To observe the effects of heterograft of glomus cells of carotid body on hemiparkinsonian rat models, rats with unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the right dopaminergic neurons of substantia nigra received intrastriatal glomus cells heterograft. Apomorphine-induced rotation was monitored for 30 min at various time points after grafting. The striata were cut and examined for dopamine content by HPLC and for immunohistochemical staining of tyrosine hydroxylase positive neurons (TH+) at the end of the experiments. The results showed that apomorphine-induced rotational behavior was significantly reduced for 12 weeks and the dopamine contents were significantly elevated after grafting (P < 0.01), and TH+ cells survived better. The present study demonstrates that intrastriatal heterograft of glomus cells within carotid body in rats with 6-OHDA-elicited lesions could reduce apomorphine-induced rotational behavior and elevate the dopamine contents and numbers of TH+ cell surviving within striatum, and can serve as a new and effective alternative for Parkinson disease.
Animals ; Carotid Body ; cytology ; transplantation ; Cell Transplantation ; Dopamine ; metabolism ; Female ; Neurons ; metabolism ; Parkinson Disease ; metabolism ; surgery ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Stereotaxic Techniques ; Transplantation, Heterologous

To observe the effects of heterograft of glomus cells of carotid body on hemiparkinsonian rat models, rats with unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the right dopaminergic neurons of substantia nigra received intrastriatal glomus cells heterograft. Apomorphine-induced rotation was monitored for 30 min at various time points after grafting. The striata were cut and examined for dopamine content by HPLC and for immunohistochemical staining of tyrosine hydroxylase positive neurons (TH+) at the end of the experiments. The results showed that apomorphine-induced rotational behavior was significantly reduced for 12 weeks and the dopamine contents were significantly elevated after grafting (P < 0.01), and TH+ cells survived better. The present study demonstrates that intrastriatal heterograft of glomus cells within carotid body in rats with 6-OHDA-elicited lesions could reduce apomorphine-induced rotational behavior and elevate the dopamine contents and numbers of TH+ cell surviving within striatum, and can serve as a new and effective alternative for Parkinson disease.
Carotid Body/*cytology ; Carotid Body/transplantation ; *Cell Transplantation ; Dopamine/*metabolism ; Neurons/metabolism ; Parkinson Disease/metabolism ; Parkinson Disease/*surgery ; Random Allocation ; Rats, Sprague-Dawley ; Stereotaxic Techniques ; Transplantation, Heterologous

The transplantation of dopaminergic neurons in the brain has been attempted in experimental animals and humans as the new treatment modality of Parkinson's disease. Before the trial of dopaminergic neuronal transplantation in human, the authors proceeded with the animal experiment of fetal dopaminergic cell transplantation in a rat Parkinson's disease model. The aims of this experiment were to confirm the availability of fetal mesencephalic cells as the donor, to compare the viability of cells according to different cell manipulation methods, and to follow up the functional recovery in the transplanted Parkinson's disease model. As a result, the authors concluded that the simple enzyme digestion method had a better cell survival rate than the multiple enzyme digestion method. Also, the transplanted mesencephalic cells could not only survive in the host animal but also promote functional recovery.
Animal ; Corpus Striatum/*physiology ; Dopamine/*metabolism ; *Fetal Tissue Transplantation ; Male ; Mesencephalon/cytology/*embryology/metabolism ; Neurons/metabolism/*transplantation ; Parkinson Disease/*surgery ; Rats ; Rats, Sprague-Dawley ; Support, Non-U.S. Gov't

For cell replacement therapy of neurodegenerative diseases such as Parkinson's disease (PD), methods for efficiently generating midbrain dopaminergic (DA) neurons from embryonic stem (ES) cells have been investigated. Two aspects of DA neuron generation are considered: genetic modification and manipulation of culture conditions. A transcription factor known as critical for development of DA neurons, Nurr1, was introduced into ES cells to see how they facilitate the generation of DA neurons from ES cells. Also, two culture procedures, the 5-stage method and stromal cell-derived inducing activity (SDIA) method, were used for ES cell differentiation. Using the 5-stage method, we and others previously demonstrated that Nurr1-overexpressing ES cells, under treatment of signaling molecules such as SHH and FGF8 followed by treatment of ascorbic acid, can differentiate into DA neurons with a high efficiency (> 60% of TH+/Tuj1+ neurons). Furthermore, using the SDIA method with treatment of signaling molecules, we found that Nurr1-overexpressing ES cells can differentiate to DA neurons with the highest efficiency ever reported (~90% of TH+/Tuj1+ neurons). Importantly, our semi-quantitative and real-time PCR analyses demonstrate that all known DA marker genes (e.g., TH, AADC and DAT) were up-regulated in Nurr1- overexpressing ES cells when compared to the na ve ES cells. These cells produced increased dopamine compared to na ve D3 cells after differentiation. In the in vivo context after transplantation, the genetically modified ES cells also showed the highly increased dopaminergic neuronal phenotypes. Thus, the combination of genetic engineering and appropriate culture conditions provides a useful tool to generate a good cell source from ES cells for cell replacement therapy of degenerative diseases such as PD.
Cell Differentiation ; Dopamine/*metabolism ; *Embryonic Induction ; Humans ; Neurons/metabolism/*transplantation ; Parkinson Disease/*surgery ; Research Support, Non-U.S. Gov't ; *Stem Cell Transplantation ; Stem Cells/*cytology

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

BACKGROUNDThere are no reports on exnografting cultured human fetal neocortical cells in this infracted cavities of adult rat brains. This study was undertaken to observe whether cultured human cortical neurons and astrocytes can survive and grow in the infarcted cavities of adult rat brains and whether they interconnect with host brains.

METHODSThe right middle cerebral artery was ligated distal to the striatal branches in 16 adult stroke-prone renovascular hypertensive rats. One week later, cultured cells from human embryonic cerebral cortexes were stereotaxically transferred to the infarcted cavity of 11 rats. The other 5 rats receiving sham transplants served as controls. For immunosuppression, all transplanted rats received intraperitoneal injection of cyclosporine A daily starting on the day of grafting. Immunohistochemistry for glial fibrillary acidic protein (GFAP), synaptophysin, neurofilament, and microtubule associated protein-2 (MAP-2) was performed on brain sections perfused in situ 8 weeks after transplantation.

RESULTSGrafts in the infarcted cavities of 6 of 10 surviving rats consisted of bands of neurons with an immature appearance, bundles of fibers, and GFAP-immunopositive astrocytes, which were unevenly distributed. The grafts were rich in synaptophysin, neurofilament, and MAP2-positive neurons with long processes. The graft/host border was diffuse with dendrites apparently bridging over to the host brain, into which neurofilament immunopositive fibers protruded.

CONCLUSIONCultured human fetal brain cells can survive and grow in the infarcted cavities of immunodepressed rats and integrate with the host brain.

Animals ; Astrocytes ; transplantation ; Brain ; pathology ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Cerebral Infarction ; metabolism ; pathology ; therapy ; Fetal Tissue Transplantation ; Glial Fibrillary Acidic Protein ; analysis ; Humans ; Microtubule-Associated Proteins ; analysis ; Neocortex ; cytology ; Neurons ; transplantation ; Rats ; Synaptophysin ; analysis

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

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

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

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