Mouse Embryonic Neural Stem Cells Research Articles

GPE Promotes the Proliferation and Migration of Mouse Embryonic Neural Stem Cells and Their Progeny In Vitro.

This study was designed to investigate a possible role of the N-terminal tripeptide of insulin-like growth factor-1 (IGF-I), Gly-Pro-Glu (GPE), physiologically generated in neurons following IGF-I-specific cleavage, in promoting neural regeneration after an injury. Primary cultures of mouse neural stem cells (NSCs), obtained from 13.5 Days post-conception (dpc) mouse embryos, were challenged with either GPE, growth hormone (GH), or GPE + GH and the effects on cell proliferation, migration, and survival were evaluated both under basal conditions and in response to a wound healing assay. The cellular pathways activated by GPE were also investigated by using specific chemical inhibitors. The results of the study indicate that GPE treatment promotes the proliferation and the migration of neural stem cells in vitro through a mechanism that involves the activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase PI3K-Akt pathways. Intriguingly, both GPE effects and the signaling pathways activated were similar to those observed after GH treatment. Based upon the results obtained from this study, GPE, as well as GH, may be useful in promoting neural protection and/or regeneration after an injury.

C-Jun Amino-Terminal Kinase is Involved in Valproic Acid-Mediated Neuronal Differentiation of Mouse Embryonic NSCs and Neurite Outgrowth of NSC-Derived Neurons

Valproic acid (VPA), an anticonvulsant and mood-stabilizing drug, can induce neuronal differentiation, promote neurite extension and exert a neuroprotective effect in central nervous system (CNS) injuries; however, comparatively little is known regarding its action on mouse embryonic neural stem cells (NSCs) and the underlying molecular mechanism. Recent studies suggested that c-Jun N-terminal kinase (JNK) is required for neurite outgrowth and neuronal differentiation during neuronal development. In the present study, we cultured mouse embryonic NSCs and treated the cells with 1 mM VPA for up to 7 days. The results indicate that VPA promotes the neuronal differentiation of mouse embryonic NSCs and neurite outgrowth of NSC-derived neurons; moreover, VPA induces the phosphorylation of c-Jun by JNK. In contrast, the specific JNK inhibitor SP600125 decreased the VPA-stimulated increase in neuronal differentiation of mouse embryonic NSCs and neurite outgrowth of NSC-derived neurons. Taken together, these results suggest that VPA promotes neuronal differentiation of mouse embryonic NSCs and neurite outgrowth of NSC-derived neurons. Moreover, JNK activation is involved in the effects of VPA stimulation.

Sex-Specific Effects of Testosterone on the Sexually Dimorphic Transcriptome and Epigenome of Embryonic Neural Stem/Progenitor Cells

The mechanisms by which sex differences in the mammalian brain arise are poorly understood, but are influenced by a combination of underlying genetic differences and gonadal hormone exposure. Using a mouse embryonic neural stem cell (eNSC) model to understand early events contributing to sexually dimorphic brain development, we identified novel interactions between chromosomal sex and hormonal exposure that are instrumental to early brain sex differences. RNA-sequencing identified 103 transcripts that were differentially expressed between XX and XY eNSCs at baseline (FDR = 0.10). Treatment with testosterone-propionate (TP) reveals sex-specific gene expression changes, causing 2854 and 792 transcripts to become differentially expressed on XX and XY genetic backgrounds respectively. Within the TP responsive transcripts, there was enrichment for genes which function as epigenetic regulators that affect both histone modifications and DNA methylation patterning. We observed that TP caused a global decrease in 5-methylcytosine abundance in both sexes, a transmissible effect that was maintained in cellular progeny. Additionally, we determined that TP was associated with residue-specific alterations in acetylation of histone tails. These findings highlight an unknown component of androgen action on cells within the developmental CNS, and contribute to a novel mechanism of action by which early hormonal organization is initiated and maintained.

Donepezil promotes differentiation of neural stem cells into mature oligodendrocytes at the expense of astrogenesis.

