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BACKGROUND: Researchers believe that hydrogen sulfide (H2S), as an important cell protective molecule, may become a new treatment method to restore the physiological function of diseased cells or organ systems through the artificial regulation of endogenous H2S biosynthesis or in vitro administration of H2S donor. ADT-OH is a slow-release donor of H2S that can improve the survival rate of hippocampal nerve cells with glutamate-induced injury, but studies on the proliferation of cerebral cortical neural precursor cells are rare. OBJECTIVE: To investigate the effect of ADT-OH on the proliferation of neural precursor cells in embryonic cerebral cortex. METHODS: Neural precursor cells from cerebral cortical ventricular zone and subventricular zone of embryonic mice at embryonic 14.5 days were isolated. Neural precursor cells from one fetal mouse were inoculated into one well (24-well plate), and cultured with the medium containing 100 μmol/L ADT-OH. The size and number of neural spheres per well were measured at 3 days after culture. The proliferation rate of cultured neural precursor cells was detected by BrdU labeling. The proliferation of the cells was further verified by immunofluorescence staining with the specific antibody Ki67. The expression of cyclin D1 was finally detected by western blot assay. RESULTS AND CONCLUSION: Our experimental results showed that ADT-OH could promote the formation of neural spheres, and further detection by BrdU and Ki67 antibody showed that ADT-OH could promote the proliferation rate of neural precursor cells. Meanwhile, the expression of cyclin D1, a proliferation-related gene, was up-regulated in neural precursor cells after ADT-OH treatment. Overall, ADT-OH may promote the proliferation of neural precursor cells by regulating the expression of cyclin D1.
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Se presenta el caso de un paciente de 20 días de nacido, procedente de Cartagena (Bolívar), hospitalizado por presentar fiebre de 6 días de evolución asociado a sintomatología respiratoria con evaluación neurológica normal. La ecografía obstétrica evidenció una microcefalia con un percentil de perímetro cefálico <2, con hipoplasia del cuerpo calloso y tomografía axial computarizada de cráneo que reportó diámetros cefálicos disminuidos, finas calcificaciones residuales en región frontal-parietal y cambios atróficos cerebrales subcorticales. Se le inició terapia antibiótica por presentar sepsis neonatal, las pruebas serológicas y la PCR para Zika resultaron positivas. Se decidió dar el alta médica al 6 día por mejoría clínica y no presentar déficit neurológico aparente. Aunque no existe un tratamiento específico, el pilar del manejo de un recién nacido con microcefalia es el seguimiento y la vigilancia futura de las posibles comorbilidades, como epilepsia, parálisis cerebral o retraso cognitivo y motor.
We present the case of a 20-day-old patient from Cartagena (Bolívar), hospitalized for presenting a 6-day fever associated with respiratory symptoms with normal neurological evaluation. The obstetric ultrasound showed a microcephaly with a percentile of cephalic perimeter <2, with hypoplasia of the corpus callosum and computed tomography of the skull that reported decreased cephalic diameters, fine residual calcifications in the frontal-parietal region and atrophic subcortical cerebral changes. Antibiotic therapy was initiated due to neonatal sepsis, the serological tests and the PCR for Zika were positive. It was decided to discharge the hospital after 6 days due to clinical improvement and for not presenting apparent neurological deficit. Although there is no specific treatment, the pillar of the management of a newborn with microcephaly is the monitoring and future surveillance of possible comorbidities, such as epilepsy, cerebral palsy or cognitive and motor retardation.
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Humans , Male , Infant, Newborn , Zika Virus , Microcephaly , Stem Cells , Pregnancy , Diagnostic Imaging , Fever , Anti-Bacterial AgentsABSTRACT
@#Objective To observe dynamic variation of neural precursor cells in dentate gyrus of hippocampus and effect of Yangxue Qingnao Granule on it in vascular dementia rats. Methods 72 Sprague-Dawley rats were randomly divided into sham group (n=24), vascular dementia group (model group, n=24) and Yangxue Qingnao Granule group (treatment group, n=24). The vascular dementia model was established with modified Pulsineli's four-vessel occlusion. The expression of Nestin was detected with Western blotting, the expression of 5-bromodeoxyuridine (BrdU) and BrdU/Nestin were detected with immunofluorescence in dentate gyrus of hippocampus 1, 2, 4 and 8 weeks after modeling. Results The expression of Nestin, BrdU and BrdU/Nestin increased in the model and treatment groups with time, peaked at 4 weeks after modeling, and it was more than that of the sham group on all the time points (P<0.01). However, it was more in the treatment group than in the model group on all the time points (P<0.01). Conclusion Yangxue Qingnao Granule promotes the proliferation of neural precursor cells in dentate gyrus of hippocampus in vascular dementia rats.
