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    02 July 2013, Volume 17 Issue 27 Previous Issue    Next Issue
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    Tissue-engineered tendon construction using bone marrow mesenchymal stem cells induced by bone morphogenetic protein 12
    Cai Rong-hui, Liu Kang
    2013, 17 (27):  4941-4950.  doi: 10.3969/j.issn.2095-4344.2013.27.001
    Abstract ( 406 )   PDF (3389KB) ( 878 )   Save

    BACKGROUND: The previous methods for the repair of tendon defect include end-to-end method, autologous tendon graft, tendon allograft or artificial tendon transplantation, but each method has its advantages and disadvantages.
    OBJECTIVE: To investigate the feasibility of constructing tissue-engineered tendon by rabbit bone marrow mesenchymal stem cells as seed cells which were induced by bone morphogenetic protein 12 and with collagen-polyglycolic acid composite as frameworks in the repair of rabbit tendon defects.
    METHODS: Bone marrow was separated from rabbit proximal femur to harvest cells, and the cells were passaged to the second generation and induced with 10 μg/L bone morphogenetic protein 12. Then the passage 2 cells were implanted into the prefabricated tissue-engineered tendon on the polyglycolic acid stitch with certain percentage together with collagenⅠsolution. The rabbits were used to establish the Achilles tendon defect models, and different methods were used to repair Achilles tendon defect: tissue-engineered tendon, collagenⅠ- polyglycolic acid stitch and end-to-end suturing in silk. Morphology, mechanics and shistopathology of the tissue-engineered tendon were observed.
    RESULTS AND CONCLUSION: Pathomorphological observation of histological section after 12 weeks showed that multiple fusiform fibroblasts were homogeneously distributed in collagen in the direction of stress mechanics. Fibrocytes increased obviously, new small vessels could be seen and collagen was found aligned compactedly. In collagen Ⅰ-polyglycolic acid stitch group, a part of fibroplasia hyperplasia accompanied by granulation tissue formation could be seen, the collage fibers were in loose filamentous network and the cells were distributed disorderly and unevenly. Granulation tissue formed around the fibrous tissue in the silk group. Biomechanics strength in bone morphogenetic protein 12+polyglycolic acid group was better than that in the collagen Ⅰ-polyglycolic acid group, and there was significant difference when compared with suture silk group. The biomechanics strength of the bone morphogenetic protein 12+polyglycolic acid group was lower than that of normal tendon. It is possible to construct a tissue-engineered tendon with autologous bone marrow mesenchymal stem cells as seed cells induced by bone morphogenetic protein 12 and with the collagen-polyglycolic acid as the framework. Constructed tissue-engineered tendon has biomechanics characteristics and can be used to repair Achilles tendon defect.

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    Chondrogenic differentiation of bone marrow mesenchymal stem cells
    Chen Liang, He Shao-ru, Zhuang Jian, Zheng Man-li, Sun Yun-xia, Liang Hui-xin, Liu Yu-mei, Sun Xin, Chen Xiao-bo
    2013, 17 (27):  4951-4957.  doi: 10.3969/j.issn.2095-4344.2013.27.002
    Abstract ( 530 )   PDF (1611KB) ( 596 )   Save

