Oligodendrocyte Progenitor Cell Transplantation Ameliorates Preterm Infant Cerebral White Matter Injury in Rats Model.

Background: Cerebral white matter injury (WMI) is the most common brain injury in preterm infants, leading to motor and developmental deficits often accompanied by cognitive impairment. However, there is no effective treatment. One promising approach for treating preterm WMI is cell replacement therapy, in which lost cells can be replaced by exogenous oligodendrocyte progenitor cells (OPCs). Methods: This study developed a method to differentiate human neural stem cells (hNSCs) into human OPCs (hOPCs). The preterm WMI animal model was established in rats on postnatal day 3, and OLIG2+/NG2+/PDGFRα+/O4+ hOPCs were enriched and transplanted into the corpus callosum on postnatal day 10. Then, histological analysis and electron microscopy were used to detect lesion structure; behavioral assays were performed to detect cognitive function. Results: Transplanted hOPCs survived and migrated throughout the major white matter tracts. Morphological differentiation of transplanted hOPCs was observed. Histological analysis revealed structural repair of lesioned areas. Re-myelination of the axons in the corpus callosum was confirmed by electron microscopy. The Morris water maze test revealed cognitive function recovery. Conclusion: Our study showed that exogenous hOPCs could differentiate into CC1+ OLS in the brain of WMI rats, improving their cognitive functions.

Intrauterine desensitization enables long term survival of human oligodendrocyte progenitor cells without immunosuppression.

Immune rejection can be reduced using immunosuppressants which are not viable for premature infants. However, desensitization can induce immune tolerance for premature infants because of underdeveloped immune system. The fetuses of Wistar rats at 15-17 days gestation were injected via hOPCs-1 into brain, muscles, and abdomen ex utero and then returned while the fetuses of control without injection. After 6 weeks of desensitization, the brain and muscles were transplanted with hOPCs-1, hNSCs-1, and hOPCs-2. After 10 and 34 weeks of desensitization, hOPCs-1 and hNSCs-1 in desensitized groups was higher than that in the control group while hOPCs-2 were rejected. Treg, CD4CD28, CD8CD28, and CD45RC between the desensitization and the control group differed significantly. Inflammatory cells in group with hOPCs-1 and hNSCs-1 was lower than that in the control group. hOPCs-1 can differentiate into myelin in desensitized groups. Wistar rats with desensitization developed immune tolerance to desensitized and transplanted cells.

Identifying Genes that Affect Differentiation of Human Neural Stem Cells and Myelination of Mature Oligodendrocytes.

Human neural stem cells (NSCs) are self-renewing, multipotent cells of the central nervous system (CNS). They are characterized by their ability to differentiate into a range of cells, including oligodendrocytes (OLs), neurons, and astrocytes, depending on exogenous stimuli. An efficient and easy directional differentiation method was developed for obtaining large quantities of high-quality of human OL progenitor cells (OPCs) and OLs from NSCs. RNA sequencing, immunofluorescence staining, flow cytometry, western blot, label-free proteomic sequencing, and qPCR were performed in OL lines differentiated from NSC lines. The changes in the positive rate of typical proteins were analyzed expressed by NSCs, neurons, astrocytes, OPCs, and OLs. We assessed Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of differentially expressed (DE) messenger RNAs (mRNAs) related to the differentiation of NSCs and the maturation of OLs. The percentage of NSCs differentiated into neurons, astrocytes, and OLs was 82.13%, 80.19%, and 90.15%, respectively. We found that nestin, PAX6, Musashi, and vimentin were highly expressed in NSCs; PDGFR-α, A2B5, NG2, OLIG2, SOX10, and NKX2-2 were highly expressed in OPCs; and CNP, GALC, PLP1, and MBP were highly expressed in OLs. RNA sequencing, western blot and qPCR revealed that ERBB4 and SORL1 gradually increased during NSC-OL differentiation. In conclusion, NSCs can differentiate into neurons, astrocytes, and OLs efficiently. PDGFR-α, APC, ID4, PLLP, and other markers were related to NSC differentiation and OL maturation. Moreover, we refined a screening method for ERBB4 and SORL1, which may underlie NSC differentiation and OL maturation. Potential unreported genes and proteins may regulate differentiation of human neural stem cells into oligodendrocyte lineage. Neural stem cells (NSCs) can differentiate into neurons, astrocytes, and oligodendrocyte (OLs) efficiently. By analyzing the DE mRNAs and proteins of NSCs and OLs lineage, we could identify reported markers and unreported markers of ERBB4 and SORL1 that may underlie regulate NSC differentiation and OL maturation.

