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1.
Article in Chinese | WPRIM | ID: wpr-828627

ABSTRACT

OBJECTIVE@#To study the effect of advanced maternal age (AMA) on the development of hippocampal neural stem cells in offspring rats.@*METHODS@#Ten 3-month-old and ten 12-month-old female Sprague-Dawley rats were housed individually with 3-month-old male rats (1:1, n=20), whose offspring rats were assigned to a control group and an AMA group. A total of 40 rats were randomly selected from each group. Immunofluorescence assay and Western blot were used to localize and determine the levels of protein expression of Nestin and doublecortin (DCX) on day 7 as well as neuronal nuclear antigen (NeuN) and glial fibrillary acidic protein (GFAP) on day 28 (n=8 for each marker). Immunofluorescence assay was also used to localize the hippocampal expression of polysialylated isoforms of neural cell adhesion molecule (PSA-NCAM) on day 14 (n=8 for each marker).@*RESULTS@#According to the Western blot results, the AMA group had significantly lower protein expression of DCX than the control group (P0.05). According to the results of immunofluorescence assay, the AMA group had significantly lower protein expression of Nestin, DCX, and PSA-NCAM in the hippocampal dentate gyrus (DG) region than the control group (P0.05). The AMA group had significantly higher expression of NeuN in the hippocampal CA1 region than the control group (P0.05). The AMA group had significantly lower expression of GFAP in the hippocampal CA1, CA3, and DG regions than the control group (P<0.05).@*CONCLUSIONS@#AMA may cause inhibition of proliferation, survival, and migration of hippocampal neural stem cells. AMA may also affect their differentiation into neurons and astrocytes, which will eventually lead to developmental disorders of hippocampal neural stem cells in offspring rats.


Subject(s)
Animals , Female , Hippocampus , Male , Maternal Age , Neural Stem Cells , Neurons , Rats , Rats, Sprague-Dawley
2.
Arq. bras. med. vet. zootec. (Online) ; 71(6): 1821-1828, Nov.-Dec. 2019. tab, graf, ilus
Article in Portuguese | LILACS, VETINDEX | ID: biblio-1055122

ABSTRACT

O objetivo deste estudo foi verificar a capacidade de diferenciação das células-tronco da polpa dentária canina em células progenitoras neurais bem como quantificar obtenção e viabilidade celular, durante três passagens em cultura. As células foram extraídas da polpa dentária de dois cadáveres caninos, com aproximadamente dez meses de idade, que foram a óbito em decorrência de traumatismo automotivo. Após três subculturas, realizou-se avaliação da viabilidade celular por quantificação em câmara de Neubauer. A partir disso, induziu-se diferenciação neural em meio de cultura neurobasal (Gibco™), com células aderidas ao plástico ou suspensas em placas tratadas com agarose. Após sete e 14 dias em cultivo indutor, observou-se morfologia e perfil imunofenotípico utilizando citometria de fluxo e imunocitoquímica fluorescente. Aos 14 dias as células apresentaram alto grau de expressão para marcadores anti-nestina e anti-glial fibrillary acidic protein (anti-GFAP). Anteriormente, obteve-se ao 25º dia, média de 18x106 células viáveis indiferenciadas oriundas do tecido pulpar. Sugere-se que as células-tronco indiferenciadas da polpa dentária canina apresentem índices satisfatórios de diferenciação em células progenitoras neurais, aderidas ou suspensas em cultura. A polpa dentária dos dentes decíduos caninos, fornece células indiferenciadas viáveis em quantidade adequada.(AU)


The objective of this study was to verify the differentiation capacity of canine tooth pulp stem cells in neural progenitor cells as well as to quantify the attainment and viability during three culture passages. The cells were extracted from the dental pulp of two canine cadavers, with approximately ten months of age, which died due to automotive trauma. After three subcultures, cell viability evaluation was performed by Neubauer chamber quantification. Neural differentiation was induced in neurobasal culture medium (Gibco ™), with cells adhered to the plastic or suspended in agarose-treated plates. After seven and 14 days in inducer culture, morphology and immunophenotypic profile were observed using flow cytometry and fluorescent immunocytochemistry. At 14 days the cells had a high degree of expression for anti-nestin and anti-glial fibrillary acidic (anti-GFAP) markers. Previously, an average of 18x106 undifferentiated viable cells from the pulp tissue were obtained on the 25th day. It is suggested that the undifferentiated canine pulp stem cells present satisfactory differentiation indices in neural progenitor cells, adhered or suspended in culture. The dental pulp of deciduous canine teeth provides viable undifferentiated cells in adequate quantity.(AU)