    BACKGROUND: Bone marrow mesenchymal stem cells are important seeded cells for construction of tissue-engineered trachea, but there is no special surface marker. Therefore, identification of bone marrow mesenchymal stem cells is mostly based on morphology, phenotype antigen and the function of differentiation.
    OBJECTIVE: To explore the feasibility of the tracheal chondrogenic differentiation of bone marrow mesenchymal stem cells under a special condition through isolation, cultivation and identification of bone marrow mesenchymal stem cells.
    METHODS: Rabbit bone marrow was acquired in the sterile environment to isolate and culture bone marrow mesenchymal stem cells to passage 2 by bone marrow adherence and screening method. Flow cytometry identified the phenotype CD44, CD45 of bone marrow mesenchymal stem cells at passages 1 and 2. Rabbit tracheal samples were acquired in the sterile environment, the tracheal chondrocytes were isolated and cultured by enzyme digestion, and toluidine blue staining was used to detect aggrecan. Bone marrow mesenchymal stem cells were co-cultured with tracheal chondrocytes by Transwell and transforming growth factor β1. Cell morphology was detected under an inverted microscope. Real-time quantitative PCR and toluidine blue staining detected the extracellular matrix components, such as type Ⅱ collagen and aggrecan.   
    RESULTS AND CONCLUSION: After isolation and culture, cells were spindle and irregular in morphology, and passaged cells thrived that were gathered into a fish-like colony growth. For passage 1 bone marrow mesenchymal stem cells, the positive rates of phenotype antigen CD44 and CD45 were respectively 96.97% and 13.72%; for passage 2 cells, the positive rates of phenotype antigen CD44 and CD45 were 99.11% and 8.54%, respectively. Tracheal chondrocytes were positive for toluidine blue staining. The morphology of induced bone marrow mesenchymal stem cells changed from long fusiform to triangular or irregular shape, indicating the chondrocytes expressed type Ⅱ collagen and aggrecan, and toluidine blue staining was positive. These results showed bone marrow adherence and screening method could acquire bone marrow mesenchymal stem cells, and the purity of passage 2 cells is higher. Under a special condition, bone marrow mesenchymal stem cells have the potential of chondrogenic differentiation, and can be selected as seed cells for construction of tissue-engineered trachea.

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    Therapeutic potential of non-adherent bone marrow-derived stem cells for acute radiation injury
    Wang Pan-jun, Fu Jin-xiang, Sun Yu, Feng Yi-zhong, Zhang Hong, Zhang Xue-guang
    2013, 17 (27):  4958-4965.  doi: 10.3969/j.issn.2095-4344.2013.27.003
    Abstract ( 402 )   PDF (2368KB) ( 507 )   Save

    BACKGROUND: Effective treatment for severed acute radiation sickness (over 8 Gy) has not been obtained at present. Mesenchymal stem cells, which are shown to secrete hematopoietic cytokines and support hematopoietic progenitors, play an important role in cute radiation sickness.
    OBJECTIVE: To investigate the therapeutic potential of non-adherent bone marrow-derived stem cells in the treatment of acute radiation injury induced by 8.5 Gy X-ray irradiation, as well as the mechanisms involved. 
    METHODS: Non-adherent marrow-derived stem cells from the long bone of fetal limbs were collected for analyzing surface antigens, cell cycle, osteogenic and adipogenic differentiation potential, and expressions of vascular endothelial growth factors and Annexin A2. After being exposed to 8.5 Gy total body irradiation, BALB/C mice were randomly assigned into transplantation group and control group. Mice in the transplantation group were given 3×106 CFDA-SE labeled human non-adherent bone marrow-derived stem cells, and those in the control group were given 0.3 mL normal saline. Then, the survival rate, peripheral white blood cells at different time, pathologic change and angiogenesis of the bone marrow were observed.
    RESULTS AND CONCLUSION: After X-ray irradiation, transplanted non-adherent mesenchymal stem cells appeared to have a homing to the site of injury. The survival rate of mice in the transplantation group was much higher than that in the control group. Compared with the control group, the white blood cells in the transplantation group decreased more slowly while recovered more rapid: the nadir appeared at day 14 after transplantation while it recovered within 30 days. The bone marrow of mice in the transplantation group regenerated more actively and had more hematopoietic islands than those in the control group on day 21. In addition, bone marrow angiogenesis of the transplantation group was more obvious than that of the control group. In conclusion, human fetal non-adherent bone marrow-derived stem cells could promote bone marrow angiogenesis in a mouse model of acute radiation injury, through which they could play an important role in tissue regeneration of acute radiation injury.

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    Bone marrow mesenchymal stem cells combined with allogeneic bone for cancellous bone defects
    Wang Feng, Fu Zhi-hou
    2013, 17 (27):  4966-4973.  doi: 10.3969/j.issn.2095-4344.2013.27.004
    Abstract ( 317 )   PDF (943KB) ( 545 )   Save

