Prospective isolation of adult neural stem cells from the mouse subependymal zone

Neural stem cells (NSCs) have the remarkable capacity to self-renew and the lifelong ability to generate neurons in the adult mammalian brain. However, the molecular and cellular mechanisms contributing to these behaviors are still not understood. Now that prospective isolation of the NSCs has become feasible, these mechanisms can be studied. Here we describe a protocol for the efficient isolation of adult NSCs, by the application of a dual-labeling strategy on the basis of their glial identity and ciliated nature. The cells are isolated from the lateral ventricular subependymal zone (SEZ) of adult hGFAP-eGFP (human glial fibrillary acidic protein-enhanced green fluorescent protein) transgenic mice by fluorescence-activated cell sorting. Staining against prominin1 (CD133) allows the isolation of the NSCs (hGFAP-eGFP(+)/prominin1(+)), which can be further subdivided by labeling with the fluorescent epidermal growth factor. This protocol, which can be completed in 7 h, allows the assessment of quantitative changes in SEZ NSCs and the examination of their molecular and functional characteristics.     

Retinoic acid promotes neural conversion of mouse embryonic stem cells in adherent monoculture

Retinoic acid (RA) plays multiple roles in the nervous system, including induction of neural differentiation, axon outgrowth and neural patterning. Previously, RA for neural differentiation of embryonic stem (ES) cells always relies on embryoid bodies (EBs) formation. Here we report an in vitro adherent monoculture system to induce mouse ES cells into neural cells accompanied with RA. RA (1 μM) treatment, during initial 2 days of differentiation, can enhance the expression of neural markers, such as Nestin, Tuj1 and MAP2, and result in an earlier neural differentiation of ES cells. Furthermore, RA promotes a significant increase in neurite elongation of ES-derived neurons. Our study also implies that RA induced to express Wnt antagonist Dickkopf-1 (Dkk-1) for neural differentiation. However, the mechanisms of RA triggering neural induction remain to be determined. Our simple and efficient strategy is proposed to provide a basis for studying RA signaling pathways in neural differentiation in vitro.     

Subventricular zone astrocytes are neural stem cells in the adult mammalian brain.

Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. This germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. Here we show that SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, we show that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. We conclude that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain.     

Genetic ablation of caveolin-1 increases neural stem cell proliferation in the subventricular zone (SVZ) of the adult mouse brain

Adult neural stem cells are self-renewing multipotent cells that have the potential to replace dysfunctional and/or dying neuronal cells at the site of brain injury or degeneration. Caveolins are well-known tumor-suppressor genes that were recently found to be involved in the regulation of stem cell proliferation. For instance, ablation of the caveolin-1 (Cav-1) gene in mice markedly increases the proliferation of intestinal and mammary stem cells. However, the roles of caveolins in the proliferation of adult neural stem cells still remain unknown. In this study, dual-label immunofluorescence analysis of the proliferation marker, Ki67, and the stem cell markers, nestin and Sox2, was performed on brains of 8 week-old wild-type (WT) and Cav-1 knockout (KO) mice. Our results demonstrate an increased number of Ki67-positive nuclei in the subventricular zone (SVZ) of Cav-1 KO brains. Importantly, our dual-label immunofluorescence analyses demonstrate increased co-localization of Ki67 with both nestin and Sox2 in the SVZ of Cav-1 KO brains. Remarkably similar results were also obtained with Cav-2 and Cav-3 KO mouse brains as well, with increased proliferation of adult neural stem cells. Thus, the SVZ of caveolin KO mouse brains displays an increased proliferation of adult neural stem cells. Caveolin proteins might represent new crucial regulators of adult neural stem cell proliferation.     

