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Lipid droplets (LDs) are dynamic lipid storage organelles. They are tightly linked to metabolism and can exert protective functions, making them important players in health and disease. Most LD studies in vivo rely on staining methods, providing only a snapshot. We therefore developed a LD-reporter mouse by endogenously labelling the LD coat protein perilipin 2 (PLIN2) with tdTomato, enabling staining-free fluorescent LD visualisation in living and fixed tissues and cells. Here we validate this model under standard and high-fat diet conditions and demonstrate that LDs are present in various cells in the healthy brain, including neurons, astrocytes, ependymal cells, neural stem/progenitor cells and microglia. Furthermore, we show that LDs are abundant during brain development and can be visualized using live-imaging of embryonic slices. Taken together, our tdTom-Plin2 mouse serves as a novel tool to study LDs and their dynamics under both physiological and diseased conditions in all tissues expressing Plin2.

SummaryIn the adult mouse brain, neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ) generate neurons and glia throughout life. microRNAs are important regulators of cell states, frequently acting in a stage- or context-dependent manner. Here, miRNA profiling of FACS-purified populations identified miR-17∼92 as highly upregulated in activated NSCs and transit amplifying cells (TACs) in comparison to quiescent NSCs. Conditional deletion of miR-17∼92 in NSCs reduced stem cell proliferation both in vitro and in vivo. In contrast, in TACs, miR-17∼92 deletion caused a selective shift from neurogenic DLX2+ TACs towards oligodendrogenic OLIG2+ TACs, resulting in increased oligodendrogenesis to the corpus callosum. miR-17∼92 deletion also decreased proliferation and maturation of intraventricular oligodendrocyte progenitor cells. Together, these findings reveal stage- and cell-type-specific functions of the miR-17∼92 cluster within adult V-SVZ neural stem cell lineages.

Neural stem cells (NSCs) in the adult mouse brain contribute to lifelong brain plasticity. NSCs in the adult ventricular-subventricular zone (V-SVZ) are heterogeneous and, depending on their location in the niche, give rise to different subtypes of olfactory bulb interneurons. Here, we show that during pregnancy multiple regionally-distinct NSCs are dynamically recruited at different times. Coordinated temporal activation of these NSC pools generates sequential waves of short-lived olfactory bulb interneuron subtypes that mature in the mother around birth and in the perinatal care period. Concomitant with neuronal addition, oligodendrocyte progenitors also transiently increase in the olfactory bulb. Thus, life experiences, such as pregnancy, can trigger transient neurogenesis and gliogenesis under tight spatial and temporal control, and may provide a novel substrate for brain plasticity in anticipation of temporary physiological demand.

Gliogenesis in the adult mouse brain Neural stem cells in the adult mouse brain can generate both neurons and glia. Exactly where each stem cell is positioned can determine what type of neurons it generates. Delgado et al. show that neural stem cells are also choosy about what sorts of glia they make and when (see the Perspective by Baldwin and Silver). Injury or selective deletion of platelet-derived growth factor receptor β (PDGFRβ) from the stem cells kicked them into overdrive and revealed their selectivity with respect to gliogenesis. An unusual type of glial progenitor cell, intraventricular oligodendrocyte progenitors, are found nestled between the cilia of ependymal cells derived from tight clusters of PDGFRβ-expressing stem cells. Science , abg8467, this issue p. 1205 ; see also abj1139, p. 1151

This protocol describes how to isolate endothelial cells and pericytes from adult mouse brain microregions using fluorescence-activated cell sorting without requirement for transgenic mice. The cells may be used for diverse downstream applications. The vasculature is emerging as a key contributor to brain function during neurodevelopment and in mature physiological and pathological states. The brain vasculature itself also exhibits regional heterogeneity, highlighting the need to develop approaches for purifying cells from different microregions. Previous approaches for isolation of endothelial cells and pericytes have predominantly required transgenic mice and large amounts of tissue, and have resulted in impure populations. In addition, the prospective purification of brain pericytes has been complicated by the fact that widely used pericyte markers are also expressed by other cell types in the brain. Here, we describe the detailed procedures for simultaneous isolation of pure populations of endothelial cells and pericytes directly from adult mouse brain microregions using fluorescence-activated cell sorting (FACS) with antibodies against CD31 (endothelial cells) and CD13 (pericytes). This protocol is scalable and takes ∼5 h, including microdissection of the region of interest, enzymatic tissue dissociation, immunostaining, and FACS. This protocol allows the isolation of brain vascular cells from any mouse strain under diverse conditions; these cells can be used for multiple downstream applications, including in vitro and in vivo experiments, and transcriptomic, proteomic, metabolomic, epigenomic, and single-cell analysis.