    BACKGROUND: Some studies have shown that bone marrow mesenchymal stem cells and allograft bone have a certain role for repairing bone defects, but the effectiveness on cancellous bone defects is seldom reported so far.
    OBJECTIVE: To observe the effectiveness of bone marrow mesenchymal stem cells combined with allogeneic bone on cancellous bone defects.
    METHODS: The models of cancellous bone defects (0.6 cm×1.2 cm) were made artificially in both condylus lateralis femoris of New Zealand white rabbits: one side served as model group implanted with combination of bone marrow mesenchymal stem cells and allogeneic bone, and the other side was considered as control group implanted with allogeneic bone.
    RESULTS AND CONCLUSION: The model group was better than the control group in new bone growth and defect repair at 4, 8, 12 weeks after implantation, which was confirmed by general observation, X-ray examination and hematoxylin-eosin staining. There was a large amount of trabecular bone formation and mature lamellar bone tissue in bone defects of model group by histological observation at 12 weeks after implantation, and bone defects of the model group were repaired basically; while there were only abundant woven bones in the control group, and bone defects in the control group were not repaired effectively. Scores on Lane-Sandhu’s X-ray combined with histological observation were higher in the model group than the control group (P < 0.05). Biomechanical test showed that the maximum pressure load of the femoral condyle and load/strain ratio in the model group were significantly higher than those in the control group at 12 weeks after implantation (P < 0.05), while the maximum strain and displacement of the model group was lower than that of the control group (P < 0.05). These findings suggest that the combination of bone marrow mesenchymal stem cells and allogeneic bone is superior to simple allogeneic bone implantation in the repair of cancellous bone defects of the femoral condyle.

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    Construction of oxalate-degrading adult bone marrow mesenchymal stem cell lines
    Yang Xu-ming, Yuan Jian, Lei Ming, Zhang Ze
    2013, 17 (27):  4974-4979.  doi: 10.3969/j.issn.2095-4344.2013.27.005
    Abstract ( 300 )   PDF (749KB) ( 637 )   Save

    BACKGROUND: High oxalic acid urine is a risk factor for stone formation. Constructing cell lines with high oxalate metabolic ability using genetic engineering and stem cell technology will become the effective method to prevent and treat calcium oxalate stone.
    OBJECTIVE: To construct the adult bone marrow mesenchymal stem cell lines that can decompose the oxalic acid, through co-transfecting the oxalic acid degradation genes Frc and Oxc of oxalobacter formigenes into the normal adult bone marrow mesenchymal stem cells. 
    METHODS: Frc and Oxc were amplified by PCR, and the eukaryotic expression vectors of pLEGFP-N1-myc-Frc and pBaBE-puro-flag-Oxc were constructed, then co-transfected into the normal adult bone marrow mesenchymal stem cells. The non-transfected bone marrow mesenchymal stem cells and the cells transfected with empty vectors were as control. Western blot was performed to detect the expression of the objective genes; the concentration of oxalate in the culture medium after transgenic was determined by ion chromatography.
    RESULTS AND CONCLUSION: Restriction enzyme digestion and sequencing results showed that the Frc and Oxc genes were successfully amplified, and the vectors of pLEGFP-N1-myc-Frc and pBaBE-puro-flag-Oxc were constructed. After tranfected into the bone marrow mesenchymal stem cells, the Western blot results showed that transfected bone marrow mesenchymal stem cells could stably express the target protein myc-formyl coenzyme A transferase enzyme and the flag-oxalyl coenzyme A decarboxylase; ion chromatography test results showed with the prolonging of the culture time, the concentration of oxalic acid in the human bone marrow mesenchymal stem cell culture medium transfected with target gene was decreased gradually. While there was no target protein expression in the non-transfected human bone marrow mesenchymal stem cells as well as the cells transfected with empty vectors. The cells had the ability of oxalate-degradation. The experiment successfully constructs the adult bone marrow mesenchymal stem cell lines that can decompose the oxalic acid, and the cell lines have the ability of oxalate-degradation and can stably express the oxalate decomposition proteins Frc and Oxc.

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    Primary culture of umbilical cord-derived mesenchymal stem cells in serum free medium
    Zhou Ting-ting, Wei Chao, Chen Xiao-dong, Li Hai, Wang Zheng
    2013, 17 (27):  4980-4987.  doi: 10.3969/j.issn.2095-4344.2013.27.006
    Abstract ( 512 )   PDF (1951KB) ( 688 )   Save