Optimization of an Intranasal Route for the Delivery of Human Neural Stem Cells to Treat a Neonatal Hypoxic-Ischemic Brain Injury Rat Model.

Objective: Stem cell administration via the intranasal route has shown promise as a new therapy for hypoxic-ischemic encephalopathy (HIE). In this study, we aimed to improve the intranasal delivery of stem cells to the brain. Methods: Human neural stem cells (hNSCs) were identified using immunofluorescence, morphological, and flow cytometry assays before transplantation, and cell migration capacity was examined using the transwell assay. Cerebral hypoxia-ischemia (HI) was induced in 7-day-old rats, followed by the intranasal transplantation of CM-Dil-labeled hNSCs. We examined various experimental conditions, including preconditioning hNSCs with hypoxia, catheter method, multiple low-dose transplantation, head position, cell appropriate concentration, and volume. Rats were sacrificed 1 or 3 days after the final intranasal administration, and parts of the nasal tissue and whole brain sections were analyzed under a fluorescence microscope. Results: The isolated hNSCs met the characteristics of neural stem cells. Hypoxia (5% O2, 24 h) enhanced the surface expression of CXC chemokine receptor 4 (CXCR4) (9.21 ± 1.9% ~ 24.76 ± 2.24%, P < 0.01) on hNSCs and improved migration (toward stromal cell-derived factor 1 [SDF-1], 0.54 ± 0.11% ~ 8.65 ± 1.76%, P < 0.001; toward fetal bovine serum, 8.36 ± 0.81% ~ 21.74 ± 0.85%, P < 0.0001). Further improvement increased the number of surviving cell distribution with increased uniformity on the olfactory epithelium and allowed the cells to stay in the nasal cavity for at least 72 h, but they did not survive for longer than 48 h. Optimization of pre-transplantation conditions augmented the success rate of intranasally delivered cells to the brain (0-41.6%). We also tentatively identified that hNSCs crossed the olfactory epithelium into the tissue space below the lamina propria, with cerebrospinal fluid entering the cribriform plate into the subarachnoid space, and then migrated toward injured areas along the brain blood vessels. Conclusion: This study offers some helpful advice and reference for addressing the problem of repeatability in the intranasal delivery of stem cells.

Transplanted Human Oligodendrocyte Progenitor Cells Restore Neurobehavioral Deficits in a Rat Model of Preterm White Matter Injury.

Background: Preterm white matter injury (PWMI) is a common brain injury and a leading cause of life-long neurological deficits in premature infants; however, no effective treatment is available yet. This study aimed to investigate the fate and effectiveness of transplanted human oligodendrocyte progenitor cells (hOPCs) in a rat model of PWMI. Methods: Hypoxia-ischemia was induced in rats at postnatal day 3, and hOPCs (6 × 105 cells/5 µL) were intracerebroventricularly transplanted at postnatal day 7. Neurobehavior was assessed 12 weeks post-transplant using the CatWalk test and Morris water maze test. Histological analyses, as well as immunohistochemical and transmission electron microscopy, were performed after transcardial perfusion. Results: Transplanted hOPCs survived for 13 weeks in PWMI brains. They were widely distributed in the injured white matter, and migrated along the corpus callosum to the contralateral hemisphere. Notably, 82.77 ± 3.27% of transplanted cells differentiated into mature oligodendrocytes, which produced myelin around the axons. Transplantation of hOPCs increased the fluorescence intensity of myelin basic protein and the thickness of myelin sheaths as observed in immunostaining and transmission electron microscopy, while it reduced white matter atrophy at the level of gross morphology. With regard to neurobehavior, the CatWalk test revealed improved locomotor function and inter-paw coordination after transplantation, and the cognitive functions of hOPC-transplanted rats were restored as revealed by the Morris water maze test. Conclusions: Myelin restoration through the transplantation of hOPCs led to neurobehavioral improvements in PWMI rats, suggesting that transplanting hOPCs may provide an effective and promising therapeutic strategy in children with PWMI.

A novel RIP1/RIP3 dual inhibitor promoted OPC survival and myelination in a rat neonatal white matter injury model with hOPC graft.

