Neural Stem Cell Cultures Research Articles

A Growth-Promoting Signaling Component Cyclin D1 in Neural Stem Cells Has Antiastrogliogenic Function to Execute Self-Renewal

Self-renewing proliferation of neural stem cells (NSCs) is intimately linked to the inhibition of neuronal and glial differentiation, however, their molecular linkage has been poorly understood. We have proposed a model previously explaining partly this linkage, in which fibroblast growth factor 2 (FGF2) and Wnt signals cooperate to promote NSC self-renewal via β-catenin accumulation, which leads to the promotion of proliferation by lymphoid enhancer factor (LEF)/T-cell factor (TCF)-mediated cyclin D1 expression and at the same time to the inhibition of neuronal differentiation by β-catenin-mediated potentiation of Notch signaling. To fully understand the mechanisms underlying NSC self-renewal, it needs to be clarified how these growth factor signals inhibit glial differentiation as well. Here, we demonstrate that cyclin D1, a NSC growth promoting signaling component and also a common component of FGF2 and Wnt signaling pathways, inhibits astroglial differentiation of NSCs. Interestingly, this effect of cyclin D1 is mediated even though its cell cycle progression activity is blocked. Forced downregulation of cyclin D1 enhances astrogliogenesis of NSCs in culture and in vivo. We further demonstrate that cyclin D1 binds to STAT3, a transcription factor downstream of astrogliogenic cytokines, and suppresses its transcriptional activity on the glial fibrillary acidic protein (Gfap) gene. Taken together with our previous finding, we provide a novel molecular mechanism for NSC self-renewal in which growth promoting signaling components activated by FGF2 and Wnts inhibit neuronal and glial differentiation.

A defined, controlled culture system for primary bovine chromaffin progenitors reveals novel biomarkers and modulators.

We present a method to efficiently culture primary chromaffin progenitors from the adult bovine adrenal medulla in a defined, serum-free monolayer system. Tissue is dissociated and plated for expansion under support by the mitogen basic fibroblast growth factor (bFGF). The cultures, although not homogenous, contain a subpopulation of cells expressing the neural stem cell marker Hes3 that also propagate. In addition, Hes3 is also expressed in the adult adrenal medulla from where the tissue is taken. Differentiation is induced by bFGF withdrawal and switching to Neurobasal medium containing B27. Following differentiation, Hes3 expression is lost, and cells acquire morphologies and biomarker expression patterns of chromaffin cells and dopaminergic neurons. We tested the effect of different treatments that we previously showed regulate Hes3 expression and cell number in cultures of fetal and adult rodent neural stem cells. Treatment of the cultures with a combination of Delta4, Angiopoietin2, and a Janus kinase inhibitor increases cell number during the expansion phase without significantly affecting catecholamine content levels. Treatment with cholera toxin does not significantly affect cell number but reduces the ratio of epinephrine to norepinephrine content and increases the dopamine content relative to total catecholamines. These data suggest that this defined culture system can be used for target identification in drug discovery programs and that the transcription factor Hes3 may serve as a new biomarker of putative adrenomedullary chromaffin progenitor cells.

An efficient and reproducible method to culture Bergmann and cortical radial glia using textured PMMA.

Radial glia cells comprise the principal population of neural stem cells (NSC) during development. Attempts to develop reproducible radial glia and NSC culture methods have met with variable results, yielding non-adherent cultures or requiring the addition of growth factors. Recent studies demonstrated that a 2-μm patterned poly-methyl methacrylate (ln2 PMMA) grooved scaffold, by mimicking the biophysical and microtopographic properties of the embryonic NSC niche, induces the de-differentiation of glial cells into functional radial glia cells. Here we describe a method for obtaining cultures of adherent Bergmann radial glia (BRG) and cortical radial glia (CRG). The growth substrate is ln2 PMMA and the addition of growth factors is not required. Postnatal glia obtained from mouse cerebellum or cerebral cortex and grown on ln2 PMMA adopted a BRG/CRG phenotype characterized by a bipolar shape, the up-regulation of progenitor markers such as nestin and Sox2, and the down-regulation of vimentin and GFAP. Neurons cultured over the BRG/CRG aligned their processes with those of the glial shafts, thus mimicking the behavior of migrating neuronal cells. The ln2 PMMA culture method offers an ideal system for analyzing both the biochemical factors controlling the neurogenic potential of BRG/CRG and neuronal migration. The ln2 PMMA method is a reproducible system to obtain immature BRG/CRG preparations in vitro. It can be used to study the properties of CNS progenitor cells as well as the interactions between radial glia and neurons, and supports cultured progenitors for use in different applications.

