Zhaoyan Wang

Effects of Neural Progenitor Cell Transplantation in Children With Severe Cerebral Palsy

Cerebral palsy (CP) is a chronic nervous system disease that severely damages the physical and developmental health of children. Traditional treatment brings about only improvement of mild to moderate CP, but severe CP still lacks effective interventions. To explore safety and efficacy of using neural progenitor cells (NPCs) to treat CP in children, we performed NPC transplantation in 45 patients with severe CP by injecting NPCs derived from aborted fetal tissue into the lateral ventricle. Gross motor function measures (GMFM), the Peabody Developmental Motor Scale-Fine Motor (PDMS-FM) test, and a unified survey questionnaire designed specifically for children with CP were used to evaluate neurological function of the patients. Motor development was significantly accelerated within the first month after cell transplantation, but the rate of improvement gradually slowed to preoperative levels. However, after 1 year, the developmental level in each functional sphere (gross motor, fine motor, and cognition) of the treatment group was significantly higher compared to the control group. No delayed complications of this therapy were noted. These results suggest that NPC transplantation is a safe and effective therapeutic method for treating children with severe CP.

Valproic acid induces apoptosis in differentiating hippocampal neurons by the release of tumor necrosis factor-α from activated astrocytes

Human studies of neurodevelopment suggest that children exposed in utero to certain antiepileptic drugs (AEDs) suffer a variety of brain-behavior sequelae, such as neural tube defects, developmental delays, cognitive deficits, etc. Valproic acid (VPA), a commonly used AED, has greater risk for these side effects compared with other AEDs. However, the detailed molecular mechanisms underlying this developmental neurotoxicity of VPA is unclear despite previous research demonstrating that VPA could induce widespread apoptotic neurodegeneration in developing brains of animal models. This study characterizes the role of astrocytes in VPA-induced neurodegeneration. In developing brains, we evaluated the developmental neurotoxicity of VPA on differentiating neurons and astrocytes from neural progenitor cells cultured from the hippocampus of human fetuses. Exposure of a neuron-enriched culture to VPA at 250μM or 500μM did not cause neuronal apoptosis, but at 1mM and 7 days exposure, a slight increase in the percentage of apoptotic cells was observed. In contrast, VPA at 250μM to 1mM, selectively induced neuronal apoptosis in a neuron-astrocyte mixed cell culture model. The VPA-treated astrocytes showed morphological changes, and the level of tumor necrosis factor-α (TNF-α) was elevated in the supernatant. Both neuronal apoptosis and TNF-α release from astrocytes increased with concentration and exposure time to VPA, suggesting a synergism between the two cell types. Treatment of the neuron-astrocyte mixed culture exposed to VPA with TNF-α antibody partly prevented neuronal apoptosis, while the addition of exogenous TNF-α induced apoptosis in both cultures. Moreover, this pro-apoptotic effect was specific to VPA, as another AED, valpromide, failed to mimic this pro-apoptotic effect, nor did an inhibitor of histone deacetylase (iHDAC), sodium butyrate (NaB). We report a novel finding that astrocytes participate in VPA induced neurodegeneration by releasing TNF-α.

Isolation and culture of human oligodendrocyte precursor cells from neurospheres

Culture of human oligodendrocyte precursor cells (OPCs) can help understand the regulatory mechanism of differentiation and myelination of oligodendrocytes. However, existing culture methods have limitations, particularly the lack of a source of human donor tissue and high cost. We sorted cells with the A2B5(+)PSA-NCAM(-) phenotype from neurospheres instead of human donor tissues through immunomagnetic sorting and subsequently cultured the isolated cells in OPC medium. Of all the isolated cells, 15.69% were of the A2B5(+)PSA-NCAM(-) phenotype. More than 90% of the isolated OPCs expressed the OPC-specific markers O4, PDGFαR, and Sox10, and less than 5% of cells expressed GFAP and Tuj-1. After induction, the isolated cells had the capacity to differentiate into oligodendrocytes. Furthermore, the OPCs could be stably passaged in vitro for at least four generations and all the cells had high expression levels of O4 and Sox10 and very low expression levels of GFAP and Tuj-1; moreover, the cells had the capacity to differentiate into oligodendrocytes. After four passages, OPCs can proliferate at least 14 times above. In addition, in the presence of B27, only one cytokine, namely, bFGF, was sufficient to maintain proliferation, and this greatly reduced the experimental cost. Cells of the A2B5(+)PSA-NCAM(-) phenotype have already been identified as OPCs. We developed and characterized a reproducible, simple, and economical method for the isolation and culture of human OPCs. This method will contribute to studying the function of OPCs in development, disease, and treatment.