BACKGROUND: The dual inhibitors of receptor interacting protein kinase-1 and -3 (RIP1 and RIP3) play an important role in cell death processes and inflammatory responses. White matter injury (WMI), a leading cause of neurodevelopmental disabilities in preterm infants, which is characterized by extensive myelination disturbances and demyelination. Neuroinflammation, leads to the loss and differentiation-inhibition of oligodendrocyte precursor cells (OPCs), represents a major barrier to myelin repair. Whether the novel RIP1/RIP3 dual inhibitor ZJU-37 can promote transplanted OPCs derived from human neural stem cells (hOPCs) survival, differentiation and myelination remains unclear. In this study, we investigated the effect of ZJU-37 on myelination and neurobehavioral function in a neonatal rat WMI model induced by hypoxia and ischemia. METHODS: In vivo, P3 rat pups were subjected to right common carotid artery ligation and hypoxia, and then treated with ZJU-37 or/and hOPCs, then OPCs apoptosis, myelination, glial cell and NLRP3 inflammasome activation together with cognitive outcome were evaluated at 12 weeks after transplantation. In vitro, the effect of ZJU-37 on NLRP3 inflammasome activation in astrocytes induced by oxygen-glucose deprivation (OGD) were examined by western blot and immunofluorescence. The effect of ZJU-37 on OPCs apoptosis induced by the conditioned medium from OGD-injured astrocytes (OGD-astrocyte-CM) was analyzed by flow cytometry and immunofluorescence. RESULTS: ZJU-37 combined with hOPCs more effectively decreased OPC apoptosis, promoted myelination in the corpus callosum and improved behavioral function compared to ZJU-37 or hOPCs treatment. In addition, the activation of glial cells and NLRP3 inflammasome was reduced by ZJU-37 or/and hOPCs treatment in the neonatal rat WMI model. In vitro, it was also confirmed that ZJU-37 can suppress NLRP3 inflammasome activation in astrocytes induced by OGD. Not only that, the OGD-astrocyte-CM treated with ZJU-37 obviously attenuated OPC apoptosis and dysdifferentiation caused by the OGD-astrocyte-CM. CONCLUSIONS: The novel RIP1/RIP3 dual inhibitor ZJU-37 may promote OPC survival, differentiation and myelination by inhibiting NLRP3 inflammasome activation in a neonatal rat model of WMI with hOPC graft.

[Tumor necrosis factor α inhibits the migration of human oligodendrocyte precursor cells in vitro].

Objective To investigate the impact of tumor necrosis factor alpha (TNF-α) on the migration ability of oligodendrocyte precursor cells (OPCs) derived from human adult neural stem cells (NSCs) for transplantation therapy. Methods Flow cytometry was performed to detect the expressions of platelet derived growth factor receptor alpha (PDGFRα) and ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 (ST8SIA1/A2B5) in human OPCs. OPCs were cultured and incubated with 0, 10, 100, 200 ng/mL TNF-α for 18 hours. OPC viability was detected by CCK-8 assay and OPC migration was detected by TranswellTM migration assay. Results OPCs derived from human adult NSC specifically expressed PDGFRα (87.9%) and A2B5 (40.0%). Treatment with 10 ng/mL TNF-α had no impact on OPC viability while both 100 ng/mL and 200 ng/mL TNF-α treatments decreased OPC viability significantly. OPC migration was reduced significantly in 10 ng/mL TNF-α treated group compared with the blank control. Conclusion TNF-α inhibits the migration of the cultured OPCs.

Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development.

BACKGROUND: Human oligodendrocyte precursor cells (hOPCs) are an important source of myelinating cells for cell transplantation to treat demyelinating diseases. Myelin oligodendrocytes develop from migratory and proliferative hOPCs. It is well known that NG2 and A2B5 are important biological markers of hOPCs. However, the functional differences between the cell populations represented by these two biomarkers have not been well studied in depth. OBJECTIVE: To study the difference between NG2 and A2B5 cells in the development of human oligodendrocyte progenitor cells. METHODS: Using cell sorting technology, we obtained NG2+/-, A2B5+/- cells. Further research was then conducted via in vitro cell proliferation and migration assays, single-cell sequencing, mRNA sequencing, and cell transplantation into shiverer mice. RESULTS: The proportion of PDGFR-α + cells in the negative cell population was higher than that in the positive cell population. The migration ability of the NG2+/-, A2B5+/- cells was inversely proportional to their myelination ability. The migration, proliferation, and myelination capacities of the negative cell population were stronger than those of the positive cell population. The ability of cell migration and proliferation of the four groups of cells from high to low was: A2B5- > NG2- > NG2+ > A2B5+. The content of PDGFR-α+ cells and the ability of cell differentiation from high to low was: NG2- > A2B5- > A2B5+ > NG2+. CONCLUSION: In summary, NG2+ and A2B5+ cells have poor myelination ability due to low levels of PDGFR-α+ cells. Therefore, hOPCs with a higher content of PDGFR-α+ cells may have a better effect in the cell transplantation treatment of demyelinating diseases.

