).