Objective To investigate the method of cultivation and the feature of differentiation of spinal cordderived stem cells in vitro.Methods The neural stemcells from spinal cord of 15 days fetal rats were harvested and cultivated in aserumfree limited medium. The stem cells were induced to differentiate and theresults were identified by cellular immunohistochemistry. Results Lots of stem cells were obtained from the spinal cord of fetal rats and the sphere of stemcells was formed about 10 days. Neural stem cells can give rise to mature neurons and astrocytes.Conclusion Epidermal growth factor/basic fibroblast growth factor serum-free limited medium can promote the proliferation activity ofthe stem cells. Spinal cord-derived stem cells can differentiate into glial cells and neurons.
Objective To observe whether Nogo-66 can inhibit the neurite outgrowth during the neuronal differentiation of the neural stem cells (NSCs) and remove such an inhibitory effect by the small interfering RNA (siRNA) mediated knockdown of the Nogo66 receptor (NgR). Methods NSCs derived from the rat spinal cord were collected, and were cultured by the suspension culture in vitro. NSCs were transfected by siRNA to knock downtheexpression of NgR. Immunofluorescence and Western blot were used to assess the knockdown efficiency. NSCs were divided into four groups and differentiated in the medium containing 10% FBS. In the control group, no intervention was applied to NSCs; in the Nogo-P4 group, NSCs were differentiated in the presence of Nogo-P4 (active segment of Nogo-66); in the siRNA group, NSCs were transfected by siRNA to knock down NgR before they were differentiated; in the siRNA and Nogo-P4 group, NSCs were transfected by siRNA to knock down NgR before they were differentiated in presence of Nogo-P4. The differentiated neurons were labeled by immunofluorescence, and the neurite length was measured by the ImagePro Plus 5.0 software. The differentiation of the neurite length was compared in each group. Results The suspension-cultured cells became the nerve bulb, which could positively expresses Nestin by immunofluorescence. At 1 week of the differentiation in the medium containing 10% FBS, the positively-labeled neuron specific enolase, the glial fibrillary acidic protein, and the myelin basic protein were observed. Both immunofluorescence and Western blot approved that the expression of NgR was knocked down by transfection of siRNA at 24 hours after the transfection. The knockdown efficiency was 90.35%±3.10%. The neurite length was 97.80±6.97 μm, 80.54±6.75 μm,92.14±7.27 μm, and 94.01±8.37 μm in the control group, the Nogo-P4 group, the siRNA group, and the siRNA and Nogo-P4 group, respectively. The Nogo-P4 group had a significant difference when compared with the otherthree groups (Plt;0.01), and the other three groups had no significant difference when compared with each other（Pgt;0.05）. ConclusionNogo-66 can inhibit the neuronal neurite outgrowth during the differentiation ofNSCs. Such an inhibitory effect can be removed by the siRNA mediated knockdown of NgR.
ObjectiveTo investigate the effect of serum on the differentiation of neural stem cells.MethodsThe neural stem cells were isolated from the embryonic hippocampus tissues of Sprague Dawley rats at 14 day of pregnancy. After culturing and passaging, the 3rd generation cells were identified by immunocytochemical staining. Then, the cells were divided into 3 groups according to the concentrations of fetal bovine serum (FBS) used in the differentiation cell culture medium: 5% (group A), 1% (group B), 0 (group C), respectively. The other components of the culture media in 3 groups were the same. Cell viability was determined by using the Live/Dead cell staining at 8 days; the expressions of glial cell marker [glial fibrillary acidic protein (GFAP)] and neuronal marker (β-Ⅲ Tubulin) were determined and analyzed by immunocytochemical staining and real-time fluorescent PCR at 4 and 8 days of culture.ResultsBased on cell morphology and immunocytochemical staining, neural stem cells were identified. Cells were growing well with no death in all groups. With decreasing FBS concentration, the expression of GFAP was significantly decreased on both protein and mRNA level, whereas the expression of β-Ⅲ Tubulin was evidently increased. The staining of each group at 8 days was more obvious than that at 4 days. There were significant differences in mRNA expressions of GFAP and β-Ⅲ Tubulin at 4 and 8 days between groups (P<0.05).ConclusionSerum can promote the differentiation of neural stem cells into glial cells. At the same time, it inhibits the differentiation of neural stem cells into neurons, the lower the serum concentration, the smaller the effect.