[Protective effect of transplantation of human oligodendrocyte precursor cells in a rat model of white matter injury].

OBJECTIVE: To study the effect of human oligodendrocyte precursor cell (hOPC) transplantation in the treatment of white matter injury (WMI). METHODS: Neonatal rats were randomly divided into a sham-operation group, a model group, and a transplantation group (n=10 each). At the age of 3 days, the rats in the model group and the transplantation group were treated with right common carotid artery ligation, followed by hypoxia for 2 hours, to prepare a rat model of WMI. hOPCs were isolated from a spontaneously aborted human fetal brain at week 11 of gestation, and then hOPCs were cultured and transplanted into the rats with WMI. At 3 months after transplantation, the water maze test was performed to evaluate neurological function, and an electron microscope was used to observe myelin sheath thickness and proliferation. RESULTS: The place navigation test using the Morris water maze showed that the model group had a significantly longer escape latency than the sham-operation group, and compared with the model group, the transplantation group had a significant reduction in escape latency (P < 0.05). To a certain degree, hOPC transplantation alleviated cognitive impairment in rats with WMI at the age of 90 days. The electron microscope images showed that hOPC transplantation promoted remyelination in the brain of WMI rats. Compared with the sham-operation group, the model group had a significant increase in the g-ratio (total axon diameter/total fiber diameter). Compared with the model group, the transplantation group had a significant reduction in the g-ratio (P < 0.05). CONCLUSIONS: Intrathecal hOPC transplantation may alleviate neurological injury and promote remyelination in a rat model of WMI.

Study on the Safety of Human Oligodendrocyte Precursor Cell Transplantation in Young Animals and Its Efficacy on Myelination.

Oligodendrocyte precursor cells (OPCs) can differentiate into myelinating oligodendrocytes during embryonic development, thereby representing an important potential source for myelin repair or regeneration. To the best of our knowledge, there are very few OPCs from human sources (human-derived OPCs [hOPCs]). In this study, we aimed to evaluate the safety and remyelination capacity of hOPCs developed in our laboratory, transplanted into the lateral ventricles of young animals. Several acute and chronic toxicity experiments were conducted in which different doses of hOPCs were transplanted into the lateral ventricles of Sprague-Dawley rats of different ages. The toxicity, biodistribution, and tumor formation ability of the injected hOPCs were examined by evaluating the rats' vital signs, developmental indicators, neural reflexes, as well as by hematology, immunology, and pathology. In addition, the hOPCs were transplanted into the corpus callosum of the shiverer mouse to verify cell myelination efficacy. Overall, our results show that transplanted hOPCs into young mice are nontoxic to their organ function or immune system. The transplanted cells engrafted in the brain and did not appear in other organs, nor did they cause tissue proliferation or tumor formation. In terms of efficacy, the transplanted hOPCs were able to form myelin in the corpus callosum, alleviate the trembling phenotype of shiverer mice, and promote normal development. The transplantation of hOPCs is safe; they can effectively form myelin in the brain, thereby providing a theoretical basis for the future clinical transplantation of hOPCs.

[Optimization of cryopreservative method for human fetal brain neural stem cells-derived oligodendrocyte precursor cells].

