Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

Parthenogenetic embryonic stem cells have pluripotent differentiation potentials,akin to fertilized embryo-derived embryonic stem cells.The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells.Before differentiation,karyotype analysis was performed,with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells.Sex chromosomes were identified as XX.Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene,Oct4,at both the mRNA and protein levels,indicating pluripotent differentiation potential of the two embryonic stem cell subtypes.Embryonic stem cells were induced with retinoic acid to form embryoid bodies,and then dispersed into single cells.Single cells were differentiated in N2 differentiation medium for 9 days.Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin,βIII-tubulin and myelin basic protein.Quantitative real-time PCR found expression of neurogenesis related genes(Sox-1,Nestin,GABA,Pax6,Zic5 and Pitx1) in both types of embryonic stem cells,and Oct4 expression was significantly decreased.Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells.Thus,our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.     

Oligomeric proanthocyanidin protects retinal ganglion cells against oxidative stress-induced apoptosis

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A ginkgo biloba extract promotes proliferation of endogenous neural stem cells in vascular dementia rats

The ginkgo biloba extract EGb761 improves memory loss and cognitive impairments in patients with senile dementia. It also promotes proliferation of neural stem cells in the subventricular zone in Parkinson’s disease model mice and in the hippocampal zone of young epileptic rats. However, it remains unclear whether EGb761 enhances proliferation of endogenous neural stem cells in the brain of rats with vascular dementia. In this study, a vascular dementia model was established by repeatedly clipping and reperfusing the bilateral common carotid arteries of rats in combination with an intraperitoneal injection of a sodium nitroprusside solution. Seven days after establishing the model, rats were intragastrically given EGb761 at 50 mg/kg per day. Learning and memory abilities were assessed using the Morris water maze and proliferation of endogenous neural stem cells in the subventricular zone and dentate gyrus were labeled by 5-bromo-2-deoxyuridine immunofluorescence in all rats at 15 days, and 1, 2, and 4 months after model establishment. The escape latencies in Morris water maze tests of rats with vascular dementia after EGb761 treatment were significantly shorter than the model group. Immunofluorescence staining showed that the number and proliferation of 5-bromo-2-deoxyuridine-positive cells in the subventricular zone and dentate gyrus of the EGb761-treated group were significantly higher than in the model group. These experimental findings suggest that EGb761 enhances proliferation of neural stem cells in the subventricular zone and dentate gyrus, and significantly improves learning and memory in rats with vascular dementia.     

Curcumin protects against ischemic spinal cord injury The pathway effect

Inducible nitric oxide synthase and N-methyI-D-aspartate receptors have been shown to participate in nerve cell injury during spinal cord ischemia. This study observed a protective effect of curcumin on ischemic spinal cord injury. Models of spinal cord ischemia were established by ligating the lumbar artery from the left renal artery to the bifurcation of the abdominal aorta. At 24 hours after model establishment, the rats were intraperitoneally injected with curcumin, Reverse transcrip- tion-polymerase chain reaction and immunohistochemical results demonstrated that after spinal cord ischemia, inducible nitric oxide synthase and N-methyI-D-aspartate receptor mRNA and protein expression significantly increased. However, curcumin significantly decreased inducible nitric oxide synthase and N-methyI-D-aspartate receptor mRNA and protein expression in the ischemic spinal cord. Tadov scale results showed that curcumin significantly improved motor function of the rat hind limb after spinal cord ischemia. The results demonstrate that curcumin exerts a neuroprotective ef- fect against ischemic spinal cord injury by decreasing inducible nitric oxide synthase and N-methyI-D-aspartate receptor expression.     