Oligodendrocytes are the myelin-forming cells of the central nervous system. Oligodendrocyte loss and failure of myelin development result in serious human disorders, including multiple sclerosis. Previously, using oligodendrocyte progenitor cells, we have shown that donepezil, which is an acetylcholinesterase inhibitor developed for the treatment of Alzheimer's disease, stimulates myelin gene expression and oligodendrocyte differentiation. Here, we aimed to analyze the effects of donepezil on primary mouse embryonic neural stem cells (NSCs). Donepezil treatment led to impaired self-renewal ability and increased apoptosis. These effects appeared to be mediated through the Akt/Bad signaling pathway. Using neurosphere differentiation analysis, we observed that donepezil leads to reduced numbers of astrocytes and increased numbers of oligodendrocytes and neurons. Consistent with this finding, mRNA and protein levels for the oligodendrocyte markers myelin-associated glycoprotein, 2', 3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and myelin basic protein, as well as the neuronal marker β-tubulin type III (Tuj1) were up-regulated. In contrast, the expression of the astrocyte marker glial fibrillary acidic protein (GFAP) was down-regulated by donepezil in a dose- and time-dependent manner. Moreover, donepezil increased oligodendrocyte differentiation, resulting in a reduction in the differentiation of NSCs into astrocytes, by suppressing the activation of signal transducer and activator of transcription 3 (STAT3), SMAD1/5/9, and the downstream target gene GFAP, even under astrocyte-inducing conditions. These results suggest that efficient differentiation of NSCs into oligodendrocytes by donepezil may indicate a novel therapeutic role for this drug in promoting repair in demyelinated lesions in addition to its role in preventing astrogenesis.

Deubiquitinating enzymes regulate Hes1 stability and neuronal differentiation.

Hairy and enhancer of split 1 (Hes1), a basic helix-loop-helix transcriptional repressor protein, regulates the maintenance of neural stem/progenitor cells by repressing proneural gene expression via Notch signaling. Previous studies showed that Hes1 expression oscillates in both mouse embryonic stem cells and neural stem cells, and that the oscillation contributes to their potency and differentiation fates. This oscillatory expression depends on the stability of Hes1, which is rapidly degraded by the ubiquitin/proteasome pathway. However, the detailed molecular mechanisms governing Hes1 stability remain unknown. We analyzed Hes1-interacting deubiquitinases purified from mouse embryonic stem cells using an Hes1-specific antibody, and identified the ubiquitin-specific protease 27x (Usp27x) as a new regulator of Hes1. We found that Hes1 was deubiquitinated and stabilized by Usp27x and its homologs ubiquitin-specific protease 22 (Usp22) and ubiquitin-specific protease 51 (Usp51). Knockdown of Usp22 shortened the half-life of Hes1, delayed its oscillation, and enhanced neuronal differentiation in mouse developing brain, whereas mis-expression of Usp27x reduced neuronal differentiation. These results suggest that these deubiquitinases modulate Hes1 protein dynamics by removing ubiquitin molecules, and thereby regulate neuronal differentiation of stem cells.

Milk fat globule-EGF factor VIII attenuates CNS injury by promoting neural stem cell proliferation and migration after cerebral ischemia.