Neural stem cells (NSCs) in specialized niches in the adult mammalian brain generate neurons throughout life. NSCs in the adult mouse ventricular-subventricular zone (V-SVZ) exhibit a regional identity and, depending on their location, generate distinct olfactory bulb interneuron subtypes. Here, we show that the hypothalamus, a brain area regulating physiological states, provides long-range regionalized input to the V-SVZ niche and can regulate specific NSC subpopulations. Hypothalamic proopiomelanocortin neurons selectively innervate the anterior ventral V-SVZ and promote the proliferation of Nkx2.1+ NSCs and the generation of deep granule neurons. Accordingly, hunger and satiety regulate adult neurogenesis by modulating the activity of this hypothalamic–V-SVZ connection. Our findings reveal that neural circuitry, via mosaic innervation of the V-SVZ, can recruit distinct NSC pools, allowing on-demand neurogenesis in response to physiology and environmental signals.

Quiescent neural stem cells (NSCs) in the adult ventricular-subventricular zone (V-SVZ) have a regional identity and undergo activation to generate neurons. The domains for gliogenesis are less explored. Here we show that Platelet-Derived Growth Factor Receptor beta (PDGFRβ) is expressed by adult V-SVZ NSCs that generate olfactory bulb interneurons and glia with slow baseline kinetics. Selective deletion of PDGFRβ in adult V-SVZ NSCs leads to their release from quiescence uncovering multiple domains in the septal wall for oligodendrocyte and astrocyte formation. Unexpectedly, we identify a novel intraventricular oligodendrocyte progenitor inside the brain ventricles. Together our findings reveal different NSC spatial domains for gliogenesis in the adult V-SVZ that are largely quiescent under homeostasis and may have key functions for brain plasticity.

Obernier et al. (2018) show that the primary mode of division of adult ventricular-subventricular zone (V-SVZ) neural stem cells is symmetric, with the majority generating two non-stem cell progeny, and a minority self-renewing. This discovery has important implications for understanding stem cell dynamics and adult neurogenesis.

International audience; The barrier between the blood and the ventricular cerebrospinal fluid (CSF) is located at the choroid plexuses. At the interface between two circulating fluids, these richly vascularized veil-like structures display a peculiar morphology explained by their developmental origin, and fulfill several functions essential for CNS homeostasis. They form a neuroprotective barrier preventing the accumulation of noxious compounds into the CSF and brain, and secrete CSF, which participates in the maintenance of a stable CNS internal environment. The CSF circulation plays an important role in volume transmission within the developing and adult brain, and CSF compartments are key to the immune surveillance of the CNS. In these contexts, the choroid plexuses are an important source of biologically active molecules involved in brain development, stem cell proliferation and differentiation, and brain repair. By sensing both physiological changes in brain homeostasis and peripheral or central insults such as inflammation, they also act as sentinels for the CNS. Finally, their role in the control of immune cell traffic between the blood and the CSF confers on the choroid plexuses a function in neuroimmune regulation and implicates them in neuroinflammation. The choroid plexuses, therefore, deserve more attention while investigating the pathophysiology of CNS diseases and related comorbidities.

Activation of the epidermal growth factor receptor (EGFR) pathway is strongly implicated in the proliferation and migration of neural/glial progenitors and gliomas. EGFR is silenced after development but becomes aberrantly overexpressed in many low-grade and most high-grade gliomas, often in the absence of gene-amplification/activating mutations. The phenotype and functional characteristics of EGFR-expressing human brain cells are not well defined, as is the relationship of EGFR expression to gliomagenesis. We recently reported on the selective retention of EGFR expression in adult human subventricular zone (SVZ) glia. Notably, we also showed a strikingly similar epigenetic landscape at the wild-type EGFR promoter between germinal matrix/SVZ and EGFR-expressing gliomas. Here we undertook a detailed characterization of EGFR-expressing cells in the developing and adult human brain using warm postmortem material, and in human glioblastomas after fresh resection, hypothesizing that EGFR-positive cells harbor stem cell-like properties in vitro, which may implicate them in gliomagenesis. To better characterize the phenotype of human EGFR-expressing brain cells in vivo, we performed detailed confocal immunofluorescence analysis with markers for glial/neuronal lineage differentiation and proliferation. We found that during neural development, many EGFR-positive cells co-express markers of oligodendrocyte lineage, such as Olig2, similarly to most glioblastomas. We then analyzed the behavior of these cells in vitro. We used our novel, human-based protocol to isolate cells directly from fresh human samples, taking advantage of the inherent binding affinity of EGFR for its natural ligand, EGF. Intriguingly, we found that EGFR-positive, but not EGFR-negative, cells isolated from germinal matrix and from glioblastomas display stem-cell characteristics in culture, including self-renewal and trilineage differentiation. Our findings suggest that EGFR expression confers stem-cell-like properties to human progenitors and glioma cells, hint that its maintenance may play a role in gliomagenesis, and provide a novel isolation platform of functionally defined EGFR+ glioma populations for further molecular analyses.