Preparation, mass diffusion, and biocompatibility analysis of porous-channel controlled calcium-alginate-gelatin hybrid microbeads for in vitro culture of NSCs.

The Ca-alginate/gelatin (CAG) microbeads were prepared and evaluated through assays for their mechanical strength, permeability, and the feasibility as a cell carrier for in vitro culture of neural stem cells. The effects of different concentrations of sodium alginate, gelatin, and calcium chloride on the mechanical strength of CAG microbeads were determined using a self-made puncture force tester. Following this, the microbeads were immersed in DMEM media for a specified period to test its decay resistance. A diffusion model including a calculation formula of diffusion coefficient was built to investigate the diffusion of glucose and bovine serum albumin (BSA) through the wall of the microbeads. Furthermore, the feasibility of the microbeads for in vitro culture was identified using neural stem cells from Kunming mouse. Through a systematic approach and comprehensive analysis, the optimal gelatin conditions for microbead preparation were determined; the final combination of parameters of 1.5% (wt%) sodium alginate (SA), 0.5% (wt%) gelatin, and 4% (wt%) CaCl2 were the best conditions for NSC cultures. This experiment demonstrated that CAG microbeads had good cytocompatibility that made it suitable for the culture and successfully maintained stemness of neural stem cells.

In vitro culture and oxygen consumption of NSCs in size-controlled neurospheres of Ca-alginate/gelatin microbead

Neural stem cells (NSCs) forming neurospheres in a conventional culture tend to develop necrotic/apoptotic centers due to mass transport limitations. In this study, the internal pore structure of calcium-alginate/gelatin (CAG) microbeads was tuned and controlled to provide a suitable three-dimensional environment supporting NSC proliferation. Direct impact of three-dimensional space availability was quantified by oxygen consumption rates of NSCs and cells were cultured in three different methods: neurospheres, single cell suspension of NSCs, and encapsulated NSCs in microbeads. Our results showed that encapsulated NSCs in CAG microbeads maintained higher cell viability than in conventional culture. In addition, NSCs encapsulated in CAG microbeads preserved their original stemness and continued to express nestin, CNPase, GFAP and β-tubulin-III post-encapsulation. Oxygen consumption rates of encapsulated NSCs in CAG microbeads were the lowest as compared to the other two culture methods. The optimal cell density supporting high cell proliferation in CAG microbeads was found to be 1.5×105cells/mL. The glucose consumption curve suggests that encapsulated NSCs in microbeads had a slower growth profile. This study presents an alternative method in hybrid microbead preparation to generate a highly favorable three-dimensional cell carrier for NSCs and was successfully applied for its effective in vitro expansion.

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Precision genome engineering

Precision genome engineering

Neural Stem Cells (NSCs) in 3D Collagen Scaffolds: developing pharmacologically monitored neuroimplants for Spinal Cord Injury (SCI)