Neural Stem/Progenitor Cell Transplantation for Cortical Visual Impairment in Neonatal Brain Injured Patients:

The purpose of this study was to investigate the clinical efficacy of neural stem/progenitor cell (NS/PC) transplantation to treat severe cortical visual impairment (CVI), a sequela of neonatal brain injury. Fifty-two patients with cerebral injury and CVI were randomly divided into two groups: the treatment group (n = 25, with the median age of 18 months) and the control group (n = 27, with the median age of 19.5 months). The treatment group received intracerebroventricular transplantation of human NS/PCs and rehabilitation training. The control group received rehabilitation only. The visual function was assessed by Holts method at various time points after transplantation. One in five patients with fundus abnormalities accompanied by blindness regained light perception. The visual functions of 75% of the patients with normal fundus were improved by one level or more in a 2-year follow-up. The median efficacy appeared 60 days posttransplantation. The total effective rate of cell transplantation on visual improvement was 64% (16 patients of 25), among which one blind patient regained light perception, five (31.2%) CVI patients improved by one level, and 10 (62.5%) improved by more than one level. Functional magnetic resonance imaging (fMRI) in a subpopulation of patients showed enhanced signals in the occipital lobe, visual pathway, and apical lobe after transplantation. In the control group, four patients with fundus abnormalities showed no improvement. Nine of 23 CVI patients with normal fundus improved visual function by more than one level. At the 2-year follow-up, no blind patients showed visual improvement. The total effective rate was 33.33% (9 of 27 patients). Among those showing visual improvement in the control group, six patients (66.67%) improved by one level, and three (33.33%) by more than one level. The median efficacy occurred in 365 days. Human NS/PC transplantation is effective to treat patients with severe CVI after neonatal brain injury. Compared with the traditional rehabilitation training, cell transplantation showed not only earlier visual improvement but also higher improvement rates and degrees. This article is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

High purity of human oligodendrocyte progenitor cells obtained from neural stem cells: Suitable for clinical application

BACKGROUND Recent studies have suggested that the transplantation of oligodendrocyte progenitor cells (OPCs) may be a promising potential therapeutic strategy for a broad range of diseases affecting myelin, such as multiple sclerosis, periventricular leukomalacia, and spinal cord injury. Clinical interest arose from the potential of human stem cells to be directed to OPCs for the clinical application of treating these diseases since large quantities of high quality OPCs are needed. However, to date, there have been precious few studies about OPC induction from human neural stem cells (NSCs). NEW METHOD Here we successfully directed human fetal NSCs into highly pure OPCs using a cocktail of basic fibroblast growth factor, platelet-derived growth factor, and neurotrophic factor-3. RESULTS These cells had typical morphology of OPCs, and 80-90% of them expressed specific OPC markers such as A2B5, O4, Sox10 and PDGF-αR. When exposed to differentiation medium, 90% of the cells differentiated into oligodendrocytes. The OPCs could be amplified in our culture medium and passaged at least 10 times. COMPARISON WITH A EXISTING METHOD Compared to a recent published method, this protocol had much higher stability and repeatability, and OPCs could be obtained from NSCs from passage 5 to 38. It also obtained more highly pure OPCs (80-90%) via simpler and more convenient manipulation. CONCLUSIONS This study provided an easy and efficient method to obtain large quantities of high-quality human OPCs to meet clinical demand.

Combined transplantation of neural precursor cells and olfactory ensheathing cells for the treatment of X-linked adrenoleukodystrophy in children

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Human Neural Stem Cells with GDNF Site-Specific Integration at AAVS1 by Using AAV Vectors Retained Their Stemness

The neural stem cells (NSCs) have the ability to self-renew, and to migrate to pathologically altered regions of the central nervous system. Glial cell derived neurotrophic factor (GDNF) could protect dopamine neurons and rescue motor neurons in vivo, which has been proposed as a promising candidate for the treatments of degenerative neurological diseases. In order to combine the advantages of neurotrophic factors and stem cells in clinical therapy, we established the modified hNSCs that has site-specific integration of GDNF gene by using recombinant adeno-associated virus (rAAV) vectors. The hNSCs were co-infected by rAAV2-EGFP-GDNF and rAAV2-SVAV2 which provide integrase to specifically integrate GDNF gene into AAVS1 site. The GDNF-hNSCs maintained their original stem cell characteristics and the ability to differentiate into neurons in vitro. In the animal model, the GDNF-hNSCs were specifically transplanted into CA1 area of hippocampi and could migrate to the dentate gyrus region and differentiate into neuronal cells while maintaining GDNF expression. hNSCs with GDNF gene site-specific integration at AAVS1 by using AAV vectors retained their stemness and effectively expressed GDNF, which indicates the potential of employing transplanted hNPCs for treatment of brain injuries and degenerative neurological diseases.

