Glutamate promotes neural stem cell proliferation by increasing the expression of vascular endothelial growth factor of astrocytes in vitro.
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The high levels of glutamate might involve in neurogenesis after brain injuries. However, the mechanisms are not fully understood. In this study, we investigated the effect of glutamate on the proliferation of rat embryonic neural stem/progenitor cells (NSCs) through regulating the vascular endothelial growth factor (VEGF) expression of astrocytes (ASTs) in vitro, and the cyclin D1 expression of NSCs. The results showed that glutamate promoted the expression and secretion of VEGF of rat astrocytes by activating group I mGluRs. Astrocyte conditioned medium-containing Glu [ACM (30%)] promoted the proliferation of embryonic NSCs compared with normal astrocyte conditioned medium+Glu [N-ACM (30%)+Glu (30 μM)] by increasing cell activity, diameter of neurospheres, bromodeoxyuridine (BrdU) incorporation and cell division; while ACM+VEGF neutralizing antibody [ACM (30%)+VEGF NAb (15 μg/ml)] significantly inhibited the proliferation of embryonic NSCs compared with ACM (30%). ACM (30%) increased the expressions of cyclin D1 and decreased cell death compared with N-ACM (30%)+Glu (30 μM). ACM (30%)+VEGF NAb (15 μg/ml) decreased the expressions of cyclin D1 and increased cell death compared with ACM (30%). These results demonstrated that glutamate could also indirectly promote the proliferation of rat embryonic NSCs through inducing the VEGF expression of ASTs in vitro, and VEGF may increase the expression of cyclin D1. These finding suggest that glutamate may be a major molecule for regulating embryonic NSC proliferation and facilitate neural repair in the process of NSC transplants after brain injuries.Keywords:
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The high levels of glutamate might involve in neurogenesis after brain injuries. However, the mechanisms are not fully understood. In this study, we investigated the effect of glutamate on the proliferation of rat embryonic neural stem/progenitor cells (NSCs) through regulating the vascular endothelial growth factor (VEGF) expression of astrocytes (ASTs) in vitro, and the cyclin D1 expression of NSCs. The results showed that glutamate promoted the expression and secretion of VEGF of rat astrocytes by activating group I mGluRs. Astrocyte conditioned medium-containing Glu [ACM (30%)] promoted the proliferation of embryonic NSCs compared with normal astrocyte conditioned medium+Glu [N-ACM (30%)+Glu (30 μM)] by increasing cell activity, diameter of neurospheres, bromodeoxyuridine (BrdU) incorporation and cell division; while ACM+VEGF neutralizing antibody [ACM (30%)+VEGF NAb (15 μg/ml)] significantly inhibited the proliferation of embryonic NSCs compared with ACM (30%). ACM (30%) increased the expressions of cyclin D1 and decreased cell death compared with N-ACM (30%)+Glu (30 μM). ACM (30%)+VEGF NAb (15 μg/ml) decreased the expressions of cyclin D1 and increased cell death compared with ACM (30%). These results demonstrated that glutamate could also indirectly promote the proliferation of rat embryonic NSCs through inducing the VEGF expression of ASTs in vitro, and VEGF may increase the expression of cyclin D1. These finding suggest that glutamate may be a major molecule for regulating embryonic NSC proliferation and facilitate neural repair in the process of NSC transplants after brain injuries.
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The question of how neural progenitor cells maintain its self-renewal throughout life is a fundamental problem in cell biology with implications in cancer, aging and neurodegenerative diseases. In this work, we have analyzed the p73 function in embryonic neural progenitor cell biology using the neurosphere (NS)-assay and showed that p73-loss has a significant role in the maintenance of neurosphere-forming cells in the embryonic brain. A comparative study of NS from Trp73−/−, p53KO, p53KO;Trp73−/− and their wild-type counterparts demonstrated that p73 deficiency results in two independent, but related, phenotypes: a smaller NS size (related to the proliferation and survival of the neural-progenitors) and a decreased capacity to form NS (self-renewal). The former seems to be the result of p53 compensatory activity, whereas the latter is p53 independent. We also demonstrate that p73 deficiency increases the population of neuronal progenitors ready to differentiate into neurons at the expense of depleting the pool of undifferentiated neurosphere-forming cells. Analysis of the neurogenic niches demonstrated that p73-loss depletes the number of neural-progenitor cells, rendering deficient niches in the adult mice. Altogether, our study identifies TP73 as a positive regulator of self-renewal with a role in the maintenance of the neurogenic capacity. Thus, proposing p73 as an important player in the development of neurodegenerative diseases and a potential therapeutic target.
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Background In the differentiation of mouse embryonic stem (ES) cells into neurons using the 5-stage method, cells in stage 4 are in general used as neural progenitors (NPs) because of their ability to give rise to neurons. The choice of stage 4 raises several questions about neural progenitors such as the type of cell types that are specifically considered to be neural progenitors, the exact time when these progenitors become capable of neurogenesis and whether neurogenesis is an independent and autonomous process or the result of an interaction between NP cells and the surrounding cells. Methodology/Principal Findings In this study, we found that the confluent monolayer cells and neural sphere like cell clusters both appeared in the culture of the first 14 days and the subsequent 6 weeks. However, only the sphere cells are neural progenitors that give rise to neurons and astrocytes. The NP cells require 14 days to mature into neural lineages fully capable of differentiation. We also found that although the confluent monolayer cells do not undergo neurogenesis, they play a crucial role in the growth, differentiation, and apoptosis of the sphere cells, during the first 14 days and long term culture, by secreted factors and direct cell to cell contact. Conclusions/Significance The sphere cells in stage 4 are more committed to developing into neural progenitors than monolayer cells. Interaction between the monolayer cells and sphere cells is important in the development of stage 4 cell characteristics.
