Objective To investigate the effects and underlying mechanisms of human placental chorionic plate-derived mesenchymal stem cells (hpcMSCs) on cognitive dysfunction, neuroinflammation, neuronal damage and synaptic plasticity in a mouse model of stroke. Methods A mouse model of middle cerebral artery occlusion (MCAO) was adopted. The mice were randomly divided into three groups: sham operation group, MCAO group and hpcMSCs treatment group, with seven mice in each group. The hpcMSCs treatment group received hpcMSCs transplantation on the 1st, 3rd and 10th day after MCAO. One month after MCAO, the cognitive ability of the mice was evaluated by Morris water maze and Y maze behavioral tests; the morphological changes and synaptic functions of hippocampal neurons were analyzed by HE staining, Nissl staining, Golgi staining and immunofluorescence staining techniques; the density and activation status of microglia was analyzed by Fluorescent labeling method; the levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6 in brain tissue were analyzed by ELISA; the expressions of phosphorylated-mitogen-activated protein kinase kinase 1 (p-MEK1), phosphorylated-extracellular regulated protein kinase (p-ERK) and phosphorylated-cAMP-response element binding protein (p-CREB) and other proteins related to neuroprotection in the signal pathways were detected by Western blotting; and electrophysiological detection was performed using hippocampal slices in vitro. Results Compared with the MCAO group, mice in the hpcMSCs treatment group showed significant improvements, including improved cognitive ability, alleviated neuroinflammation (demonstrated by reduced microglial activation and decreased levels of inflammatory factors TNF-α, IL-1β and IL-6), and increased neuronal density with normalized morphology of neurons in the hippocampal CA1 region. The treatment group also demonstrated a significantly increased number of Nissl-positive cells and density of dendritic spines of hippocampal neurons, along with restored frequency of miniature excitatory postsynaptic potential (mEPSP). Moreover, hpcMSCs treatment significantly increased the expression levels of p-MEK1, p-ERK and p-CREB in the hippocampus. Conclusion Transplantation of hpcMSCs ameliorates cognitive dysfunction and hippocampal neuronal injury in stroke mice through the reduction of neuroinflammation, restoration of hippocampal neuronal function, promotion of synaptic plasticity and activation of the MEK/ERK/CREB signaling pathway. These findings suggest a new potential therapeutic approach for post-stroke neural repair.
Animals ; Hippocampus/physiopathology* ; Mice ; Cognitive Dysfunction/etiology* ; Mesenchymal Stem Cell Transplantation ; Male ; Neurons/metabolism* ; Stroke/metabolism* ; Humans ; Neuroinflammatory Diseases/therapy* ; Female ; Cyclic AMP Response Element-Binding Protein/metabolism* ; Disease Models, Animal ; Mesenchymal Stem Cells/cytology* ; Mice, Inbred C57BL

OBJECTIVETo explore the effects of umbilical cord blood mononuclear cells (UCBMC) transplantation on the neuronal apoptosis and the expression of Bcl-2 and Bax proteins in neonatal rats with hypoxic-ischemic brain damage (HIBD).

METHODSSeven-day-old Sprague-Dawley neonatal rats were randomly divided into normal control (N)+normal saline (NS), HIBD+NS, N+UCBMC, and HIBD+UCBMC groups. HIBD model was prepared using the classical Rice-Vannucci method. Twenty-four hours after HIBD, UCBMC were transplanted in the N+UCBMC and HIBD+UCBMC groups. Seven days after transplantation, NeuN/Cleaved-Caspase-3 double immunofluorescence staining and TUNEL methods were used to observe neural apoptosis in the cortex. The expression levels of Bax and Bcl-2 proteins were examined by Western blot analysis.

RESULTSThere were more NeuNcleaved Caspase-3DAPIand TUNELDAPIcells in the HIBD+NS group compared with the N+NS and N+UCBMC groups (P<0.01). There were less NeuNcleaved Caspase-3DAPIand TUNELDAPIcells in the HIBD+UCBMC group compared with the HIBD+NS group (P<0.01). The concentration of Bax protein was higher and that of Bcl-2 proteins was lower in the HIBD+NS group compared with the N+NS and N+UCBMC groups (P<0.01). The concentration of Bax protein in HIBD+UCBMC group was lower than that in the HIBD+NS group (P<0.01). The concentration of Bcl-2 protein was higher compared with the HIBD+NS, N+NS and N+UCBMC groups (P<0.05).

CONCLUSIONSUCBMC transplantation via lateral ventricle can upregulate the expression of Bcl-2 protein and down-regulate the expression of Bax protein, thus alleviating brain neural apoptosis in neonatal rats with HIBD.

