Hepatocyte growth factor enhances the ability of dental pulp stem cells to ameliorate atherosclerosis in apolipoprotein E-knockout mice

BACKGROUND Atherosclerosis(AS),a chronic inflammatory disease of blood vessels,is a major contributor to cardiovascular disease.Dental pulp stem cells(DPSCs)are capable of exerting immunomodulatory and anti-inflammatory effects by secreting cytokines and exosomes and are widely used to treat autoimmune and inflam-mation-related diseases.Hepatocyte growth factor(HGF)is a pleiotropic cytokine that plays a key role in many inflammatory and autoimmune diseases.AIM To modify DPSCs with HGF(DPSC-HGF)and evaluate the therapeutic effect of DPSC-HGF on AS using an apolipoprotein E-knockout(ApoE-/-)mouse model and an in vitro cellular model.METHODS ApoE-/-mice were fed with a high-fat diet(HFD)for 12 wk and injected with DPSC-HGF or Ad-Null modified DPSCs(DPSC-Null)through tail vein at weeks 4,7,and 11,respectively,and the therapeutic efficacy and mechanisms were analyzed by histopathology,flow cytometry,lipid and glucose measurements,real-time reverse transcription polymerase chain reaction(RT-PCR),and enzyme-linked immunosorbent assay at the different time points of the experiment.An in vitro inflammatory cell model was established by using RAW264.7 cells and human aortic endothelial cells(HAOECs),and indirect co-cultured with supernatant of DPSC-Null(DPSC-Null-CM)or DPSC-HGF-CM,and the effect and mechanisms were analyzed by flow cytometry,RT-PCR and western blot.Nuclear factor-κB(NF-κB)activators and inhibitors were also used to validate the related signaling pathways.RESULTS DPSC-Null and DPSC-HGF treatments decreased the area of atherosclerotic plaques and reduced the expression of inflammatory factors,and the percentage of macrophages in the aorta,and DPSC-HGF treatment had more pronounced effects.DPSCs treatment had no effect on serum lipoprotein levels.The FACS results showed that DPSCs treatment reduced the percentages of monocytes,neutrophils,and M1 macrophages in the peripheral blood and spleen.DPSC-Null-CM and DPSC-HGF-CM reduced adhesion molecule expression in tumor necrosis factor-αstimulated HAOECs and regulated M1 polarization and inflammatory factor expression in lipopolysaccharide-induced RAW264.7 cells by inhibiting the NF-κB signaling pathway.CONCLUSION This study suggested that DPSC-HGF could more effectively ameliorate AS in ApoE-/-mice on a HFD,and could be of greater value in stem cell-based treatments for AS.

Microvesicles derived from mesenchymal stem cells inhibit acute respiratory distress syndrome-related pulmonary fibrosis in mouse partly through hepatocyte growth factor

BACKGROUND Pulmonary fibrosis is one of the main reasons for the high mortality rate among acute respiratory distress syndrome(ARDS)patients.Mesenchymal stromal cell-derived microvesicles(MSC-MVs)have been shown to exert antifibrotic effects in lung diseases.AIM To investigate the effects and mechanisms of MSC-MVs on pulmonary fibrosis in ARDS mouse models.METHODS MSC-MVs with low hepatocyte growth factor(HGF)expression(siHGF-MSC-MVs)were obtained via lentivirus transfection and used to establish the ARDS pulmonary fibrosis mouse model.Following intubation,respiratory mechanics-related indicators were measured via an experimental small animal lung function tester.Homing of MSC-MVs in lung tissues was investigated by near-infrared live imaging.Immunohistochemical,western blotting,ELISA and other methods were used to detect expression of pulmonary fibrosis-related proteins and to compare effects on pulmonary fibrosis and fibrosis-related indicators.RESULTS The MSC-MVs gradually migrated and homed to damaged lung tissues in the ARDS model mice.Treatment with MSC-MVs significantly reduced lung injury and pulmonary fibrosis scores.However,low expression of HGF(siHGF-MSC-MVs)significantly inhibited the effects of MSC-MVs(P<0.05).Compared with the ARDS pulmonary fibrosis group,the MSC-MVs group exhibited suppressed expression of type I collagen antigen,type III collagen antigen,and the proteins transforming growth factor-βandα-smooth muscle actin,whereas the siHGF-MVs group exhibited significantly increased expression of these proteins.In addition,pulmonary compliance and the pressure of oxygen/oxygen inhalation ratio were significantly lower in the MSC-MVs group,and the effects of the MSC-MVs were significantly inhibited by low HGF expression(all P<0.05).CONCLUSION MSC-MVs improved lung ventilation functions and inhibited pulmonary fibrosis in ARDS mice partly via HGF mRNA transfer.