    BACKGROUND: The application of mesenchymal stem cells derived from umbilical cord cultured in serum-containing medium has some obstacles.
    OBJECTIVE: To establish serum free medium system for primary culture of umbilical cord-derived mesenchymal stem cells.
    METHODS: Wharton’s Jelly was isolated from umbilical cord, minced, 1-3 mm3, and subsequently incubated in either serum containing medium or serum free medium. Some cells were harvested on days 11, 14 and 17 for some detection. Based on the minimal criteria established by the International Society for Cellular Therapy in 2006, mesenchymal stem cells were assayed with colony forming unit-fibroblast.
    RESULTS AND CONCLUSION: The mesenchymal stem cells grew rapidly in serum free medium condition compared with serum containing medium. On day 11, the number of colonies was larger in serum free medium condition than that in serum containing medium. Thus, serum free medium could maintain properties of mesenchymal stem cells and provide possibility a credible alternative to serum containing medium for primary culture of umbilical cord-derived mesenchymal stem cells.

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    Expression of specific genes of cardiomyocytes differentiated from human umbilical cord mesenchymal stem cells
    Tang Xin, Wang Yan, Yi Hai-bo, Pang Tian-shu
    2013, 17 (27):  4988-4991.  doi: 10.3969/j.issn.2095-4344.2013.27.007
    Abstract ( 306 )   PDF (1103KB) ( 557 )   Save

    BACKGROUND: Intramyocardial transplantation of autologous umbilical cord-derived mesenchymal stem cells for repair of myocardial tissue damage is paid increasing attention in the cardiovascular field.  
    OBJECTIVE: Human umbilical cord mesenchymal stem cells were isolated and cultured. Passage 2 human umbilical cord mesenchymal stem cells were treated with various concentratins of 5-azacytidine (2.5, 5, 10, 20, 40, 80 μmol/L) for 24 hours . After removal of 5-azacytidine, cells were cultured for another 4 weeks.
    RESULTS AND CONCLUSION: Before 5-azacytidine treatment, filament-like structures or particles were not observed in the cells, but the amount of cytoplasm was less and uniform, nuclear/cytoplasm ratio was high, cells exhibited typical fusiform appearance and grew in a swirl-like manner, and nucleolus was obvious. After treatment with 5-azacytidine for 24 hours, some cells died in each group, and typical fusiform appearance turned into stick-like or column-like appearance, especially in the 40 and 80 μmol/L 5-azacytidine groups. Reverse transcription-PCR results showed that atrial natriuretic peptide and α-skeletal actin gene expression levels were detected on human umbilical cord mesenchymal stem cells after treatment with 2.5 or 40 μmol/L 5-azacytidine for 4 weeks or with 5, 10, 20 μmol/L 5-azacytidine for 1, 2, 3 and 4 weeks. These findings suggest that 5-azacytidine-induced human umbilical cord mesenchymal stem cells express the specific gene of myocardiocytes.

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    Comparison of biological characteristics of adipose-derived stem cells from different parts
    Lin Li-xin, Huang Yong, Wang Yu-ting, Wang Peng, Wang Xue-ming, Jiang Lei, Lin Guan-yu
    2013, 17 (27):  4992-4997.  doi: 10.3969/j.issn.2095-4344.2013.27.008
    Abstract ( 379 )   PDF (2122KB) ( 1050 )   Save

    BACKGROUND: Whether the differences exist between adipose-derived stem cells isolated from different parts of rats when cultured in vitro has been poorly understood.
    OBJECTIVE: To compare the growth characteristics and adipogenic ability of adipose-derived stem cells isolated from different parts of rats.
    METHODS: Freshly isolated adipose-derived stem cells were obtained from 5 mL inguinal groove and greater omentum adipose tissue of F344 rats using type Ⅰ collagenase digestion method. Then, adipose-derived stem cells were counted and cultured in vitro. Morphological and growth characteristics of adipose-derived stem cells derived from the two sites were observed. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was utilized to examine the doubling time of adipose-derived stem cells from different parts. The passage 2 adipose-derived stem cells were induced adipogenically. Fourteen days after being induced, the differentiated cells were stained with oil red O and the positive cells were counted. The adipogenic differentiation ability of adipose-derived stem cells from the different parts was assessed.
    RESULTS AND CONCLUSION: (1) The number of adipose-derived stem cells from the greater omentum fat tissue in the same group was (281±10)×107/L, which was significantly higher than that from the inguinal groove fat tissue [(85±10)×107/L] (P < 0.01). Adipose-derived stem cells from the greater omentum and inguinal groove fat tissue achieved the exponential growth period on days 5 and 6, respectively, and achieved the platform period on days 9 and 10, respectively. The corresponding doubling time was 50 hours and 60 hours, respectively. After being passaged, adipose-derived stem cells grew in fibroblast-like shape actively. The adipogenic differentiation rate of adipose-derived stem cells from the greater omentum fat tissue was higher than that from the inguinal groove fat tissue [(38.90±2.86)% vs. (35.30±3.29)%, P < 0.01]. This shows that the number and the adipogenic differentiation ability of adipose-derived stem cells from different parts of the same F344 rat are different.

