AIM:We investigated the survival,migration and differentiation abilities of human oligodendro-cyte precursor cells(hOPC)in the brains of mice with vascular dementia(VaD),the effects of hOPC on cerebral white matter,and the underlying mechanisms.METHODS:Mouse VaD model was constructed using the bilateral common ca-rotid artery stenosis method,and the mice were randomly divided into sham,VaD and hOPC groups.Eight weeks after model establishment,the mice in VaD and hOPC groups received equal volume of vehicle(PBS)and hOPC solution,re-spectively,through the corpus callosum.Survival,migration and differentiation of hOPC in the brain were observed by im-munofluorescence staining at 4 and 12 weeks after transplantation.Twelve weeks after transplantation,the effects of hOPC on mouse brain white matter were detected by immunofluorescence staining of myelin basic protein(MBP),myelin-associ-ated glycoprotein(MAG),neurofilament protein 200(NF200)and non-phosphorylated neurofilament H(using monoclo-nal antibody SMI32),and by water maze experiments.Paracrine signaling by hOPC was explored using immunofluores-cence staining for vascular endothelial growth factor(VEGF).RESULTS:The hOPC survived in the brains of VaD mice for 12 weeks,migrated to damaged white matter areas,and partially differentiated into mature oligodendrocytes(approxi-mately 64%).Twelve weeks after transplantation,hOPC significantly increased the fluorescence intensity of MBP,MAG,and NF200(P<0.05 or P<0.01)and decreased the fluorescence intensity of SMI32(P<0.01).The VEGF expression in hOPC-treated mice was significantly higher than that in sham and VaD groups(P<0.01).The difference in water maze test performance between hOPC and sham groups was not statistically significant(P>0.05).The mice in hOPC group had a shorter latency than those in VaD group(P<0.05 or P<0.01),and performed more platform crossings than those in VaD group(P<0.05).CONCLUSION:The hOPC can survive,migrate and differentiate in the brains of VaD mice,attenuate cerebral white matter lesions,and improve cognitive function.These improvements may be attributed to cell replacement and paracrine effects.

BACKGROUND:Oligodendrocyte precursor cells are seed cells for the treatment of white matter damage diseases.Establishing an efficient and stable in vitro differentiation method is an important prerequisite for clinical translational research.OBJECTIVE:To investigate the effect of fibronectin on biological characteristics such as proliferation,migration,and differentiation of oligodendrocyte precursor cells derived from human neural stem cells.METHODS:Human neural stem cells cultured in suspension were digested into single cells using Accutase.The expression of specific markers Nestin,Sox2,Vimentin,CD133,and Musashi was detected by flow cytometry.The single cells of human neural stem cells were resuspended in oligodendrocyte precursor cell medium and seeded in six-well plates coated with different concentrations of fibronectin(0,1,2.5,5,and 10 μg/mL).Accutase digestion was performed after 7 days of culture.Cells were counted by trypan staining.Fibronectin-coated group with the strongest amplification ability and the oligodendrocyte precursor cells without fibronectin-coated group were selected for further tests.The migration ability of the two groups of cells was detected by Transwell.Flow cytometry was used to detect the expression of Olig2,Sox10,and PDGFR-α.Oligodendrocyte precursor cells were induced to differentiate into oligodendrocytes for 3 weeks,and the expression of Galc in differentiated cells was detected by immunofluorescence staining.RESULTS AND CONCLUSION:(1)H uman neural stem cells grew in suspension spheres.Flow cytometry showed that human neural stem cells highly expressed Nestin,Sox2,Vimentin,CD133,and Musashi.(2)The cell bodies of oligodendrocyte precursor cells induced by human neural stem cells were round or oval,with strong refractive nature and bipolar or tertiary protrusions.Compared with the 0 μg/mL fibronectin coating group,there was a significant difference in the amplification ability of oligodendrocyte precursor cells in the 2.5,5,and 10 μg/mL fibronectin coating groups(P＜0.05).The amplification ability of oligodendrocyte precursor cells was the strongest when the fibronectin concentration was 10 μg/mL.(3)Flow cytometry results showed that the oligodendrocyte precursor cell markers 0Iig2,Sox10,and PDGFR-α were highly expressed in the 0 and 10 μg/mL fibronectin coating groups,and there was no significant difference between the two groups(P＞0.05).(4)Transwell chamber assay results showed that compared with the 0 μg/mL fibronectin-coated group,the migration ability of oligodendrocyte precursor cells in the 10 μg/mL fibronectin-coated group was increased(P＜0.01).(5)After 3 weeks of differentiation into oligodendrocytes,oligodendrocyte precursor cells showed complex morphology with multiple branches,grids or membrane sheets.Immunofluorescence staining results showed that there was no statistical difference in the Galc positive rate of oligodendrocytes between the two groups(P＞0.05).These findings indicate that when the concentration of fibronectin coated well plate is 10 μg/mL,the proliferation and migration of oligodendrocyte precursor cells are the strongest,but it does not affect the expression of oligodendrocyte precursor cells-specific markers Olig2,Sox10,and PDGFR-α and their differentiation into oligodendrocytes.