The mediators in activating neural stem cells during the regenerative process of neurogenesis following stroke have not been fully identified. Milk fat globule-EGF Factor VIII (MFG-E8), a secreted glycoprotein serves several cellular functions by binding to its receptor, αv β3-integrin. However, its role in regulating neural stem cells after stroke has not been determined yet. We therefore, aim to reveal whether MFG-E8 promotes neural stem cell proliferation and migration during stroke. Stroke was induced in wild-type (Wt) and MFG-E8-deficinet (Mfge8-/-) mice by transient middle cerebral artery occlusion (tMCAO). Commercially available recombinant mouse MFG-E8 (rmMFG-E8) was used for mechanistic assays in neural stem cell line, while the in house prepared recombinant human MFG-E8 (rhMFG-E8) was used for in vivo administration into rats with tMCAO. The in vitro effects of recombinant rmMFG-E8 for the neural stem cell proliferation and migration were determined by BrdU and transwell migration assay, respectively. The expression of cyclin D2, p53 and netrin-1, was analyzed by qPCR. We report that the treatment of rhMFG-E8 significantly improved the neurological deficit score, body weight lost and neural stem cell proliferation in a rat model of tMCAO. Conversely, decreased neural stem cell proliferation was observed in Mfge8-/- mice in comparison with the Wt counterparts underwent tMCAO. rmMFG-E8 stimulated the proliferation of mouse embryonic neural stem cells via upregulation of cyclin D2 and downregulation of p53, which is mediated by αv β3-integrin. rmMFG-E8 also promoted mouse embryonic neural stem cell migration via αv β3-integrin dependent manner in upregulating netrin-1. Our findings suggest MFG-E8 to promote neural stem cell proliferation and migration, which therefore establishes a promising therapeutic strategy for cerebral ischemia.

Transcriptomic study of mouse embryonic neural stem cell differentiation under ethanol treatment.

Neural stem cells (NSCs) can be differentiated into one of three cell lineages: neurons, astrocytes or, oligodendrocytes. Some neurotoxins have the ability to deregulate this dynamic process. NSC cell fate can be altered by ethanol as reported previously. Our aim was to investigate the alteration of genes by ethanol during NSC differentiation and to explore the molecular mechanism underlying this phenomenon. Here, mouse fetal forebrain derived NSCs were differentiated for 2 days with or without of ethanol (50 mM). We performed a comparative microarray analysis at day two using GeneChip(®) Mouse Genome 430A 2.0 arrays. Microarray analysis showed that the expressions of 496 genes were altered by ethanol (56 and 440 were up- and down-regulated, respectively). Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed the association of the following altered genes in the Wnt signaling pathway: Wnt5a, Csnk2a1, Tcf7l2, Ccnd2, Nlk, Tbl1x, Tbl1xr1, Rac2 and Nfatc3. Quantitative real time PCR analysis also demonstrated the relative expression levels of these genes. As Wnt signaling is a player of brain development, ethanol-induced alterations may contribute to improper development of the brain. Our data could be a useful resource for elucidating the mechanism behind the ethanol neurotoxicity in developing brain.

The histone deacetylase inhibitor trichostatin A induces neurite outgrowth in PC12 cells via the epigenetically regulated expression of the nur77 gene

Histone deacetylase (HDAC) inhibitors induce histone acetylation and gene expression by changing local chromatin structures. They can thereby influence various cells to proliferate or differentiate. It has been reported that trichostatin A (TSA) or valproic acid (VPA) can induce the neuronal differentiation of mouse embryonic neural stem cells and rat cerebellar granule cells. It is unclear however which gene is responsible for the neuronal differentiation induced by HDAC inhibitors. In this study, we investigated the contribution of immediate early gene (IEG) nur77 to the neuronal differentiation induced by TSA. We report that TSA induces neurite outgrowth in PC12 cells, and C646, an inhibitor of HAT (histone acetyl transferase) (p300), prevents TSA-induced neurite formation. The acetylation of the Lys14 residue of histone H3, and mRNA and protein expression of nur77 gene were found to be stimulated after treatment with TSA, but not in the presence of C646. A knock-down of nur77 inhibits the neurite outgrowth induced by TSA. Furthermore, the ectopic expression of nur77 significantly elicits neurite formation in PC12 cells. These results suggest that the expression of nur77, which is up-regulated via the TSA-induced acetylation of Lys14 on histone H3, is essential for the neuronal differentiation in TSA-induced PC12 cells.

Global transcriptome profiling of genes that are differentially regulated during differentiation of mouse embryonic neural stem cells into astrocytes.