Subject(s)
Animals , Dogs , Dental Pulp/ultrastructure , Neural Stem Cells , Cell- and Tissue-Based Therapy/veterinary , Demyelinating Diseases/veterinary , Flow Cytometry/veterinary
3.
Article in English | WPRIM | ID: wpr-719638

ABSTRACT

Cell therapeutic agents for treating degenerative brain diseases using neural stem cells are actively being developed. However, few systems have been developed to monitor in real time whether the transplanted neural stem cells are actually differentiated into neurons. Therefore, it is necessary to develop a technology capable of specifically monitoring neuronal differentiation in vivo. In this study, we established a system that expresses cell membrane-targeting red fluorescent protein under control of the Synapsin promoter in order to specifically monitor differentiation from neural stem cells into neurons. In order to overcome the weak expression level of the tissue-specific promoter system, the partial 5′ UTR sequence of Creb was added for efficient expression of the cell surface-specific antigen. This system was able to track functional neuronal differentiation of neural stem cells transplanted in vivo, which will help improve stem cell therapies.


Subject(s)
Antigens, Surface , Brain Diseases , Neural Stem Cells , Neurons , Stem Cells
4.
Article in English | WPRIM | ID: wpr-719412

ABSTRACT

Neural stem cells (NSCs) can proliferate and differentiate into multiple cell types that constitute the nervous system. NSCs can be derived from developing fetuses, embryonic stem cells, or induced pluripotent stem cells. NSCs provide a good platform to screen drugs for neurodegenerative diseases and also have potential applications in regenerative medicine. Natural products have long been used as compounds to develop new drugs. In this review, natural products that control NSC fate and induce their differentiation into neurons or glia are discussed. These phytochemicals enable promising advances to be made in the treatment of neurodegenerative diseases.


Subject(s)
Biological Products , Embryonic Stem Cells , Fetus , Induced Pluripotent Stem Cells , Nervous System , Neural Stem Cells , Neurodegenerative Diseases , Neurogenesis , Neuroglia , Neurons , Neuroprotection , Phytochemicals , Regenerative Medicine
5.
Article in Chinese | WPRIM | ID: wpr-773505

ABSTRACT

OBJECTIVE@#To observe the effects of on the expression of β-tubulin Ⅲ and glial fibrillary acidic protein (GFAP) and the proliferation and differentiation of murine neural stem cells (NSCs) .@*METHODS@#An immortalized murine NSC line was divided into model control (MC) group, 10% drug-containing serum group (NLXT group), and 10% Naoluoxintong drug-containing serum with inhibitor Y27632 group (Y-27632 group) with corresponding treatments. The activity of the NSCs was detected after the treatments using MTT assay, and the migration of the cells was observed with Transwell assay. The expressions of β-tubulin Ⅲ, GFAP and MAP-2 proteins in the cells were detected with immunoblotting, and the expressions of DCX, NEUN, and β-tubulin Ⅲ were also detected with immunofluorescence assay.@*RESULTS@#Compared with that in MC group, the number of migrated cells in NLXT group and Y-27632 group increased significantly at 1 day and 3 days after induction ( < 0.05). The survival rate and the number of migrated cells in NLXT group and Y-27632 group increased significantly on day 7 ( < 0.01). Compared with those in MC group, the expressions of β-tubulin Ⅲ, MAP2 and GFAP protein in NLXT group and Y-27632 group were significantly increased on days 3 ( < 0.01) and 7 ( < 0.05). The numbers of β-tubulinⅢ/ GFAP, BrdU/DCX, and BrdU/NEUN labeled cells in the NLXT group and Y-27632 group were significantly greater than those in the MC group.@*CONCLUSIONS@# promotes the proliferation and differentiation of murine NSCs by regulating the expressions of β-tubulinⅢ/GFAP.


Subject(s)
Animals , Cell Differentiation , Cell Proliferation , Glial Fibrillary Acidic Protein , Mice , Neural Stem Cells , Tubulin
6.
Article in English | WPRIM | ID: wpr-772943

ABSTRACT

N-methyladenosine (mA), catalyzed by the methyltransferase complex consisting of Mettl3 and Mettl14, is the most abundant RNA modification in mRNAs and participates in diverse biological processes. However, the roles and precise mechanisms of mA modification in regulating neuronal development and adult neurogenesis remain unclear. Here, we examined the function of Mettl3, the key component of the complex, in neuronal development and adult neurogenesis of mice. We found that the depletion of Mettl3 significantly reduced mA levels in adult neural stem cells (aNSCs) and inhibited the proliferation of aNSCs. Mettl3 depletion not only inhibited neuronal development and skewed the differentiation of aNSCs more toward glial lineage, but also affected the morphological maturation of newborn neurons in the adult brain. mA immunoprecipitation combined with deep sequencing (MeRIP-seq) revealed that mA was predominantly enriched in transcripts related to neurogenesis and neuronal development. Mechanistically, mA was present on the transcripts of histone methyltransferase Ezh2, and its reduction upon Mettl3 knockdown decreased both Ezh2 protein expression and consequent H3K27me3 levels. The defects of neurogenesis and neuronal development induced by Mettl3 depletion could be rescued by Ezh2 overexpression. Collectively, our results uncover a crosstalk between RNA and histone modifications and indicate that Mettl3-mediated mA modification plays an important role in regulating neurogenesis and neuronal development through modulating Ezh2.