Differentiation of human adipose-derived stem cells into neuron-like cells by Radix Angelicae Sinensis

Human adipose tissues are an ideal source of stem cells. It is important to find inducers that can safely and effectively differentiate stem cells into functional neurons for clinical use. In this study, we investigate the use of Radix Angelicae Sinensis as an inducer of neuronal differentiation. Primary human adipose-derived stem cells were obtained from adult subcutaneous fatty tissue, then pre-induced with 10% Radix Angelicae Sinensis injection for 24 hours, and incubated in serum-free Dulbecco＇s modified Eagle＇s medium/Nutrient Mixture F-12 containing 40% Radix Angelicae Sinensis to induce its differentiation into neuron-like cells. Butylated hydroxyanisole, a common in- ducer for neuronal differentiation, was used as the control. After human adipose-derived stem cells differentiated into neuron-like cells under the induction of Radix Angelicae Sinensis for 24 hours, the positive expression of neuron-specific enolase was lower than that of the butylated hydroxyani- sole-induced group, and the expression of glial fibrillary acidic protein was negative. Alter they were induced for 48 hours, the positive expression of neuron specific enolase in human adipose-derived stem cells was significantly higher than that of the butylated hydroxyanisole-induced group. Our experimental findings indicate that Radix Angelicae Sinensis can induce human adipose-derived stem cell differentiation into neuron-like cells and produce less cytotoxicity.     

Growth and differentiation of neural stem cells in a three-dimensional collagen gel scaffold

Collagen protein is an ideal scaffold material for the transplantation of neural stem cells. In this study, rat neural stem cells were seeded into a three-dimensional collagen gel scaffold, with suspension cultured neural stem cells being used as a control group. Neural stem cells, which were cultured in medium containing epidermal growth factor and basic fibroblast growth factor, actively expanded and formed neurospheres in both culture groups. In serum-free medium conditions, the processes extended from neurospheres in the collagen gel group were much longer than those in the suspension culture group. Immunofluorescence staining showed that neurospheres cultured in collagen gels were stained positive for nestin and differentiated cells were stained positive for the neuronal marker βIII-tubulin, the astrocytic marker glial fibrillary acidic protein and the oligodendrocytic marker 2’,3’-cyclic nucleotide 3’-phosphodiesterase. Compared with neurospheres cultured in suspension, the differentiation potential of neural stem cells cultured in collagen gels increased, with the formation of neurons at an early stage. Our results show that the three-dimensional collagen gel culture system is superior to suspension culture in the proliferation, differentiation and process outgrowth of neural stem cells.     

Similarity on neural stem cells and brain tumor stem cells in transgenic brain tumor mouse models

Although it is believed that glioma is derived from brain tumor stem cells, the source and molecular signal pathways of these cells are still unclear. In this study, we used stable doxycycline-inducible transgenic mouse brain tumor models (c-myc⁺/SV40Tag⁺/Tet-on⁺) to explore the malignant trans-formation potential of neural stem cells by observing the differences of neural stem cells and brain tumor stem cells in the tumor models. Results showed that chromosome instability occurred in brain tumor stem cells. The numbers of cytolysosomes and autophagosomes in brain tumor stem cells and induced neural stem cells were lower and the proliferative activity was obviously stronger than that in normal neural stem cells. Normal neural stem cells could differentiate into glial fibrillary acidic protein-positive and microtubule associated protein-2-positive cells, which were also negative for nestin. However, glial fibrillary acidic protein/nestin, microtubule associated protein-2/nestin, and glial fibrillary acidic protein/microtubule associated protein-2 double-positive cells were found in induced neural stem cells and brain tumor stem cells. Results indicate that induced neural stem cells are similar to brain tumor stem cells, and are possibly the source of brain tumor stem cells.     

Influence of endogenous ciliary neurotrophic factor on neural differentiation of adult rat hippocampal progenitors

Ciliary neurotrophic factor is the only known neurotrophic factor that can promote differentiation of hippocampal neural progenitor cells to glial cells and neurons in adult rats. This process is similar to spontaneous differentiation. Therefore, ciliary neurotrophic factor may be involved in spontaneous differentiation of neural stem cells. To verify this hypothesis, the present study isolated neural progenitor cells from adult male rats and cultured them in vitro. Results showed that when neural progenitor cells were cultured in the absence of mitogen fibroblast growth factor-2 or epidermal growth factor, they underwent spontaneous differentiation into neurons and glial cells. Western blot and immunocytochemical staining showed that exogenous ciliary neurotrophic factor strongly induced adult hippocampal progenitor cells to differentiate into neurons and glial cells. Moreover, passage 4 adult hippocampal progenitor cells expressed high levels of endogenous ciliary neurotrophic factor, and a neutralizing antibody against ciliary neurotrophic factor prevented the spontaneous neuronal and glial differentiation of adult hippocampal progenitor cells. These results suggest that the spontaneous differentiation of adult hippocampal progenitor cells is mediated partially by endogenous ciliary neurotrophic factor.     

