Abstract:In adult mammals, the subventricular zone of the lateral ventricles (SVZ) and the subgranular zone of the dentate gyrus (DG) demonstrate ongoing neurogenesis, and multipotent neural stem/progenitor cells (NSCs) in these two regions exhibit different intrinsic properties. However, investigation of the mechanisms underlying such differences has been limited by a lack of efficient methods for isolating NSCs, particularly from the adult DG. Here we describe a protocol that enables us to isolate self-renewing and m… Show more
“…To obtain a single cell suspension, a mechanical dissociation was performed by passing the tissue fragments through a glass pipette. Single cells were seeded in a six-well plate in serum-free neurobasal medium (Miltenyi Biotech, Germany) supplemented with B27 and 2 mM glutamine (Gibco, Germany), 2 lg/ml heparin (Sigma, Germany), 20 ng/ml epidermal growth factor (R&D Systems, Germany) and 20 ng/ml basic fibroblast growth factor (CellSystems, Germany), 100 lg/ml penicillin, and 100 U/ ml streptomycin (Guo et al 2012). After 3 days of cultivation, the medium was changed.…”
Neural stem/progenitor cells (NSPCs) have the potential to self-renew and to generate all neural lineages as well as to repopulate damaged areas in the brain. Our previous targeting strategies have indicated precursor cell heterogeneity between different brain regions that warrants the development of NSPC-specific delivery vehicles. Here, we demonstrate a target-specific adenoviral vector system for the in vivo manipulation of progenitor cells in the subventricular zone of the adult mouse brain. For this purpose, we identified a series of peptide ligands via phage display. The peptide with the highest affinity, SNQLPQQ, was expressed in conjunction with a bispecific adaptor molecule. To verify the targeting potential of the specific peptide, green fluorescent protein-expressing Ad vectors were coupled with the adaptor molecule and injected into the subventricular region of adult mice by stereotaxic surgery. An efficient and selective transduction of NSPCs in the SVZ was achieved, whereas hippocampal NSPCs were negative. Our results offer an expeditious and simple tool to produce retargeted viral vectors for a specific and direct in vivo manipulation of these progenitor cells. This powerful technique provides an opportunity to develop innovative strategies and express therapeutic genes in specific types of neural progenitor cells to allow success in treatment of brain disorders.
“…To obtain a single cell suspension, a mechanical dissociation was performed by passing the tissue fragments through a glass pipette. Single cells were seeded in a six-well plate in serum-free neurobasal medium (Miltenyi Biotech, Germany) supplemented with B27 and 2 mM glutamine (Gibco, Germany), 2 lg/ml heparin (Sigma, Germany), 20 ng/ml epidermal growth factor (R&D Systems, Germany) and 20 ng/ml basic fibroblast growth factor (CellSystems, Germany), 100 lg/ml penicillin, and 100 U/ ml streptomycin (Guo et al 2012). After 3 days of cultivation, the medium was changed.…”
Neural stem/progenitor cells (NSPCs) have the potential to self-renew and to generate all neural lineages as well as to repopulate damaged areas in the brain. Our previous targeting strategies have indicated precursor cell heterogeneity between different brain regions that warrants the development of NSPC-specific delivery vehicles. Here, we demonstrate a target-specific adenoviral vector system for the in vivo manipulation of progenitor cells in the subventricular zone of the adult mouse brain. For this purpose, we identified a series of peptide ligands via phage display. The peptide with the highest affinity, SNQLPQQ, was expressed in conjunction with a bispecific adaptor molecule. To verify the targeting potential of the specific peptide, green fluorescent protein-expressing Ad vectors were coupled with the adaptor molecule and injected into the subventricular region of adult mice by stereotaxic surgery. An efficient and selective transduction of NSPCs in the SVZ was achieved, whereas hippocampal NSPCs were negative. Our results offer an expeditious and simple tool to produce retargeted viral vectors for a specific and direct in vivo manipulation of these progenitor cells. This powerful technique provides an opportunity to develop innovative strategies and express therapeutic genes in specific types of neural progenitor cells to allow success in treatment of brain disorders.
“…The primary aNPCs were prepared as previously described (Guo et al, 2012; Pan et al, 2013) from the SGZ of the DG from 6-7 week-old male C57BL/6J mice (Taconic, Hudson, NY). The solutions and media used during the aNPC isolation were filter sterilized.…”
Adult hippocampal neurogenesis is the process whereby adult neural precursor cells (aNPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate adult-born, functional neurons in the hippocampus. This process is modulated by various extracellular and intracellular stimuli, and the adult-born neurons have been implicated in hippocampus-dependent learning and memory. However, studies on how neurotoxic agents affect this process and the underlying mechanisms are limited. The goal of this study was to determine whether lead, a heavy metal, directly impairs critical processes in adult neurogenesis and to characterize the underlying signaling pathways using primary cultured SGZ-aNPCs isolated from adult mice. We report here that lead significantly increases apoptosis and inhibits proliferation in SGZ-aNPCs. In addition, lead significantly impairs spontaneous neuronal differentiation and maturation. Furthermore, we found that activation of the c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase signaling pathways are important for lead cytotoxicity. Our data suggest that lead can directly act on adult neural stem cells and impair critical processes in adult hippocampal neurogenesis, which may contribute to its neurotoxicity and adverse effects on cognition in adults.
“…Adult NSPCs can be isolated from neurogenic niches of adult CNSsubventricular zone (SVZ) or subgranular zone of hippocampal dentate gyrus [17]. In this case, the source availability is also the main limiting factor, which is further complicated by the difficulty to isolate and successfully maintain these cells in vitro [18][19][20].…”
Ischemic stroke is caused by occlusion of a cerebral artery, which gives rise to focal ischemia with irreversible injury in a core region and partially reversible damage in the surrounding penumbra zone. Stroke leads to neural death and consequently neurological impairments. Therapeutic intervention of stroke comprises thrombolysis and thrombectomy by chemical or surgical means, respectively. If done in time, these treatments may improve stroke outcome. However, many stroke patients cannot get sufficient degree of such treatment due to incompatibility or delay with admission to the clinic and suffer chronic neurological impairments. This has raised the need to develop effective treatments to improve poststroke recovery. Induced brain plasticity and cell replacement using neural stem cells are two promising strategies for therapy for stroke. This review will discuss the potential of such therapy as well as the factors that need to be taken into account for successful development of new therapy. Neural stem cells are multipotent with the capacity to self-renew and generate mature cells of the nervous system. They can be obtained from embryonic, fetal, or adult central nervous system, as well as through genetic reprogramming of somatic cells. Neural stem cell transplantation has proved to be effective in rodent studies. However, to translate these results into the clinical application, the variety of intrinsic and external factors must be carefully evaluated. This includes accurate stroke outcome predictions, choice of neural stem cell sources and evaluation of the risk of malignant transformation, selection of cell implantation paradigms and criteria for suitable patients.
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