Many genes are associated with the differentiation of neural stem cells (NSCs) into astrocytes, the most abundant and functionally diverse population of glial cells in the central nervous system, particularly in the brain. In the present study, we differentiated NSCs from the forebrain of embryonic day 14.5 mouse embryos into astrocytes over 1 and 7 days. We identified transcriptomes of NSCs and astrocytes using RNA sequencing and analyzed enriched gene networks, signal pathways, and ontology. To identify important regulators of differentiation, we performed gene clustering according to expression patterns and promoter CG types. Our data show that genes related to system development, including Fbln2, Bcan, Ncam1, Itih3, Tnr, and Vcan, regulate NSC differentiation through WNT/beta-catenin and epithelial to mesenchymal transition pathways. We identified many CG-rich promoter genes related to basic cellular maintenance such as transcription, translation, and structural components and CG-poor promoter genes that are highly associated with cell-type-specific functions or play important roles during development. Our study provides a foundation for further research on NSC differentiation and the future application of stem cells.

MicroRNA miR-124 Controls the Choice between Neuronal and Astrocyte Differentiation by Fine-tuning Ezh2 Expression

Polycomb group protein Ezh2 is a histone H3 Lys-27 histone methyltransferase orchestrating an extensive epigenetic regulatory program. Several nervous system-specific genes are known to be repressed by Ezh2 in stem cells and derepressed during neuronal differentiation. However, the molecular mechanisms underlying this regulation remain poorly understood. Here we show that Ezh2 levels are dampened during neuronal differentiation by brain-enriched microRNA miR-124. Expression of miR-124 in a neuroblastoma cells line was sufficient to up-regulate a significant fraction of nervous system-specific Ezh2 target genes. On the other hand, naturally elevated expression of miR-124 in embryonic carcinoma cells undergoing neuronal differentiation correlated with down-regulation of Ezh2 levels. Importantly, overexpression of Ezh2 mRNA with a 3'-untranslated region (3'-UTR) lacking a functional miR-124 binding site, but not with the wild-type Ezh2 3'-UTR, hampered neuronal and promoted astrocyte-specific differentiation in P19 and embryonic mouse neural stem cells. Overall, our results uncover a molecular mechanism that allows miR-124 to balance the choice between alternative differentiation possibilities through fine-tuning the expression of a critical epigenetic regulator.

Nardosinone Improves the Proliferation, Migration and Selective Differentiation of Mouse Embryonic Neural Stem Cells

In this study, we investigated the impact of Nardosinone, a bioactive component in Nardostachys root, on the proliferation and differentiation of neural stem cells. The neural stem cells were isolated from cerebrums of embryonic day 14 CD1 mice. The proliferation of cells was monitored using the cell counting kit-8 assay, bromodeoxyuridine incorporation and cell cycle analysis. Cell migration and differentiation were investigated with the neurosphere assay and cell specific markers, respectively. The results showed that Nardosinone promotes cells proliferation and increases cells migration distance in a dose-dependent manner. Nardosinone also induces the selective differentiation of neural stem cells to neurons and oligodendrocytes, as indicated by the expression of microtubule-associated protein-2 and myelin basic protein, respectively. Nardosinone also increases the expression of phospho-extracellular signal-regulated kinase and phospho-cAMP response element binding protein during proliferation and differentiation. In conclusion, this study reveals the regulatory effects of Nardosinone on neural stem cells, which may have significant implications for the treatment of brain injury and neurodegenerative diseases.

Effects of mitogens on mouse embryonic stem cell-derived neural stem cells

Effects of mitogens on mouse embryonic stem cell-derived neural stem cells

Analysis of Epigenetic Factors in Mouse Embryonic Neural Stem Cells Exposed to Hyperglycemia