Migration capacity of human umbilical cord mesenchymal stem cells towards glioma in vivo*

High-grade glioma is the most common malignant primary brain tumor in adults.The poor prognosis of glioma,combined with a resistance to currently available treatments,necessitates the development of more effective tumor-selective therapies.Stem cell-based therapies are emerging as novel cell-based delivery vehicle for therapeutic agents.In the present study,we successfully isolated human umbilical cord mesenchymal stem cells by explant culture.The human umbilical cord mesenchymal stem cells were adherent to plastic surfaces,expressed specific surface phenotypes of mesenchymal stem cells as demonstrated by flow cytometry,and possessed multi-differentiation potentials in permissive induction media in vitro.Furthermore,human umbilical cord mesenchymal stem cells demonstrated excellent glioma-specific targeting capacity in established rat glioma models after intratumoral injection or contralateral ventricular administration in vivo.The excellent glioma-specific targeting ability and extensive intratumoral distribution of human umbilical cord mesenchymal stem cells indicate that they may serve as a novel cellular vehicle for delivering therapeutic molecules in glioma therapy.     

Jiaweisinisan facilitates neurogenesis in the hippocampus after stress damage

The traditional Chinese medicine Jiaweisinisan has antidepressant effects, and can inhibit hypothalamus-pituitary-adrenal gland axis hyperactivity in stress-induced depression. In this study, rat hippocampal neural precursor cells were cultured in serum-free medium in vitro and a stress damage model was established with 120 μM corticosterone. Cells were treated with 10% (v/v) Jiaweisinisan drug-containing serum and the corticosterone antagonist RU38486. Results of the 3-(4,5-dimethylthiazol-2-yl)-3,5-di-phenytetrazoliumromide assay showed that both Jiaweisinisan drug-containing serum and RU38486 promoted the proliferation of neural precursor cells after corticosterone exposure. Immunofluorescence detection showed that after Jiaweisinisan drug-containing serum and RU38486 treatment, the 5-bromo-2-deoxyuridine/terminal deoxynucleotidyl transferase dUTP nick end labeling ratio in hippocampal neural precursor cells significantly increased, and the apoptotic rates of glial cells reduced, and neuron-like cell differentiation from neural precursor cells significantly increased. Our experimental findings indicate that Jiaweisinisan promotes hippocampal neurogenesis after stress damage.     

Emodin prevents hypoxic-ischemic neuronal injury Involvement of the activin A pathway

Emodin, an extract of dried rhizomes and the root of the Rhizoma Polygoni Cuspidati, can protect neurons from hypoxic-ischemic brain damage. This study aimed to verify the underlying mechanism. After PC12 cells had differentiated into neuron-like cells under the induction of mouse nerve growth factor, cells were subjected to oxygen-glucose deprivation and treated with emodin. Results showed that the viability of neuron-like cells cultured under an ischemia-hypoxia environment decreased, while the expression of activin A and caspase-3 in cells increased. Emodin raised the survival rate of oxygen-glucose deprived neuron-like cells, increased activin A expression, and decreased caspase-3 expression. Experimental findings indicate that emodin can inhibit neuronal apoptosis and alleviate the injury of nerve cells after oxygen-glucose deprivation through the activin A pathway.     