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    Estrogen effects on adipogenic differentiation of cryopreserved adipose-derived mesenchymal stem cells from the adipose capsule of kidney
    Zhang Hui, Zheng Hong-guang, Zhang De-wei, Mei Yu-ming
    2013, 17 (27):  4998-5004.  doi: 10.3969/j.issn.2095-4344.2013.27.009
    Abstract ( 351 )   PDF (2368KB) ( 640 )   Save

    BACKGROUND: Estrogen exerts a negative regulatory role in adipogenic differentiation of adipose stem cells, but there is no report concerning estrogen effects on adipogenic differentiation of adipose stem cells from the adipose capsule of kidney after long-term cryopreservation.
    OBJECTIVE: To explore the impact of estrogen on adipogenic differentiation of fresh adipose-derived mesenchymal stem cells from the kidney adipose capsule or those after long-term cryopreservation.
    METHODS: Passage 3 long-term cryopreserved and fresh adipose-derived mesenchymal stem cells from the kidney adipose capsule were divided into four groups, all of which were induced to adipogenic cells by induced fluid: fresh cells + 10-7 mol/L estrogen, cryopreserved cells + 10-7 mol/L estrogen, fresh cells and cryopreserved cells groups. Oil red O staining and adipogenic quantitative detection were performed at 14 days after induced adipogenic differentiation.
    RESULTS AND CONCLUSION: There were no differences in the morphology and arrangement between the cryopreserved and fresh cells. Both cryopreserved and fresh cells expressed CD29 and CD 44, but did not express CD31. Intracellular lipid droplets were observed after adipogenic differentiation by oil red O staining, and the cells were positive for oil red O staining. The adipogenic volume comparison among the four groups was detected on day 14 after adipogenic differentiation, and the absorbance values showed significant difference between the fresh cells and fresh cells + estrogen groups, as well as between the cryopreserved cells and cryopreserved cells + estrogen groups, but no difference between the fresh and cryopreserved cells groups. It is proved that low-dose estrogen can inhibit the adipogenic differentiation of long-term cryopreserved adipose-derived mesenchymal stem cells from the kidney adipose capsule; however, there is no significant difference between passage 3 long-term cryopreserved and fresh adipose-derived mesenchymal stem cells from the kidney adipose capsule in adipogenic differentiation.

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    Human umbilical cord blood mesenchymal stem cell transplantation improves the
    liver function of liver cirrhosis rats
    Liao Jin-mao, Hu Xiao-xuan, Li Zhuo-ri
    2013, 17 (27):  5005-5011.  doi: 10.3969/j.issn.2095-4344.2013.27.010
    Abstract ( 410 )   PDF (2018KB) ( 607 )   Save

