Studies using model organisms ranging from worms and flies to mammals have GDC-0941 revealed that the insulin/insulin-like growth factor1 signalling pathway is a highly conserved mechanism that influences lifespan. It was first demonstrated in C. elegans that mutation in age-1, a homologue of the mammalian phosphatidylinositol-3 kinase extended lifespan. Subsequently it was found that a mutation in daf-2,a homologue of the mammalian insulin and IGF1 receptors , also dramatically prolonged the lifespan of C. elegans. Similarly, reduced insulin signalling was demonstrated to extend lifespan in Drosophila. The TNF family member B cell activating factor and its cognate receptor BAFF-R are required for generation and maintenance of the mature B cell pool. It has been reported that signals from BAFF-R activate the alternative NF-kB pathway via processing of the NF-kB2 protein p100 to p52. As neurospheres grow, the cells from the core are exposed to lower concentration of growth factors, leading to an increased cell death. This may be due to the fact that neurospheres deprived of growth factors in suspension are able to produce cytokines and growth factors that, at the same time, can induce differentiation and prevent cell death. It could be argued that, owing to the fact that EGF and FGF-2 are known to induce self-renewal of NPC, the effects found after removal of these factors on cell differentiation is predictable and expected based on published literature. Yet, Caldwell and colleagues showed that the combination of cell–cell interactions during differentiation and growth factor administration, can increase the number of generated neurons. This is only one example of the importance of cell-cell interaction and of the aspects associated to its three-dimensional structure for neurosphere plasticity. Correspondingly, western blot analysis of control B cell lysates demonstrated that BAFF induced processing of p100 to p52 but did not upregulate p100 or RelB in control B cells, suggesting specific activation of the alternative NF-kB pathway. Consistent with previous reports , CD40 ligation induced processing of NF-kB2 as well as upregulation of p100 and RelB, indicating the activation of both canonical and alternative pathways, whereas LPS treatment induced only the canonical pathway. As shown in Fig. 5b, B cells from TRAF6-DB mice exhibited normal processing of p100 to p52 in response to BAFF and anti-CD40 Ab. However, upregulation of p100 and RelB was obviously impaired in response to both LPS and anti-CD40 Ab, suggesting defective canonical NF-kB activation. Staining for GFAP, b-tubulin III and Nestin in hNPC from the CTR and MFM groups cultured in suspension revealed two major findings. Removal of growth factors modifies the distribution and proportions of b-tubulin III, GFAP and Nestin positive cells in whole neurospheres. In the CTR group, GFAP was found across the whole neurosphere, b-tubulin III was preferably expressed in the neurosphere core and Nestin was found in the border of the sphere.