Inflammatory events drive neural stem cell migration by elevating stromal-derived factor 1 alpha

Micropatterned stretching system for the investigation of mechanical tension on neural stem cells behavior

To investigate the effects of crocin on proliferation and migration of endogenous neural stem cells and the Notch1 signalling pathway in rats after cerebral ischemia reperfusion. SD rats were randomly divided into the sham operation group, model group and administration group (crocin). Middle cerebral artery occlusion (MCAO/R) was used to establish the focal cerebral ischemia reperfusion model in rat. After surgical treatment, the treatment group was treated with crocin. Quantitative polymerase chain reaction (qPCR) was used to detect the changes in the expression of Notch1, Bax and bcl-2 proteins in rat endogenous neural stem cells after cerebral ischemia reperfusion. ELISA was used to detect changes in inflammatory factors. Neural stem cells were cultured in vitro, which were divided into: the normal control group, the hypoglycaemic deprivation/reoxygenation group, hypoglycaemic deprivation/reoxygenation group with alow concentration of crocin, and hypoglycaemic deprivation/reoxygenation group with ahigh concentration of crocin. The cell proliferation assay detects cell activity. The cell migration assay tests the cell migration ability. And flow cytometry was used to determine cell apoptosis. Compared with the sham group, the Notch1 signalling pathway was activated in the model group. The expression of Notch1 in the crocin group was increased compared to the model group. Crocin can inhibit the release of inflammatory factors. The results of our experiments showed that crocin could induce the proliferation and migration of neural stem cells and inhibit the apoptosis of neural stem cells in the hypoglycaemic/reoxygenation model group. Crocin sufficiently promotes the proliferation and migration of neural stem cells and inhibits the apoptosis of these cells in rats after ischemia-reperfusion by manipulating the Notch signalling pathway.

We investigated the migration of endogenous neural stem cells (NSCs) toward an infarct lesion in a photo-thrombotic stroke model. The lesions produced by using rose bengal dye (20 mg/kg) with cold light in the motor cortex of Sprague-Dawley rats were also evaluated with sequential magnetic resonance imaging (MRI) from 30 minutes through 8 weeks. Migration of NSCs was identified by immunohistochemistry for nestin monoclonal antibody in the lesion cortex, subventricular zone (SVZ), and corpus callosum (CC). The contrast to noncontrast ratio (CNR) on MRI was greatest at 12 hours in DWI and decreased over time. By contrast, T1-weighted and T2-weighted images showed a constant CNR from the beginning through 8 weeks. MRI of the lesional cortex correlated with histopathologic findings, which could be divided into three stages: acute (edema and necrosis) within 24 hours, subacute (acute and chronic inflammatory cell infiltration) at 2 to 7 days, and chronic (gliofibrosis) at 2 to 4 weeks. The volume of the infarct was significantly reduced by reparative gliofibrosis. The number of nestin+ NSCs in the contralateral SVZ was similar to that of the ipsilateral SVZ in each group. However, the number of nestin+ NSCs in the ipsilateral cortex and CC increased at 12 hours to 3 days compared with the contralateral side (p<0.01) and was reduced significantly by 7 days (p<0.01). Active emigration of internal NSCs from the SVZ toward the infarct lesion may also contribute to decreased volume of the infarct lesion, but the self-repair mechanism by endogenous NSCs is insufficient to treat stroke causing extensive neuronal death. Further studies should be focused on amplification technologies of NSCs to enhance the collection of endogenous or transplanted NSCs for the treatment of stroke.

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.

There has been tremendous progress in understanding neural stem cell (NSC) biology, with genetic and cell biological methods identifying sequential gene expression and molecular interactions guiding NSC specification into distinct neuronal and glial populations during development. Data has emerged on the possible exploitation of NSC-based strategies to repair adult diseased brain. However, despite increased information on lineage specific transcription factors, cell-cycle regulators and epigenetic factors involved in the fate and plasticity of NSCs, understanding of extracellular cues driving the behavior of embryonic and adult NSCs is still very limited. Knowledge of factors regulating brain development is crucial in understanding the pathogenetic mechanisms of brain dysfunction. Since injury-activated repair mechanisms in adult brain often recapitulate ontogenetic events, the identification of these players will also reveal novel regenerative strategies. Here, we highlight the purinergic system as a key emerging player in the endogenous control of NSCs. Purinergic signalling molecules (ATP, UTP and adenosine) act with growth factors in regulating the synchronized proliferation, migration, differentiation and death of NSCs during brain and spinal cord development. At early stages of development, transient and time-specific release of ATP is critical for initiating eye formation; once anatomical CNS structures are defined, purinergic molecules participate in calcium-dependent neuron-glia communication controlling NSC behaviour. When development is complete, some purinergic mechanisms are silenced, but can be re-activated in adult brain after injury, suggesting a role in regeneration and self-repair. Targeting the purinergic system to develop new strategies for neurodevelopmental disorders and neurodegenerative diseases will be also discussed.