Subject(s)
Adenosine , Metabolism , Adult Stem Cells , Cell Biology , Metabolism , Animals , Brain , Metabolism , Cell Differentiation , Genetics , Cell Proliferation , Enhancer of Zeste Homolog 2 Protein , Metabolism , Gene Expression Regulation , Methyltransferases , Metabolism , Mice, Inbred C57BL , Neural Stem Cells , Cell Biology , Metabolism , Neurogenesis , Genetics , Neurons , Cell Biology , Metabolism , RNA, Messenger , Genetics , Metabolism
7.
Article in English | WPRIM | ID: wpr-763116

ABSTRACT

PURPOSE: In the present study, human neural stem cells (hNSCs) with tumor-tropic behavior were used as drug delivery vehicle to selectively target melanoma. A hNSC line (HB1.F3) was transduced into two types: one expressed only the cytosine deaminase (CD) gene (HB1.F3. CD) and the other expressed both CD and human interferon-β (IFN-β) genes (HB1.F3.CD. IFN-β). MATERIALS AND METHODS: This study verified the tumor-tropic migratory competence of engineered hNSCs on melanoma (A375SM) using a modified Boyden chamber assay in vitro and CM-DiI staining in vivo. The antitumor effect of HB1.F3.CD and HB1.F3.CD.IFN-β on melanoma was also confirmed using an MTT assay in vitro and xenograft mouse models. RESULTS: A secreted form of IFN-β from the HB1.F3.CD.IFN-β cells modified the epithelial-mesenchymal transition (EMT) process and metastasis of melanoma. 5-Fluorouracil treatment also accelerated the expression of the pro-apoptotic protein BAX and decelerated the expression of the anti-apoptotic protein Bcl-xL on melanoma cell line. CONCLUSION: Our results illustrate that engineered hNSCs prevented malignant melanoma cells from proliferating in the presence of the prodrug, and the form that secreted IFN-β intervened in the EMT process and melanoma metastasis. Hence, neural stem cell-directed enzyme/prodrug therapy is a plausible treatment for malignant melanoma.


Subject(s)
Animals , Cell Line , Cytosine Deaminase , Epithelial-Mesenchymal Transition , Flucytosine , Fluorouracil , Heterografts , Humans , In Vitro Techniques , Melanoma , Mental Competency , Mice , Neoplasm Metastasis , Neural Stem Cells , Stem Cells
8.
Article in English | WPRIM | ID: wpr-759978

ABSTRACT

OBJECTIVE: Astrocyte dysfunctions are related to several central nervous system (CNS) pathologies. Transcriptomic profiling of human mRNAs to investigate astrocyte functions may provide the basic molecular-biological data pertaining to the cellular activities of astrocytes. METHODS: Human Primary astrocytes (HPAs) and human neural stem cell line (HB1.F3) were used for differential digital gene analysis. In this study, a massively parallel sequencing platform, next-generation sequencing (NGS), was used to obtain the digital gene expression (DGE) data from HPAs. A comparative analysis of the DGE from HPA and HB1.F3 cells was performed. Sequencing was performed using NGS platform, and subsequently, bioinformatic analyses were implemented to reveal the identity of the pathways, relatively up- or down-regulated in HPA cells. RESULTS: The top, novel canonical pathways up-regulated in HPA cells than in the HB1.F3 cells were “Cyclins and cell cycle regulation,” “Integrin signaling,” “Regulation of eIF4 and p70S6K signaling,” “Wnt/β-catenin signaling,” “mTOR signaling,” “Aryl hydrocarbon receptor signaling,” “Hippo signaling,” “RhoA signaling,” “Signaling by Rho family GTPases,” and “Glioma signaling” pathways. The down-regulated pathways were “Cell cycle: G1/S checkpoint regulation,” “eIF2 signaling,” “Cell cycle: G2/M DNA damage checkpoint regulation,” “Telomerase signaling,” “RhoGDI signaling,” “NRF2-mediated oxidative stress response,” “ERK/MAPK signaling,” “ATM signaling,” “Pancreatic adenocarcinoma signaling,” “VEGF signaling,” and “Role of CHK proteins in cell cycle checkpoint control” pathways. CONCLUSION: This study would be a good reference to understand astrocyte functions at the molecular level, and to develop a diagnostic test, based on the DGE pattern of astrocytes, as a powerful, new clinical tool in many CNS diseases.