AIM:We investigated the survival,migration and differentiation abilities of human oligodendro-cyte precursor cells(hOPC)in the brains of mice with vascular dementia(VaD),the effects of hOPC on cerebral white matter,and the underlying mechanisms.METHODS:Mouse VaD model was constructed using the bilateral common ca-rotid artery stenosis method,and the mice were randomly divided into sham,VaD and hOPC groups.Eight weeks after model establishment,the mice in VaD and hOPC groups received equal volume of vehicle(PBS)and hOPC solution,re-spectively,through the corpus callosum.Survival,migration and differentiation of hOPC in the brain were observed by im-munofluorescence staining at 4 and 12 weeks after transplantation.Twelve weeks after transplantation,the effects of hOPC on mouse brain white matter were detected by immunofluorescence staining of myelin basic protein(MBP),myelin-associ-ated glycoprotein(MAG),neurofilament protein 200(NF200)and non-phosphorylated neurofilament H(using monoclo-nal antibody SMI32),and by water maze experiments.Paracrine signaling by hOPC was explored using immunofluores-cence staining for vascular endothelial growth factor(VEGF).RESULTS:The hOPC survived in the brains of VaD mice for 12 weeks,migrated to damaged white matter areas,and partially differentiated into mature oligodendrocytes(approxi-mately 64%).Twelve weeks after transplantation,hOPC significantly increased the fluorescence intensity of MBP,MAG,and NF200(P<0.05 or P<0.01)and decreased the fluorescence intensity of SMI32(P<0.01).The VEGF expression in hOPC-treated mice was significantly higher than that in sham and VaD groups(P<0.01).The difference in water maze test performance between hOPC and sham groups was not statistically significant(P>0.05).The mice in hOPC group had a shorter latency than those in VaD group(P<0.05 or P<0.01),and performed more platform crossings than those in VaD group(P<0.05).CONCLUSION:The hOPC can survive,migrate and differentiate in the brains of VaD mice,attenuate cerebral white matter lesions,and improve cognitive function.These improvements may be attributed to cell replacement and paracrine effects.