ObjectiveTo establish the system of isolation, cultivation, and identification of the neural stem cells (NSCs) from subventricular zone (SVZ) of neonatal mice so as to seek for the appropriate seed cells for potential therapeutic interventions of neurological disorders. MethodsNSCs were isolated enzymatically and mechanically from SVZ of neonatal mice and cultured. The cellular morphology was observed by inverted microscopy. Immunocytochemical stainings of anti-Nestin and anti-SOX-2 were used to identify NSCs of passage 3. To study the differentiation of NSCs, NSCs were plated into 24-wells in the medium supplemented without epidermal growth factor (EGF) and basic fibroblastic growth factor (bFGF) for 3 or 7 days. To compare the differentiation and proliferation potential of NSCs with different cultivation time, the BrdU pulse-labeling method and MTT test were used. To identify neurons and astrocytes, the anti-β-tubulin Ⅲ (Tuj-1) and anti-glial fibrillary acidic protein (GFAP) staining were used. ResultsThe cells of the SVZ can be isolated and cultured in vitro, and these cells began to form neurospheres after cultured for 3 days at primary passage. While cultured for 7 days, these cells formed more neurospheres, and the volume of the neurospheres became bigger than neurospheres cultured for 3 days. In addition, after cultured for 7 days, the phenomena of fusion of neurospheres and adherent differentiation of neurospheres were observed under inverted microscope. These cells were provided with the typical phenotype of NSCs. The immunofluorescence staining results revealed that these cells showed positive immunoreactivity to Nestin and SOX-2. During the 4 hours BrdU pulse, the number of proliferated NSCs cultured for 3 days (75.817±2.961) was significantly higher than that of NSCs cultured for 7 days (56.600±4.881) (t=3.366, P=0.028). The results of MTT assay revealed that the absorbance (A) value of NSCs cultured for 3 days (0.478±0.025) was significantly higher than that of NSCs which were cultured for 7 days (0.366±0.032)(t=2.752, P=0.011). After cultivated without EGF and bFGF, the percentage of Tuj-1 and GFAP positive cells in NSCs was 23.1%±3.7% and 23.7%±3.8% for 3 days and was 40.1%±3.6% and 37.1%±4.5% for 7 days, respectively, all showing significant differences (t=3.285, P=0.030; t=3.930, P=0.017). ConclusionThe NSCs from SVZ of neonatal mice have potentials of self-renewal and multipotential differentiation in vitro. With different cultivation time, the potentials of proliferation and differentiation of NSCs are different.
Endogenous adult neural stem cells are closely related to the normal physiological functions of the brain and many neurodegenerative diseases. Neurons are affected by factors such as extracellular microenvironment and intracellular signaling. In recent years, some specific signaling pathways have been found that affect the occurrence of neural stem cells in adult neural networks, including proliferation, differentiation, maturation, migration, and integration with host functions. In this paper, we summarize the signals and their molecular mechanisms, including the related signaling pathways, neurotrophic factors, neurotransmitters, intracellular transcription factors and epigenetic regulation of neuronal differentiation from both the extracellular and intracellular aspects, providing basic theoretical support for the treatment of central nervous system diseases through neural stem cells approach.
Spinal cord injury (SCI), especially the complete SCI, usually results in complete paralysis below the level of the injury and seriously affects the patient’s quality of life. SCI repair is still a worldwide medical problem. In the last twenty years, Professor DAI Jianwu and his team pioneered complete SCI model by removing spinal tissue with varied lengths in rodents, canine, and non-human primates to verify therapeutic effect of different repair strategies. Moreover, they also started the first clinical study of functional collagen scaffold on patients with acute complete SCI on January 16th, 2015. This review mainly focusses on the possible mechanisms responsible for complete SCI. In common, recovery of some sensory and motor functions post complete SCI include the following three contributing reasons. ① Regeneration of long ascending and descending axons throughout the lesion site to re-connect the original targets; ② New neural circuits formed in the lesion site by newly generated neurons post injury, which effectively re-connect the transected stumps; ③ The combined effect of ① and ②. The numerous studies have confirmed that neural circuits rebuilt across the injury site by newborn neurons might be the main mechanisms for functional recovery of animals from rodents to dogs. In many SCI model, especially the complete spinal cord transection model, many studies have convincingly demonstrated that the quantity and length of regenerated long descending axons, particularly like CST fibers, are too few to across the lesion site that is millimeters in length to realize motor functional recovery. Hence, it is more feasible in guiding neuronal relays formation by bio-scaffolds implantation than directing long motor axons regeneration in improving motor function of animals with complete spinal cord transection. However, some other issues such as promoting more neuronal relays formation, debugging wrong connections, and maintaining adequate neural circuits for functional recovery are urgent problems to be addressed.
Objective To investigate the memory amelioration of the Alzheimer disease (AD)model rat after being transplanted the single neural stem cells(NSC) and NSC modified with human brain-derived neurotrophic factor（hBDNF） gene. Methods Forty SD rats were divided evenly into 4 groups randomly. The AD model rats were made by cutting unilaterallythe fibria fornix of male rats. Ten to twelve days after surgery, the genetically modified and unmodified NSC were implanted into the lateral cerebral ventricle of group Ⅲ and group Ⅳ respectively. Two weeks after transplantation, theamelioration of memory impairment of the rats was detected by Morris water maze. Results The average escaping latency of the group Ⅲ and group Ⅳ (41.84±21.76 s,25.23±17.06 s respectively) was shorter than that of the group Ⅱ(70.91±23.67 s) (Plt;0.01). The percentage of swimming distance inthe platform quadrant in group Ⅲ (36.9%) and in group Ⅳ(42.0%) was higherthan that in the group Ⅱ(26.0%) (Plt;0.01). More marginal and random strategies were used in group Ⅱ.The percentage of swimming distance in the platform quadrant in group Ⅳ was also greater than that in group Ⅲ(Plt;0.05). There were no significant differences in the average escaping latency, the percentage of swimming distance in the platform quadrant and the probe strategy between group Ⅳ and group Ⅰ(Pgt;0.05).More lineal and oriented strategies were used in group Ⅳ. Conclusion The behavioral amelioration of AD model rat was obtained by transplanting single NSC and hBDNF-gene-modified NSC. The effect of the NSC group modified with hBDNF gene is better than that of the groupⅢ.