Subject(s)
Adenocarcinoma , Astrocytes , Cell Cycle , Cell Cycle Checkpoints , Central Nervous System , Central Nervous System Diseases , Computational Biology , Diagnostic Tests, Routine , DNA Damage , Gene Expression , High-Throughput Nucleotide Sequencing , Humans , Neural Stem Cells , Oxidative Stress , Pathology , Ribosomal Protein S6 Kinases, 70-kDa , RNA, Messenger
9.
Neonatal Medicine ; : 1-16, 2019.
Article in Korean | WPRIM | ID: wpr-741669

ABSTRACT

Neonatal hypoxic-ischemic (HI) brain injury is a major cause of neonatal mortality and long-term neurodevelopmental disabilities. Although promising neuroprotective interventions have been studied, the current management of HI brain injury has been limited to supportive measures and induced hypothermia. In addition to engrafting, migrating toward the damage sites and differentiating into multiple lineages, multipotent neural stem/progenitor cells (NSPCs) also provide trophic/immunomodulatory factors and integrate into the host neurons upon implantation into an HI-injured brain. However, NSPC-based therapies have shown poor cell survival and integration, poor differentiation or restricted differentiation into the glial lineages. Furthermore, to achieve full functional recovery following brain injury, the optimization of cell therapy is needed to recapitulate the precise migration of stem cells to the region of interest and the neural rewiring present in the brain microenvironment. Therefore, the efficacy of NSPCs in the treatment of CNS injury is currently insufficient. Human NSPCs (hNSPCs) were isolated from the forebrain of an aborted fetus at 13 weeks of gestation with full parental consent and the approval of the Institutional Review Board of the Yonsei University College of Medicine. Here, to enhance the regenerative ability of hNSPCs in HI brain injury, cells were either pretreated with pharmacological agents or engineered to serve as vehicles for gene delivery. Furthermore, when combined with a poly (glycolic acid)-based synthetic scaffold, hNSPCs provide a more versatile treatment for neonatal HI brain injury. Finally, hNSPCs transfected with zinc-doped ferrite magnetic nanoparticles for controlling both cell migration and differentiation offer a simple and smart tool for cell-based therapies.


Subject(s)
Aborted Fetus , Brain Injuries , Brain , Cell Movement , Cell Survival , Cell- and Tissue-Based Therapy , Ethics Committees, Research , Genetic Therapy , Humans , Hypothermia, Induced , Hypoxia-Ischemia, Brain , Infant , Infant Mortality , Nanoparticles , Neural Stem Cells , Neurons , Parental Consent , Pregnancy , Prosencephalon , Stem Cells , Translational Medical Research
10.
Experimental Neurobiology ; : 229-246, 2019.
Article in English | WPRIM | ID: wpr-739542

ABSTRACT

Neural stem cells (NSCs) have the ability to self-renew and differentiate into neurons, oligodendrocytes, and astrocytes. Highly dynamic nature of NSC differentiation requires the intimate involvement of catabolic processes such as autophagy. Autophagy is a major intracellular degradation pathway necessary for cellular homeostasis and remodeling. Autophagy is important for mammalian development and its role in neurogenesis has recently drawn much attention. However, little is known about how autophagy is associated with differentiation of NSCs into other neural lineages. Here, we report that autophagy plays a critical role in differentiation of adult rat hippocampal neural stem (HCN) cells into astrocytes. During differentiation, autophagy flux peaked at early time points, and remained high. Pharmacological or genetic suppression of autophagy by stable knockdown of Atg7, LC3 or CRISPR-Cas9-mediated knockout (KO) of p62 impaired astrogenesis, while reintroduction of p62 recovered astrogenesis in p62 KO HCN cells. Taken together, our findings suggest that autophagy plays a key role in astrogenesis in adult NSCs.


Subject(s)
Adult Stem Cells , Adult , Animals , Astrocytes , Autophagy , Cell Differentiation , Homeostasis , Humans , Neural Stem Cells , Neurogenesis , Neurons , Oligodendroglia , Rats , Suppression, Genetic
11.
Acta Physiologica Sinica ; (6): 431-438, 2019.
Article in Chinese | WPRIM | ID: wpr-777170