Adult neurogenic niches harbor quiescent neural stem cells; however, their in vivo identity has been elusive. Here, we prospectively isolate GFAP(+)CD133(+) (quiescent neural stem cells [qNSCs]) and GFAP(+)CD133(+)EGFR(+) (activated neural stem cells [aNSCs]) from the adult ventricular-subventricular zone. aNSCs are rapidly cycling, highly neurogenic in vivo, and enriched in colony-forming cells in vitro. In contrast, qNSCs are largely dormant in vivo, generate olfactory bulb interneurons with slower kinetics, and only rarely form colonies in vitro. Moreover, qNSCs are Nestin negative, a marker widely used for neural stem cells. Upon activation, qNSCs upregulate Nestin and EGFR and become highly proliferative. Notably, qNSCs and aNSCs can interconvert in vitro. Transcriptome analysis reveals that qNSCs share features with quiescent stem cells from other organs. Finally, small-molecule screening identified the GPCR ligands, S1P and PGD2, as factors that actively maintain the quiescent state of qNSCs.

Direct isolation of human neural and glioma stem cells from fresh tissues permits their biological study without prior culture and may capture novel aspects of their molecular phenotype in their native state. Recently, we demonstrated the ability to prospectively isolate stem cell populations from fresh human germinal matrix and glioblastoma samples, exploiting the ability of cells to bind the Epidermal Growth Factor (EGF) ligand in fluorescence-activated cell sorting (FACS). We demonstrated that FACS-isolated EGF-bound neural and glioblastoma populations encompass the sphere-forming colonies in vitro, and are capable of both self-renewal and multilineage differentiation. Here we describe in detail the purification methodology of EGF-bound (i.e., EGFR+) human neural and glioma cells with stem cell properties from fresh postmortem and surgical tissues. The ability to prospectively isolate stem cell populations using native ligand-binding ability opens new doors for understanding both normal and tumor cell biology in uncultured conditions, and is applicable for various downstream molecular sequencing studies at both population and single-cell resolution.

Summary Characterization of non-neoplastic and malignant human stem cell populations in their native state can provide new insights into gliomagenesis. Here we developed a purification strategy to directly isolate EGFR+/− populations from human germinal matrix (GM) and adult subventricular zone autopsy tissues, and from de novo glioblastoma (GBM) resections, enriching for cells capable of binding EGF ligand (LBEGFR+), and uniquely compared their functional and molecular properties. LBEGFR+ populations in both GM and GBM encompassed all sphere-forming cells and displayed proliferative stem cell properties in vitro. In xenografts, LBEGFR+ GBM cells showed robust tumor initiation and progression to high-grade, infiltrative gliomas. Whole-transcriptome sequencing analysis confirmed enrichment of proliferative pathways in both developing and neoplastic freshly isolated EGFR+ populations, and identified both unique and shared sets of genes. The ability to prospectively isolate stem cell populations using native ligand-binding capacity opens new doors onto understanding both normal human development and tumor cell biology., Graphical Abstract, Highlights • Prospective purification of EGFR+ cells from fresh human neural tissues using EGF • In vitro stem cell properties are restricted to EGFR+ germinal matrix and GBM cells • EGFR+ GBM cells are tumor-initiating and re-form high-grade gliomas in mice • RNA-seq defines developmentally shared pathways in EGFR+ GBM cells, Understanding human brain development and gliomagenesis requires deeper analysis of stem cells in their native state. Here, Tsankova and colleagues employ EGF ligand to prospectively isolate proliferative EGFR+ stem-like populations from fresh germinal matrix and glioblastoma tissues, capturing sphere-forming and tumor-initiating cells in uncultured state. Comparative transcriptome analysis of these populations provides molecular insight into the dysregulated developmental pathways in GBM.

