Migration of Neural Stem Cells in Hippocampal Slices in Hypoxia Modeling

MCP-1 induces migration of adult neural stem cells

Purpose: To investigate the effect of miR-9 on the proliferation, differentiation and migration of human neural stem cells (NSCs).Methods: The expression of miR-9 was investigated by quantitative real-time polymerase chain reaction (RT-PCR). Cell proliferation was assessed by cell counting kit-8 (CCK8) assay, while cell migration was studied by Transwell assay. The effect of miR-9 on differentiation of NSCs was investigated by western blot analysis of key differentiation marker proteins. Protein expression was determined by western blotting.Results: Transfection and over-expression of miR-9 in NSCs significantly enhanced the proliferation of NSCs (p < 0.05) in a time-dependent manner, as was evident from CCK8 assay data. MiR-9 overexpression caused down-regulation of Nestin and SOX-2, and up-regulation of Tuj-1 and MAP-2. The migration of NSCs was 37 % in the cells transfected with empty vector, compared to 68 % in the cells transfected with miR-9. This effect of miR-9 on cell migration was accompanied by up-regulation of matrix metallopeptidase 9 (MMP-9) and matrix metallopeptidase 2 (MMP-2).Conclusion: These results show that miR-9 promotes the proliferation, differentiation and migration of NSCs, and thus may be an important drug target for the generation of NSCs.Keywords: Neural stem cells, MicroRNA, Mir-9, Migration, Differentiation, Proliferation

ProBDNF inhibits proliferation, migration and differentiation of mouse neural stem cells

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.

目的 研究细胞外基质成份纤维连接蛋白(Fn)对人神经干细胞迁移的作用.方法用Fn包被培养孔,于细胞球转种后的24 h、48 h、72 h、96 h、120 h、144 h、168 h和240 h各时间点动态观察神经球细胞的迁移变化,测量细胞迁移的最远距离,计算迁移速率,用荧光免疫技术检测迁移细胞的特异性表型标志,计算一定视野内nestin+细胞所占的百分比,同时,平行观察含胎牛血清(FBS)培养诱导的迁移.设立多聚赖氨酸(Ply)包被组以及空白组作为实验对照.结果Ply包被组以及空白组均无细胞迁移发生,FBS组和n组的细胞球有细胞从球体向外迁移,二者迁移速率均在前48 h达最高峰,但迁移方式不同.Fn组迁移的细胞分化不明显,细胞分裂相多见,nestin+细胞占(30.60±8.06)%,而FBS组迁移出来的细胞成进行性分化梯度,偶见有nestin+细胞,外周边缘区域GFAP+星形胶质细胞是其主要细胞群.结论Fn在体外可促进人胎脑神经干细胞的迁移。

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.

Electrical Guidance of Human Stem Cells in the Rat Brain

Many in vivo and in vitro studies have demonstrated the targeted migration of neural stem cells (NSC) to infiltrating brain tumors, including malignant glioma, highlighting a potential therapeutic approach. However, there is not enough information to apply this approach to clinical therapy. The most important things in stem cell therapy for brain tumors involve selecting the appropriate neural progenitor type and optimizing the efficiency of the cell engraftment. By histological analysis using two different live-dyes, human NSCs were shown to migrate away from the transplanted site in the direction of the expanding C6 glioma and to intermix with the tumor bed, especially with the tumor core. This intermixing occurred within 7 days when NSCs were implanted into glioma model. The time course of migratory HB1.F5 with the greatest mobility of three NSC lines was as follows. As early as 3 days after transplantation, several NSCs were found leaving the implant site, primarily approaching microsatellites and frontier cells located near the site of NSC implantation. Through 7 days post-transplantation, massive numbers of NSCs continued to be attracted to and interspersed with C6 glioma, and were finally distributed extensively throughout the whole tumor bed, including the core and penumbra of the tumor mass. However, NSCs appeared to penetrate into the tumor mass very well, whereas normal fibroblast cells could not migrate. These findings strengthen the potential for human NSCs as attractive vehicles to improve therapeutic gene delivery to cancer or glioma if they are optimized to selectively kill neoplastic cells.