Animals ; Animals, Newborn ; Apoptosis ; Caspase 3 ; metabolism ; Cord Blood Stem Cell Transplantation ; methods ; Female ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; therapy ; Male ; Neurons ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; analysis

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry(+) NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.
Animals ; Cell Differentiation ; Cell Line ; Embryonic Stem Cells ; cytology ; metabolism ; transplantation ; Gene Knock-In Techniques ; Genes, Reporter ; Homologous Recombination ; Humans ; Luminescent Proteins ; genetics ; Mice ; Mice, SCID ; Nestin ; genetics ; Neural Stem Cells ; cytology ; metabolism ; Neurons ; cytology ; metabolism ; Teratoma ; pathology

Neural progenitor cells (NPs) have shown several promising benefits for the treatment of neurological disorders. To evaluate the therapeutic potential of human neural progenitor cells (hNPs) in amyotrophic lateral sclerosis (ALS), we transplanted hNPs or growth factor (GF)-expressing hNPs into the central nervous system (CNS) of mutant Cu/Zn superoxide dismutase (SOD(1G93A)) transgenic mice. The hNPs were engineered to express brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), VEGF, neurotrophin-3 (NT-3), or glial cell-derived neurotrophic factor (GDNF), respectively, by adenoviral vector and GDNF by lentiviral vector before transplantation. Donor-derived cells engrafted and migrated into the spinal cord or brain of ALS mice and differentiated into neurons, oligodendrocytes, or glutamate transporter-1 (GLT1)-expressing astrocytes while some cells retained immature markers. Transplantation of GDNF- or IGF-1-expressing hNPs attenuated the loss of motor neurons and induced trophic changes in motor neurons of the spinal cord. However, improvement in motor performance and extension of lifespan were not observed in all hNP transplantation groups compared to vehicle-injected controls. Moreover, the lifespan of GDNF-expressing hNP recipient mice by lentiviral vector was shortened compared to controls, which was largely due to the decreased survival times of female animals. These results imply that although implanted hNPs differentiate into GLT1-expressing astrocytes and secrete GFs, which maintain dying motor neurons, inadequate trophic support could be harmful and there is sexual dimorphism in response to GDNF delivery in ALS mice. Therefore, additional therapeutic approaches may be required for full functional recovery.
Adenoviridae/genetics ; Amyotrophic Lateral Sclerosis/metabolism/mortality/*therapy ; Animals ; Astrocytes/metabolism ; Brain/*embryology ; Cell Differentiation ; Disease Models, Animal ; Excitatory Amino Acid Transporter 2/metabolism ; Female ; Fetal Stem Cells/*metabolism ; Genetic Vectors ; Humans ; Immunoenzyme Techniques ; Male ; Mice ; Mice, Transgenic ; Motor Neurons/*physiology ; Nerve Growth Factors/*metabolism ; *Stem Cell Transplantation ; Superoxide Dismutase/genetics ; Transfection ; Vascular Endothelial Growth Factor A/genetics/metabolism

BACKGROUNDNeural stem cells (NSCs) transplantation and gene therapy have been widely investigated for treating the cerebullar and myelonic injuries, however, studies on the ophthalmology are rare. The aim of this study was to investigate the migration and differentiation of brain-derived neurotrophic factor (BDNF) gene transgenic NSCs transplanted into the normal rat retinas.

METHODSNSCs were cultured and purified in vitro and infected with recombinant retrovirus pLXSN-BDNF and pLXSN respectively, to obtain the BDNF overexpressed NSCs (BDNF-NSCs) and control cells (p-NSCs). The expression of BDNF genes in two transgenic NSCs and untreated NSCs were measured by fluorescent quantitative polymerase chain reaction (FQ-PCR) and enzyme-linked immunosorbent assay (ELISA). BDNF-NSCs and NSCs were infected with adeno-associated viruses-enhanced green fluorescent protein (AAV-EGFP) to track them in vivo and served as donor cells for transplantation into the subretinal space of normal rat retinas, phosphated buffer solution (PBS) served as pseudo transplantation for a negative control. Survival, migration, and differentiation of donor cells in host retinas were observed and analyzed with Heidelberg retina angiograph (HRA) and immunohistochemistry, respectively.

RESULTSNSCs were purified successfully by limiting dilution assay. The expression of BDNF gene in BDNF-NSCs was the highest among three groups both at mRNA level tested by FQ-PCR (P < 0.05) and at protein level measured by ELISA (P < 0.05), which showed that BDNF was overexpressed in BDNF-NSCs. The results of HRA demonstrated that graft cells could survive well and migrate into the host retinas, while the immunohistochemical analysis revealed that transplanted BDNF-NSCs differentiated into neuron more efficiently compared with the control NSCs 2 months after transplantation.