ABSTRACT

The present study was aimed to investigate the effects and mechanisms of electro-acupuncture (EA) on proliferation and differentiation of neural stem cells in the hippocampus of C57 mice exposed to different doses of X-ray radiation. Thirty-day-old C57BL/6J mice were randomly divided into control, irradiation, and EA groups. The control group was not treated with irradiation. The irradiation groups were exposed to different doses of X-ray (4, 8 or 16 Gy) for 10 min. The EA groups were electro-acupunctured at Baihui, Fengfu and bilateral Shenyu for 3 courses of treatment after X-ray radiation. Immunohistochemistry was used to evaluate proliferation and differentiation of the hippocampal neural stem cell. RT-PCR and Western blot were used to detect mRNA and protein expressions of Notch1 and Mash1 in the hippocampus, respectively. The results showed that, compared with the control group, the numbers of BrdU positive cells (4, 8 Gy subgroup) and BrdU/NeuN double-labeling positive cells (3 dose subgroups) were decreased significantly in the irradiation group, but the above changes could be reversed by EA. Compared with the control group, the number of BrdU/GFAP double-labeling positive cells in each dose subgroup of irradiation group was decreased significantly, while EA could reverse the change of 4 and 8 Gy dose subgroups. In addition, compared with the control group, the expression levels of Notch1 mRNA and protein in hippocampus were up-regulated, and the expression levels of Mash1 mRNA and protein were significantly decreased in each dose subgroup of irradiation group. Compared with irradiation group, the expression levels of Notch1 mRNA and protein in hippocampus of EA group were decreased significantly in each dose subgroup, and the expression levels of Mash1 mRNA and protein were increased significantly in 4 and 8 Gy subgroups. These results suggest that irradiation affects the proliferation and differentiation of neural stem cells in hippocampus of mice, whereas EA may significantly increase the proliferation and differentiation of hippocampal neural stem cells via the regulation of Notch signaling pathway.


Subject(s)
Animals , Basic Helix-Loop-Helix Transcription Factors , Metabolism , Cell Differentiation , Cell Proliferation , Electroacupuncture , Hippocampus , Cell Biology , Radiation Effects , Mice, Inbred C57BL , Neural Stem Cells , Cell Biology , Radiation Effects , Random Allocation , Receptor, Notch1 , Metabolism , X-Rays
12.
Acta Physiologica Sinica ; (6): 454-462, 2019.
Article in Chinese | WPRIM | ID: wpr-777168

ABSTRACT

Neural stem cell therapy, as a new therapeutic method for neural diseases, has aroused a wide concern for over 20 years since neural stem cells were first found in 1992. Ischemic stroke is highly concerned because of its high incidence, mortality and disability rates. Because the brain has a limited ability to repair itself, to improve neural function and promote neural regeneration may help to prevent occurrence and development of neurological diseases. It is noteworthy that some stroke patients showed an ability to repair brain several months after the stroke happened, suggesting an existence of endogenous nerve repair in these patients. The research advances in functions of endogenous neural stem cells in neural regeneration and the related regulators after ischemic stroke are summarized in this review to provide new views of the mechanism of neural functional recovery after ischemic stroke.


Subject(s)
Brain Ischemia , Therapeutics , Humans , Nerve Regeneration , Neural Stem Cells , Cell Biology , Stroke , Therapeutics
13.
Article in Chinese | WPRIM | ID: wpr-776519

ABSTRACT

OBJECTIVE@#To investigate the effects of optical genetic techniques on new neurons through the Wnt/β-Catenin pathway.@*METHODS@#Neural stem cells (ESCs)were extracted from the cerebral cortex of fetal rat and transfected by lentivirus carrying DCX-ChR2-EGFP gene and the expression of DCX of newborn neurons differentiated from neural stem cells were observed. All cells were divided into 3 groups(n=9): control group, NSCs+EGFP and NSCs+ChR2 groups. The control group was normal cultured NSCs (NSCs group); the neural stem cells in NSCs+EGFP group were transfected with lentivirus carrying EGFP gene. The neural stem cells in NSCs+ChR2 group were infected with lentivirus carrying DCX-ChR2-EGFP gene. After 48 hours of lentivirus infection, 470 nm blue laser irradiation was performed for 3 consecutive days. NeuN positive cell density(the maturation of neural stem cells)and the ratio of NeuN/Hoechst in each group were observed. Western blot was used to detect the expression levels of MAP2, NeuN, Neurog2, NeuroD1 and GluR2. Western blot was used to detect the expressions of β-catenin and TCF4 associated with Wnt/β-catenin signaling channel. Verapamil (100 μmol/L, L-type calcium channel blockers) and Dkk1 (50 μg/ml, β-catenin inhibitor) were used to treat stem cells of the NSCs+ChR2 group and then the expressions of MAP2, NeuN, Neurog2, NeuroD1 and GluR were detected by Western blot.@*RESULTS@#After 3 days of 470 nm blue laser irradiation, NeuN positive cell density(the maturation of neural stem cells)and the ratio of NeuN/Hoechst, the expression levels of the protein MAP2, NeuN, Neurog2, NeuroD1, GluR and the protein β-catenin and TCF4 associated with Wnt/β-catenin signaling channel detected by Western blot were significantly increased in the group of NSCs+ChR2, compared with NSCs and NSCs+EGFP groups. The expressions of MAP2, NeuN, Neurog2, NeuroD1 and GluR were remarkably decreased after treated by verapamil and Dkk1 in the group of NSCs+ChR2. It was proved that the opening of ChR2 channel producing cationic influx promoted the maturation of neural stem cells and induced by the Wnt/β-catenin signaling pathway.@*CONCLUSION@#Optical genetic promoted the maturation of newborn neurons through the Wnt/β-catenin signaling pathway.


