OBJECTIVEThis study aimed to investigate the expression pattern and function of Nuclear receptor subfamily 2 group E member 1 (Nr2e1) in retinoic acid (RA)-induced brain abnormality.

METHODSThe mouse model of brain abnormality was established by administering 28 mg/kg RA, and neural stem cells (NSCs) were isolated from the mouse embryo and cultured in vitro. Nr2e1 expression was detected by whole mount in situ hybridization, RT-PCR, and Western blotting. Nr2e1 function was determined by transducing Nr2e1 shRNA into NSCs, and the effect on the sonic hedgehog (Shh) signaling pathway was assessed in the cells. In addition, the regulation of Nr2e1 expression by RA was also determined in vitro.

RESULTSNr2e1 expression was significantly downregulated in the brain and NSCs of RA-treated mouse embryos, and knockdown of Nr2e1 affected the proliferation of NSCs in vitro. In addition, a similar expression pattern of Nr2e1 and RA receptor (RAR) α was observed after treatment of NSCs with different concentrations of RA.

CONCLUSIONOur study demonstrated that Nr2e1 could be regulated by RA, which would aid a better understanding of the mechanism underlying RA-induced brain abnormality.

Animals ; Brain ; cytology ; embryology ; Cell Proliferation ; Down-Regulation ; Gene Expression Regulation ; Gene Expression Regulation, Developmental ; drug effects ; Mice ; Mice, Inbred C57BL ; Neural Stem Cells ; drug effects ; physiology ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Tretinoin ; pharmacology

The present paper is aimedto investigate the effect of basic fibroblast growth factor (bFGF) on proliferation, migration and differentiation of endogenous neural stem cell in rat cerebral cortex with global brain ischemia-reperfusion. A global brain ischemia-reperfusion model was established. Immunohistochemistry was used to observe the pathological changes and the expression of BrdU and Nestin in cerebral cortex. RT-PCR was used to measure the NSE mRNA in brain tissue. The results of measurements indicated that in sham operation group, there was no positive cell in cerebral cortex, and the content of NSE mRNA did not change. In the operation group, the expression of BrdU and Nestin increased significantly at the end of the 3rd day, and peaked on the 7th day. NSE mRNA expression did not significantly increase. In bFGF group, compared with sham operation group and model group, the number of BrdU-positive and Nestin-positive cells increased significantly at each time point (P<0. 05), and peaked at the end of the 11th day, and the content of NSE mRNA increased significantly (P<0. 05). This research demonstrated that the proliferation of endogenous neural stem cells in situ could be induced by global cerebral ischemia and reperfu- sion, and could be promoted and extended by bFGF. In additiion, bFGF might promote endogenous neural stem cells differentiated into neurons.
Animals ; Brain Ischemia ; pathology ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Cerebral Cortex ; cytology ; metabolism ; pathology ; Fibroblast Growth Factor 2 ; pharmacology ; Nestin ; metabolism ; Neural Stem Cells ; drug effects ; Rats ; Reperfusion Injury

OBJECTIVETo investigate the effects and underlying molecular mechanisms of icariin (ICA) on self-renewal and differentiation of neural stem cells (NSCs).

METHODSNSCs were derived from forebrains of mice embryos by mechanical dissociation into single cell suspension. The self-renewal of NSCs was measured by neurosphere formation assay. The proliferation of NSCs was detected by water-soluble tetrazolium (WST) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Protein expression of neuron-specific marker tubulin-βIII(TuJ1) and astrocyte-specific marker glial fibrillary acidic protein (GFAP) were measured by immunofluorescence and Western blotting. Using microarray, the differentially expressed genes (DEGs) were screened between NSCs with or without ICA treatment. The signaling pathways enriched by these DEGs and their role in mediating effects of ICA were analyzed.

RESULTSICA significantly promoted neurosphere formation of NSCs cultured in growth protocol in a dose-dependent manner and achieved the maximum effects at 100 nmol/L. ICA also increased optical absorbance value and EdU incorporation into nuclei of NSCs. ICA had no significant effects on the percentage of TuJ1 or GFAP-positive cells, and TuJ1 or GFAP protein expression in NSCs cultured in differentiation protocol. A total of 478 genes were found to be differentially regulated. Among signaling pathways significantly enriched by DEGs, mitogen activated protein kinase (MAPK) pathway was of interest. Blockade of extracellular signal-regulated kinase (ERK)/MAPK, other than p38/MAPK subfamily pathway partially abolished effects of ICA on neurosphere formation and EdU incorporation of NSCs.