Objective To explore the impact of various conditions during cryopreservation on the survival rate of oligodendrocyte precursor cells (OPCs) derived from human fetal neural stem cells. Methods We compared the cell viability of oligodendrocyte precursors harvested with or without digestion. Then we tested the impact of 3 factors during cryopreservation, freezing solutions (solution with 70 mL/L DMSO and 930 mL/L FBS; solution with 70 mL/L DMSO, 300 mL/L FBS and OPC culture medium; solution with 70 mL/L DMSO, 300 mL/L FBS, 0.2 mol/mL trehalose and OPC culture medium; solution with 70 mL/L DMSO, 300 mL/L FBS, 300 mL/L HES and OPC culture medium), freezing methods (the step-wised freezing or rapid freezing within liquid nitrogen) and storage durations for the better survival rate of OPCs. The optimized method with the best survival rate of OPCs was implemented and at day 7 after recovery, the viability, OPCs specific markers [platelet derived growth factor receptor alpha (PDGFRα), ST8 alpha-N-acetyl-neuraminide alpha-2, 8-sialyltransferase 1 (ST8SIA1/A2B5), chondroitin sulfate proteoglycan 4 (CSPG4/NG2), ki67 were tested and compared with immunofluorescent cytochemical staining. Results Harvesting with digestion contributed to higher OPCs survival rate. OPCs of rapid freezing had survival rates less than 30% and couldn't be re-cultured. The step-wised freezing group showed higher recovery rate. Harvesting with digestion, preservation solution with trehalose, using 2.0×106/mL of cell number, step-wised freezing, contributed to the highest OPCs survival rate reaching (75.73±6.66)%. Compared with the fresh cultured group, cell proliferation, ki67 antigen, PDGFRα, A2B5 and NG2 expression of OPCs were similar in the recovered cells. Storage duration didn't affect OPCs survival rate. Conclusion Harvesting with digestion, step-wised freezing, preservation solution with trehalose contribute to higher OPCs survival rate during cryopreservation and cell-thawing. Storage time doesn't affect phenotypes and viability of OPCs.

[Transplantation of adipose-derived mesenchymal stem cell improves cardiac function in rats with acute myocardial infarction].

Objective To investigate the effects of the transplantation of autologous and allogeneic adipose-derived mesenchymal stem cells (ADMSCs) on the cardiac function in rats with acute myocardial infarction (AMI). Methods Firstly, ADMSCs were isolated from BN and Lewis rats, and the third generation were labeled with CM-DiI. Then 45 male Lewis rats were randomly divided into 3 groups (control group, autologous cell transplantation group, and allogeneic cell transplantation group). The AMI model was established by ligating the rats' left anterior descending artery, and then the infarcted myocardium as well as the peripheral parts was injected by the labeled ADMSCs via the endocardium. At 7 days after ADMSC transplantation, the infiltration of CD4+ T lymphocytes, CD8+ T lymphocytes and CD68+ macrophages were detected by immunofluorescence technique. At 7, 14 and 28 days after transplantation, survival rate of the transplanted cells was compared. Then echocardiography was used to detect the rats' cardiac function at 28 days after transplantation. And the expression of α-smooth muscle actin (α-SMA) was detected by immunofluorescence technique for determining the angiogenesis near the transplanted cells. Results The positive rate of the cells labeled with CM-DiI was nearly 100%. At 7 days after transplantation, lymphocyte infiltration and macrophages were observed around the transplanted cells in the allogeneic cell transplantation group. No lymphocyte infiltration and macrophages were observed in the autologous cell transplantation group. Compared with allogeneic cell transplantation, the survival rate of the transplanted cells increased significantly. Moreover, the left ventricular short axis shortening rate (LVFS) and the diastolic left ventricular anterior wall thickness (LVAWTd) increased at 7, 14 and 28 days after transplantation. Meanwhile, the number of angiogenesis around the infarcted myocardium also significantly increased. Conclusion Autologous ADMSC transplantation is significantly better at improving cardiac function in AMI rats than allogeneic ADMSC transplantation.

[Comparison of immunofluorescence cytochemical staining results in frozen sections of different-thickness neurospheres].