Protective effect of miconazole on rat myelin sheaths following premature infant cerebral white matter injury

The aim of the present study was to investigate the protective effects of miconazole on myelin sheaths following cerebral white matter damage (WMD) in premature infant rats. Sprague Dawley rats (3-days-old) were randomly divided into four groups (n=30 each) as follows: Sham surgery group, WMD model group, 10 mg/kg/day treatment group and 40 mg/kg/day treatment group. A cerebral white matter lesion model was created by ligating the right common carotid artery for 80 min. Treatment groups were administered with 10 or 40 mg/kg miconazole at 4–8 days following birth (early treatment group) or 5–11 days following birth (late treatment group). Rats in the model group received the same concentration of dimethylsulfoxide. Myelin basic protein (MBP) immunohistochemical staining and western blotting were used to detect the expression of cerebral white matter-specific MBP, and changes in myelin structure were observed using transmission electron microscopy. No swelling or necrosis was observed in the corpus callosum of the sham group rats, whereas rats in the model group demonstrated edema, loose structure, fiber disorder, inflammatory gliocytes and selective white matter lesions. Following treatment with miconazole, MBP expression in the corpus callosum was significantly higher compared with the model group. Furthermore, in the model group, myelin sheaths in the corpus callosum were loose with small vacuoles, there was a marked decrease in thickness and structural damage was observed. Conversely, a marked improvement in myelination was observed in the treatment group. The results of the present study suggest that miconazole is able to promote formation of the myelin sheath to ameliorate premature cerebral white matter lesions caused by ischemia or hypoxia in rats.

Clinical observation of electroencephalographic changes and risk of convulsion occurrence in children receiving neural precursor cell transplantation

PURPOSE This study was intended to observe electroencephalographic (EEG) changes and convulsion attacks in children receiving neural precursor cell transplantation, and to explore the possibility of electrophysiological changes and risk of convulsion occurrence after cell transplantation. METHOD 228 children were included in this study who received neural precursor cell transplantation in our hospital between March 2008 and July 2012. No history of convulsion attacks was elicited before cell transplantation. Data about EEG change and convulsion occurrence before and after cell transplantation were analyzed statistically. RESULTS Of the 228 pediatric patients, EEG improvement, deterioration and no significant change were observed in 60, 45 and 122 patients, respectively. One month after transplantation, four (1.76%) patients experienced new convulsions. Of the 227 patients, 25 showed increased and/or abnormal discharges on EEG. Of these, 19 underwent EEG re-examination six months post-operation. Except the convulsive cases mentioned above, there were no new cases of convulsions in the remaining patients. Of the 27 patients including those with abnormal discharge, increased discharge and convulsion attacks, 17 achieved varying degrees of therapeutic efficacy. CONCLUSION Intraventricular transplantation of neural precursor cells is associated with EEG changes in some children and clinical convulsion attacks in individual patients. However, these abnormal changes do not last long and usually return to normal levels within 1-6 months after surgery, along with disappearance of convulsions. Simultaneous occurrence of EEG changes and convulsions do not appear to affect therapeutic efficacy.

Growth Factor Changes in Cerebrospinal Fluid of Children with Mental Retardation before and after Neural Precursor Cell Transplantation

OBJECTIVE To investigate growth factor changes in cerebrospinal fluid (CSF) of children with mental retardation (MR) before and after neural precursor cell transplantation (NPCT), in an attempt to provide experimental support for the clinical treatment of MR with NPCT. METHODS The study comprised of 28 MR children who received twice NPCT in our hospital. CSF was collected at both times of NPCT to assess growth factors by ELISA. In addition, the content of insulinlike growth factor 1 (IGF-1) in CSF was assayed to determine possible correlations between IGF-1 changes and the short-term therapeutic effect of NPCT. RESULTS Of all the growth factors detected in CSF, only IGF-1 was increased significantly after NPCT (P<0.05). Fifteen of the twenty-eight MR children achieved short-term therapeutic efficacy, whereby the content of IGF-1 after NPCT was significantly higher than that before NPCT (P<0.05). There was no difference in IGF-1 content before and after NPCT in the remaining 13 MR children without shortterm therapeutic effect (P=0.657). There was a significant difference in IGF-change between the two groups of patients (P<0.05). CONCLUSION IGF-1 may be one of the mechanisms contributing to the therapeutic effect of NPCT.