Neural stem cells (NSCs) reside in specialized niches in the adult mammalian brain. The ventricular-subventricular zone (V-SVZ), adjacent to the lateral ventricles, gives rise to olfactory bulb (OB) neurons, and some astrocytes and oligodendrocytes throughout life. In vitro assays have been widely used to retrospectively identify NSCs. However, cells that behave as stem cells in vitro do not reflect the identity, diversity, and behavior of NSCs in vivo. Novel tools including fluorescence activated cell sorting, lineage-tracing, and clonal analysis have uncovered multiple layers of adult V-SVZ NSC heterogeneity, including proliferation state and regional identity. In light of these findings, we reexamine the concept of adult NSCs, considering heterogeneity as a key parameter for analyzing their dynamics in vivo. V-SVZ NSCs form a mosaic of quiescent (qNSCs) and activated cells (aNSCs) that reside in regionally distinct microdomains, reflecting their regional embryonic origins, and give rise to specific subtypes of OB interneurons. Prospective purification and transcriptome analysis of qNSCs and aNSCs has illuminated their molecular and functional properties. qNSCs are slowly dividing, have slow kinetics of neurogenesis in vivo, can be recruited to regenerate the V-SVZ, and only rarely give rise to in vitro colonies. aNSCs are highly proliferative, undergo rapid clonal expansion of the neurogenic lineage in vivo, and readily form in vitro colonies. Key open questions remain about stem cell dynamics in vivo and the lineage relationship between qNSCs and aNSCs under homeostasis and regeneration, as well as context-dependent plasticity of regionally distinct adult NSCs under different external stimuli. WIREs Dev Biol 2016, 5:640-658. doi: 10.1002/wdev.248 For further resources related to this article, please visit the WIREs website.

Sphere-forming assays have been widely used to retrospectively identify stem cells based on their reported capacity to evaluate self-renewal and differentiation at the single cell level in vitro. The discovery of markers that allow the prospective isolation of stem cells and their progeny from their in vivo niche allows the functional properties of purified populations to be defined. We provide an historical perspective of the evolution of the neurosphere assay, and highlight limitations in the use of sphere-forming assays, in the context of neurospheres. We discuss theoretical and technical considerations of experimental design and interpretation that surround the use of this assay with any tissue.

A major question in studying adult neurogenesis is the source and identity of molecules that regulate stem cells. In this issue of Neuron, Delgado et al. (2014) uncover that endothelial-derived NT-3 acts as a mediator of quiescence in the V-SVZ adult neural stem cell niche.

We investigated the expression and role of the dopamine receptor 3 (D3R) in postnatal mouse subventricular zone (SVZ). In situ hybridization detected selective D3R mRNA expression in the SVZ. Fluorescence activated cell sorting (FACS) of adult SVZ subtypes using hGFAP-GFP and Dcx-GFP mice showed that transit amplifying progenitor cells and niche astrocytes expressed D3R whereas stem cell-like astrocytes and neuroblasts did not. To determine D3R's role in SVZ neurogenesis, we administered U-99194A, a D3R preferential antagonist, and bromodeoxyuridine in postnatal mice. In vivo D3R antagonism decreased the numbers of newborn neurons reaching the core and the periglomerular layer of the olfactory bulb. Moreover, it decreased progenitor cell proliferation but did not change the number of label-retaining (stem) cells, commensurate with its expression on transit amplifying progenitor cells but not SVZ stem cell-like astrocytes. Collectively, this study suggests that dopaminergic stimulation of D3R drives proliferation via rapidly amplifying progenitor cells to promote murine SVZ neurogenesis.

The inference of transcriptional networks that regulate transitions into physiological or pathological cellular states remains a central challenge in systems biology. A mesenchymal phenotype is the hallmark of tumour aggressiveness in human malignant glioma, but the regulatory programs responsible for implementing the associated molecular signature are largely unknown. Here we show that reverse-engineering and an unbiased interrogation of a glioma-specific regulatory network reveal the transcriptional module that activates expression of mesenchymal genes in malignant glioma. Two transcription factors (C/EBPbeta and STAT3) emerge as synergistic initiators and master regulators of mesenchymal transformation. Ectopic co-expression of C/EBPbeta and STAT3 reprograms neural stem cells along the aberrant mesenchymal lineage, whereas elimination of the two factors in glioma cells leads to collapse of the mesenchymal signature and reduces tumour aggressiveness. In human glioma, expression of C/EBPbeta and STAT3 correlates with mesenchymal differentiation and predicts poor clinical outcome. These results show that the activation of a small regulatory module is necessary and sufficient to initiate and maintain an aberrant phenotypic state in cancer cells.

The subventricular zone (SVZ) is the largest neurogenic niche in the adult mammalian brain. We found that the brain-enriched microRNA miR-124 is an important regulator of the temporal progression of adult neurogenesis in mice. Knockdown of endogenous miR-124 maintained purified SVZ stem cells as dividing precursors, whereas ectopic expression led to precocious and increased neuron formation. Furthermore, blocking miR-124 function during regeneration led to hyperplasias, followed by a delayed burst of neurogenesis. We identified the SRY-box transcription factor Sox9 as being a physiological target of miR-124 at the transition from the transit amplifying cell to the neuroblast stage. Sox9 overexpression abolished neuronal differentiation, whereas Sox9 knockdown led to increased neuron formation. Thus miR-124-mediated repression of Sox9 is important for progression along the SVZ stem cell lineage to neurons.