BackgroundMaternal diabetes alters gene expression leading to neural tube defects (NTDs) in the developing brain. The mechanistic pathways that deregulate the gene expression remain unknown. It is hypothesized that exposure of neural stem cells (NSCs) to high glucose/hyperglycemia results in activation of epigenetic mechanisms which alter gene expression and cell fate during brain development.Methods and FindingsNSCs were isolated from normal pregnancy and streptozotocin induced-diabetic pregnancy and cultured in physiological glucose. In order to examine hyperglycemia induced epigenetic changes in NSCs, chromatin reorganization, global histone status at lysine 9 residue of histone H3 (acetylation and trimethylation) and global DNA methylation were examined and found to be altered by hyperglycemia. In NSCs, hyperglycemia increased the expression of Dcx (Doublecortin) and Pafah1b1 (Platelet activating factor acetyl hydrolase, isoform 1b, subunit 1) proteins concomitant with decreased expression of four microRNAs (mmu-miR-200a, mmu-miR-200b, mmu-miR-466a-3p and mmu-miR-466 d-3p) predicted to target these genes. Knockdown of specific microRNAs in NSCs resulted in increased expression of Dcx and Pafah1b1 proteins confirming target prediction and altered NSC fate by increasing the expression of neuronal and glial lineage markers.Conclusion/InterpretationThis study revealed that hyperglycemia alters the epigenetic mechanisms in NSCs, resulting in altered expression of some development control genes which may form the basis for the NTDs. Since epigenetic changes are reversible, they may be valuable therapeutic targets in order to improve fetal outcomes in diabetic pregnancy.

Huperzine A promotes hippocampal neurogenesis in vitro and in vivo

Huperzine A (Hup A) is a lycopodium alkaloid from Huperzia serrata, which has been used as a therapeutic agent in several neurological disorders. Despite the diverse pharmacological activities Hup A has, its role in hippocampal neurogenesis remains to be established. This study showed that Hup A not only promoted the proliferation of cultured mouse embryonic hippocampal neural stem cells (NSCs), but also increased the newly generated cells in the subgranular zone (SGZ) of the hippocampus in adult mice. Furthermore, the in vitro findings indicated that low concentrations of Hup A stimulated the proliferation of cultured NSCs, whereas extremely high concentration of it decreased the cell proliferation. Hup A activated mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, which was a well-known regulator of biological processes including cell proliferation and differentiation. ERK inhibitor dramatically inhibited the proliferative effect of Hup A on NSCs. Administration of Hup A to adult mice significantly enhanced the cell proliferation in dentate gyrus of hippocampus, and increased the remaining newborn cells 4 weeks after the drug administration. Moreover, the newly generated BrdU+/NeuN+ neurons were also increased by Hup A treatment. These findings suggest a novel role of Hup A in neurogenesis and provide a new insight into its therapeutic effects in neurological disorders via a neurogenesis-related mechanism.

Embryonic Stem Cell‐Derived Neural Stem Cells Fuse with Microglia and Mature Neurons

Transplantation of neural stem cells (NSCs) is a novel strategy to restore function in the diseased brain, acting through multiple mechanisms, for example, neuronal replacement, neuroprotection, and modulation of inflammation. Whether transplanted NSCs can operate by fusing with microglial cells or mature neurons is largely unknown. Here, we have studied the interaction of a mouse embryonic stem cell-derived neural stem (NS) cell line with rat and mouse microglia and neurons in vitro and in vivo. We show that NS cells spontaneously fuse with cocultured cortical neurons, and that this process requires the presence of microglia. Our in vitro data indicate that the NS cells can first fuse with microglia and then with neurons. The fused NS/microglial cells express markers and retain genetic and functional characteristics of both parental cell types, being able to respond to microglia-specific stimuli (LPS and IL-4/IL-13) and to differentiate to neurons and astrocytes. The NS cells fuse with microglia, at least partly, through interaction between phosphatidylserine exposed on the surface of NS cells and CD36 receptor on microglia. Transplantation of NS cells into rodent cortex results in fusion with mature pyramidal neurons, which often carry two nuclei, a process probably mediated by microglia. The fusogenic role of microglia could be even more important after NSC transplantation into brains affected by neurodegenerative diseases associated with microglia activation. It remains to be elucidated how the occurrence of the fused cells will influence the functional outcome after NSC transplantation in the diseased brain.