BACKGROUND:Oligodendrocyte precursor cells are seed cells for the treatment of white matter damage diseases.Establishing an efficient and stable in vitro differentiation method is an important prerequisite for clinical translational research.OBJECTIVE:To investigate the effect of fibronectin on biological characteristics such as proliferation,migration,and differentiation of oligodendrocyte precursor cells derived from human neural stem cells.METHODS:Human neural stem cells cultured in suspension were digested into single cells using Accutase.The expression of specific markers Nestin,Sox2,Vimentin,CD133,and Musashi was detected by flow cytometry.The single cells of human neural stem cells were resuspended in oligodendrocyte precursor cell medium and seeded in six-well plates coated with different concentrations of fibronectin(0,1,2.5,5,and 10 μg/mL).Accutase digestion was performed after 7 days of culture.Cells were counted by trypan staining.Fibronectin-coated group with the strongest amplification ability and the oligodendrocyte precursor cells without fibronectin-coated group were selected for further tests.The migration ability of the two groups of cells was detected by Transwell.Flow cytometry was used to detect the expression of Olig2,Sox10,and PDGFR-α.Oligodendrocyte precursor cells were induced to differentiate into oligodendrocytes for 3 weeks,and the expression of Galc in differentiated cells was detected by immunofluorescence staining.RESULTS AND CONCLUSION:(1)H uman neural stem cells grew in suspension spheres.Flow cytometry showed that human neural stem cells highly expressed Nestin,Sox2,Vimentin,CD133,and Musashi.(2)The cell bodies of oligodendrocyte precursor cells induced by human neural stem cells were round or oval,with strong refractive nature and bipolar or tertiary protrusions.Compared with the 0 μg/mL fibronectin coating group,there was a significant difference in the amplification ability of oligodendrocyte precursor cells in the 2.5,5,and 10 μg/mL fibronectin coating groups(P＜0.05).The amplification ability of oligodendrocyte precursor cells was the strongest when the fibronectin concentration was 10 μg/mL.(3)Flow cytometry results showed that the oligodendrocyte precursor cell markers 0Iig2,Sox10,and PDGFR-α were highly expressed in the 0 and 10 μg/mL fibronectin coating groups,and there was no significant difference between the two groups(P＞0.05).(4)Transwell chamber assay results showed that compared with the 0 μg/mL fibronectin-coated group,the migration ability of oligodendrocyte precursor cells in the 10 μg/mL fibronectin-coated group was increased(P＜0.01).(5)After 3 weeks of differentiation into oligodendrocytes,oligodendrocyte precursor cells showed complex morphology with multiple branches,grids or membrane sheets.Immunofluorescence staining results showed that there was no statistical difference in the Galc positive rate of oligodendrocytes between the two groups(P＞0.05).These findings indicate that when the concentration of fibronectin coated well plate is 10 μg/mL,the proliferation and migration of oligodendrocyte precursor cells are the strongest,but it does not affect the expression of oligodendrocyte precursor cells-specific markers Olig2,Sox10,and PDGFR-α and their differentiation into oligodendrocytes.

Objective To assess the clinical efficacy of human neural stem cell (hNSC) transplantation in the treatment of severe cerebral palsy (CP) in children.Methods hNSCs were obtained from the forebrain of 10 to 12-week-fetus.Forty children with CP were voluntarily received hNSC transplantation that were injected into cerebral ventricle.The development of motor and fine motor functions were evaluated by GMFM and PDMS-FM 1 month before hNSC transplantation.as well as 3 and 6 months after hNSC transplantation.Results Twenty six (65％) cases displayed improvement from day 5 to month 6 after hNSC transplantation.GMFM assessment showed that the percentage was (4.52±2.50) ％ 1 month before hNSC transplantation,(7.74±2.94) ％ 3 months after hNSC transplantation and (13.01±6.71)％ 6 months after hNSC transplantation,indicating a significant improvement by the treatment of hNSC transplantation(P＜0.05).The percentage in PDMS-FM evaluation was (15.01± 12.00)％,(20.34± 11.91) ％ and (30.02± 12.50) ％ one month before hNSC transplantation,3 and 6 months after hNSC transplantation,respectively,also suggesting a significant improvement induced by hNSC transplantation treatment (P＜0.05).Moreover,the developmental improvement was the most prominent among 1-3 months post hNSC transplantation.Then the development slowed down.Significantly,patients received no hNSC transplantation experienced serious adverse events or complications.Conclusions hNSC transplantation is an effective and safer therapy for severe CP.Future observations are needed to evaluate long-term clinical efficacy of the therapy.

Objective To investigate the effects of Chaishao Chengqi Decotion on intestinal microbiota and intestinal mucosal lesion in severe acute pancreatitis(SAP).Methods 46 Patients with SAP were randomized into two groups.Routine medical management was initiated in two groups.The treatment group received Chaishao Chengqi Decotion for 1 week.The plasma concentrations ofDiamine Oxidase (DAO),D-lactate were detected and changes of microflora were evaluated by bacterial culture.Results The levels of DAO in the treatment group (4.65 ±0.82)U/ml were significantly lower than those in the control group (5.66 ±2.17) U/ml (P ＜ 0.05) 7 days after treatment.The levels of D-lactate in the treatment group (10.65 ± 5.24)mg/L were significantly higher than those in the control group (5.42±2.13)mg/L (P＜0.05).Bacterial culture revealed that the amount of bifidobaterium (6.02± 1.42)In/g and lactobacilli (7.21 ± 2.02) In/g were significantly increased when compared to those before treatment (3.74± 1.71)In/g and (4.03 ± 1.79)In/g respectively,while there was imbalance of intestinal microflora to some extent in the control group.Conelusien Chaishao Chengqi Decotion exerted the protective effects on gut barrier function by alleviating the damage of intestinal mucosa and balancing the intestinal microbiota following severe acute pancreatitis.