Low-temperature 3D-printed collagen/chitosan scaffolds loaded with exosomes derived from neural stem cells pretreated with insulin growth factor-1 enhance neural regeneration after traumatic brain injury

There are various clinical treatments for traumatic brain injury,including surgery,drug therapy,and rehabilitation therapy;howeve r,the therapeutic effects are limited.Scaffolds combined with exosomes represent a promising but challenging method for improving the repair of traumatic brain injury.In this study,we determined the ability of a novel 3D-printed collagen/chitosan scaffold loaded with exosomes derived from neural stem cells pretreated with insulin-like growth factor-1(3D-CC-INEXOS) to improve traumatic brain injury repair and functional recove ry after traumatic brain injury in rats.Composite scaffolds comprising collagen,chitosan,and exosomes derived from neural stem cells pretreated with insulin-like growth fa ctor-1(INEXOS) continuously released exosomes for 2weeks.Transplantation of 3D-CC-INExos scaffolds significantly improved motor and cognitive functions in a rat traumatic brain injury model,as assessed by the Morris water maze test and modified neurological seve rity scores.In addition,immunofluorescence staining and transmission electron microscopy showed that3D-CC-INExos implantation significantly improved the recove ry of damaged nerve tissue in the injured area.In conclusion,this study suggests that transplanted3D-CC-INExos scaffolds might provide a potential strategy for the treatment of traumatic brain injury and lay a solid foundation for clinical translation.

Culture and identification of neonatal rat brain-derived neural stem cells

BACKGROUND Timing of passaging,passage number,passaging approaches and methods for cell identification are critical factors influencing the quality of neural stem cells(NSCs)culture.How to effectively culture and identify NSCs is a continuous interest in NSCs study while these factors are comprehensively considered.AIM To establish a simplified and efficient method for culture and identification of neonatal rat brain-derived NSCs.METHODS First,curved tip operating scissors were used to dissect brain tissues from new born rats(2 to 3 d)and the brain tissues were cut into approximately 1 mm^(3)sections.Filter the single cell suspension through a nylon mesh(200-mesh)and culture the sections in suspensions.Passaging was conducted with TrypLTM Express combined with mechanical tapping and pipetting techniques.Second,identify the 5th generation of passaged NSCs as well as the revived NSCs from cryopreservation.BrdU incorporation method was used to detect self-renew and proliferation capabilities of cells.Different NSCs specific antibodies(anti-nestin,NF200,NSE and GFAP antibodies)were used to identify NSCs specific surface markers and muti-differentiation capabilities by immunofluorescence staining.RESULTS Brain derived cells from newborn rats(2 to 3 d)proliferate and aggregate into spherical-shaped clusters with sustained continuous and stable passaging.When BrdU was incorporated into the 5th generation of passaged cells,positive BrdU cells and nestin cells were observed by immunofluorescence staining.After induction of dissociation using 5%fetal bovine serum,positive NF200,NSE and GFAP cells were observed by immunofluorescence staining.CONCLUSION This is a simplified and efficient method for neonatal rat brain-derived neural stem cell culture and identification.