Lewis X-carrying N-Glycans Regulate the Proliferation of Mouse Embryonic Neural Stem Cells via the Notch Signaling Pathway

Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into brain-forming cells. Several signaling pathways have been shown to be involved in the fate determination process of NSCs, but the molecular mechanisms underlying the maintenance of neural cell stemness remain largely unknown. Our previous study showed that human natural killer carbohydrate epitopes expressed specifically by mouse NSCs modulate the Ras-MAPK pathway, raising the possibility of regulatory roles of glycoprotein glycans in the specific signaling pathways involved in NSC fate determination. To address this issue, we performed comparative N-glycosylation profiling of NSCs before and after differentiation in a comprehensive and quantitative manner. We found that Lewis X-carrying N-glycans were specifically displayed on undifferentiated cells, whereas pauci-mannose-type N-glycans were predominantly expressed on differentiated cells. Furthermore, by knocking down a fucosyltransferase 9 with short interfering RNA, we demonstrated that the Lewis X-carrying N-glycans were actively involved in the proliferation of NSCs via modulation of the expression level of Musashi-1, which is an activator of the Notch signaling pathway. Our findings suggest that Lewis X carbohydrates, which have so far been characterized as undifferentiation markers, actually operate as activators of the Notch signaling pathway for the maintenance of NSC stemness during brain development.

Thyroid Hormone Promotes Neuronal Differentiation of Embryonic Neural Stem Cells by Inhibiting STAT3 Signaling Through TRα1

A deficiency of maternal thyroid hormones (THs) during pregnancy may have severe impacts on fetal brain development. However, the cellular targets of THs and their underlying mechanisms are still unclear. In this study, we found that maternal hypothyroidism during pregnancy in mice inhibited neurogenesis in the embryonic telencephalon and caused learning and memory impairment in the offspring. To explore the underlying mechanisms, we treated cultured mouse embryonic neural stem cells (eNSCs) with a physiological level of 3, 5, 3'-triiodo-L-thyronine (T3). We found that T3 promoted the neuronal differentiation of eNSCs, while inhibiting astrocytic differentiation. In addition, the proliferation and maintenance of eNSCs were inhibited by T3. Furthermore, the TH receptor alpha 1 (TRα1) was detected in the eNSCs both in vivo and in vitro. Silencing TRα1 protein expression with specific siRNA eliminated the effects of T3 on eNSCs. We also found that T3 decreased STAT3 phosphorylation and STAT3-DNA binding activity through TRα1. The over expression of STAT3 attenuated the promotive effects of T3 on neuronal differentiation of eNSCs. Taken together, these results suggest that T3 promotes the neuronal differentiation of eNSCs by inhibiting STAT3 signaling activity through TRα1 and contributes to early neurogenesis in the embryonic telencephalon. Our studies reveal the physiological effects of TH in regulating eNSCs differentiation and suggest that eNSCs are one of the major cellular targets in the central nervous system by which TH influences early brain development. These findings also provide new insights into the mechanisms of neurological deficits caused by TH deficiency during embryogenesis.

Enhanced cellular uptake and long-term retention of chitosan-modified iron-oxide nanoparticles for MRI-based cell tracking

Tracking cells after therapeutic transplantation is imperative for evaluation of implanted cell fate and function. In this study, ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) were surface functionalized with water-soluble chitosan, a cationic polysaccharide that mediates enhanced endocytic uptake, endosomal escape into the cytosol, and subsequent long-term retention of nanoparticles. NP surface and chitosan were independently fluorescently labeled. Our NPs enable NP trafficking studies and determination of fate beyond uptake by fluorescence microscopy as well as tracking of labeled cells as localized regions of hypointensity in T2*-weighted magnetic resonance imaging (MRI) images. Adult rat neural stem cells (NSCs) were labeled with NPs, and assessment of NSC proliferation rates and differentiation potential revealed no significant differences between labeled and unlabeled NSCs. Significantly enhanced uptake of chitosan NPs in comparison to native NPs was confirmed by transmission electron microscopy, nuclear magnetic resonance (NMR) spectroscopy and in vitro cellular MRI at 11.7 Tesla. While only negligible fractions of native NPs enter cells, chitosan NPs appear within membranous vesicles within 2 hours of exposure. Additionally, chitosan-functionalized NPs escaped from membrane-bound vesicles within days, circumventing NP endo-lysosomal trafficking and exocytosis and hence enabling long-term tracking of labeled cells. Finally, our labeling strategy does not contain any NSC-specific reagents. To demonstrate general applicability across a variety of primary and immortalized cell types, embryonic mouse NSCs, mouse embryonic stem cells, HEK 293 kidney cells, and HeLa cervical cancer cells were additionally exposed to chitosan-USPIO NPs and exhibited similarly efficient loading as verified by NMR relaxometry. Our efficient and versatile labeling technology can support cell tracking with close to single cell resolution by MRI in vitro, for example, in complex tissue models not optically accessible by confocal or multi-photon fluorescence microscopy, and potentially in vivo, for example, in animal models of human disease or injury.