Hu protein as an early marker of neuronal phenotypic differentiation by subependymal zone cells of the adult songbird forebrain

The avian forebrain exhibits neurogenesis in adulthood, with neuronal production from ependymal/subependymal zone (SZ) precursor cells. To follow the commitment of newborn cells to neuronal lineage, we used their expression of the Hu family of neuronal RNA-binding proteins to identify them before their migration from the SZ. Adult canaries were injected with [³H]thymidine as a marker of DNA replication, sacrificed after varying intervals, stained for Hu, and autoradiographed. We found that Hu was not expressed by premitotic precursor cells, but rather appeared within hours in their neuronal progeny, which did not embark on parenchymal migration until 4 to 7 days later. Hu was expressed by all neurons, but not glia, both in vivo and in vitro, as determined by ultrastructural analysis as well as co-localization of Hu and cell-type selective antigens. In addition, co-staining for Hu and N-cadherin, whose expression is down-regulated on neuronal emigration from the SZ, revealed their initial co-expression by neuronal daughter cells still within the SZ. These results suggest that Hu expression may be used as a very early indicator of neuronal differentiation by SZ cells. Furthermore, the data indicate that in the adult avian brain, neuronal phenotype is established within hours of precursor mitosis, even though the neuronal daughter cells do not initiate parenchymal migration for at least 4 days thereafter, following their down-regulation of N-cadherin. © 1995 John Wiley & Sons, Inc.     

Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions

The aim of this study was to compare the in vitro effects of glial cell line-derived neurotrophic factor, stem cell factor, granulocyte macrophage-colony stimulating factor, and co-culture with Sertoli cells on the efficiency of adult mouse spermatogonial stem cells colony formation. For these purpose, both Sertoli and spermatogonial cells were isolated from adult mouse testes. The identity of the cells was confirmed through analysis of alkaline phosphatase activity, immunocytochemistry against OCT-4, c-kit, and vimentin, and also by transplantation of these cells in the recipient testes. The isolated spermatogonial cells were treated either with various concentrations of the above mentioned factors or co-cultured with Sertoli cells for 3 wk. The spermatogonial cells of the resulting colonies were transplanted via rete testis into the mouse testes, which were irradiated with 14 Gy. The results indicated that glial cell line-derived neurotrophic factor is the most appropriate factor for in vitro colonization of adult mice spermatogonial cells compared with other cytokines and growth factors. A short-term co-culture with Sertoli cells showed a significant increase in the number and diameter of the colonies compared with the treated growth factors and the control group. We have also demonstrated that mouse spermatogonial stem cells in the colonies after co-culturing with Sertoli cells could induce spermatogenesis in the recipient testes after transplantation.     

EphB signaling controls lineage plasticity of adult neural stem cell niche cells

Stem cells remain in specialized niches over the lifespan of the organism in many organs to ensure tissue homeostasis and enable regeneration. How the niche is maintained is not understood, but is probably as important as intrinsic stem cell self-renewal capacity for tissue integrity. We here demonstrate a high degree of phenotypic plasticity of the two main niche cell types, ependymal cells and astrocytes, in the neurogenic lateral ventricle walls in the adult mouse brain. In response to a lesion, astrocytes give rise to ependymal cells and ependymal cells give rise to niche astrocytes. We identify EphB2 forward signaling as a key pathway regulating niche cell plasticity. EphB2 acts downstream of Notch and is required for the maintenance of ependymal cell characteristics, thereby inhibiting the transition from ependymal cell to astrocyte. Our results show that niche cell identity is actively maintained and that niche cells retain a high level of plasticity.     

Development of neural stem cell in the adult brain

New neurons are continuously generated in the dentate gyrus of the mammalian hippocampus and in the subventricular zone of the lateral ventricles throughout life. The origin of these new neurons is believed to be from multipotent adult neural stem cells. Aided by new methodologies, significant progress has been made in the characterization of neural stem cells and their development in the adult brain. Recent studies have also begun to reveal essential extrinsic and intrinsic molecular mechanisms that govern sequential steps of adult neurogenesis in the hippocampus and subventricular zone/olfactory bulb, from proliferation and fate specification of neural progenitors to maturation, navigation, and synaptic integration of the neuronal progeny. Future identification of molecular mechanisms and physiological functions of adult neurogenesis will provide further insight into the plasticity and regenerative capacity of the mature central nervous system.     