Event Abstract Back to Event Neural Stem Cells (NSCs) in 3D Collagen Scaffolds: developing pharmacologically monitored neuroimplants for Spinal Cord Injury (SCI) Alexandra Kourgiantaki1* 1 University of Crete, Medicine, Greece Spinal cord injury, a traumatic disease characterised by a massive degeneration of neural tissue, was recently targeted for neuroregenerative interventions. Our approach is the development of pharmacologically pulsed neuroimplants using 3D collagen scaffolds hosting NSCs. We aim to monitor the properties of NSCs ex vivo and in vivo, using synthetic small molecules with neuroprotective and neurogenic properties. Synthetic, highly lipophilic CNS bioavailable small molecules, synthesized by our group (microneurotrophins), bind to neurotrophins receptors (Gravanis et al, Science Signaling, 2012, Calogeropoulou et al., J Med Chem., 2009). BNN27 can specifically interact with TrkA and p75NTR receptors activating specific signalling pathways controlling neuronal cell survival and neurogenesis (Charalampopoulos et al, PNAS, 2004, Lazaridis et al., PLoS Biol., 2011). We are seeding embryonic and adult mouse NSC on collagen 3D scaffolds of different composition (collagen, chondroitin-6-sulphate and gelatin) and construction (size of pores and stiffness), testing cell behaviour (survival, proliferation or differentiation) in basal conditions or pulsed with neurotrophins and/or microneurotrophins. Using the knock in sox2-egfp mice strain and fluorescence activated cell sorting (FACS) analysis, we obtain NSCs cultures with a sox2-positive population more than 90% pure. We evaluate specific markers of proliferation (ki67) and/or differentiation (GFAP for glial cells, Tuj1 for mature neurons and O4 for oligodendrocytes): we are currently testing the possible effect of BNN27 on proliferation of cortical NSCs in 2D cultures (increased numbers of ki67 positive cells up to 12%). The composition and the structure of 3D scaffolds seem to play a significant functional role: scaffolds with a combined composition such as 50% collagen/50% gelatin and 92% collagen/8% chondroitin-6-sulphate support NSC survival since they sustain sox2 expression and propagate neurosphere formation. On the other hand, sensory neurons (Dorsal Root Ganglia, DRGs) grew better in pure collagen scaffolds with less rigid structure. Our preliminary findings suggest that microneurotrophin BNN27 enhances the proliferation of NSCs in culture. Furthermore, the structure of collagen scaffolds heavily affects cellular properties, like stemness or differentiation. Keywords: Neural Stem Cells, Collagen scaffolds, synthetic neurotrophins, Spinal Cord Injuries, differentiation and proliferation Conference: 4th NAMASEN Training Workshop - Dendrites 2014, Heraklion, Greece, 1 Jul - 4 Jul, 2014. Presentation Type: Poster presentation Topic: functional or structural plasticity and homeostasis Citation: Kourgiantaki A (2014). Neural Stem Cells (NSCs) in 3D Collagen Scaffolds: developing pharmacologically monitored neuroimplants for Spinal Cord Injury (SCI). Front. Syst. Neurosci. Conference Abstract: 4th NAMASEN Training Workshop - Dendrites 2014. doi: 10.3389/conf.fnsys.2014.05.00003 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 May 2014; Published Online: 12 Jun 2014. * Correspondence: Ms. Alexandra Kourgiantaki, University of Crete, Medicine, Heraklion, Greece, a.kourgiantaki@med.uoc.gr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Alexandra Kourgiantaki Google Alexandra Kourgiantaki Google Scholar Alexandra Kourgiantaki PubMed Alexandra Kourgiantaki Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Stimulation of Neural Stem Cell Proliferation by Inhibition of Phosphodiesterase 5