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Objective To observe dynamic variation of neural precursor cells in dentate gyrus of hippocampus and effect of Yangxue Qingnao Granule on it in vascular dementia rats. Methods 72 Sprague-Dawley rats were randomly divided into sham group (n=24), vascular dementia group (model group, n=24) and Yangxue Qingnao Granule group (treatment group, n=24). The vascular dementia model was estab-lished with modified Pulsineli's four-vessel occlusion. The expression of Nestin was detected with Western blotting, the expression of 5-bro-modeoxyuridine (BrdU) and BrdU/Nestin were detected with immunofluorescence in dentate gyrus of hippocampus 1, 2, 4 and 8 weeks af-ter modeling. Results The expression of Nestin, BrdU and BrdU/Nestin increased in the model and treatment groups with time, peaked at 4 weeks after modeling, and it was more than that of the sham group on all the time points (P<0.01). However, it was more in the treatment group than in the model group on all the time points (P<0.01). Conclusion Yangxue Qingnao Granule promotes the proliferation of neural precursor cells in dentate gyrus of hippocampus in vascular dementia rats.
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Objective To study the effect of neuronal Nogo-66 receptor (NgR1) antagonist,soluble Nogo-66 receptor (sNgR1-Fc),on promoting the endogenous neural precursor cells (NPCs) differentiating into neurons in order to clarify the mechanism.Methods The cortical infarction was induced by photochemistry,named photothrombotic cortical injury (PCI).Twelve Sprague Dawley rats were randomly divided (random number) into three groups:Sham-operated group,PBS group,and sNgR1-Fc group.PBS (PBS group) or sNgR1-Fc (sNgR1-Fc group) was injected into the lateral ventricle of brain with a minipump.BrdU (Bromodeoxyuridine) was injected into the peritoneal cavity 4-6 days after PCI.The subdentate gyrus zone (SGZ) of brain from sacrificed rat was harvested for Immunohistochemistry to observe the ratio of NeuN +/BrdU + cells 35 days after PCI.Proteins including Nestin、Notch1 and Mash1 were detected by Western Blot.Results The cortical infarction in rat was successfully induced by photochemistry.Thirty-five days after PCI,the BrdU + cells number and theratio of NeuN +/BrdU + in the SGZ of the ipsilateral cerebrum hemisphere with PCI were significantly higher in sNgR1-Fc group than those in PBS group (P ＜ 0.05).The levels of Notch1,Mash1 and Neuro D in the sNgR1-Fc group were significantly higher than those in the PBS group (P ＜ 0.05),which were significantly higher than those in the Sham-operated group.Conclusions sNgR1-Fc could promote the endogenous NPCs differentiating into neurons in a cortical infarction model.The mechanisms may be attributed to the Notch/bHLH (proneural basic helix-loop-helix genes) signaling way.
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The transplantation of neural precursor cells (NPCs) is known to be a promising approach to ameliorating behavioral deficits after stroke in a rodent model of middle cerebral artery occlusion (MCAo). Previous studies have shown that transplanted NPCs migrate toward the infarct region, survive and differentiate into mature neurons to some extent. However, the spatiotemporal dynamics of NPC migration following transplantation into stroke animals have yet to be elucidated. In this study, we investigated the fates of human embryonic stem cell (hESC)-derived NPCs (ENStem-A) for 8 weeks following transplantation into the side contralateral to the infarct region using 7.0T animal magnetic resonance imaging (MRI). T2- and T2*-weighted MRI analyses indicated that the migrating cells were clearly detectable at the infarct boundary zone by 1 week, and the intensity of the MRI signals robustly increased within 4 weeks after transplantation. Afterwards, the signals were slightly increased or unchanged. At 8 weeks, we performed Prussian blue staining and immunohistochemical staining using human-specific markers, and found that high percentages of transplanted cells migrated to the infarct boundary. Most of these cells were CXCR4-positive. We also observed that the migrating cells expressed markers for various stages of neural differentiation, including Nestin, Tuj1, NeuN, TH, DARPP-32 and SV38, indicating that the transplanted cells may partially contribute to the reconstruction of the damaged neural tissues after stroke. Interestingly, we found that the extent of gliosis (glial fibrillary acidic protein-positive cells) and apoptosis (TUNEL-positive cells) were significantly decreased in the cell-transplanted group, suggesting that hESC-NPCs have a positive role in reducing glia scar formation and cell death after stroke. No tumors formed in our study. We also performed various behavioral tests, including rotarod, stepping and modified neurological severity score tests, and found that the transplanted animals exhibited significant improvements in sensorimotor functions during the 8 weeks after transplantation. Taken together, these results strongly suggest that hESC-NPCs have the capacity to migrate to the infarct region, form neural tissues efficiently and contribute to behavioral recovery in a rodent model of ischemic stroke.