Mapping transplanted stem cell migration after a stroke: a serial, in vivo magnetic resonance imaging study

Background/Aims: Neural stem cells (NSCs) reside in a hypoxic environment, and hypoxia plays an important role in their development and differentiation. This study aimed to explore the underlying mechanisms by which hypoxia affects NSC behavior. Methods: In the current study, we downloaded the gene expression dataset GSE68572 and identified the differentially expressed genes (DEGs) by analyzing high-throughput gene expression in hypoxic and normoxic NSCs. Subsequently, we analyzed these data using a combined bioinformatics approach and predicted the microRNAs (miRNAs) targeting the key gene using miRNA databases. Quantitative real-time PCR (qRT-PCR) was used to validate the expression of the top five DEGs. Results: In total, 1347 genes were identified as DEGs. We identified the predominant gene ontology categories and Kyoto Encyclopedia of Genes and Genomes pathways that were significantly over-represented in the hypoxic NSCs. A protein–protein interaction network he identification of miRNAs and their putative targets may offer new diagnostic and therapeutic strategies for liver cancer the top 10 core genes. Vascular endothelial growth factor A (VEGFA) had the highest degree and may be the key gene concerning NSC behavior under hypoxia. Further validation of the top five DEGs by qRT-PCR demonstrated that four DEGs were significantly higher and one DEG was significantly lower in the hypoxic group than in the control group. Seven miRNAs were predicted and proved to target VEGFA. Conclusion: This preliminary study can prompt the understanding of the molecular mechanisms by which hypoxia has an impact on NSC behavior and can help to optimize stem cell therapies for central nervous system injuries and diseases.

Neurogenesis involves generation of new neurons through finely tuned multistep processes, such as neural stem cell (NSC) proliferation, migration, differentiation, and integration into existing neuronal circuitry in the dentate gyrus of the hippocampus and subventricular zone. Adult hippocampal neurogenesis is involved in cognitive functions and altered in various neurodegenerative disorders, including Alzheimer disease (AD). Ethosuximide (ETH), an anticonvulsant drug is used for the treatment of epileptic seizures. However, the effects of ETH on adult hippocampal neurogenesis and the underlying cellular and molecular mechanism(s) are yet unexplored. Herein, we studied the effects of ETH on rat multipotent NSC proliferation and neuronal differentiation and adult hippocampal neurogenesis in an amyloid β (Aβ) toxin-induced rat model of AD-like phenotypes. ETH potently induced NSC proliferation and neuronal differentiation in the hippocampus-derived NSC in vitro. ETH enhanced NSC proliferation and neuronal differentiation and reduced Aβ toxin-mediated toxicity and neurodegeneration, leading to behavioral recovery in the rat AD model. ETH inhibited Aβ-mediated suppression of neurogenic and Akt/Wnt/β-catenin pathway gene expression in the hippocampus. ETH activated the PI3K·Akt and Wnt·β-catenin transduction pathways that are known to be involved in the regulation of neurogenesis. Inhibition of the PI3K·Akt and Wnt·β-catenin pathways effectively blocked the mitogenic and neurogenic effects of ETH. In silico molecular target prediction docking studies suggest that ETH interacts with Akt, Dkk-1, and GSK-3β. Our findings suggest that ETH stimulates NSC proliferation and differentiation in vitro and adult hippocampal neurogenesis via the PI3K·Akt and Wnt·β-catenin signaling.

Herein, we investigated the effect of electrospun polycaprolactone fumarate (PCLF) nanofibers on neural stem cell (NSC) behavior in the in vitro setting. Murine NSCs were isolated from adult mice subventricular zone and immunophenotyped by flow cytometry assay and immunofluorescence staining. Cells were cultured on the plastic surface, laminin-coated surface, and electrospun PCLF nanofibers. Cell morphology, attachment, and spreading were evaluated by scanning electron microscopy analysis. Cell viability and proliferation rates were evaluated by MTT assay. The proliferation of plated cells was investigated by monitoring Ki-67-positive cells using flow cytometry analysis. The protein levels of Map-2 and GFAP were detected by using immunofluorescence staining to show neural and astrocyte differentiation capacity. Scanning electron microscopy images revealed an extensive distribution, morphological adaptation, and cell-to-cell connectivity in NSCs upon culture on the PCLF surface. MTT analysis showed that the NSCs had more survival rates on the PCLF surface compared to the laminin and control groups over time (p < 0.05). In contrast to the laminin group, Ki-67 analysis showed a decrease of proliferating cells in the PCLF group. Immunofluorescence staining revealed the prominent increase of Map-2 and GFAP reduction in NSCs from the PCLF group compared to the laminin and control groups, showing the stimulatory effect of PCLF on targeted maturation of NSCs (p < 0.05). In brief, PCLF based construct promotes NSCs morphological adaptation and neuronal differentiation, suggesting PCLF as an appropriate and applicable substrate in neural tissue engineering.