CONCLUSIONICA can promote the selfrenewal of NSCs at least partially through ERK/MAPK signaling pathway.

Animals ; Cell Aggregation ; drug effects ; genetics ; Cell Differentiation ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; genetics ; Deoxyuridine ; analogs & derivatives ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Flavonoids ; pharmacology ; Gene Expression Regulation ; drug effects ; MAP Kinase Signaling System ; drug effects ; genetics ; Mice ; Neural Stem Cells ; cytology ; drug effects ; enzymology

BACKGROUNDReplacement of spiral ganglion neurons would be one prioritized step in an attempt to restore sensory neuronal hearing loss. However, the possibility that transplanted neurons could regenerate new synaptic connections to hair cells has not been explored. The objective of this study was to test whether neural stem cell (NSC)-derived neurons can form synaptic connections with hair cells in vitro.

METHODSNSCs were mechanically separated from the hippocampus in SD rat embryos (E12-E14) and cultured in a serum-free medium containing basic fibroblast growth factor and epidermal growth factor. Rat NSCs were co-cultured with explants of cochlea sensory epithelia obtained from postnatal Day 3 rats under transway filter membrane.

RESULTSAt Day 3, the NSCs began to show chemotactic differentiation and grew toward cochlea sensory epithelia. After 9-day co-culture, neurites of NSC-derived neurons predominantly elongated toward hair cells. Immunohistochemical analyses revealed the fibers overlapped with synapsin and hair cells, indicating the formation of new synaptic connections. After 14-day culture, triple staining revealed the fibers overlapped with PSD95 (postsynaptic density) which is juxtaposed with CtBP2 (presynaptic vesicle), indicating the formation of new ribbon synapse.

CONCLUSIONSNSC-derived neurons can make synaptic connections with hair cells and provide a model for studying synaptic plasticity and regeneration. Whether the newly forming synapse is functional merits further electrophysiological study.

Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Epidermal Growth Factor ; pharmacology ; Fibroblast Growth Factor 2 ; pharmacology ; Hair Cells, Auditory ; cytology ; drug effects ; ultrastructure ; Neural Stem Cells ; cytology ; drug effects ; ultrastructure ; Neurons ; cytology ; drug effects ; ultrastructure ; Rats ; Rats, Sprague-Dawley ; Synapses ; drug effects ; metabolism

Alzheimer's disease (AD) is the most common cause of age-related dementia. The neuropathological hallmarks of AD include extracellular deposition of amyloid-beta peptides and neurofibrillary tangles that lead to intracellular hyperphosphorylated tau in the brain. Soluble amyloid-beta oligomers are the primary pathogenic factor leading to cognitive impairment in AD. Neural stem cells (NSCs) are able to self-renew and give rise to multiple neural cell lineages in both developing and adult central nervous systems. To explore the relationship between AD-related pathology and the behaviors of NSCs that enable neuroregeneration, a number of studies have used animal and in vitro models to investigate the role of amyloid-beta on NSCs derived from various brain regions at different developmental stages. However, the Abeta effects on NSCs remain poorly understood because of conflicting results. To investigate the effects of amyloid-beta oligomers on human NSCs, we established amyloid precursor protein Swedish mutant-expressing cells and identified cell-derived amyloid-beta oligomers in the culture media. Human NSCs were isolated from an aborted fetal telencephalon at 13 weeks of gestation and expanded in culture as neurospheres. Human NSCs exposure to cell-derived amyloid-beta oligomers decreased dividing potential resulting from senescence through telomere attrition, impaired neurogenesis and promoted gliogenesis, and attenuated mobility. These amyloid-beta oligomers modulated the proliferation, differentiation and migration patterns of human NSCs via a glycogen synthase kinase-3beta-mediated signaling pathway. These findings contribute to the development of human NSC-based therapy for AD by elucidating the effects of Abeta oligomers on human NSCs.
Amyloid beta-Peptides/*pharmacology ; Animals ; Apoptosis ; Cell Aging ; Cell Movement ; Cell Proliferation ; Culture Media, Conditioned/chemistry/pharmacology ; Fetus/cytology ; Glycogen Synthase Kinase 3/*metabolism ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred C57BL ; Neural Stem Cells/*drug effects/metabolism/physiology ; Signal Transduction ; Telomere Shortening

OBJECTIVETo observe the effects of ginseng and Ligustrazine drug containing serum on the proliferation, vitality, and extracellular-signal-responsive kinase (ERK) pathway in neural stem cells undergoing in vitro oxygen-glucose deprivation/reoxygenation culture.