Stem cell-derived neural stem/progenitor cell supporting factor is an autocrine/paracrine survival factor for adult neural stem/progenitor cells

Recent evidence suggests that adult neural stem/progenitor cells (ANSCs) secrete autocrine/paracrine factors and that these intrinsic factors are involved in the maintenance of adult neurogenesis. We identified a novel secretory molecule, stem cell-derived neural stem/progenitor cell supporting factor (SDNSF), from adult hippocampal neural stem/progenitor cells by using the signal sequence trap method. The expression of SDNSF in adult central nervous system was localized to hippocampus including dentate gyrus, where the neurogenesis persists throughout life. In induced neurogenesis status seen in ischemically treated hippocampus, the expression of SDNSF was up-regulated. As functional aspects, SDNSF protein provided a dose-dependent survival effect for ANSC following basic fibroblast growth factor 2 (FGF-2) withdrawal. ANSCs treated by SDNSF also retain self-renewal potential and multipotency in the absence of FGF-2. However, SDNSF did not have mitogenic activity, nor was it a cofactor that promoted the mitogenic effects of FGF-2. These data suggested an important role of SDNSF as an autocrine/paracrine factor in maintaining stem cell potential and lifelong neurogenesis in adult central nervous system.     

室管膜下区神经干细胞与神经发生的研究进展

室管膜下区(subventricular zone,SVZ)存在着神经干细胞(nueral stem cells,NSCs),是成年哺乳动物脑内重要的神经发生区域。神经发生过程极为复杂,包括一系列的生物学事件。在病理状态下,SVZ区的细胞增殖,新生的神经细胞迁移到病灶处,取代或修复受损的细胞,起到保护脑组织的作用。该文就SVZ区的神经干细胞、神经发生过程及病理状态下神经发生的相关研究做一综述。     

Architecture and cell types of the adult subventricular zone: In search of the stem cells

Neural stem cells are maintained in the subventricular zone (SVZ) of the adult mammalian brain. Here, we review the cellular organization of this germinal layer and propose lineage relationships of the three main cell types found in this area. The majority of cells in the adult SVZ are migrating neuroblasts (type A cells) that continue to proliferate. These cells form an extensive network of tangentially oriented pathways throughout the lateral wall of the lateral ventricle. Type A cells move long distances through this network at high speeds by means of chain migration. Cells in the SVZ network enter the rostral migratory stream (RMS) and migrate anteriorly into the olfactory bulb, where they differentiate into interneurons. The chains of type A cells are ensheathed by slowly proliferating astrocytes (type B cells), the second most common cell type in this germinal layer. The most actively proliferating cells in the SVZ, type C, form small clusters dispersed throughout the network. These foci of proliferating type C cells are in close proximity to chains of type A cells. We discuss possible lineage relationships among these cells and hypothesize which are the neural stem cells in the adult SVZ. In addition, we suggest that interactions between type A, B, and C cells may regulate proliferation and initial differentiation within this germinal layer. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 234–248, 1998     

Taurine stimulates proliferation and promotes neurogenesis of mouse adult cultured neural stem/progenitor cells

This study reports an effect of taurine (1-10 mM) increasing markedly (120%) the number of neural precursor cells (NPCs) from adult mouse subventricular zone, cultured as neurospheres. This effect is one of the highest reported for adult neural precursor cells. Taurine-containing cultures showed 73-120% more cells than controls, after 24 and 96 h in culture, respectively. Taurine effect is due to enhanced proliferation as assessed by BrdU incorporation assays. In taurine cultures BrdU incorporation was markedly higher than controls from 1.5 to 48 h, with the maximal difference found at 1.5 h. This effect of taurine reproduced at every passage with the same window time. Taurine effects are not mimicked by glycine, alanine or GABA. Clonal efficiency values of 3.6% for taurine cultures and 1.3% for control cultures suggest a taurine influence on both, progenitor and stem cells. Upon differentiation, the proportion of neurons in control and taurine cultures was 3.1% (±0.5) and 10.2% (±0.8), respectively. These results are relevant for taurine implication in brain development as well as in adult neurogenesis. Possible mechanisms underlying taurine effects on cell proliferation are discussed.