Construction and functional identification of siRNA adenoviral vector targeting HIF-1α in mouse

Objective To construct an adenoviral vector targeting hypoxic inducible factor-1α (HIF-1α) gene in mouse and to identify the effect of the adenoviral vector,AdHIF-1α-shRNA-EGFP,on the expression of HIF-1α in cultured mouse embryonic neural stem cells,and provide a basis for investigating the contributiou of HIF-1 signaling pathway in neural stem cell biology.Methods The HIF-1α template DNA sequence was designed by using bioinformatics.DNA recombinant technique was used to construct recombinant adenoviral vector,AdHIF-1α-shRNA-EGFP.Polymerase chain reaction(PCR) and sequencing were performed.The neural stem cells from frontal cortex of the mouse were transfected with AdHIF-1α-shRNA-EGFP in vitro.The efficiency of transfection was detected by flow cytometry.The transfected cells were cultured under hypoxia condition.Double immunofluorescent staining of nestin and HIF-α was performed,and the expression of HIF-1α was quantified by reverse transcription - polymerase chain reaction (RT-PCR) and Western blot analysis.Results The sequence cloned into the adenoviral vector was identical with the one synthesized chemically.The neural stem cells were transfeeted effectively by AdHIF-1α-shRNA-EGFP.The results from RT-PCR and Western blot analysis showed that the expression of HIF-1α gene and protein in the neural stem cells transfected with AdHIF-1α-shRNA-EGFP decreased by 52.05％ and 67.27％ under hypoxia condition,respectively.Conclusion AdHIF-1α -shRNA-EGFP could effectively inhibit hypoxia - induced expression of HIF-1α gene and protein in the mouse neural stem cells in vitro. Key words: Neural stem cells; Mouse; Hypoxic inducible factor-1α; Small interference RNA; Adenoviral vector

Severe Hypoxia: Consequences to Neural Stem Cells and Neurons

Multiple neurological diseases result from a pathological hypoxia in the brain, resulting in various motor, sensory or cognitive sequelae. Understanding the response of neural stem cells (NSCs) and differentiated neurons to hypoxia will help better treat such diseases. We exposed mouse embryonic primary neurons (PN) and neural stem cells to 1% O2 in vitro. Both cell types survived and retained their immunocyto-chemical markers, and neurons showed no obvious morphological changes. Microarray analysis showed that the number of genes with significantly altered expression levels was almost five-fold higher in NSCs compared to PN. NSCs displayed a clear block in G1/S phase of the cell cycle and a number of down-regulated cytokine genes. Various growth factors (e.g. neural growth factor, prolactin), involved in survival and proliferation, genes of the Notch pathway, and genes involved in glial differentiation, and cell-matrix adhesion were up-regulated. PN displayed a down-regulation of a number of genes involved in neuron-specific functions, in particular, transmitter-related (e.g. synaptic transmission, neurotransmitter transport and release, learning, adult behavior). We conclude that hypoxia 1-down-regulates genes involved in multiple neuronal functions which can negatively impact learning and memory; 2-induces a cell cycle block in NSCs; 3-can precondition NSC towards a particular differentiation potential while maintaining them fully undifferentiated.