3aicalin inhibits colistin sulfate-induced apoptosis of PC12 cells

Baicalin, a type of flavonoid extracted from the dried root of Scutellaria baicalensis georgi, has been shown to effectively inhibit cell apoptosis. Therefore, we assumed that baicalin would suppress colistin sulfate-induced neuronal apoptosis. PC12 cells exposed to colistin sulfate （62.5-500 μg/mL） for 24 hours resulted in PCl2 cell apoptosis. In addition, caspase-3 activity, lactate dehydrogenase level and free radical content increased in a dose-dependent manner. Subsequently, PC12 cells were pretreated with baicalin （25, 50 and 100 pg/mL）, and exposed to 125 pg/mL colistin sulfate. Cell morphology markedly changed, and cell viability increased. Moreover, caspase-3 activity, lac- tate dehydrogenase level and free radical content decreased. Results indicated that baicalin inhib- ited colistin sulfate-induced PC12 cell apoptosis by suppressing free radical injury, and reducing caspase-3 activity and lactate dehydrogenase activity.     

Protective effect of oxysophoridine on cerebral ischemia/reperfusion injury in mice

Oxysophoridine, a new alkaloid extracted from Sophora alopecuroides L., has been shown to have a protective effect against ischemic brain damage. In this study, a focal cerebral ischemia/reperfusion injury model was established using middle cerebral artery occlusion in mice. Both 62.5, 125, and 250 mg/kg oxysophoridine, via intraperitoneal injection, and 6 mg/kg nimodipine, via intragastric administration, were administered daily for 7 days before modeling. After 24 hours of reperfusion, mice were tested for neurological deficit, cerebral infarct size was assessed and brain tissue was collected. Results showed that oxysophoridine at 125, 250 mg/kg and 6 mg/kg nimodipine could reduce neurological deficit scores, cerebral infarct size and brain water content in mice. These results provided evidence that oxysophoridine plays a protective role in cerebral ischemia/reperfusion injury. In addition, oxysophoridine at 62.5, 125, and 250 mg/kg and 6 mg/kg nimodipine increased adenosine-triphosphate content, and decreased malondialdehyde and nitric oxide content. These compounds enhanced the activities of glutathione-peroxidase, superoxide dismutase, catalase, and lactate dehydrogenase, and decreased the activity of nitric oxide synthase. Protein and mRNA expression levels of N-methyl-D-aspartate receptor subunit NR1 were markedly inhibited in the presence of 250 mg/kg oxysophoridine and 6 mg/kg nimodipine. Our experimental findings indicated that oxysophoridine has a neuroprotective effect against cerebral ischemia/reperfusion injury in mice, and that the effect may be due to its ability to inhibit oxidative stress and expression of the N-methyl-D-aspartate receptor subunit NR1.     

Two outward potassium current types are expressed during the neural differentiation of neural stem cells

The electrophysiological properties of potassium ion channels are regarded as a basic index for determining the functional differentiation of neural stem cells. In this study, neural stem cells from th...     

Optimal time for subarachnoid transplantation of neural progenitor cells in the treatment of contusive spinal cord injury

This study aimed to identify the optimal neural progenitor cell transplantation time for spinal cord injury in rats via the subarachnoid space. Cultured neural progenitor cells from 14-day embryonic rats, constitutively expressing enhanced green fluorescence protein, or media alone, were injected into the subarachnoid space of adult rats at 1 hour (acute stage), 7 days (subacute stage) and 28 days (chronic stage) after contusive spinal cord injury. Results showed that grafted neural progenitor cells migrated and aggregated around the blood vessels of the injured region, and infiltrated the spinal cord parenchyma along the tissue spaces in the acute stage transplantation group. However, this was not observed in subacute and chronic stage transplantation groups. O4- and glial fibrillary acidic protein-positive cells, representing oligodendrocytes and astrocytes respectively, were detected in the core of the grafted cluster attached to the cauda equina pia surface in the chronic stage transplantation group 8 weeks after transplantation. Both acute and subacute stage transplantation groups were negative for O4 and glial fibrillary acidic protein cells. Basso, Beattie and Bresnahan scale score comparisons indicated that rat hind limb locomotor activity showed better recovery after acute stage transplantation than after subacute and chronic transplantation. Our experimental findings suggest that the subarachnoid route could be useful for transplantation of neural progenitor cells at the acute stage of spinal cord injury. Although grafted cells survived only for a short time and did not differentiate into astrocytes or neurons, they were able to reach the parenchyma of the injured spinal cord and improve neurological function in rats. Transplantation efficacy was enhanced at the acute stage in comparison with subacute and chronic stages.     