METHODSThe cultured neural stem cells were randomly divided into 5 groups, i.e., the normal control group (Group A), the oxygen-glucose deprivation/reoxygenation group (Group B), the oxygen-glucose deprivation/reoxygenation +ginseng serum group (Group C), the oxygen-glucose deprivation/reoxygenation + Ligustrazine serum group (Group D), and oxygen-glucose deprivation/reoxygenation +ginseng and Ligustrazine drug serum group (Group E).The protein expression levels of ERK and phosphorylated ERK (p-ERK) were observed using immunoblotting. The proliferation of neural stem cells was observed using 5-bromodeoxyuridine (BrdU) incorporation assay. The vitality of neural stem cells was detected using methyl thiazolyl tetrazolium (MTT) colorimetry.

RESULTSThe p-ERK level increased transiently at 10 min and 30 min after reoxygenation, but it decreased to the normal level at 4 h, 6 h, and 1 day, respectively. Compared with Group B, the p-ERK level at 6 h after reoxygenation could be elevated in Group C, D, and E. The proliferation and the vitality of neural stem cells at 1 day after reoxygenation could be enhanced. Furthermore, the effects of combination of ginseng and Ligusticum were better than those of using ginseng or Ligusticum alone.

CONCLUSIONSCombination of ginseng and Ligusticum could promote the proliferation and vitality of rats' neural stem cells undergoing oxygen-glucose deprivation/reoxygenation culture through ERK signal pathway. Its effects was better than that of using ginseng or Ligusticum alone.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Ligusticum ; chemistry ; MAP Kinase Signaling System ; drug effects ; Male ; Neural Stem Cells ; cytology ; drug effects ; metabolism ; Panax ; chemistry ; Phosphorylation ; Pyrazines ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo screen out main molecular target promoting human neural stem cells (NSCs) of ginsenoside Rg1 by using the gene chip technology.

METHODFirst, MTT assay was adopted to screen out the optimal concentration of Rg1-promoted NSC proliferation (120 mg x L(-1)). Then, on the 7th day after the Rg1-promoted NSC proliferation, the expression of target genes was observed by the gene chip technology. The most important target gene and signal transduction pathways were screened out through the data calculations.

RESULTOn the 7th day after the Rg1-promoted NSC proliferation, obtained 440 differential genes, 266 significantly upregulated genes and 174 significantly down-regulated genes. HES1 gene, CAMP (cyclic adenosine monophosphate)-PKA (protein kinase A) and PI3K (phosphatidylinositol 3 kinase)-AKT signal transduction pathways were closely related to the NSC proliferation.

CONCLUSIONThe differentially expressed genes screened out by gene chip may provide new clues for studies on molecular mechanism of ginsenoside Rg1-promoted NSCs proliferation.

Cell Proliferation ; drug effects ; Ginsenosides ; pharmacology ; Humans ; Neural Stem Cells ; cytology ; drug effects ; metabolism ; Oligonucleotide Array Sequence Analysis ; RNA ; genetics ; isolation & purification

OBJECTIVETo investigate effects of paraquat on the mRNA expression of key elements of Notch signaling (Notch1, Jagged1 and DTX1) during differentiation process of human neural stem cells (hNSCs).

METHODShNSCs exposed to PQ at the concentrations 0.10, 1.00, 10.00 M. Cell proliferation ability was assessed using MTT assay and mRNA expressions of Notch1, Jagged1 and DTX1 were detected by Real-time RT-PCR at 2, 4, 8, 12 d of differentiation.