Role of brahma-related gene 1/brahma-associated factor subunits in neural stem/progenitor cells and related neural developmental disorders

Different fates of neural stem/progenitor cells(NSPCs)and their progeny are determined by the gene regulatory network,where a chromatin-remodeling complex affects synergy with other regulators.Here,we review recent research progress indicating that the BRG1/BRM-associated factor(BAF)complex plays an important role in NSPCs during neural development and neural developmental disorders.Several studies based on animal models have shown that mutations in the BAF complex may cause abnormal neural differentiation,which can also lead to various diseases in humans.We discussed BAF complex subunits and their main characteristics in NSPCs.With advances in studies of human pluripotent stem cells and the feasibility of driving their differentiation into NSPCs,we can now investigate the role of the BAF complex in regulating the balance between self-renewal and differentiation of NSPCs.Considering recent progress in these research areas,we suggest that three approaches should be used in investigations in the near future.Sequencing of whole human exome and genome-wide association studies suggest that mutations in the subunits of the BAF complex are related to neurodevelopmental disorders.More insight into the mechanism of BAF complex regulation in NSPCs during neural cell fate decisions and neurodevelopment may help in exploiting new methods for clinical applications.

Early expressions of hypoxia-inducible factor 1alpha and vascular endothelial growth factor increase the neuronal plasticity of activated endogenous neural stem cells after focal cerebral ischemia

Endogenous neural stem cells become ＂activated＂ after neuronal injury, but the activation sequence and fate of endogenous neural stem cells in focal cerebral ischemia model are little known. We evaluated the relationships between neural stem cells and hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a photothromobotic rat stroke model using immunohistochemistry and western blot analysis. We also evaluated the chronological changes of neural stem cells by 5-bromo-2′-deoxyuridine（BrdU） incorporation. Hypoxia-inducible factor-1α expression was initially increased from 1 hour after ischemic injury, followed by vascular endothelial growth factor expression. Hypoxia-inducible factor-1α immunoreactivity was detected in the ipsilateral cortical neurons of the infarct core and peri-infarct area. Vascular endothelial growth factor immunoreactivity was detected in bilateral cortex, but ipsilateral cortex staining intensity and numbers were greater than the contralateral cortex. Vascular endothelial growth factor immunoreactive cells were easily found along the peri-infarct area 12 hours after focal cerebral ischemia. The expression of nestin increased throughout the microvasculature in the ischemic core and the peri-infarct area in all experimental rats after 24 hours of ischemic injury. Nestin immunoreactivity increased in the subventricular zone during 12 hours to 3 days, and prominently increased in the ipsilateral cortex between 3–7 days. Nestin-labeled cells showed dual differentiation with microvessels near the infarct core and reactive astrocytes in the peri-infarct area. BrdU-labeled cells were increased gradually from day 1 in the ipsilateral subventricular zone and cortex, and numerous BrdU-labeled cells were observed in the peri-infarct area and non-lesioned cortex at 3 days. BrdU-labeled cells rather than neurons, were mainly co-labeled with nestin and GFAP. Early expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor after ischemia made up the microenvironment to increase the neuronal plasticity of activated endogenous neural stem cells. Moreover, neural precursor cells after large-scale cortical injury could be recruited from the cortex nearby infarct core and subventricular zone.

Transplantation of vascular endothelial growth factor-modified neural stem/progenitor cells promotes the recovery of neurological function following hypoxic-ischemic brain damage

Neural stem/progenitor cell （NSC） transplantation has been shown to effectively improve neurological function in rats with hypoxic-isch- emic brain damage. Vascular endothelial growth factor （VEGF） is a signaling protein that stimulates angiogenesis and improves neural regeneration. We hypothesized that transplantation of VEGF-transfected NSCs would alleviate hypoxic-ischemic brain damage in neo- natal rats. We produced and transfected a recombinant lentiviral vector containing the VEGF165gene into cultured NSCs. The transfected NSCs were transplanted into the left sensorimotor cortex of rats 3 days after hypoxic-ischemic brain damage. Compared with the NSCs group, VEGF mRNA and protein expression levels were increased in the transgene NSCs group, and learning and memory abilities were significantly improved at 30 days. Furthermore, histopathological changes were alleviated in these animals. Our findings indicate that transplantation of VEGF-transfected NSCs may facilitate the recovery of neurological function, and that its therapeutic effectiveness is better than that of unmodified NSCs.