Effects of neurotrophin-3 on the differentiation of neural stem cells into neurons and oligodendrocytes

In this study,cells from the cerebral cortex of fetal rats at pregnant 16 days were harvested and cultured with 20 μg/L neurotrophin-3.After 7 days of culture,immunocytochemical staining showed that,22.4% of cells were positive for nestin,10.5% were positive for β-III tubulin(neuronal marker),and 60.6% were positive for glial fibrillary acidic protein,but no cells were positive for O4(oligodendrocytic marker).At 14 days,there were 5.6% nestin-,9.6% β-III tubulin-,81.1% glial fibrillary acidic protein-,and 2.2% O4-positive cells.In cells not treated with neurotrophin-3,some were nestin-positive,while the majority showed positive staining for glial fibrillary acidic protein.Our experimental findings indicate that neurotrophin-3 is a crucial factor for inducing neural stem cells differentiation into neurons and oligodendrocytes.     

Hyperbaric oxygen treatment promotes neural stem cell proliferation in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage

Hyperbaric oxygen therapy for the treatment of neonatal hypoxic-ischemic brain damage has been used clinically for many years, but its effectiveness remains controversial. In addition, the mechanism of this potential neuroprotective effect remains unclear. This study aimed to investigate the influence of hyperbaric oxygen on the proliferation of neural stem cells in the subventricular zone of neonatal Sprague-Dawley rats (7 days old) subjected to hypoxic-ischemic brain damage. Six hours after modeling, rats were treated with hyperbaric oxygen once daily for 7 days. Immunohistochemistry revealed that the number of 5-bromo-2′-deoxyuridine positive and nestin positive cells in the subventricular zone of neonatal rats increased at day 3 after hypoxic-ischemic brain damage and peaked at day 5. After hyperbaric oxygen treatment, the number of 5-bromo-2′- deoxyuridine positive and nestin positive cells began to increase at day 1, and was significantly higher than that in normal rats and model rats until day 21. Hematoxylin-eosin staining showed that hyperbaric oxygen treatment could attenuate pathological changes to brain tissue in neonatal rats, and reduce the number of degenerating and necrotic nerve cells. Our experimental findings indicate that hyperbaric oxygen treatment enhances the proliferation of neural stem cells in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage, and has therapeutic potential for promoting neurological recovery following brain injury.     

Neuroprotective effects of rutaecarpine on cerebral ischemia reperfusion injury**

Rutaecarpine,an active component of the traditional Chinese medicine Tetradium ruticarpum,has been shown to improve myocardial ischemia reperfusion injury.Because both cardiovascular and cerebrovascular diseases are forms of ischemic vascular disease,they are closely related.We hypothesized that rutaecarpine also has neuroprotective effects on cerebral ischemia reperfusion injury.A cerebral ischemia reperfusion model was established after 84,252 and 504 μg/kg rutaecarpine were given to mice via intraperitoneal injection,daily for 7 days.Results of the step through test,2,3,5-triphenyl tetrazolium chloride dyeing and oxidative stress indicators showed that rutaecarpine could improve learning and memory ability,neurological symptoms and reduce infarction volume and cerebral water content in mice with cerebral ischemia reperfusion injury.Rutaecarpine could significantly decrease the malondialdehyde content and increase the activities of superoxide dismutase and glutathione peroxidase in mouse brain.Therefore,rutaecarpine could improve neurological function following injury induced by cerebral ischemia reperfusion,and the mechanism of this improvement may be associated with oxidative stress.These results verify that rutaecarpine has neuroprotective effects on cerebral ischemia reperfusion in mice.