Involvement of SHP2 in focal adhesion, migration and differentiation of neural stem cells

Small direct current (DC) electric fields (EFs) guide neurite growth and migration of rodent neural stem cells (NSCs). However, this could be species dependent. Therefore, it is critical to investigate how human NSCs (hNSCs) respond to EF before any possible clinical attempt. Aiming to characterize the EF-stimulated and guided migration of hNSCs, we derived hNSCs from a well-established human embryonic stem cell line H9. Small applied DC EFs, as low as 16 mV/mm, induced significant directional migration toward the cathode. Reversal of the field polarity reversed migration of hNSCs. The galvanotactic/electrotactic response was both time and voltage dependent. The migration directedness and distance to the cathode increased with the increase of field strength. (Rho-kinase) inhibitor Y27632 is used to enhance viability of stem cells and has previously been reported to inhibit EF-guided directional migration in induced pluripotent stem cells and neurons. However, its presence did not significantly affect the directionality of hNSC migration in an EF. Cytokine receptor [C-X-C chemokine receptor type 4 (CXCR4)] is important for chemotaxis of NSCs in the brain. The blockage of CXCR4 did not affect the electrotaxis of hNSCs. We conclude that hNSCs respond to a small EF by directional migration. Applied EFs could potentially be further exploited to guide hNSCs to injured sites in the central nervous system to improve the outcome of various diseases.

Objective To investigate the effects of electrical stimulation of olfactory bulb( OB) on the proliferation, migration and differentiation of neural stem cell ( NSC ) in subventricular zone. Method Forty - eight adult female Sprague - Dawley(SD) rats were randomly divided into control group, sham stimulation group and stimulation group including 6 time points(1 day,3 day,7 day, 14 day,21 day, 28 day). The rats were injected intraperitoneally with bromodeoxyuridine( BrdU) to detect the proliferation of NSC by immunohistochemistry staining. Another 18 rats were randomly divided into control group, sham stimulation group and stimulation group. Four weeks after BrdU injection, the rats were sacrificed and immunohistochemistry and immunofluorescence were used to investigate the migration and differentiation of NSC in OB. Results In SVZ, BrdU - positive cells began to increase 1 d after stimulation (17. 67 ± 1.03, P <0.01) .reached to the maximum level at 1 week(28. 50 ± 1. 87, P <0. 01) ,then decreased to normal at 3 week. Four weeks after injection of BrdU,the BrdU -positive cells significantly increased in RMS(67. 33 ±3.50, P <0.01) and OB(44.33 ±5.47, P <0.01) in stimulation group. Fluorescence double staining showed that the stimulation of OB had no effect on the differentiation of NSC into neurons or gliocytes. Conclusions Electrical stimulation of OB could promote proliferation, migration of NSC in SVZ, but it has no effect on the differentiation of NSC into neurons or gliocytes. Key words: Electrical stimulation; Olfactory bulb; Neurogenesis; Rats

It has long been asserted that failure to recover from central nervous system diseases is due to the system's intricate structure and the regenerative incapacity of adult neurons. Yet over recent decades, numerous studies have established that endogenous neurogenesis occurs in the adult central nervous system, including humans'. This has challenged the long-held scientific consensus that the number of adult neurons remains constant, and that new central nervous system neurons cannot be created or renewed. Herein, we present a comprehensive overview of the alterations and regulatory mechanisms of endogenous neurogenesis following central nervous system injury, and describe novel treatment strategies that target endogenous neurogenesis and newborn neurons in the treatment of central nervous system injury. Central nervous system injury frequently results in alterations of endogenous neurogenesis, encompassing the activation, proliferation, ectopic migration, differentiation, and functional integration of endogenous neural stem cells. Because of the unfavorable local microenvironment, most activated neural stem cells differentiate into glial cells rather than neurons. Consequently, the injury-induced endogenous neurogenesis response is inadequate for repairing impaired neural function. Scientists have attempted to enhance endogenous neurogenesis using various strategies, including using neurotrophic factors, bioactive materials, and cell reprogramming techniques. Used alone or in combination, these therapeutic strategies can promote targeted migration of neural stem cells to an injured area, ensure their survival and differentiation into mature functional neurons, and facilitate their integration into the neural circuit. Thus can integration replenish lost neurons after central nervous system injury, by improving the local microenvironment. By regulating each phase of endogenous neurogenesis, endogenous neural stem cells can be harnessed to promote effective regeneration of newborn neurons. This offers a novel approach for treating central nervous system injury.