Human umbilical cord mesenchymal stem cells derivedexosomes on VEGF-A in hypoxic-induced mice retinal astrocytes and mice model of retinopathy of prematurity

AIM:To observe the effect of human umbilical cord mesenchymal stem cells(hUCMSCs)secretions on the relevant factors in mouse retinal astrocytes,and to investigate the effect of hUCMSCs on the expression of vascular endothelial growth factor-A(VEGF-A)and to observe the therapeutic effect on the mouse model of retinopathy of prematurity(ROP).METHODS:Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group.MTT assay,flow cytometry,reverse transcription-polymerase chain reaction(RT-PCR)and Western blot were used to detect related indicators.Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored.At last,the efficacy of exosomes of UCMSCs in a mouse ROP model was explored.Graphpad6 was used to comprehensively process data information.RESULTS:The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation.Reactive oxygen species(ROS)and hypoxia inducible factor-1α(HIF-1α)of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased,and the ROP cell model was established after 6h of hypoxia.The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α,the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent.Compared with the ROP cell model group,the expression of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signal pathway related factors in the hUCMSCs exocrine group is significantly decreased.The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1αin ROP model tissues.HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion.CONCLUSION:In a hypoxia induced mouse retinal astrocyte model,hUCMSCs exosomes are found to effectively reduce the expression of HIF-1αand VEGF-A,which are positively correlated with the concentration of hUCMSCs exosomes.HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1αand VEGF-A proteins in ROP mice,and are positively correlated with drug dosage.Besides,they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.

Potential plausible role of Wharton’s jelly mesenchymal stem cells for diabetic bone regeneration

This letter addresses the review titled“Wharton’s jelly mesenchymal stem cells:Future regenerative medicine for clinical applications in mitigation of radiation injury”.The review highlights the regenerative potential of Wharton’s jelly mesenchymal stem cells(WJ-MSCs)and describes why WJ-MSCs will become one of the most probable stem cells for future regenerative medicine.The potential plausible role of WJ-MSCs for diabetic bone regeneration should be noticeable,which will provide a new strategy for improving bone regeneration under diabetic conditions.

Response of the sensorimotor cortex of cerebral palsy rats receiving transplantation of vascular endothelial growth factor 165-transfected neural stem cells

Neural stem cells are characterized by the ability to differentiate and stably express exogenous ge- nes. Vascular endothelial growth factor plays a role in protecting local blood vessels and neurons of newborn rats with hypoxic-ischemic encephalopathy. Transplantation of vascular endothelial growth factor-transfected neural stem cells may be neuroprotective in rats with cerebral palsy. In this study, 7-day-old Sprague-Dawley rats were divided into five groups： （1） sham operation （control）, （2） cerebral palsy model alone or with （3） phosphate-buffered saline, （4） vascular en- dothelial growth factor 165 ＋ neural stem cells, or （5） neural stem cells alone. The cerebral palsy model was established by ligating the left common carotid artery followed by exposure to hypox- ia. Phosphate-buffered saline, vascular endothelial growth factor ＋ neural stem cells, and neural stem cells alone were administered into the sensorimotor cortex using the stereotaxic instrument and microsyringe. After transplantation, the radial-arm water maze test and holding test were performed. Immunohistochemistry for vascular endothelial growth factor and histology using hematoxylin-eosin were performed on cerebral cortex. Results revealed that the number of vas- cular endothelial growth factor-positive cells in cerebral palsy rats transplanted with vascular endothelial growth factor-transfected neural stem cells was increased, the time for finding water and the finding repetitions were reduced, the holding time was prolonged, and the degree of cell degeneration or necrosis was reduced. These findings indicate that the transplantation of vascu- lar endothelial growth factor-transfected neural stem cells alleviates brain damage and cognitive deficits, and is neuroprotective in neonatal rats with hypoxia ischemic-mediated cerebral palsy.