CONCLUSIONSThe seed cells of NSCs highly secreting BDNF were established. BDNF can promote NSCs to migrate and differentiate into neural cells in the normal host retinas.

Animals ; Brain-Derived Neurotrophic Factor ; genetics ; metabolism ; Cell Differentiation ; physiology ; Cell Movement ; physiology ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Enzyme-Linked Immunosorbent Assay ; Immunohistochemistry ; Neurons ; cytology ; Rats ; Retina ; cytology ; metabolism ; Stem Cell Transplantation

OBJECTIVETo observe the effects of neural stem cells (NSCs) transplantation on the brain derived neurotrophic factor (BDNF) after the spinal cord injury (SCI) of rats, and to investigate the mechanism of repairing the SCI by NSCs transplantation.

METHODSNeural stem cells were cultured from the hippocampus of rats' embryo and identified by immunocytochemistry. Seven days after the operation of SCI, the NSCs were transplanted into the injured site. Sixty adult Wistar rats were randomly divided into three groups: SCI cured with NSCs transplantation (group A), SCI received DMEM solution (group B), control group (group C). Then the expression of BDNF of the lesion and neighbor areas were examined by reverse transcsription polymerase chain reaction (RT-PCR) and immunohistochemistry, so as to investigated the mechanism of repairing the SCI after NSCS transplantation.

RESULTSAccording the RT-PCR results analysis, the expression of BDNF mRNA of group A enhanced higher than that of group B on the 1st, 3rd, 5th day after transplantation of NSCs. According the immunohistochemistry results analysis, the expression of BDNF mRNA of group A enhanced higher than that of group B on the 7th, 14th, 28th day similarly.

CONCLUSIONThe transplantation of NSCs can change the tiny-entironment by upregulating the expression of BDNF. It maybe one of the mechanism of repairing the SCI by NSCs transplantation.

Animals ; Brain-Derived Neurotrophic Factor ; genetics ; metabolism ; Cells, Cultured ; Disease Models, Animal ; Gene Expression ; Humans ; Male ; Mesenchymal Stem Cell Transplantation ; Neurons ; metabolism ; transplantation ; Random Allocation ; Rats ; Rats, Wistar ; Spinal Cord Injuries ; genetics ; metabolism ; surgery ; therapy ; Up-Regulation

OBJECTIVETo observe D(2) receptor expression on human neural progenitor cell line hNPC-TERT before and after transplantation into rabbit central nervous system.

METHODSD(2) receptor expression on cultured hNPC-TERT cells was verified and quantitatively analyzed with immunofluorescence assay and receptor radio ligand binding assay, respectively. 3 x 10(6) hNPC-TERT cells were implanted in the spinal cord of New Zealand rabbit with HeLa cells as the control. Two days after implantation, positron-emission tomography (PET) scan with (11)C-raclopride as the radiotracer was performed in the living animals or for the isolated spinal cords, and cryosections of the spinal cord containing the implanted cells were prepared for immunofluorescence assay.

RESULTSCultured hNPC-TERT cells showed high expression of D(2) receptor (Bmax=8 x 10(4)). PET scans of the rabbits identified visible radioactive accumulations at the site where hNPC-TERT cells were implanted but not at the site of HeLa cell implantation. Region of interest analysis showed a significant difference between the two cells in the maximal standard uptake value at the cell implantation sites. The results were further confirmed with ex vivo PET imaging of the spinal cord and tissue immunofluorescence assay.

CONCLUSIONHuman neural progenitor cells hNPC-TERT highly express dopamine D(2) receptors and retain this capacity after implantation into the spinal cord, suggesting their potential for treatment of such nerve system disease as Parkinson syndrome.

Animals ; Cell Line, Transformed ; Female ; Fetal Stem Cells ; cytology ; metabolism ; transplantation ; Fluorescent Antibody Technique ; HeLa Cells ; Humans ; Neurons ; cytology ; metabolism ; transplantation ; Positron-Emission Tomography ; Rabbits ; Radioligand Assay ; Receptors, Dopamine D2 ; metabolism ; Spinal Cord ; metabolism ; surgery ; Stem Cell Transplantation ; methods ; Telomerase ; genetics ; Transplantation, Heterologous

OBJECTIVETo investigate a possibility of repairing damaged brain by intracerebroventricular transplantation of neural stem cells (NSCs) in the adult mice subjected to glutamate-induced excitotoxic injury.