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Animals , Humans , Male , Rats , Apoptosis , Cell Differentiation , Cell Movement , Embryonic Stem Cells/cytology , Glial Fibrillary Acidic Protein/genetics , Infarction, Middle Cerebral Artery/metabolism , Neural Stem Cells/cytology , Psychomotor Performance , Rats, Sprague-Dawley , Receptors, CXCR4/geneticsABSTRACT
BACKGROUND:Whether embryonic stem cel transplantation can be an effective method for treatment of cerebrovascular disease has become a hot spot. OBJECTIVE:To investigate the effect and feasibility of embryonic stem cel-differentiated neural precursor cel transplantation for the treatment of cerebrovascular disease. METHODS:The rat models of Parkinson’s disease, ischemic brain injury and vascular dementia were established and the embryonic stem cells were in vitro cultured and induced to differentiate into the neural precursor cells. The embryonic stem cel-differentiated neural precursor cells were transplanted into the rat brains with corresponding cerebrovascular disease, and then the rotational behavior changes, pathological changes in the brain tissue as wel as changes in the hippocampal structure and the number of nerve cells in the rats with cerebrovascular disease were observed. RESUTLS AND CONCLUSION:After the embryonic stem cel-differentiated neural precursor cells transplanted into the rat brains with Parkinson’s disease, the frequency of apomorphine-induced rotation was decreased and in the downward trend, while the striatal dopamine levels were significantly increased. After transplantation of embryonic stem cel-differentiated neural precursor cells into the rat brains with ischemic brain injury, the cells could survive for a long time, and then migrated and distributed in the injured hippocampus to form the hippocampal structure;the cells could differentiate into neurons, and the number of nerve cells in the injured hippocampus was significantly increased. The results indicate that the transplanted glial cel line-derived neurotrophic factor gene-modified embryonic stem cells can improve the learning and memory function of vascular dementia rats, enhance neural plasticity, and induce self-directed migration and differentiation into mature neurons.
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Objective To reseach the time point of the highest percentage of neural precursor cells derived from adipose stromal cells (ADSCs) in vitro, and to observe the ultrastructure features of neural precursor cells. Methods Used the β-mercaptoethanol to induce ADSCs to differentiate into neural precursor cells and neuron-like cells. The morphology of the uninductedcells and inducted cells were observed with inverted phase contrast microscope. The expression of nestin which was the marker of neural precursor cell in each group was detected using immunofluorescence staining method. The ultrastructural feature of cells which was induced for 3 hours were observed. Results The highest ratio of positive expression of nestin was 3 hours following induction,with the ratio ( 86.25 ± 4.82) %. There were many protuberance on the cell membrane under transmission electron microscopy.There were plenty of organelles in the neural precursor cells. The neural precursor cells had a large size nucleus,large nucleoplasmic index, much extended chromatin,and less condensed chromatin. The nucleus had double-layer nuclear envelope, more nuclear pore on the nuclear envelope. Conclusion The time point of the highest percentage of neural precursor cells derived from ADSCs is 3 hours,and the ultrastructral feature of induced neural precursor cells confirm that cells at this time point are in a state of split active period.
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Cell therapy for neurological disorders has advanced, and neural precursor cells (NPC) may become the ideal candidates for neural transplantation in a wide range of diseases. However, additional work has to be done to determine either the ideal culture environment for NPC expansion in vitro, without altering their plasticity, or the FGF-2 and EGF mechanisms of cell signaling in neurospheres growth, survival and differentiation. In this work we evaluated mouse neurospheres cultured with and without FGF-2 and EGF containing medium and showed that those growth factors are responsible for NPC proliferation. It is also demonstrated that endogenous production of growth factors shifts from FGF-2 to IGF-1/PDGFb upon EGF and FGF-2 withdrawal. Mouse NPC cultured in suspension showed different patterns of neuronal localization (core versus shell) for both EGF and FGF-2 withdrawal and control groups. Taken together, these results show that EGF and FGF-2 removal play an important role in NPC differentiation and may contribute to a better understanding of mechanisms of NPC differentiation. Our findings suggest that depriving NPC of growth factors prior to grafting might enhance their chance to effectively integrate into the host.