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Abstract Cytomegalovirus (CMV) is the most common infectious cause of congenital anomalies of the CNS in humans. We recently reported that the murine cytomegalovirus (MCMV) immediate‐early (IE) gene promoter directs astrocyte‐specific expression in adult transgenic mice. In the present study, we analyzed the activation of the MCMV IE promoter in developing transgenic mouse brains and compared the activation with that of the Musashi 1 (Msi1) gene, which is expressed in neural progenitor cells, including neural stem cells. During the early phase of neurogenesis, the transgene was expressed predominantly in endothelial cells of the vessels, but not in neuroepithelial cells in which Msi1 was expressed. During later stages of gestation, expression of the transgene was largely restricted to the ventricular zone (VZ) in the CNS, similar to the expression of Msi1. In neurosphere cultures from transgenic embryos in the late phase of neurogenesis, the transgene was expressed in some cells of neurospheres expressing Msi1 and nestin. In neural precursor cells induced to differentiate from stem cells, expression of the transgene was detected in glial progenitor cells, expressing GFAP, nestin, and Msi1, but not in cells expressing MAP2 or MAG. In postnatal development, persistent expression of the transgene was observed in astrocyte lineage cells as was Msi1. These spatiotemporal changes of the MCMV IE promoter activity during development of transgenic mice correlated with susceptible sites in congenital HCMV infection. Moreover, this transgenic mouse model may provide useful model for analysis of the regulation of the switching of neuronal and astrocyte differentiation, and the maintenance of the astrocyte lineage. GLIA 35:41–52, 2001. © 2001 Wiley‐Liss, Inc.
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Objective To investigate the culture,proliferation and differentiation of human neural progenitor cells derived from embryonic brains of different age.Methods The free-floating cells were cultured as 6-9 weeks embryonic whole brain group,14-17 weeks embryonic whole brain group,14-17 weeks embryonic striatum group.The expression of Nestin,self-renewal and multipotential properties of the cell clusters were identified.The growth and proliferation of the cell aggregations of each group were observed.After the neurospheres from each group were induced,the percentage of neuron or astrocyte was investigated by immunocytochemical staining.Results The cell aggregations of each group were positive of Nestin and Brdu immunochemical stainings,and could differentiate into MAP2 or GFAP positive cells.One week in vitro,there were the most neurospheres in 14-17 weeks embryonic striatum group,and a few in 6-9 weeks embryonic whole brain group,and fewer in 14-17 weeks embryonic whole brain group.Single cell from 14-17 weeks embryonic striatum group could form cmone.After differentiation of neural progenitor cells from each group,the percentage of MAP2 or GFAP positive cells had no significant difference among the three groups.Conclusion Neural progenitor cells can be derived from embryonic brains of different age.With gestational age increasing,the neural progenitor cells become more difficult to be isolated from the whole brains.Different regions should be obtained as primary culture according to the different embryo age.The percentage of differentiated neuron or astrocyte is coincident in neural progenitor cells from embryonic brains of different age.
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Background and purpose In adult brain, it has been reported newly generated neural stem / progenitor cells survived and differentiated to mature neurons in the severely damaged area after stroke. In neonatal brain, however, it was demonstrated that neurogenesis was stimulated after hypoxia ischemia (HI) and provided neuronal progenitors to the infarct area, but only few new neurons could survive under an unfavorable microenvironment and lack of appropriate trophic support. Recently, neural stem / progenitor cells were isolated from the infarct area in adult mouse brain and elucidated its character as a neural stem / progenitor cell. However, it is still unclear whether the infarct area in neonatal brain has neural stem / progenitor cells as same feature as adult brain. Therefore, the present study was conducted to investigate whether neural stem / progenitor cells could be isolated from infarct area in neonatal rat brain, and to clear its feature. Methods HI brain injury was induced in 7-days-old rat pups by the left common carotid artery occlusion followed by 120 minutes exposure to 8% oxygen. Three days after HI, the brain is removed and sliced at 2 mm thickness, and then six columns are punched out from various infarct areas for cell culture using neurosphere method. The cell clusters are analyzed by immunocytochemistry and mRNA expression for neuron, astrocyte and oligodendrocyte. Also, the cell clusters are investigated the ability of induction for neuron, astrocyte and oligodendrocyte. Results The numbers of neurospere-like cell clusters from infarct areas at 3 days after HI are dramatically increased compared with those from sham control animals. Four and 30 days incubation of the cell clusters provided various types of cells positive for BrdU, Nestin, NG2, β III tubulin, GFAP, O4, Vimentin or Iba1. These cell clusters expressed mRNA such as Nanog, Sox2, Oct3/4 and Rex1 as same as ES cell. Also these cell clusters could be differentiated into neurons, astrocytes or oligodendrocytes, and expressed mRNA such as Nestin, βIIItubulin, GFAP and MBP. Conclusion We have isolated injury-induced neural stem / progenitor cells form ischemic area after neonatal HI. This cell has a potential to use as a source for new neurons driving CNS repair after neonatal HI.
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