The involvement of nitric oxide (NO) and cyclic GMP (cGMP) in neurogenesis has been progressively unmasked over the last decade. Phosphodiesterase 5 (PDE5) specifically degrades cGMP and is highly abundant in the mammalian brain. Inhibition of cGMP hydrolysis by blocking PDE5 is a possible strategy to enhance the first step of neurogenesis, proliferation of neural stem cells (NSC). In this work, we have studied the effect on cell proliferation of 3 inhibitors with different selectivity and potency for PDE5, T0156, sildenafil, and zaprinast, using subventricular zone-(SVZ-) derived NSC cultures. We observed that a short- (6 h) or a long-term (24 h) treatment with PDE5 inhibitors increased SVZ-derived NSC proliferation. Cell proliferation induced by PDE5 inhibitors was dependent on the activation of the mitogen-activated protein kinase (MAPK) and was abolished by inhibitors of MAPK signaling, soluble guanylyl cyclase, and protein kinase G. Moreover, sildenafil neither activated ERK1/2 nor altered p27Kip1 levels, suggesting the involvement of pathways different from those activated by T0156 or zaprinast. In agreement with the present results, PDE5 inhibitors may be an interesting therapeutic approach for enhancing the proliferation stage of adult neurogenesis.

Direct reprogramming of porcine fibroblasts to neural progenitor cells

Direct reprogramming of porcine fibroblasts to neural progenitor cells

Extremely low‐frequency electromagnetic fields enhance the survival of newborn neurons in the mouse hippocampus

In recent years, much effort has been devoted to identifying stimuli capable of enhancing adult neurogenesis, a process that generates new neurons throughout life, and that appears to be dysfunctional in the senescent brain and in several neuropsychiatric and neurodegenerative diseases. We previously reported that in vivo exposure to extremely low-frequency electromagnetic fields (ELFEFs) promotes the proliferation and neuronal differentiation of hippocampal neural stem cells (NSCs) that functionally integrate in the dentate gyrus. Here, we extended our studies to specifically assess the influence of ELFEFs on hippocampal newborn cell survival, which is a very critical issue in adult neurogenesis regulation. Mice were injected with 5-bromo-2'-deoxyuridine (BrdU) to label newborn cells, and were exposed to ELFEFs 9 days later, when the most dramatic decrease in the number of newly generated neurons occurs. The results showed that ELFEF exposure (3.5 h/day for 6 days) enhanced newborn neuron survival as documented by double staining for BrdU and doublecortin, to identify immature neurons, or NeuN labeling of mature neurons. The effects of ELFEFs were associated with enhanced spatial learning and memory. In an in vitro model of hippocampal NSCs, ELFEFs exerted their pro-survival action by rescuing differentiating neurons from apoptotic cell death. Western immunoblot assay revealed reduced expression of the pro-apoptotic protein Bax, and increased levels of the anti-apoptotic protein Bcl-2, in the hippocampi of ELFEF-exposed mice as well as in ELFEF-exposed NSC cultures, as compared with their sham-exposed counterparts. Our results may have clinical implications for the treatment of impaired neurogenesis associated with brain aging and neurodegenerative diseases.

MRNA Transfection of Mouse and Human Neural Stem Cell Cultures

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

Chemically modified electrospun silica nanofibers for promoting growth and differentiation of neural stem cells.

In this study, pure silica nanofibers (SNFs) were fabricated by the electrospinning technique. Subsequently, the as-prepared SNFs were modified with (3-aminopropyl) trimethoxysilane (APTS) for applications in neural tissue engineering. The structure and properties of the as-prepared SNFs and the modified SNFs (SNF-APxM, x = 1-3) were evaluated with FTIR, TGA, nitrogen adsorption/desorption isotherms, and SEM. It was found that the surface hydrophilicity of SNF-APxM was lowered upon increasing the amino alkyl group content. The SEM and confocal images revealed that neural stem cells (NSCs) cultured on the electrospun SNFs and SNF-APxM substrates were elongated along the fibers in comparison to poly-d-lysine-coated (PDL-coated) substrate. In addition, a higher degree of proliferation and more responsive cells were observed for the NSCs cultured on the SNF-AP3M substrate than those on the SNFs and the PDL-coated substrates. The results indicated that the APTS-modified silica nanofibers can be potential substrates for regulating growth and differentiation of NSCs in culture.