As terapias celulares para doenças neurológicas têm avançado e células precursoras neurais (NPC) surgem como candidatas ideais para o transplante de células neurais em muitas doenças. No entanto, trabalhos adicionais devem ser feitos para determinar o ambiente de cultivo ideal para a expansão in vitro das NPC, sem alterar sua plasticidade, e os mecanismos de sinalização celular do fator de crescimento epidérmico (EGF) e fator de crescimento de fibroblasto 2 (FGF-2) no crescimento, sobrevivência e diferenciação da neuroesfera. Nesse trabalho avaliamosNPCcultivadas na presença e na ausência de FGF-2 e EGF e mostramos que esses fatores de crescimento são responsáveis pela proliferação das NPC. Também foi demonstrado que a produção endógena de fatores de crescimento alterna de FGF-2 a fator de crescimento de insulina 1 (IGF-1) e fator de crescimento derivado de plaquetas b (PDGFb) após remoção de EGF e FGF-2. NPC de camundongo cultivadas em suspensão mostraram padrões de localização neuronal distintos (centro versus borda) tanto no grupo controle como no grupo sem EGF e FGF-2. Juntos, esses resultados mostram que a remoção de EGF e FGF-2 exerce importante ação na diferenciação de NPC e possivelmente contribui para melhor compreensão dos mecanismos envolvidos na diferenciação. Nossos achados sugerem que, privando as NPC de fatores de crescimento antes do transplante, talvez aumente as chances de que as células efetivamente se integrem ao hospedeiro.
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Animals , Mice , Cell Differentiation/drug effects , Epidermal Growth Factor/pharmacology , /pharmacology , Neuronal Plasticity/physiology , Neurons/drug effects , Stem Cells/drug effects , Cell Culture Techniques/methods , Cell Differentiation/physiology , Neurons/cytology , Neurons/physiology , Stem Cells/cytology , Stem Cells/physiologyABSTRACT
The differentiation of neural precursor cells (NPCs) into neurons and astrocytes is a process that is tightly controlled by complicated and ill-defined gene networks. To extend our knowledge to gene networks, we performed a temporal analysis of gene expression during the differentiation (2, 4, and 8 days) of spinal cord-derived NPCs using oligonucleotide microarray technology. Out of 32,996 genes analyzed, 1878 exhibited significant changes in expression level (fold change>2, p<0.05) at least once throughout the differentiation process. These 1878 genes were classified into 12 groups by k-means clustering, based on their expression patterns. K-means clustering analysis revealed that the genes involved in astrogenesis were categorized into the clusters containing constantly upregulated genes, whereas the genes involved in neurogenesis were grouped to the cluster showing a sudden decrease in gene expression on Day 8. Functional analysis of the differentially expressedgenes indicated the enrichment of genes for Pax6- NeuroD signaling-TGFb-SMAD and BMP-SMAD-which suggest the implication of these genes in the differentiation of NPCs and, in particular, key roles for Nova1 and TGFBR1 in the neurogenesis/astrogenesis of mouse spinal cord.
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Animals , Mice , Astrocytes , Gene Expression , Gene Regulatory Networks , Neurogenesis , Neurons , Oligonucleotide Array Sequence Analysis , Spinal CordABSTRACT
Objective:To remove murine embryonic stem cells(mESC)from the differentiating cell culture and purify the differentiated cells by Magnetic Activated Cell Sorting(MACS).Methods:Neural differentiation of mESC was induced by a 5-stage method.The specific cell surface marker,SSEA-1,was used to identify ES cells in the differentiating cells.The optimal dilutions of mouse anti mouse SSEA-1 IgM primary antibody and FITC conjugated goat anti mouse secondary antibody were determined before the flow cytometry test.The incubation time and incubation temperature of primary antibody were all optimized to make the cytometry test accurate.After the optimization,stage 4 cells were dissociated into single cell suspension,incubated with antibody of SSEA-1 and microbeads conjugated goat anti mouse IgM,and then sorted through the magnetic field.The rate of SSEA-1 positive cells in pre-and post-separation groups was assessed by flow cytometry,and the viability of cells was evaluated by trypan blue staining counting under light microscopy.Results:The proportion of SSEA-1 positive cells in the separated cells can be reduced from(7.19?1.36)% to(1.34?0.80)%.The survival rate of sorted cells was more than 92%,similar to that of pre-separation cells.Conclusions:The MACS system we used can effectively remove mESC from the differentiated cells.The sorted cells will be well provided for the subsequent studies about transplantation therapy.
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Aim To study the effects of agmatine on the proliferation ability of neural progenitor cells from neonatal rat hippocampus.Methods Hippocampus of neonatal rat was isolated and made into single-cell suspension,which was cultured in serum-free medium and then using CCK-8 kit assay,the survival rate of neurospheres incubated with various concentrations of agmatine and efaroxan.Furthermore,the effect of these drugs was measured with()~3H-thymidine incorporation assay.Results Cells we cultured could continuously proliferate and cultured as floating neurospheres.CCK-8 kit assay revealed that agmatine 1 ?mol?L~(-1) and 10 ?mol?L~(-1) enhanced the survival rate of neural stem cell,and the result alike with()~3H-thymidine incorporation assay.Efaroxan 10 ?mol?L~(-1) inhibit proliferation effect of agmatine on neural stem cell.Conclusion Agmatine was found to increase the proliferation of neural stem cell and efaroxan can block the proliferation effect.It suggested that imidazolineⅠ receptor may be related to the proliferation effect.