SummaryStem cells (SC) exhibit a unique capacity for self-renewal in an undifferentiated state. It is unclear whether the self-renewal of pluripotent embryonic SC (ESC) and of tissue-specific adult SC such as hematopoietic SC (HSC) is controlled by common mechanisms. The deletion of transcription factor Zfx impaired the self-renewal but not the differentiation capacity of murine ESC; conversely, Zfx overexpression facilitated ESC self-renewal by opposing differentiation. Furthermore, Zfx deletion abolished the maintenance of adult HSC but did not affect erythromyeloid progenitors or fetal HSC. Zfx-deficient ESC and HSC showed increased apoptosis and SC-specific upregulation of stress-inducible genes. Zfx directly activated common target genes in ESC and HSC, as well as ESC-specific target genes including ESC self-renewal regulators Tbx3 and Tcl1. These studies identify Zfx as a shared transcriptional regulator of ESC and HSC, suggesting a common genetic basis of self-renewal in embryonic and adult SC.

Several signaling pathways important for the proliferation and growth of brain cells are pathologically dysregulated in gliomas, including the epidermal growth factor receptor (EGFR). Expression of EGFR is high in neural progenitors during development and in gliomas but decreases significantly in most adult brain regions. Here we show that EGFR expression is maintained in the astrocyte ribbon of the adult human subventricular zone. The transcriptional regulation of EGFR expression is poorly understood. To investigate the role of epigenetics on EGFR regulation in the contexts of neural development and gliomagenesis, we measured levels of DNA methylation and histone H3 modifications at the EGFR promoter in human brain tissues, glioma specimens, and EGFR-expressing neural cells, acutely isolated from their native niche. While DNA was constitutively hypomethylated in non-neoplastic and glioma samples, regardless of their EGFR-expression status, the activating histone modifications H3K27ac and H3K4me3 were enriched only when EGFR is highly expressed (developing germinal matrix and gliomas). Conversely, repressive H3K27me3 marks predominated in adult white matter where EGFR is repressed. Furthermore, the histone methyltransferase core enzyme ASH2L was bound at EGFR in the germinal matrix and in gliomas where levels of H3K4me3 are high, and the histone acetyltransferase P300 was bound in samples with H3K27ac enrichment. Our studies use human cells and tissues undisturbed by cell-culture artifact, and point to an important, locus-specific role for chromatin remodeling in EGFR expression in human neural development that may be dysregulated during gliomagenesis, unraveling potential novel targets for future drug therapy.

Adult neural stem cells reside in specialized niches. In the ventricular-subventricular zone (V-SVZ), quiescent neural stem cells (qNSCs) become activated (aNSCs), and generate transit amplifying cells (TACs), which give rise to neuroblasts that migrate to the olfactory bulb. The vasculature is an important component of the adult neural stem cell niche, but whether vascular cells in neurogenic areas are intrinsically different from those elsewhere in the brain is unknown. Moreover, the contribution of pericytes to the neural stem cell niche has not been defined. Here, we describe a rapid FACS purification strategy to simultaneously isolate primary endothelial cells and pericytes from brain microregions of nontransgenic mice using CD31 and CD13 as surface markers. We compared the effect of purified vascular cells from a neurogenic (V-SVZ) and non-neurogenic brain region (cortex) on the V-SVZ stem cell lineagein vitro. Endothelial and pericyte diffusible signals from both regions differentially promote the proliferation and neuronal differentiation of qNSCs, aNSCs, and TACs. Unexpectedly, diffusible cortical signals had the most potent effects on V-SVZ proliferation and neurogenesis, highlighting the intrinsic capacity of non-neurogenic vasculature to support stem cell behavior. Finally, we identify PlGF-2 as an endothelial-derived mitogen that promotes V-SVZ cell proliferation. This purification strategy provides a platform to define the functional and molecular contribution of vascular cells to stem cell niches and other brain regions under different physiological and pathological states.