RESULTSCompared with control group, NOTCH1, JAG1 mRNA expression levels exposed to PQ at the concentration of 0.10 M significantly reduced at 2, 4, 8 d and significantly went up at 12d (P < 0.01). Compared with control group, NOTCH1, JAG1 and DTX1 mRNA expression levels exposed to PQ at the concentration of 10.00 M significantly reduced at 2, 8, 12 d (P < 0.01). PQ could down-regulate Notch1, Jagged1 and DTX1 mRNA expressions at the early stage of differentiation, then up-regulate Notch1 mRNA expression, and down-regulate Notch1, Jagged1 and DTX1 mRNA expressions at the end of differentiation.

CONCLUSIONNotch signaling pathway may be involved in differentiation of neural stem cell exposed to PQ.

Calcium-Binding Proteins ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; drug effects ; metabolism ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Jagged-1 Protein ; Membrane Proteins ; metabolism ; Neural Stem Cells ; cytology ; drug effects ; metabolism ; Paraquat ; pharmacology ; Receptor, Notch1 ; metabolism ; Serrate-Jagged Proteins ; Signal Transduction ; drug effects ; Ubiquitin-Protein Ligases ; metabolism

Neural stem cells (NSCs) have been suggested as a groundbreaking solution for stroke patients because they have the potential for self-renewal and differentiation into neurons. The differentiation of NSCs into neurons is integral for increasing the therapeutic efficiency of NSCs during inflammation. Apoptosis signal-regulating kinase 1 (ASK1) is preferentially activated by oxidative stress and inflammation, which is the fundamental pathology of brain damage in stroke. ASK1 may be involved in the early inflammation response after stroke and may be related to the differentiation of NSCs because of the relationship between ASK1 and the p38 mitogen-activated protein kinase pathway. Therefore, we investigated whether ASK1 is linked to the differentiation of NSCs under the context of inflammation. On the basis of the results of a microarray analysis, we performed the following experiments: western blot analysis to confirm ASK1, DCX, MAP2, phospho-p38 expression; fluorescence-activated cell sorting assay to estimate cell death; and immunocytochemistry to visualize and confirm the differentiation of cells in brain tissue. Neurosphere size and cell survival were highly maintained in ASK1-suppressed, lipopolysaccharide (LPS)-treated brains compared with only LPS-treated brains. The number of positive cells for MAP2, a neuronal marker, was lower in the ASK1-suppressed group than in the control group. According to our microarray data, phospho-p38 expression was inversely linked to ASK1 suppression, and our immunohistochemistry data showed that slight upregulation of ASK1 by LPS promoted the differentiation of endogenous, neuronal stem cells into neurons, but highly increased ASK1 levels after cerebral ischemic damage led to high levels of cell death. We conclude that ASK1 is regulated in response to the early inflammation phase and regulates the differentiation of NSCs after inflammatory-inducing events, such as ischemic stroke.
Animals ; Cell Death ; Infarction, Middle Cerebral Artery/*metabolism ; Lipopolysaccharides/pharmacology ; MAP Kinase Kinase Kinase 5/genetics/*metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Microtubule-Associated Proteins/genetics/metabolism ; Neural Stem Cells/cytology/drug effects/*metabolism ; *Neurogenesis ; Neuropeptides/genetics/metabolism ; p38 Mitogen-Activated Protein Kinases/genetics/metabolism

Human embryonic stem cells (hESCs) are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages, including neural stem (NS) cells. Diverse regulatory signaling pathways of neural stem cells differentiation have been discovered, and this will be of great benefit to uncover the mechanisms of neuronal differentiation in vivo and in vitro. However, the limitations of hESCs resource along with the religious and ethical concerns impede the progress of ESCs application. Therefore, the induced pluripotent stem cells (iPSCs) via somatic cell reprogramming have opened up another new territory for regenerative medicine. iPSCs now can be derived from a number of lineages of cells, and are able to differentiate into certain cell types, including neurons. Patient-specifi c iPSCs are being used in human neurodegenerative disease modeling and drug screening. Furthermore, with the development of somatic direct reprogramming or lineage reprogramming technique, a more effective approach for regenerative medicine could become a complement for iPSCs.
Cell Differentiation ; Cell Lineage ; Cell Transdifferentiation ; Cellular Reprogramming ; drug effects ; Embryonic Stem Cells ; cytology ; Humans ; Induced Pluripotent Stem Cells ; cytology ; transplantation ; Neural Stem Cells ; cytology ; transplantation ; Neurodegenerative Diseases ; therapy ; Regenerative Medicine ; Transcription Factors ; genetics ; metabolism