To study the effects of total ginsenosides (TG) extract from Panax ginseng on neural stem cell (NSC) proliferation and differentiation and their underlying mechanisms. The migration of NSCs after treatment with various concentrations of TG extract (50, 100, or 200 µ g/mL) were monitored. The proliferation of NSCs was examined by a combination of cell counting kit-8 and neurosphere assays. NSC differentiation mediated by TG extract was evaluated by Western blotting and immunofluorescence staining to monitor the expression of nestin and microtubule associated protein 2 (MAP2). The GSK-3β/β-catenin pathway in TG-treated NSCs was examined by Western blot assay. The NSCs with constitutively active GSK-3β mutant were made by adenovirus-mediated gene transfection, then the proliferation and differentiation of NSCs mediated by TG were further verified. TG treatment significantly enhanced NSC migration (P<0.01 or P<0.05) and increased the proliferation of NSCs (P<0.01 or P<0.05). TG mediation also significantly upregulated MAP2 expression but downregulated nestin expression (P<0.01 or P<0.05). TG extract also significantly induced GSK-3β phosphorylation at Ser9, leading to GSK-3β inactivation and, consequently, the activation of the GSK-3β/β-catenin pathway (P<0.01 or P<0.05). In addition, constitutive activation of GSK-3β in NSCs by the transfection of GSK-3β S9A mutant was found to significantly suppress TG-mediated NSC proliferation and differentiation (P<0.01 or P<0.05). TG promoted NSC proliferation and neuronal differentiation by inactivating GSK-3β.

Brain damage due to stroke is a leading cause of death and long‐term disability in the US. Treatment options are limited, necessitating a better understanding of reparative and pathological processes after stroke. Ischemia stimulates the proliferation and migration of neural stem cells (NSCs) from the subventricular zone toward the ischemic lesion in mice. During this time, the migrating NSCs differentiate into GFAP positive cells and pro‐reparative glia. While the importance of this process is not fully elucidated, it is clear that limiting NSC response increases lesion size and worsens behavioral outcomes after ischemic stroke. We tested the effect of low‐density lipoprotein receptor related protein 1 (LRP1) on modulating NSC response and differentiation, and found that virtually every aspect of NSC physiology we tested was altered after deletion of LRP1; increased glial differentiation, proliferation, and self‐renewal were measured. After middle cerebral artery occlusion in mice, we observed a marked reduction in the migration of neuroblasts toward the site of the lesion, which correlated with increased gliosis and worsened behavioral outcomes. Loss of LRP1 also caused reduction in expression of the chemokine receptor CXCR4, which could account for the loss of migration. Ongoing studies involve elucidating the mechanism by which LRP1 regulates CXCR4 expression as well as understanding the effect on pro‐reparative processes after stroke.Support or Funding InformationThis research is supported by a pilot grant from the William and Ella Owen's Medical Research Foundation, and a Veteran's Health Administration Career Development Award K2BX003240.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

20th National Meeting of the British Neuroscience Association

The subventricular zone (SVZ) of the brain constitutes a niche for neural stem and progenitor cells that can initiate repair after central nervous system (CNS) injury. In a relapsing-remitting model of experimental autoimmune encephalomyelitis (EAE), the neural stem cells (NSCs) become activated and initiate regeneration during acute disease, but lose this ability during the chronic phases of disease. We hypothesized that chronic microglia activation contributes to the failure of the NSC repair potential in the SVZ. Using bromodeoxyuridine injections at different time points during EAE, we quantified the number of proliferating and differentiating progenitors, and evaluated the structure of the SVZ by electron microscopy. In vivo minocycline treatment during EAE was used to address the effect of microglia inactivation on SVZ dysfunction. In vivo treatment with minocycline, an inhibitor of microglia activation, increases stem cell proliferation in both naive and EAE animals. Minocycline treatment decreases cortical and periventricular pathology in the chronic phase of EAE, improving the proliferation of Sox2 stem cells and NG2 oligodendrocyte precursors cells originating in the SVZ and their differentiation into mature oligodendrocytes. These data suggest that failure of repair observed during chronic EAE correlates with microglia activation and that treatments targeting chronic microglial activation have the potential for enhancing repair in the CNS.

The twitcher mouse is a model of human Krabbe's disease caused by a mutation in the galacto-cerebrosidase gene. As a result of deficient catabolism of myelin, death of oligodendrocytes and demyelination occur widely in the central and peripheral nervous system, making it an ideal model for investigation of myelin repair strategies. Here we describe the use of mouse neural stem cells (NSCs) expressing enhanced green fluorescence protein (eGFP) for transplantation in neonatal normal and twitcher mice. Normal and twitcher mice in all age groups (20, 30, and 45 days old) showed engraftment and differentiation of injected cells. The engrafted cells were found in the ventricles and a wide range of regions in the brain parenchyma. There was no significant difference in the total number of cells engrafted and the pattern of engraftment between 30-day-old normal and twitcher mice. The average number of engrafted cells in the brain of a 30-day-old mouse was 964 +/- 281 (n = 8). Engrafted cells with the morphology of neurons, astrocytes, and oligodendrocytes were identified. Differentiation into oligodendrocytes was confirmed by immunohistochemical staining using a cell-type-specific marker. There was a higher percentage of cells engrafted in the grey matter than in the white matter (p < 0.01) in both normal and twitcher mouse brain. This study indicates that the environment of demyelination in 30-day-old twitcher mouse brain has not significantly altered the engraftment and distribution patterns of NSCs after neonatal transplantation.

Early stem cell aging in the mature brain.