The lateral walls of the forebrain lateral ventricles are the richest source of stem cells in the adult mammalian brain. These stem cells give rise to new olfactory neurons that are renewed throughout life. The neurons originate in the subventricular zone (SVZ), migrate within the rostral extension (RE) of the SVZ along the rostral migratory stream (RMS) within tube-like structures formed of glial cells, to eventually reach the olfactory bulb (OB). We demonstrate that, contrary to the current view, multipotential (neuronal-astroglial-oligodendroglial) precursors with stem cell features can be isolated not only from the SVZ but also from the entire RE, including the distal portion within the OB. Specifically, these stem cells do not derive from the migratory neuroblasts coming from the SVZ. Interestingly, stem cells isolated from the proximal RE generate significantly more oligodendrocytes, and those from the distal RE proliferate significantly more slowly than stem cells derived from the SVZ and other RE regions. These findings demonstrate that stem cells are not confined to the forebrain periventricular region and indicate that stem cells endowed with different functional characteristics occur at different levels of the SVZ-RE pathway.

Adult neurogenesis is a widespread phenomenon occurring in many species, including humans. The functional and therapeutic implications of this form of brain plasticity are now beginning to be realized. Comparative approaches to adult neurogenesis will yield important clues about brain repair. Here, we compare adult neurogenesis in birds and mammals. We review recent studies on the glial identity of stem cells that generate new neurons, the different modes of migration used by the newly generated neurons to reach their destinations, and how these systems respond to experimentally induced cell death. We integrate these findings to address how comparative analysis at the molecular level might be used for brain repair.

Neuronal precursors reside in the subventricular zone (SVZ) of adult mammals. This region is composed of a network of chains of migrating neuroblasts ensheathed by astrocytes and juxtaposed by clusters of immature precursors (type C cells). Here we show that after antimitotic treatment with cytosine-β- d -arabinofuranoside, neuroblasts and type C cells are eliminated but some astrocytes remain. Remarkably, the SVZ network rapidly regenerates. Soon after cytosine-β- d -arabinofuranoside treatment astrocytes divide. Two days later, type C cells reappear, followed at 4.5 days by migrating neuroblasts. By 10 days the SVZ network is fully regenerated, and the orientation and organization of chains of migrating neuroblasts resemble that of normal mice. This regeneration reveals an unexpected plasticity in the adult central nervous system and should provide a model system to study the early stages of neurogenesis in the adult 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.

Given their capacity to regenerate cells lost through injury or disease, stem cells offer new vistas into possible treatments for degenerative diseases and their underlying causes. As such, stem cell biology is emerging as a driving force behind many studies in the field of regenerative medicine. This review focuses on our current understanding of the applications of stem cells in treating ailments of the human brain, with an emphasis on neurodegenerative diseases. Two types of neural stem cells are discussed: endogenous neural stem cells residing within the adult brain, and pluripotent stem cells capable of forming neural cells in culture. Endogenous neural stem cells give rise to neurons throughout life, but they are restricted to specialized regions in the brain. Elucidating the molecular mechanisms regulating these cells is key in determining their therapeutic potential, as well as finding mechanisms to activate dormant stem cells outside of these specialized microdomains. In parallel, patient-derived stem cells can be used to generate neural cells in culture, providing new tools for disease modeling, drug testing and cell-based therapies. Turning these technologies into viable treatments will require the integration of basic science with clinical skills in rehabilitation.

Stem cells persist in specialized niches in the adult mammalian brain. Emerging findings highlight the complexity and heterogeneity of different compartments in the niche, as well as the presence of local signaling microdomains. Stem cell quiescence and activation are regulated not only by anchorage to the niche and diffusible signals, but also by biophysical properties, including fluid dynamics. Importantly, the adult neural stem cell niche integrates both local and systemic changes, reflecting the physiological state of the organism. Moreover niche signaling is bidirectional, with stem cells and their progeny and niche cells dynamically interacting with each other during homeostasis, regeneration and aging.

Cells in the brains of adult mammals continue to proliferate in the subventricular zone (SVZ) throughout the lateral wall of the lateral ventricle. Here we show, using whole mount dissections of this wall from adult mice, that the SVZ is organized as an extensive network of chains of neuronal precursors. These chains are immunopositive to the polysialylated form of NCAM, a molecule present at sites of plasticity, and TuJ1, an early neuronal marker. The majority of the chains are oriented along the rostrocaudal axis and many join the rostral migratory stream that terminates in the olfactory bulb. Using focal microinjections of DiI and transplantation of SVZ cells carrying a neuron-specific reporter gene, we demonstrate that cells originating at different rostrocaudal levels of the SVZ migrate rostrally and reach the olfactory bulb where they differentiate into neurons. Our results reveal an extensive network of pathways for the tangential chain migration of neuronal precursors throughout the lateral wall of the lateral ventricle in the adult mammalian brain.