Objective To explore the optimal frozen section thickness of neurospheres for immunofluorescence cytochemical staining. Methods We selected the neurospheres of 10-12-day suspension culture to make frozen sections of varying thickness: 4, 7 and 10 µm, and then performed immunofluorescent staining to compare the expression and location of nestin. Results The diameters of the neurospheres cultured for 10-12 days were among 200-250 µm. The neurospheres were in a good condition and had a strong refractivity. The cells were spherical in shape, with burrs in the peripheral area. Practically, it was hard to make the 4 µm frozen sections which were wrapped around themselves easily. The advantages were that, their cells had a clear-cut structure, uniform staining and practical density, thus making it easier to calculate the cell number. The imaging was clear and the nuclei were distinct, too. It could be seen that much of the whole cytoplasm, with the nestins presenting positive staining, wrapped itself around the nuclei in which the DAPI also presented positive staining, and intercellular details could be observed meanwhile. Comparatively, the 7 µm frozen sections were not that hard to get. They were quite smooth and showed rather uniform staining. The cell number turned out to be larger than that of the 4 µm frozen sections, but the cell structure was not that clear-cut, so it was difficult to make an accurate estimate of the cell number. Besides, to be in full focus could not be achieved while using fluorescent photography. Hence, only part of the entire cell morphology could be seen. The frozen slice of 10 µm is relatively easy to make, the slice is flat, but did not have a clear-cut cell structure, either. There was a serious piling phenomenon and no clear imaging. Consequently, it was hard to see the complete cell morphology. Conclusion The 4 µm frozen sections of neurospheres are more conducive to the observation and analysis of immunofluorescent staining results than those of other thicknesses.

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.

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.

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.

[Long-term effect of oligodendrocyte precursor cell transplantation on a rat model of white matter injury in the preterm infant].

OBJECTIVE: To investigate the long-term effect of oligodendrocyte precursor cell (OPC) transplantation on a rat model of white matter injury (WMI) in the preterm infant. METHODS: A total of 80 Sprague-Dawley rats aged 3 days were randomly divided into sham-operation group, model control group, 5-day ventricular/white matter transplantation group, 9-day ventricular/white matter transplantation group, 14-day ventricular/white matter transplantation group (n=10 each). All groups except the sham-operation group were treated with right common carotid artery ligation and hypoxia for 80 minutes to establish a rat model of WMI in the preterm infant. OPCs were prepared from the human fetal brain tissue (10-12 gestational weeks). At 5, 9, and 14 days after modeling, 3×105 OPCs were injected into the right lateral ventricle or white matter in each transplantation group, and myelin sheath and neurological function were evaluated under an electron microscope at ages of 60 and 90 days. RESULTS: Electron microscopy showed that at an age of 60 days, each transplantation group had a slight improvement in myelin sheath injury compared with the model control group; at an age of 90 days, each transplantation group had significantly thickened myelin sheath and reduced structural damage compared with the model control group, and the 14-day transplantation groups had the most significant changes. There were no significant differences in the degree of myelin sheath injury between the ventricular and white matter transplantation groups at different time points. At an age of 60 or 90 days, the transplantation groups had a significantly higher modified neurological severity score (mNSS) than the sham-operation group and a significantly lower mNSS than the model control group (P<0.05). CONCLUSIONS: OPC transplantation may have a long-term effect in the treatment of WMI in the preterm infant, and delayed transplantation may enhance its therapeutic effect.

Analysis of Adverse Events Related to 720 Cases of Neural Progenitor Cell Transplantation.

BACKGROUND: Cell therapies have shown to be able to improve neurological functions to some extent for patients with refractory central nervous system (CNS) diseases or damages. Meanwhile, increasing attention has been drawn to the operation-related and (or) cell-related adverse events when performing cell therapy. Our study is to explore the safety issue from 720 cases of neural progenitor cell (NPC) transplantation based on clinic manifestations and examinations. METHOD: A retrospective analysis of all adverse events associated with 720 cases of NPC transplantation by administering the cells into the ventricles was done. RESULTS: One hundred and sixty-six cases had postoperative crying and irritability, 69 with vomiting and 84 with fever. None of them had CNS infection, but 4 cases presented intracranial hemorrhage. One month after cell therapy, 568 cases did EEG test, in which 153 patients showed improvement, 74 had abnormal changes in and 341 cases had no changes in; two patients developed new-onset convulsions and 3 had recurrent convulsions; 6 cases had intracranial hemorrhage, but no other CNS sequelae left from the primary diseases. 180 patients were able to follow-up for their clinical evaluation and head MRI or CT examination 2 years after transplantation. All patients didn't show signs of tumorigenesis and no serious and irreversible operation- or cell-related adverse events. IN CONCLUSION: there are mild adverse reactions and reversible adverse events following cell transplantation, our study indicated that NPC transplantation is a safe therapy in clinical treatment. Further clinical trials are necessary to establish the safety of this therapy.

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.