Subject(s)
Animals , Cells, Cultured , Neural Stem Cells , Cell Biology , Neurons , Cell Biology , Optogenetics , Rats , Transfection , Wnt Signaling Pathway
14.
Article in Chinese | WPRIM | ID: wpr-776518

ABSTRACT

OBJECTIVE@#To study the effect of exendin-4(Ex-4) on the differentiation of neural stem cells(NSCs) in adult mouse subventricular zone(SVZ)and its mechanism .@*METHODS@#NSCs in the SVZ were derived from 5-week C57BL/6J mice and the expression of nestin was detected by immunofluorescence. The cell morphology was observed after the cells treatmed with 100 nmol/L Ex-4 for 14 days.The expressions of nestin and glucagon-like peptide-1 receptor (GLP-1R) were detected by immunofluorescence. GLP-1R was knocked down by using shRNA and the study was divided into four groups: control group, Ex-4 group, GLP-1R knockdown group, GLP-1R knockdown + Ex-4 group. After treatment with 100 nmol/L Ex-4 for 14 d, β-tublin III and glial fibrillary acidic protein (GFAP) were labeled by immunofluorescence and then the proportion of β-tublin III positive cells were counted. Western blot was used to detect the activation of cAMP-response element binding protein (CREB) in NSCs. In order to further study the effects of Ex-4 on mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-hydroxy kinase (PI3K) pathways, the cells were pretreated with MAPK inhibitor U0126 at a concentration of 0.07 μmol/L for 30 min or PI3K inhibitor LY294002 at 50 μmol for 2 h, respectively. The study was divided into six groups: control group, Ex-4 group, U0126 group, U0126 + Ex-4 group, LY294002 group, LY294002 + Ex-4 group. The activation of CREB in each group was detected by Western blot. The experiment was repeated three times independently.@*RESULTS@#NSCs were successfully extracted from SVZ of C57BL/6J mice. Immunofluorescence showed that nestin and GLP-1R were positive in NSCs. Compared with the control group, the proportion of neurons differentiated from Ex-4 group was higher. The percentage of neurons in GLP-1R knockdown + Ex-4 group was basically the same as that in control group (P<0.01). The positive cells of beta-tublin III showed positive activation of GLP-1R and CREB. Western blot showed that CREB was significantly activated in the Ex-4 group, and knockdown of GLP-1R abolished its activation (P<0.01). U0126 did not affect Ex-4-mediated CERB activation, and LY294002 significantly reduced Ex-4-mediated CREB activation (P<0.01).@*CONCLUSION@#Ex-4 promotes the differentiation of NSCs into neurons in SVZ of adult mice through GLP-1R receptor, which may be achieved through PI3K/CREB pathway.


Subject(s)
Animals , Cell Differentiation , Cells, Cultured , Cyclic AMP Response Element-Binding Protein , Metabolism , Exenatide , Pharmacology , Gene Knockdown Techniques , Glucagon-Like Peptide-1 Receptor , Genetics , Metabolism , Lateral Ventricles , Cell Biology , Mice , Mice, Inbred C57BL , Neural Stem Cells , Cell Biology , Phosphatidylinositol 3-Kinases
15.
Article in Chinese | WPRIM | ID: wpr-776000

ABSTRACT

To compare the biological functions of astrocytes cultured by two methods. Methods The primary astrocytes were cultured from rodent neonatal brain,whereas the differentiated astrocytes were prepared by differentiating neural stem cells with fetal bovine serum.The morphologies of these two different types of astrocytes were observed under microscope and the expression of glial fibrillary acidic protein(GFAP),an astrocyte-specific marker,was detected by immunofluorescence staining after treatment with 10 cytokines.Changes in GFAP,glutamate synthetase(GS),glutamate-aspartic acid transporter(xCT),neuregulin-1(NRG),N-methyl-D-aspartic acid receptor(NMDA),lipoprotein lipase(LPL)were detected and compared. Results The morphologies and GFAP expression differed between these two astrocyte types.Microarray showed that the expressions of GFAP,GS,xCT,NRG,NMDA,and LPL were significantly higher in primary astrocytes than in differentiated astrocytes.None of these 10 cytokines increased the expression of GFAP in primary astrocytes,whereas treatment with transforming growth factor-β(TGF-β)significantly increased the expression of GFAP in the differentiated astrocytes. Conclusion Compared with the differentiated astrocytes,the primary astrocytes are more similar to reactive astrocytes,and TGF-β can promote the transition of differentiated cells to reactive cells.


