In the adult brain, continual neurogenesis of olfactory neurons is sustained by the existence of neural stem cells (NSCs) in the subependymal niche. Elimination of the cyclin-dependent kinase inhibitor 1A (p21) leads to premature exhaustion of the subependymal NSC pool, suggesting a relationship between cell cycle control and long-term self-renewal, but the molecular mechanisms underlying NSC maintenance by p21 remain unexplored. Here we identify a function of p21 in the direct regulation of the expression of pluripotency factor Sox2, a key regulator of the specification and maintenance of neural progenitors. We observe that p21 directly binds a Sox2 enhancer and negatively regulates Sox2 expression in NSCs. Augmented levels of Sox2 in p21 null cells induce replicative stress and a DNA damage response that leads to cell growth arrest mediated by increased levels of p19(Arf) and p53. Our results show a regulation of NSC expansion driven by a p21/Sox2/p53 axis.

Relative quiescence and self renewal are defining features of adult stem cells, but their potential coordination remains unclear. Subependymal neural stem cells (NSCs) lacking cyclin-dependent kinase (CDK) inhibitor (CKI) 1a (p21) exhibit rapid expansion that is followed by their permanent loss later in life. Here we demonstrate that transcription of the gene encoding bone morphogenetic protein 2 (Bmp2) in NSCs is under the direct negative control of p21 through actions that are independent of CDK. Loss of p21 in NSCs results in increased levels of secreted BMP2, which induce premature terminal differentiation of multipotent NSCs into mature non-neurogenic astrocytes in an autocrine and/or paracrine manner. We also show that the cell-nonautonomous p21-null phenotype is modulated by the Noggin-rich environment of the subependymal niche. The dual function that we describe here provides a physiological example of combined cell-autonomous and cell-nonautonomous functions of p21 with implications in self renewal, linking the relative quiescence of adult stem cells to their longevity and potentiality.

Safe and efficient gene delivery vectors will enhance the prospects for polynucleotide-based therapies. Herein a new approach toward structurally optimized gene vector design based on the preparation of clickable poly(allylamino-phosphazene)s that can be converted to several cationic and anionic derivatives via thiol-ene addition is described. Simultaneous co-incubation of alkylamine-and alkylcarboxylate-poly(phosphazenes) with polynucleotide generates binary polyelectrolyte nanoparticles. Screening of a series of these complexes for transfection in glioblastoma cells shows that the inclusion of 6-mercaptohexanoic acid substituted poly(phosphazene)s in the complexes results in six-fold and 19-fold higher luciferase expression in U87MG cells and GBM1 primary cells, respectively. This effect is attributed to the specific ionization properties of these materials that improved polyplex intracellular trafficking. Transfection in 3D-spheroid models and subcutaneous xenograft U87MG tumors confirms higher transgene expression for the binary cationic/anionic poly(phosphazene) complexes compared to the related polycation-pDNA complexes and to PEI-pDNA complexes. The data also indicate a notable capacity of the mixed complexes to deliver genes to the inner cores of tumor spheroids. Extension of this approach to siRNA delivery shows that the mixed poly(phosphazene) complexes can silence DYRK1A, a gene implicated in glioblastoma initiation and progression, reducing U87MG cell renewal in vitro and delaying tumor growth in vivo.