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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.

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.

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.