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.

Neural stem cells (NSCs) have widely been used in the treatment of human neurological disorders as cell therapy via intracerebral or intraventricular infusion. However, the migration mechanism required for NSCs homing and recruitment remains to be elucidated. Recently, SDF-1/CXCR4 axis was shown to be responsible for in cell migration and differentiation during the neural development stage and involved in the pathophysiological process of neurological disorders. In this study, we investigated the effect of SDF-1 in migration of NSCs in vitro and in vivo. The expression of CXCR4 receptor was examined by immunocytochemistry and RT-PCR. The migratory ability of NSCs induced by SDF-1 was assessed by transwell chemotaxis assay. The traumatic brain injury rat model was well established, and the recruitment of NSCs and expression of SDF-1 were investigated in vivo. Our findings demonstrated that SDF-1, in vitro, significantly induced the migratory of NSCs in a dose-dependent manner. An overexpression of neural stem cell marker Nestin in the hippocampus was observed after TBI, and the expressions of SDF-1 surrounding the lesion areas were significantly increased. Our results suggested that the migration of NSCs was activated by chemotactic effect of SDF-1. It was also proved the relevance of SDF-1 in the migration of endogenous NSCs after brain injury. Taken together, these results demonstrated that SDF-1/CXCR4 axis may play crucial role in the migration of Nestin-positive cell after brain injury.

Objective To observe the migration of neural stem cells (NSCs) to glioma stem cells (GSC), and to investigate the chemotactic mechanism. Methods GSCs were cultured and expanded in stem cell medium from human glioma cell line U251 and surgically resected specimens of human gliomas. All the GSCs were identified by using flow cytometry and Western blotting. Migrations of NSCs to GSCs,differentiated cells and concentration gradient fluid of growth factors were detected by Transwell assay. Concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secreted by GSCs and differentiated cells were determined by using enzyme linked immunosorbent assay (ELISA). The in vitro migration of NSCs (marked by Dio) to GSCs (marked by Dil) was observed by using fluorescent microscopy. Results Neurospheres of GSCs, with high expression of stemness markers (CD133: 11.02％-33.55％ ), were cultured in stem cell medium and could be differentiated in serumcontaining medium. After differentiation, the expression of stemness markers was decreased, while the marker of glial fibrillary acidic protein (GFAP) was increased ( P ＜ 0.05). Meanwhile, the concentrations of VEGF and bFGF secreted by GSCs were higher than those by differentiated cells. Both GSCs and differentiated cells could induce the migration of NSCs, while GSCs showed significantly stronger chemotactic effect (P ＜ 0. 05). The migratory ability of NSCs might be positively correlated with the concentration of growth factors in chemotactic fluid ( P ＜ 0. 05 ). NSCs could migrate to GSCs and surround the neurosphere of GSCs, which displays cytostatic effect in vitro. Conclusion Gliona stem cells could induce the migration of NSCs and show enhanced chemotaxis compared with the differentiated cells. This chemotactic mechanism might be related to high concentrations of growth factors secreted by GSCs. Migration of NSCs to GSCs displays cytostatic effect in vitro. Key words: Neural stem cells; Glioma stem cells; Migration; Chemotactic factors