Cloning of the Eukaryotic Expression Vector with Nerve Growth Factor in Rats and Its Effects on Proliferation and Differentiation of Mesencephal Neural Stem Cells of Fetal Rats

The eukaryotic expression vector containing full-length cDNA sequence of rate nerve growth factor （NGF） β subunit was constructed and its effects on proliferation and differentiation of neural stem cells were observed. By using PCR, full-length cDNA sequence of NGF β subunit in rats was cloned and ligated into the eukaryotic expression vector pEGFP-N1-NGF. The recombinant plasmid pEGFP-N1-NGF was transfected into the mesencephal neural stem cells of embryonic rats by Lipofectamin and transiently expressed. MTT method was used to determine the effects of NGF on proliferation of neural stem cells, and under phase-contrast microscopy, the effects of NGF on growth of nervous processes following differentiation of neural stem cells were observed. Sequence analysis indicated that the cloned full-length cDNA sequence of rat NGF β was identical to that of published sequence encoding NGF in gene GeneBank. The transfection of recombinant plasmid pEGFP-N1-NGF into mesencephal neural stem cells of embryonic rats could obviously promote proliferation of neural stem cells and faciliate the growth of neural stem cells-derived nerve cells. It was suggested that neural stem cells could be used as a vehicle of gene transfer, and the expression of NGF β subunit in the neural stem cells could promote the growth of nerve cells derived from neural stem cells.

Repair of spinal cord injury by neural stem cells transfected with brain-derived neurotrophic factor-green fluorescent protein in rats A double effect of stem cells and growth factors?

Brain-derived neurotrophic factor（BDNF）can significantly promote nerve regeneration and repair.High expression of the BDNF-green fluorescent protein（GFP）gene persists for a long time after transfection into neural stem cells.Nevertheless,little is known about the biological characteristics of BDNF-GFP modified nerve stem cells in vivo and their ability to induce BDNF expression or repair spinal cord injury.In the present study,we transplanted BDNF-GFP transgenic neural stem cells into a hemisection model of rats.Rats with BDNF-GFP stem cells exhibited significantly increased BDNF expression and better locomotor function compared with stem cells alone.Cellular therapy with BDNF-GFP transgenic stem cells can improve outcomes better than stem cells alone and may have therapeutic potential for spinal cord injury.

Prolonged propagation of rat neural stem cells relies on inhibiting autocrine/paracrine bone morphogenetic protein and platelet derived growth factor signals

Continuous expansion of rat neural stem cell lines has not been achieved due to proliferation arrest and spontaneous differentiation in vitro. In the current study, neural precursor cells derived from the subventricular zone of adult rats spontaneously underwent astroglial and oligodendroglial differentiation after limited propagation. This differentiation was largely induced by autocrine or paracrine bone morphogenetic protein and platelet derived growth factor signals. The results showed that, by inhibiting bone morphogenetic protein and platelet derived growth factor signals, adult rat neural precursor cells could be extensively cultured in vitro as tripotent stem cell lines. In addition to adult rat neural stem cells, we found that bone morphogenetic protein antagonists can promote the proliferation of human neural stem cells. Therefore, the present findings illustrated the role of autocrine or paracrine bone morphogenetic protein and platelet derived growth factor signaling in determining neural stem cell self-renewal and differentiation. By antagonizing both signals, the long-term propagation of rat neural stem cell lines can be achieved.

Effects of basic fibroblast growth factor on beta-catenin protein and mRNA expression during the proliferation of endogenous neural stem cells following focal cerebral ischemia