Molecular features of neural stem cells enable their enrichment using pharmacological inhibitors of survival‐promoting kinases

Isolating a pure population of neural stem cells (NSCs) has been difficult since no exclusive surface markers have been identified for panning or FACS purification. Moreover, additional refinements for maintaining NSCs in culture are required, since NSCs generate a variety of neural precursors (NPs) as they proliferate. Here, we demonstrate that post-natal rat NPs express low levels of pro-apoptotic molecules and resist phosphatidylinositol 3'OH kinase and extracellular regulated kinase 1/2 inhibition as compared to late oligodendrocyte progenitors. Furthermore, maintaining subventricular zone precursors in LY294002 and PD98059, inhibitors of PI3K and ERK1/2 signaling, eliminated lineage-restricted precursors as revealed by enrichment for Nestin(+)/SOX-2(+) cells. The cells that survived formed neurospheres and 89% of these neurospheres were tripotential, generating neurons, astrocytes, and oligodendrocytes. Without this enrichment step, less than 50% of the NPs were Nestin(+)/SOX-2(+) and 42% of the neurospheres were tripotential. In addition, neurospheres enriched using this procedure produced 3-times more secondary neurospheres, supporting the conclusion that this procedure enriches for NSCs. A number of genes that enhance survival were more highly expressed in neurospheres compared to late oligodendrocyte progenitors. Altogether, these studies demonstrate that primitive neural precursors can be enriched using a relatively simple and inexpensive means that will facilitate cell replacement strategies using stem cells as well as other studies whose goal is to reveal the fundamental properties of primitive neural precursors.

CXCR4 prevents dispersion of granule neuron precursors in the adult dentate gyrus

Neurogenesis in the adult dentate gyrus (DG) generates new granule neurons that differentiate in the inner one-third of the granule cell layer (GCL). The migrating precursors of these neurons arise from neural stem cells (NSCs) in the subgranular zone (SGZ). Although it is established that pathological conditions, including epilepsy and stroke, cause dispersion of granule neuron precursors, little is known about the factors that regulate their normal placement. Based on the high expression of the chemokine CXCL12 in the adult GCL and its role in guiding neuronal migration in development, we addressed the function of the CXCL12 receptor CXCR4 in adult neurogenesis. Using transgenic reporter mice, we detected Cxcr4-GFP expression in NSCs, neuronal-committed progenitors, and immature neurons of adult and aged mice. Analyses of hippocampal NSC cultures and hippocampal tissue by immunoblot and immunohistochemistry provided evidence for CXCL12-promoted phosphorylation/activation of CXCR4 receptors in NSCs in vivo and in vitro. Cxcr4 deletion in NSCs of the postnatal or mature DG using Cre technology reduced neurogenesis. Fifty days after Cxcr4 ablation in the mature DG, the SGZ showed a severe reduction of Sox2-positive neural stem/early progenitor cells, NeuroD-positive neuronal-committed progenitors, and DCX-positive immature neurons. Many immature neurons were ectopically placed in the hilus and inner molecular layer, and some developed an aberrant dendritic morphology. Only few misplaced cells survived permanently as ectopic neurons. Thus, CXCR4 signaling maintains the NSC pool in the DG and specifies the inner one-third of the GCL as differentiation area for immature granule neurons.

Role of cyclic nucleotide-gated channels in the modulation of mouse hippocampal neurogenesis.

Neural stem cells generate neurons in the hippocampal dentate gyrus in mammals, including humans, throughout adulthood. Adult hippocampal neurogenesis has been the focus of many studies due to its relevance in processes such as learning and memory and its documented impairment in some neurodegenerative diseases. However, we are still far from having a complete picture of the mechanism regulating this process. Our study focused on the possible role of cyclic nucleotide-gated (CNG) channels. These voltage-independent channels activated by cyclic nucleotides, first described in retinal and olfactory receptors, have been receiving increasing attention for their involvement in several brain functions. Here we show that the rod-type, CNGA1, and olfactory-type, CNGA2, subunits are expressed in hippocampal neural stem cells in culture and in situ in the hippocampal neurogenic niche of adult mice. Pharmacological blockade of CNG channels did not affect cultured neural stem cell proliferation but reduced their differentiation towards the neuronal phenotype. The membrane permeant cGMP analogue, 8-Br-cGMP, enhanced neural stem cell differentiation to neurons and this effect was prevented by CNG channel blockade. In addition, patch-clamp recording from neuron-like differentiating neural stem cells revealed cGMP-activated currents attributable to ion flow through CNG channels. The current work provides novel insights into the role of CNG channels in promoting hippocampal neurogenesis, which may prove to be relevant for stem cell-based treatment of cognitive impairment and brain damage.

Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence.

The majority of neural stem cells (NSCs) in the adult brain are quiescent, and this fraction increases with aging. Although signaling pathways that promote NSC quiescence have been identified, the transcriptional mechanisms involved are mostly unknown, largely due to lack of a cell culture model. In this study, we first demonstrate that NSC cultures (NS cells) exposed to BMP4 acquire cellular and transcriptional characteristics of quiescent cells. We then use epigenomic profiling to identify enhancers associated with the quiescent NS cell state. Motif enrichment analysis of these enhancers predicts a major role for the nuclear factor one (NFI) family in the gene regulatory network controlling NS cell quiescence. Interestingly, we found that the family member NFIX is robustly induced when NS cells enter quiescence. Using genome-wide location analysis and overexpression and silencing experiments, we demonstrate that NFIX has a major role in the induction of quiescence in cultured NSCs. Transcript profiling of NS cells overexpressing or silenced for Nfix and the phenotypic analysis of the hippocampus of Nfix mutant mice suggest that NFIX controls the quiescent state by regulating the interactions of NSCs with their microenvironment.

36: Herpes simplex virus type 1 selectively blocks development of neuronal precursors derived from neural progenitor cell

Herpes simplex virus type 1 (HSV-1) is a common human pathogen and one of the leading causes of encephalitis (HSE). HSE patients that survive encephalitis often display severe and permanent neurological impairment. Our previous results in an animal model of HSE revealed HSV-1 infects cells in the subventricular zone (SVZ) where neural stem cells (NSCs) reside. We hypothesize the neurological sequelae observed in encephalitic patients is the result of altering the course of NSC differentiation as a consequence of HSV-1 infection of these cells. In order to test this hypothesis, mouse GFP-expressing NSCs were cultured and infected with different concentrations of HSV-1 (McKrae strain). NSCs were susceptible to HSV-1 as shown by plaque assay and immunohistochemistry. HSV-1 infection of NSC cultures resulted in the specific loss of the neuronal precursors (doublecortin+) with no significant changes in the percentage of astrocytes (GFAP+) or oligodendrocytes (GALC+). The addition of microglia or microglia/NSC conditioned media to the NSC cultures prevented the loss of neurons. Analysis of a number of transcription factors from these cultures identified phosphorylated Stat3 as correlative with protection. Suspension array analysis or ELISA of analytes including IL-1α, IL-1β, IL-6, IL-10, TNF-α, CXCL10, IFN-α, IFN-β, CSF, and IGF-1 detected IL-6, TNF-α, and CXCL10 in the conditioned media. The addition of neutralizing antibody to IL-6 was found to block the protective effect of the microglia in the NSC cultures. In vivo, HSV-1 infected mice presenting with encephalitis showed a 30–50% reduction in NSCs levels in the SVZ as determined by flow cytometry. We conclude NSCs are susceptible to HSV-1 which impacts specifically on neuronal survival without affecting the differentiation process of other NSC-derived cells. It is tempting to speculate that this loss contributes to cognitive impairment in the host.

An interview with François Guillemot

François Guillemot heads the Division of Molecular Neurobiology at the MRC National Institute of Medical Research (NIMR) in London. His research focuses on the transcriptional control of neurogenesis, adult neurogenesis and the epigenetic regulation of gene expression in neural development. François recently became an editor for Development, and we asked him about his research and career, as well as his lab’s future move to the Crick Institute.