Subject(s)
Animals , Animals, Newborn , Astrocytes , Cell Biology , Cell Differentiation , Cells, Cultured , Glial Fibrillary Acidic Protein , Metabolism , Neural Stem Cells , Cell Biology , Rodentia , Transforming Growth Factor beta , Pharmacology
16.
Neuroscience Bulletin ; (6): 216-224, 2019.
Article in English | WPRIM | ID: wpr-775435

ABSTRACT

Diffuse intrinsic pontine glioma (DIPG) is the main cause of brain tumor-related death among children. Until now, there is still a lack of effective therapy with prolonged overall survival for this disease. A typical strategy for preclinical cancer research is to find out the molecular differences between tumor tissue and para-tumor normal tissue, in order to identify potential therapeutic targets. Unfortunately, it is impossible to obtain normal tissue for DIPG because of the vital functions of the pons. Here we report the human fetal hindbrain-derived neural progenitor cells (pontine progenitor cells, PPCs) as normal control cells for DIPG. The PPCs not only harbored similar cell biological and molecular signatures as DIPG glioma stem cells, but also had the potential to be immortalized by the DIPG-specific mutation H3K27M in vitro. These findings provide researchers with a candidate normal control and a potential medicine carrier for preclinical research on DIPG.


Subject(s)
Animals , Brain Stem Neoplasms , Genetics , Metabolism , Pathology , Cell Line, Tumor , Cellular Senescence , Female , Glioma , Genetics , Metabolism , Pathology , Histones , Genetics , Humans , Mice, Inbred NOD , Mice, SCID , Neoplasm Transplantation , Neoplastic Stem Cells , Metabolism , Pathology , Neural Stem Cells , Metabolism , Pathology , Pons , Embryology , Metabolism , Pathology , Primary Cell Culture
17.
Neuroscience Bulletin ; (6): 486-496, 2019.
Article in English | WPRIM | ID: wpr-775423

ABSTRACT

MicroRNA-132 (miR-132), a small RNA that regulates gene expression, is known to promote neurogenesis in the embryonic nervous system and adult brain. Although exposure to psychoactive substances can increase miR-132 expression in cultured neural stem cells (NSCs) and the adult brain of rodents, little is known about its role in opioid addiction. So, we set out to determine the effect of miR-132 on differentiation of the NSCs and whether this effect is involved in opioid addiction using the rat morphine self-administration (MSA) model. We found that miR-132 overexpression enhanced the differentiation of NSCs in vivo and in vitro. Similarly, specific overexpression of miR-132 in NSCs of the adult hippocampal dentate gyrus (DG) during the acquisition stage of MSA potentiated morphine-seeking behavior. These findings indicate that miR-132 is involved in opioid addiction, probably by promoting the differentiation of NSCs in the adult DG.


Subject(s)
Animals , Cell Differentiation , Cell Line, Tumor , Dentate Gyrus , Metabolism , Gene Expression Regulation , Male , MicroRNAs , Metabolism , Neural Stem Cells , Metabolism , Opioid-Related Disorders , Metabolism , Rats, Sprague-Dawley
18.
Article in Chinese | WPRIM | ID: wpr-775098