To explore the expression of Wnt-1 during the process of inducing neural stem cells (NSCs) into neurons by using all-trans-retinoic acid (ATRA) in vitro and the effect of Wnt-1 on NSCs differentiation. Methods NSCs isolated from cerebral cortex of SD rat embryo (12-16 days’ gestation) were cultured. The concentration of cells at passage 3 were adjusted to 1 × 106 cells /mL and treated with ATRA at 0.5, 1.0, 5.0 and 10.0 μmol/L, respectively. Differentiation ratio of NSCsinto neurons in each group was detected by double-labelling immunofluorescence technique and flow cytometry, and 1.0 μmol/ L was selected as the best concentration for ATRA to promote NSCs differentiation. In experimental group, NSCs at passage 3 were cultured with ATRA at 1.0 μmol/L in vitro, and expression of Wnt-1 was detected by immunocytochemistry staining, realtime flurescent quantitive PCR and Western blot at 3, 5, 7 and 9 days after culture, respectively. The cells at passage 3 receiving no ATRA served as control group. Results Immunocytochemistry staining: in the control group, there was l ittle Wnt-1 protein expression; in the experimental group, peak expression of Wnt-1 and numerous positive cells occurred at 3 days after culture, the positive expression of Wnt-1 was still evident at 5 days after culture, and there was significant difference between two groups in integrated absorbance (IA) value at 3 and 5 days after culture(P lt; 0.05), obvious decrease of positive expression of Wnt-1 was evident, and no significant difference was evident between two groups in IA value at 7 and 9 days (P gt; 0.05). Real-time fluorescence quantitative PCR: the relative expression of Wnt-1 mRNA in the control group was 0.021 7 ± 0.072 1; the relative expression of Wnt-1 mRNA in the experimental group at 3, 5, 7 and 9 days was 0.512 2 ± 0.280 0, 0.216 4 ± 0.887 0, 0.038 5 ± 0.299 4 and 0.035 5 ± 0.309 5, respectively, indicating the value decreased over time, and there were significant difference between two groups at 3 and 5 days (P lt; 0.05), and no significant difference at 7 and 9 days (P gt; 0.05) . Western blot detection: specific and visible staining band was noted; in the control group, Wnt-1 protein expression was 0.005 1 ± 0.558 3; in the experimental group, Wnt-1 protein expression at 3, 5, 7 and 9 days was 0.451 7 ± 0.071 3, 0.311 7 ± 0.080 5, 0.007 3 ± 0.052 7 and 0.004 7 ± 0.931 4, respectively, suggesting the value decreased over time; there were significant differences between two groups at 3 and 5 days (P lt; 0.05), and no significant differences at 7 and 9 days (P gt; 0.05). Conclusion With the induction of ATRA at 1.0 μmol/L, Wnt-1 and NSCs differentiation in early stage are positively correlated. Its possible mechanism may rely on the activation of such signals as classic Wnt-1 signal pathway, indicating Wnt-1 relates to the differentation of NSCs into neurons.
Seeding cells play an important role in the peripheral nerve damage repair. Seeding cells studied consequently in peripheral nerve are Schwann cells, bone marrow mesenchymal stem cells and neural stem cells. Schwann cells, the first seeding cells, are various unique glial cells in the peripheral nervous system, which can form the myelin sheath for insulation and package of the neuron projecting axons in the peripheral nervous system so that the conduction velocity of the nerve signal was accelerated. It can be proved that Schwann cells played an important role in the maintenance of peripheral nerve function and in the regeneration process after peripheral nerve injury. The second, bone marrow mesenchymal stem cells are the various mesenchymal stem cells mainly exist in the systemic connective tissues and organs. These functional stem cells are often studied at present, and it has been found that they have exuberant proliferation and differentiation potentials. Neural stem cells, mentioned the third in sequence, are the kind of pluripotent cells with multi-directional differentiation, which could conduct the self-renewal function, and generate and differentiate neurons, astrocytes and oligodendrocytes through asymmetric cell division. These three types of seed cells are discussed in this paper.
ObjectiveTo review the research progress of neural regulation mechanism of vasculogenesis. MethodsThe relevant literature on neural regulation mechanism of vasculogenesis was extensively reviewed. ResultsNeural regulation of vasculogenesis depends on synergistic effect among various cells of neurovascular unit, and co-participation of multiple cytokines, and it is closely related to a variety of repair mechanism, such as nerve regeneration and synaptic plasticity, but the specific mechanism need to be further investigated. ConclusionThe research of the neural regulation mechanism of vasculogenesis will contribute to further understanding repair mechanism of nerves and vessels injuries.