METHODSMouse NSCs were isolated from the brains of embryos at 15-day postcoitum (dpc). The expression of nestin, a special antigen for NSC, was detected by immunocytochemistry. Immunofluorescence staining was carried out to observe the survival and location of transplanted NSCs. The animals in the MSG + NSCs group received intracerebroventricular transplantation of NSCs (approximately 1.0 x 10(5) cells) separately on day 1 and day 10 after 10-d MSG exposure (4.0 g/kg per day). The mice in control and MSG groups received intracerebroventricular injection of Dulbecco's minimum essential medium (DMEM) instead of NSCs. On day 11 after the last NSC transplantation, the test of Y-maze discrimination learning was performed, and then the histopathology of the animal brains was studied to analyze the MSG-induced functional and morphological changes of brain and the effects of intracerebroventricular transplantation of NSCs on the brain repair.

RESULTSThe isolated cells were Nestin-positive. The grafted NSCs in the host brain were region-specifically survived at 10-d post-transplantation. Intracerebroventricular transplantation of NSCs obviously facilitated the brain recovery from glutamate-induced behavioral disturbances and histopathological impairs in adult mice.

CONCLUSIONIntracerebroventricular transplantation of NSCs may be feasible in repairing diseased or damaged brain tissue.

Animals ; Cell Count ; Disease Models, Animal ; Embryo, Mammalian ; Glutamic Acid ; toxicity ; Injections, Intraventricular ; methods ; Intermediate Filament Proteins ; metabolism ; Mice ; Mice, Inbred Strains ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurons ; physiology ; Neurotoxicity Syndromes ; etiology ; pathology ; surgery ; Stem Cell Transplantation ; methods ; Stem Cells ; physiology ; Time Factors

OBJECTIVETo observe the migration and differentiation of the neural precursor cells (NPCs) that derived from murine embryonic stem cells (ESCs) when they were transplanted into amyloid beta (A beta)-treated rat hippocampus.

METHODSMESPU35, a murine ESC cell line that express the enhanced green fluorescent protein (EGFP), was induced differentiation into nestin-positive NPCs by modified serum-free methods. The A beta plaques and the differentiation of the grafted cells were observed by immunofluorescent staining.

RESULTSComparing 16 weeks with 4 weeks post-transplantation, the migration distance increased about 5 times; the rate of migratory NPCs differentiating into glial fibrillary acidic protein (GFAP)-positive cells kept rising from (30.41+/-1.45) % to (49.25+/-1.23) %, and the rate of NPCs differentiating into neurofilament 200 (NF200) positive cells increased from (16.68+/-0.95) % to (27.94+/-1.21) %. Meanwhile, the GFAP-positive cells targeting to the ipsilateral side of A beta plaques increased from 60.2% to 81.3%, while the NF200-positive cells increased from 61.3% to 84.1%. The migration distance had significant positive linear correlations to the neuronal differentiation rate (r = 0.991) and to the astrocytic differentiation rate (r = 0.953).

CONCLUSIONEngrafted NPCs migrate targetedly to the A beta injection site and differentiate into neurons and astrocytes.

Amyloid beta-Peptides ; administration & dosage ; metabolism ; Animals ; Cell Differentiation ; Cell Movement ; Embryonic Stem Cells ; cytology ; physiology ; transplantation ; Fluorescent Antibody Technique ; Glial Fibrillary Acidic Protein ; metabolism ; Green Fluorescent Proteins ; metabolism ; Hippocampus ; cytology ; physiology ; Injections, Intraventricular ; Male ; Neurons ; cytology ; physiology ; transplantation ; Rats ; Rats, Wistar ; Stem Cell Transplantation

OBJECTIVETo explore the feasibility to construct genetic engineering human neural stem cells (hNSCs) mediated by lentivirus to express multigene in order to provide a graft source for further studies of spinal cord injury (SCI).

METHODSHuman neural stem cells from the brain cortex of human abortus were isolated and cultured, then gene was modified by lentivirus to express both green fluorescence protein (GFP) and rat neurotrophin-3 (NT-3); the transgenic expression was detected by the methods of fluorescence microscope, dorsal root ganglion of fetal rats and slot blot.

RESULTSGenetic engineering hNSCs were successfully constructed. All of the genetic engineering hNSCs which expressed bright green fluorescence were observed under the fluorescence microscope. The conditioned medium of transgenic hNSCs could induce neurite flourishing outgrowth from dorsal root ganglion (DRG). The genetic engineering hNSCs expressed high level NT-3 which could be detected by using slot blot.

CONCLUSIONSGenetic engineering hNSCs mediated by lentivirus can be constructed to express multigene successfully.

Animals ; Cell Differentiation ; Cells, Cultured ; Feasibility Studies ; Gene Expression ; Genetic Engineering ; methods ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry ; Lentivirus ; genetics ; Microscopy, Fluorescence ; Neurons ; metabolism ; Rats ; Stem Cell Transplantation ; Stem Cells ; metabolism ; Transgenes