Objective To investigate the clinical effect of human neural stem cells transplantation on severe visual disability infants after cerebral palsy. Methods Cells obtained from the forebrain of an 11-week-old abortive fetus were cultured and expanded for 15 days, then injected into cerebral ventricle of 7 patients. Results Their vision of 4 patients improved, as well as changes of flash visual evoked potential and functional magnetic resonance imaging in a few days after transplantation. Conclusion Neural stem cells transplantation may benefit in some CP children with severe visual disability.

Objective To isolate and culture the neural stem cells (NSCs) from the cerebral cortex of newborn rats, and investigate the cell-replace responses of the NSCs transplanted into the sibling rats with focal cerebral cortex ischemic lesion. Methods The serum-free medium DMEM/F12 (1∶1) containing basic fibroblast growth factor (bFGF or FGF2) and epidermal growth factor (EGF) was used to culture the neural stem cell spheres. The NSCs were identified by detecting the neural stem cell marker nestin with enzyme immune assay and inducing neural stem cell spheres to differentiation. The NSCs which would be used in the transplantation experiment were labeled by BrdU incorporation when cultured in vitro. The focal ischemic models were made by opening the skulls and removing the cerebral menings of 4-day-old rats to stop the blood supply for the neopallium. The BrdU-labeled NSCs were transplanted into the cerebral lesion boundary zones of the focal ischemic sibling rat models. The experiment rats were divided into lesion-transplantation group, lesion-control group and sham-operation-control group. Recipients were killed and the brains were examined by detecting the BrdU-labeled cells with enzyme immunohistochemistry at 4,7,14,30 days postgrafting, indicating the grafts living and migration in the host. Results The neural stem cell spheres, which floated and grew in medium, expressed nestin, as well as gave rise to neurons and astrocytes, could be obtained through culturing the cells derived from the cerebral cortex of newborn rats in vitro for a week. In the transplantation of the NSCs, the grafts were easy to migrate along the boundary zones of the focal ischemic lesions, and promoted the restore of the tissue structures in the damaged areas, the damage recovered well through the cell-replace responses. The BrdU-labeled positive cells in the lesion areas were full of the visual fields under microscope, the greatest density of the positive cells were focused in the granular layer of the injured cerebral cortex and not found in remote sites from the lesion. The number of BrdU-labeled cells gradually decreased in the brains of the sham-operation-control rats, only a few positive cells were found when examined at 14 days postgrafting, significantly less than that in the lesion-transplantation rats. Conclusions NSCs exist in the cerebral cortex of newborn rats. The ischemia can promote proliferation and graft of NSCs. The grafted NSCs play an important role in the recovery of focal cerebral cortex ischemic lesion.

Objective: To investigate if bFGF can penetrate placental barrier. Methods: Sixteen day pregnant Wistar rats were selected. bFGF labeled with 125 I was injected peritoneally into the rats. The radioactivity of bFGF in different organs were determined in 30 min. Results: (1) 125 I bFGF was detected in the brain, heart, liver,lung and spleen. (2)With the same dose of 125 I bFGF, the concentration of it in the brain was at lowest level of all other organs.(3) In the range of safe dose, the permeability of bFGF through placental barrier was increased obviously. Conclusion: bFGF may penetrate placental barrier into rat's brain, which makes possible for the therapeutic intervention of bFGF in feotus. [

0.05) . No significant differences were observed between the expansion folds of hNSC derived from various fetal brain samples when cultured under the same conditions. LIF played great roles on cell proliferation,in LIF + groups, hNSC cell number increased ranging from 4 000-8 400 folds, no cell differentiation occurred; and in LIF" groups,only 43 to 96 folds.The differentiation phenomenons were watched when cultured more than two months. In the course of cell culturing, observed that the effects of LIF on hNSC expansion were obviously demonstrated 50-60 days after inoculation.The number of neurons and astrocytes differentiated from the cultured hNSC were respectively identified by means of Immuno-cytochemical fluorescent assay, and the percentages of neurons(as a proportion of neuron and astrocyte number) were calculated,which were ranging from 12% to 83% in LIF+ cultures, significantly higher than 8% to 23% in LIF- ones(P