ABSTRACT

OBJECTIVE@#To study the effects of different melatonin treatment regimens on the proliferation of neural stem cells (NSCs) and long-term histopathology in neonatal rats with hypoxic-ischemic brain damage (HIBD), and to identify better melatonin treatment regimens.@*METHODS@#A total of 96 Sprague-Dawley rats aged 7 days were randomly divided into normal control, HIBD, single-dose immediate melatonin treatment (SDIT), and 7-day continuous melatonin treatment (7DCT) groups, with 24 rats in each group. The rat model of HIBD was prepared by isolation and electrocoagulation of the right common carotid artery as well as hypoxic treatment in a hypoxic chamber (oxygen concentration 8.00% ± 0.01%) for 2 hours. On day 7 after modeling, proliferating cell nuclear antigen/Nestin double-labeling immunofluorescence was used to measure the proliferation of endogenous NSCs in the subventricular zone (SVZ) and the hippocampal dentate gyrus (DG) region in 8 rats in each group, and Western blot was used to measure the protein expression of Nestin in brain. On day 28 after modeling, hematoxylin-eosin (HE) staining and Nissl staining were used to observe the changes in the histopathology and the number of pyramidal cells in the hippocampal CA1 region in 8 rats in each group.@*RESULTS@#Immunofluorescent staining showed that compared with the HIBD group, the SDIT and 7DCT groups had a significant increase in the number of PCNA+Nestin+DAPI+ cells, and the 7DCT group had a significantly higher number than the SDIT group (P<0.01). Western blot showed that the SDIT and 7DCT groups had significantly higher protein expression of Nestin than the HIBD group, and the 7DCT group had significantly higher expression than the SDIT group (P<0.05). HE staining showed that the SDIT and 7DCT groups had alleviated cell injury, and Nissl staining showed that compared with the HIBD group, the SDIT and 7DCT groups had a significant increase in the number of pyramidal cells, and the 7DCT group had a significantly higher number than the SDIT group (P<0.01).@*CONCLUSIONS@#Both single-dose immediate melatonin treatment and 7-day continuous melatonin treatment can promote the proliferation of endogenous NSCs and alleviate long-term histological injury in the brain of neonatal rats with HIBD. A 7-day continuous melatonin treatment has a better effect than single-dose immediate melatonin treatment.


Subject(s)
Animals , Animals, Newborn , Brain , Cell Proliferation , Hypoxia-Ischemia, Brain , Melatonin , Neural Stem Cells , Neurons , Rats , Rats, Sprague-Dawley
19.
Article in English | WPRIM | ID: wpr-785827

ABSTRACT

PSMD10(Gankyrin), a proteasome assembly chaperone, is a widely known oncoprotein which aspects many hall mark properties of cancer. However, except proteasome assembly chaperon function its role in normal cell function remains unknown. To address this issue, we induced PSMD10(Gankyrin) overexpression in HEK293 cells and the resultant large-scale changes in gene expression profile were analyzed. We constituted networks from microarray data of these differentially expressed genes and carried out extensive topological analyses. The overrecurring yet consistent theme that appeared throughout analysis using varied network metrics is that all genes and interactions identified as important would be involved in neurogenesis and neuronal development. Intrigued we tested the possibility that PSMD10(Gankyrin) may be strongly associated with cell fate decisions that commit neural stem cells to differentiate into neurons. Overexpression of PSMD10(Gankyrin) in human neural progenitor cells facilitated neuronal differentiation via β-catenin Ngn1 pathway. Here for the first time we provide preliminary and yet compelling experimental evidence for the involvement of a potential oncoprotein – PSMD10(Gankyrin), in neuronal differentiation.


Subject(s)
HEK293 Cells , Humans , Neural Stem Cells , Neurogenesis , Neurons , Proteasome Endopeptidase Complex , Stem Cells , Transcriptome
20.
Article in English | WPRIM | ID: wpr-764063

ABSTRACT

BACKGROUND AND OBJECTIVES: Genomic imprinting modulates growth and development in mammals and is associated with genetic disorders. Although uniparental embryonic stem cells have been used to study genomic imprinting, there is an ethical issue associated with the destruction of human embryos. In this study, to investigate the genomic imprinting status in human neurodevelopment, we used human uniparental induced pluripotent stem cells (iPSCs) that possessed only maternal alleles and differentiated into neural cell lineages. METHODS: Human somatic iPSCs (hSiPSCs) and human parthenogenetic iPSCs (hPgiPSCs) were differentiated into neural stem cells (NSCs) and named hSi-NSCs and hPgi-NSCs respectively. DNA methylation and gene expression of imprinted genes related neurodevelopment was analyzed during reprogramming and neural lineage differentiation. RESULTS: The DNA methylation and expression of imprinted genes were altered or maintained after differentiation into NSCs. The imprinting status in NSCs were maintained after terminal differentiation into neurons and astrocytes. In contrast, gene expression was differentially presented in a cell type-specific manner. CONCLUSIONS: This study suggests that genomic imprinting should be determined in each neural cell type because the genomic imprinting status can differ in a cell type-specific manner. In addition, the in vitro model established in this study would be useful for verifying the epigenetic alteration of imprinted genes which can be differentially changed during neurodevelopment in human and for screening novel imprinted genes related to neurodevelopment. Moreover, the confirmed genomic imprinting status could be used to find out an abnormal genomic imprinting status of imprinted genes related with neurogenetic disorders according to uniparental genotypes.


Subject(s)
Alleles , Astrocytes , Cell Lineage , DNA Methylation , Embryonic Stem Cells , Embryonic Structures , Epigenomics , Ethics , Gene Expression , Genomic Imprinting , Genotype , Growth and Development , Humans , In Vitro Techniques , Induced Pluripotent Stem Cells , Mammals , Mass Screening , Neural Stem Cells , Neurons
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