BACKGROUND： The Wnt/β-catenin signaling pathway plays an important role in neural development. ,β-catenin is an important component of the Wnt/β-catenin signaling pathway. The Wnt signaling pathway has been shown to regulate the interaction of neural stem cells with the extracellular matrix. OBJECTIVE： To investigate the effects of basic fibroblast growth factor （bFGF） on β-catenin protein and mRNA expression, and on hippocampal neural stem cell proliferation in a rat model of cerebral ischemia/reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, neurobiology experiment was performed in Shenyang Medical College between August 2006 and August 2008. MATERIALS： A total of 72 healthy male Wistar rats, aged 3 months, were used in this study. bFGF was provided by Beijing SL Pharmaceutical Co.,Ltd., China. METHODS： Rats were randomly divided into 3 groups： sham-operated, ischemia/reperfusion, and bFGF-treated （n = 24 per group）. Focal cerebral ischemia/reperfusion was induced in rats from the ischemia/reperfusion group and the bFGF-treated group by 2 hour right middle cerebral artery occlusion and 2 hour restoration of blood flow using the suture method. The ischemia/reperfusion and bFGF-treated groups were intraperitoneally administered 500 IU/mL of bFGF, or the same volume of physiological saline, once a day at postoperative days 1 3, and once every 3 days thereafter. Simultaneously, the sham-operated group underwent experimental procedures identical to the ischemia/reperfusion and bFGF-treated groups, with the exception of ischemia/reperfusion induction and drug administration. At 2 hours, 2, 6, 13, and 20 days after ischemiaJreperfusion induction, 50 mg/kg bromodeoxyuridine （BrdU） was administered to each group, twice daily, to label proliferating neural stem cells. MAIN OUTCOME MEASURES： The effects of bFGF on BrdU labeling, and ,8 -catenin mRNA and protein expression, in neural stem cells were examined by immunohistochemistry, Western blot, RT-PCR, and in situ hybridization techniques. RESULTS： In the sham-operated group, only a few BrdU-immunoreactive neural stem cells were found. In the ischemia/reperfusion group, BrdU-immunoreactive cells began to increase from 3 days after ischemia/reperfusion induction, reached a peak level at 7 days, and gradually reduced from 21 days. At 3, 7, 14, and 21 days after ischemia/reperfusion induction, the numbers of BrdU-immunoreactive cells were significantly greater in the bFGF-treated group than in the ischemia/reperfusion group. The sham-operated group exhibited slight expression of β-catenin and β-catenin mRNA. In the ischemia/reperfusion group, the expression of β-catenin and β-catenin mRNA gradually increased with reperfusion time, peaked at 14 days after reperfusion, and gradually decreased thereafter; by 21 days, the expression was markedly lower. Following bFGF injection, the expression of hippocampal BrdU, β-catenin, and β-catenin mRNA had apparently increased in each group. CONCLUSION： bFGF promotes neural stem cell proliferation, and the expression of β-catenin and β-catenin mRNA in the ischemic brain tissue. These findings indicate that bFGF promotion of neural stem cell proliferation may be mediated by Wnt/β-catenin signaling pathway.

Lentiviral-mediated vascular endothelial growth factor 165 gene transfer into neural stem cells promotes proliferation

We constructed a lentiviral vector carrying vascular endothelial growth factor 165, which was used to transfect neural stem cells. The transfection rate was approximately 50%, as determined by flow cytometry. Vascular endothelial growth factor protein was detected in neural stem cells and promoted proliferation.

Nerve growth factor promotes in vitro proliferation of neural stem cells from tree shrews

Neural stem cells promote neuronal regeneration and repair of brain tissue after injury,but have limited resources and proliferative ability in vivo.We hypothesized that nerve growth factor would promote in vitro proliferation of neural stem cells derived from the tree shrews,a primate-like mammal that has been proposed as an alternative to primates in biomedical translational research.We cultured neural stem cells from the hippocampus of tree shrews at embryonic day 38,and added nerve growth factor（100 μg/L） to the culture medium.Neural stem cells from the hippocampus of tree shrews cultured without nerve growth factor were used as controls.After 3 days,fluorescence microscopy after DAPI and nestin staining revealed that the number of neurospheres and DAPI/nestin-positive cells was markedly greater in the nerve growth factor-treated cells than in control cells.These findings demonstrate that nerve growth factor promotes the proliferation of neural stem cells derived from tree shrews.

Overexpression of vascular endothelial growth factor enhances the neuroprotective effects of bone marrow mesenchymal stem cell transplantation in ischemic stroke

Although bone marrow mesenchymal stem cells(BMSCs)might have therapeutic potency in ischemic stroke,the benefits are limited.The current study investigated the effects of BMSCs engineered to overexpress vascular endothelial growth factor(VEGF)on behavioral defects in a rat model of transient cerebral ischemia,which was induced by middle cerebral artery occlusion.VEGF-BMSCs or control grafts were injected into the left striatum of the infarcted hemisphere 24 hours after stroke.We found that compared with the stroke-only group and the vehicle-and BMSCs-control groups,the VEGF-BMSCs treated animals displayed the largest benefits,as evidenced by attenuated behavioral defects and smaller infarct volume 7 days after stroke.Additionally,VEGF-BMSCs greatly inhibited destruction of the blood-brain barrier,increased the regeneration of blood vessels in the region of ischemic penumbra,and reducedneuronal degeneration surrounding the infarct core.Further mechanistic studies showed that among all transplant groups,VEGF-BMSCs transplantation induced the highest level of brain-derived neurotrophic factor.These results suggest that BMSCs transplantation with vascular endothelial growth factor has the potential to treat ischemic stroke with better results than are currently available.