We investigated the expression and role of the dopamine receptor 3 (D3R) in postnatal mouse subventricular zone (SVZ). In situ hybridization detected selective D3R mRNA expression in the SVZ. Fluorescence activated cell sorting (FACS) of adult SVZ subtypes using hGFAP-GFP and Dcx-GFP mice showed that transit amplifying progenitor cells and niche astrocytes expressed D3R whereas stem cell-like astrocytes and neuroblasts did not. To determine D3R’s role in SVZ neurogenesis, we administered U-99194A, a D3R preferential antagonist, and bromodeoxyuridine (BrdU) in postnatal mice. In vivo D3R antagonism decreased the numbers of newborn neurons reaching the core and the periglomerular layer of the olfactory bulb. Moreover, it decreased progenitor cell proliferation but did not change the number of label-retaining (stem) cells, commensurate with its expression on transit amplifying progenitor cells but not SVZ stem cell-like astrocytes. Collectively, this study suggests that dopaminergic stimulation of D3R drives proliferation via rapidly amplifying progenitor cells to promote murine SVZ neurogenesis.

The ability to prospectively isolate adult neural stem cells and their progeny is crucial to study their biology and therapeutic potential. Stem cells in adult mammalian neurogenic niches are a subset of astrocytes. A major limitation in the field has been the inability to distinguish stem cell astrocytes from niche astrocytes. Here, we show that epidermal growth factor receptor (EGFR)-positive subventricular-zone (SVZ) astrocytes are activated stem cells that are eliminated by antimitotic treatment. We developed a simple strategy to simultaneously purify cells at different stages of the adult SVZ stem cell lineage by using FACS. This method combines the use of fluorescent EGF ligand, CD24, and GFP expression in GFAP::GFP transgenic mice and allows the simultaneous purification of activated stem cell astrocytes (GFP + EGFR + CD24 − ), niche astrocytes (GFP + EGFR − CD24 − ), transit amplifying cells (GFP − EGFR + CD24 − ), and neuroblasts (GFP − EGFR − CD24 low ). One in three EGFR + astrocytes gives rise to neurospheres in vitro, a 20-fold enrichment over unsorted cells. Importantly, these cells constitute the neurosphere-forming population among SVZ astrocytes. This approach will be of great utility for future functional and molecular studies of the SVZ stem cell lineage.

The complexity and cellular diversity of the adult brain arises from the proliferation and differentiation of a small number of stem cells. The intrinsic state of stem cells depends on their spatial and temporal history and affects their responsiveness to extrinsic signals from the microenvironment. Stem cell self-renewal and differentiation along neuronal and glial lineages are defined by the dynamic interplay between transcription, epigenetic control, and posttranscriptional regulators, including microRNAs, whose key role in stem cell biology is just emerging.

Adult neurogenesis occurs in most species and is regulated by a wide variety of environmental and pharmacological challenges. The functional integration of neurons generated in the adult was first demonstrated in songbirds more than two decades ago. In the adult mammalian brain, neurons are continuously generated in two structures, the olfactory bulb and the hippocampus. Current evidence suggests that adult-born immature neurons have distinct electrophysiological properties from old neurons, and proposed roles in a variety of functions including olfaction, learning and mood regulation.

The identification of adult brain regions harboring neural stem cells and of their continual generation of new neurons throughout life challenges traditional views of the adult brain’s germinal potential and suggests new alternatives for brain repair. Surprisingly, cells with characteristics of mature astrocytes have been shown to function as the primary precursors of new neurons in vivo. This observation goes against the generalized assumption that the rare cells with undifferentiated appearance are neural stem cells. It suggests that neural stem cells in vivo have important structural functions in addition to their role as primary progenitors. Surprisingly, neurospheres - a commonly used model of neural stem cells in culture - are not necessarily derived from the in vivo primary precursors but from transit amplifying cells. We suggest that neural stem cells, when activated to become transit amplifying cells, upregulate the expression of growth factor receptors on their surface, allowing their isolation as neurospheres.

The adult mammalian brain harbors multipotent stem cells, which reside and participate in specialized niches that support self-renewal and differentiation. The first cellular and molecular elements of the stem cell niche in the adult brain have been identified and include cell-cell interactions and somatic cell signaling, the vasculature, the extracellular matrix and basal lamina. Furthermore, regulation at the epigenetic level via chromatin modification and remodeling is an integral aspect of stem cell biology. Understanding the in vivo stem cell niche will provide a framework for the elucidation of stem cell function in the adult brain.

Glia are the most numerous cells in the brain, and their many diverse functions highlight their essential role in the nervous system. Recent studies have revealed an unexpected new role for glia in a wide variety of species, that of stem cells/progenitors in the adult and embryonic brain. Differentiation along the glial lineage may be a default state of development reflected in the progression of stem cells along the neuroepithelial-->radial glia-->astrocyte lineage.

