SummaryIn vivo and in vitro studies indicate that a subpopulation of human marrow-derived stromal cells (MSCs, also known as mesenchymal stem cells) has potential to differentiate into multiple cell types, including osteoblasts.In this study, we tested the hypothesis that there are intrinsic effects of age in human MSCs (17-90 years). We tested the effect of age on senescence-associated β β β β -galactosidase, proliferation, apoptosis, p53 pathway genes, and osteoblast differentiation in confluent monolayers by alkaline phosphatase activity and osteoblast gene expression analysis. There were fourfold more human bone MSCs (hMSCs) positive for senescence-associated β β β β -galactosidase in samples from older than younger subjects ( P < 0.001; n = 17). Doubling time of hMSCs was 1.7-fold longer in cells from the older than the younger subjects, and was positively correlated with age ( P = 0.002; n = 19). Novel age-related changes were identified. With age, more cells were apoptotic ( P = 0.016; n = 10). Further, there were age-related increases in expression of p53 and its pathway genes, p21 and BAX . Consistent with other experiments, there was a significant age-related decrease in generation of osteoblasts both in the STRO-1 + cells ( P = 0.047; n = 8) and in adherent MSCs ( P < 0.001; n = 10). In sum, there is an age-dependent decrease in proliferation and osteoblast differentiation, and an increase in senescence-associated β β β β -galactosidase-positive cells and apoptosis in hMSCs. Up-regulation of the p53 pathway with age may have a critical role in mediating the reduction in both proliferation and osteoblastogenesis of hMSCs. These findings support the view that there are intrinsic alterations in human MSCs with aging that may contribute to the process of skeletal aging in humans.
UNC-6/Netrin and its receptor UNC-40/DCC are conserved regulators of growth cone guidance. By directly observing developing neurons in vivo, we show that UNC-6 and UNC-40 also function during axon formation to initiate, maintain and orient asymmetric neuronal growth. The immature HSN neuron of Caenorhabditis elegans breaks spherical symmetry to extend a leading edge toward ventral UNC-6. In unc-6 and unc-40 mutants, leading edge formation fails, the cell remains symmetrical until late in development and the axon that eventually forms is misguided. Thus netrin has two activities: one that breaks neuronal symmetry and one that guides the future axon. As the axon forms, UNC-6, UNC-40 and the lipid modulators AGE-1/phosphoinositide 3-kinase (PI3K) and DAF-18/ PTEN drive the actin-regulatory pleckstrin homology (PH) domain protein MIG-10/lamellipodin ventrally in HSN to promote asymmetric growth. The coupling of a directional netrin cue to sustained asymmetric growth via PI3K signaling is reminiscent of polarization in chemotaxing cells.Early in its development, a neuron extends long neurites tipped by dynamic, actin-rich growth cones. These neurites may become axons or dendrites, depending on the activity of an intrinsic neuronal polarity program that is regulated by the cytoskeleton, PAR polarity proteins, and lipid and protein kinases 1-7 . Although the ability to form long neurites is a defining feature of neurons, it is unclear what causes a neuron to break spherical symmetry and elongate processes. Studies in vitro have suggested that symmetry breaking is an active process that can be stimulated by neurotrophins or extracellular matrix components 6,8 , but little is known about the mechanisms that initiate and orient asymmetric growth in vivo 7 .Scattered studies of axonogenesis in vivo suggest that neurons usually extend their first stable process in the exact direction that will be taken by the eventual axon. Both the initial exploratory filopodia and the first microtubule-containing process of the grasshopper Ti1 neuron emerge from the cell body in the direction of its future axon 9 . Developing vertebrate retinal ganglion cells (RGCs) send their axons directly toward the optic nerve head, a trajectory marked before the growth cone forms by a small thickening of the RGC membrane 10 . These observations suggest that guidance information might direct the earliest stage of asymmetric neuronal growth.Axon formation has conceptual similarities to the polarization of cells during chemotaxis 11, 12 . In both cases, a symmetrical cell generates an asymmetric domain, the growth cone or
Planarian flatworms regenerate every organ after amputation. Adult pluripotent stem cells drive this ability, but how injury activates and directs stem cells into the appropriate lineages is unclear. Here we describe a single-organ regeneration assay in which ejection of the planarian pharynx is selectively induced by brief exposure of animals to sodium azide. To identify genes required for pharynx regeneration, we performed an RNAi screen of 356 genes upregulated after amputation, using successful feeding as a proxy for regeneration. We found that knockdown of 20 genes caused a wide range of regeneration phenotypes and that RNAi of the forkhead transcription factor FoxA, which is expressed in a subpopulation of stem cells, specifically inhibited regrowth of the pharynx. Selective amputation of the pharynx therefore permits the identification of genes required for organ-specific regeneration and suggests an ancient function for FoxA-dependent transcriptional programs in driving regeneration.DOI: http://dx.doi.org/10.7554/eLife.02238.001
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