Directional induction of dopaminergic neurons from neural stem cells using substantia nigra homogenates and basic fibroblast growth factor

To date, complex components of available reagents have been used for directional induction of neural stem cells into dopaminergic neurons, resulting in a poor ability to repeat experiments. This study sought to investigate whether a homogenate of the substantia nigra of adult rats and/or basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons. Tyrosine hydroxylase-positive cells were observed exclusively after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor for 48 hours in vitro. However, in the groups treated with homogenate supernatant or basic fibroblast growth factor alone, tyrosine hydroxylase expression was not observed. Moreover, the content of dopamine in the culture medium of subventricular zone neurons was significantly increased at 48 hours after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor. Experimental findings indicate that the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons in the substantia nigra with the ability to secrete dopamine.

Basic fibroblast growth factor increases the numbe of endogenous neural stem cells and inhibits the expression of amino methyl isoxazole propionic acid receptors in amyotrophic lateral sclerosis mice

This study aimed to investigate the number of amino methyl isoxazole propionic acid （AMPA） receptors and production of endogenous neural stem cells in the SOD1 G93AG1H transgenic mouse model of amyotrophic lateral sclerosis, at postnatal day 60 following administration of basic fibroblast growth factor （FGF-2）. A radioligand binding assay and immunohistochemistry were used to estimate the number of AMPA receptors and endogenous neural stem cells respectively. Results showed that the number of AMPA receptors and endogenous neural stem cells in the brain stem and sensorimotor cortex were significantly increased, while motor function was significantly decreased at postnatal days 90 and 120. After administration of FGF-2 into mice, numbers of endogenous neural stem cells increased, while expression of AMPA receptors decreased, whilst motor functions were recovered. At postnatal day 120, the number of AMPA receptors was negatively correlated with the number of endogenous neural stem cells in model mice and FGF-2-treated mice. Our experimental findings indicate that FGF-2 can inhibit AMPA receptors and increase the number of endogenous neural stem cells, thus repairing neural injury in amyotrophic lateral sclerosis mice.

Superparamagnetic Iron Oxide Labeling of Spinal Cord Neural Stem Cells Genetically Modified by Nerve Growth Factor-β

This study established superparamagnetic iron oxide （SPIO）-labeled nerve growth fac-tor-β （NGF-β） gene-modified spinal cord-derived neural stem cells （NSCs）. The El4 rat embryonic spinal cord-derived NSCs were isolated and cultured. The cells of the third passage were transfected with plasmid pcDNA3-hNGFβ by using FuGENE HD transfection reagent. The expression of NGFβ was measured by immunocytochemistry and Western blotting. The positive clones were selected, allowed to proliferate and then labeled with SPIO, which was mediated by FuGENE HD transfection reagent. Prussian blue staining and transmission electron microscopy （TEM） were used to identify the SPIO particles in the cells. The distinctive markers for stem cells （nestin）, neuron （β-Ⅲ-tubulin）, oligodendrocyte （CNPase） and astrocyte （GFAP） were employed to evaluate the differentiation ability of the labeled cells. The immunocytochemistry and western blotting showed that NGF-β was expressed in spinal cord-derived NSCs. Prussian blue staining indicated that numerous blue-stained particles appeared in the cytoplasma of the labeled cells. TEM showed that SPIO particles were found in vacuolar structures of different sizes and the cytoplasma. The immunocytochemistry demonstrated that the labeled cells were nestin-positive. After differentiation, the cells expressed β-Ⅲ-tubulin, CNPase and GFAP. It was concluded that the SPIO-labeled NGF-β gene-modified spinal cord-derived NSC were successfully established, which are multipotent and capable of self-renewal.