Neural stem cells in the subventricular zone (SVZ) continue to generate new neurons in the adult brain. SVZ cells exposed to EGF in culture grow to form neurospheres that are multipotent and self-renewing. We show here that the majority of these EGF-responsive cells are not derived from relatively quiescent stem cells in vivo, but from the highly mitotic, Dlx2+, transit-amplifying C cells. When exposed to EGF, C cells downregulate Dlx2, arrest neuronal production, and become highly proliferative and invasive. Killing Dlx2+ cells dramatically reduces the in vivo response to EGF and neurosphere formation in vitro. Furthermore, purified C cells are 53-fold enriched for neurosphere generation. We conclude that transit-amplifying cells retain stem cell competence under the influence of growth factors.

Neurogenesis continues into adult life in restricted germinal layers. The identification of the neural stem cells that give rise to these new neurons has important clinical implications and provides fundamental information to understand the origins of the new neurons. Work in adult birds and rodents yielded a surprising result: the neural stem cells appear to have characteristics of glia. In adult birds, the primary neuronal precursors are radial glia. In adult mammals, the primary neuronal precursors have properties of astrocytes. Radial glial cells have previously been shown to transform into astrocytes; both cell types are classically considered part of a committed astroglial lineage. Instead, we propose that neural stem cells are contained within this astroglial lineage. These findings in adult vertebrate brain, together with recent work in the developing mammalian cerebral cortex, force us to reexamine traditional concepts about the origin of neurons and glia in the central nervous system. In particular, neural stem cells possess a surprisingly elaborate structure, suggesting that in addition to their progenitor role, they have important structural and metabolic support functions. The very same cells that give birth to new neurons also seem to nurture their maturation and support their function.

An unexpected form of plasticity in the adult brain is the constant generation of new neurons throughout adult life. The addition of thousands of new neurons each day may be a means of sculpting brain circuitry. This turnover has important implications not only for brain function but also for brain repair and pathological conditions such as tumors. This review will cover neurogenesis and the identity of stem cells in the adult brain. The adult brain contains two main types of cells, neurons and macroglia. Macroglia comprise oligodendrocytes and astrocytes. Oligodendrocytes are the cells that form myelin sheaths around the axons of neurons and allow the rapid conduction of electrical impulses. Astrocytes are mysterious cells that have multiple functions, including maintaining homeostasis, absorbing neurotransmitters released by neurons, and providing trophic support. Astrocytes are also induced to divide in response to lesions of the brain resulting in the formation of glial scars. Almost all neurons and glia in the brain are derived from multipotent stem cells located in the germinal layers next to the ventricular cavities of the developing brain. As development proceeds, precursors become progressively more restricted

The subventricular zone (SVZ) of the lateral ventricles, the largest remaining germinal zone of the adult mammalian brain, contains an extensive network of neuroblasts migrating rostrally to the olfactory bulb. Little is known about the endogenous proliferation signals for SVZ neural stem cells or guidance cues along the migration pathway. Here we show that the receptor tyrosine kinases EphB1-3 and EphA4 and their transmembrane ligands, ephrins-B2/3, are expressed by cells of the SVZ. Electron microscopy revealed ephrin-B ligands associated with SVZ astrocytes, which function as stem cells in this germinal zone. A three-day infusion of the ectodomain of either EphB2 or ephrin-B2 into the lateral ventricle disrupted migration of neuroblasts and increased cell proliferation. These results suggest that Eph/ephrin signaling is involved in the migration of neuroblasts in the adult SVZ and in either direct or indirect regulation of cell proliferation.

Neurons continue to be born in the subventricular zone (SVZ) of the lateral ventricles of adult mice. These cells migrate as a network of chains through the SVZ and the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into mature neurons. The OB is the only known target for these neuronal precursors. Here, we show that, after elimination of the OB, the SVZ and RMS persist and become dramatically larger. The proportion of dividing [bromodeoxyuridine (BrdU)-labeled] or dying (pyknotic or terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeled) cells in the RMS was not significantly affected at 3 d or 3 weeks after bulbectomy (OBX). However, by 3 months after OBX, the percentage of BrdU-labeled cells in the RMS decreased by half and that of dying cells doubled. Surprisingly, the rostral migration of precursors continued along the RMS after OBX. This was demonstrated by focal microinjections of BrdU and grafts of SVZ cells carrying LacZ under the control of a neuron-specific promoter gene. Results indicate that the OB is not essential for proliferation and the directional migration of SVZ precursors.