    BACKGROUND: The feasibility and the mechanism of human umbilical cord blood mesenchymal stem cell transplantation for the treatment of liver cirrhosis need to be discussed in-depth.
    OBJECTIVE: To observe the effect of human umbilical cord blood mesenchymal stem cell transplantation through portal vein on the liver function and tissue pathological changes of the rats with liver cirrhosis.
    METHODS: Carbon tetrachloride was used to prepare rat model of liver cirrhosis. After the success of modeling, the rats in the cell transplantation group received portal vein injection of 1 mL 5-bromo-2-deoxyuridine -labeled human umbilical cord blood mesenchymal stem cells (5×106), the model group was injected with the same volume of PBS; the normal rats received 1 mL human umbilical cord blood mesenchymal stem cell transplantation via the portal vein were as the control group. At 4 weeks after transplantation, the rat tail vein blood and liver tissue were obtained for testing.
    RESULTS AND CONCLUSION: At 4 weeks after cell transplantation, compared with the model group, levels of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin in the cell transplantation group were significantly decreased, while the albumin level was increased significantly (P < 0.01); the liver cell inflammatory necrosis, steatosis and liver fibrosis were improved significantly (P < 0.05 or P < 0.01). Immunohistochemistry and immunofluorescence staining showed that human umbilical cord blood mesenchymal stem cell colonization could be seen in the rat liver tissues of the cell transplantation group and control group, but the number of 5-bromo-2-deoxyuridine-positive cells in cell transplantation group was significantly larger than that in the control group. Reverse transcription-PCR test result showed that the expressions of cytokeratin 18 and albumin mRNA could be observed in the rat liver tissue of the cell transplantation group, but no expression could be seen in the control group. It is visible that human umbilical cord mesenchymal stem cells can improve liver 
    function and pathological damage of liver cirrhosis rats in a certain extent, which may relate with the intrahepatic homing colonization and hepatocyte-like cell differentiation of the transplanted cells in the liver cirrhosis rats.

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    Myogenetic potential of rabbit adipose-derived stromal cells
    Liu Jing, Lin Wan-jun, Liu Yin-Zhong, Li Xiao-mian, Guo Hong-gang
    2013, 17 (27):  5012-5018.  doi: 10.3969/j.issn.2095-4344.2013.27.011
    Abstract ( 325 )   PDF (736KB) ( 464 )   Save

    BACKGROUND: Myogenetic seed cells are the fundamental for construction of tissue-engineered smooth muscle, and optimizing the amplification of seed cells is the key in further clinical application.
    OBJECTIVE: To analyze the myogenetic potential of rabbit adipose-derived stromal cells induced with the combination of MyoD, transforming growth factor β1 and 5-azacytidine, and to investigate a new way to induce cells.
    METHODS: The rabbit abdominal fats were isolated and then the adipose-derived stromal cells were separated by collagenase digestion method for myogenetic induction with different methods: 5-azacytidine induction group, MyoD+transforming growth factor β1 induction group, 5-azacytidine+MyoD+transforming growth factor β1 combination induction group. The blank control group was set. The morphological characteristics of cells were observed at 1, 4, 8, 12, 16, 20, 24 and 28 days after induction, and the collagen type Ⅲ level were detected with 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry and immunohistochemistry.
    RESULTS AND CONCLUSION: Compared with the other groups, the cell activity was higher in the combination induction group and reached peak at 16 days after induction, the number and volume of myotubes were increased at 20 days with regular arrangement, and desmin showed strong expression. Cell cycle showed the ratio of adipose-derived stromal cells in the DNA replication phase was increased in the combination induction group, the ratio of cells in the clearance period was decreased; the level of collagen type Ⅲ was increased significantly, and the difference was significant. The results indicate that 5-azacytidine combined with multiple factors can promote the differentiation of adipose-derived stromal cells into myoblasts with significant cell proliferation, which is considered as the ideal method to in vitro induction of myoblasts.

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    Mesenchymal stem cells for the treatment of spinocerebellar ataxia
    Hu Jing-qiong, Ouyang Wei-xiang, Li Hui-yu, Wang Jun-feng, Lu Cong, Zhang Lan-nan, Xu Hai-bo, Chen Li-li, Huang Shi-ang
    2013, 17 (27):  5019-5025.  doi: 10.3969/j.issn.2095-4344.2013.27.012
    Abstract ( 690 )   PDF (890KB) ( 950 )   Save

    BACKGROUND: Spinocerebellar ataxia is a common neurodegenerative disease characterized by slowly progressive movement incoordination of the limbs. It responds badly to common medication.
    OBJECTIVE: To observe the clinical effect of autologous bone marrow mesenchymal stem cells and allogeneic umbilical cord mesenchymal stem cells tranfusion in the treatment of spinocerebellar ataxia.
    METHODS: A total of 27 spinocerebellar ataxia patients treated with mesenchymal stem cells treatment were included for comprehensive statistical analysis. Among these patients, six patients received autologous bone marrow mesenchymal stem cells lumbar puncture treatment and 21 patients received allogeneic umbilical cord mesenchymal stem cells lumbar puncture treatment combined with intravenous infusion. The neurologic function of the patients in the two groups was evaluated with International Cooperative Ataxia Rating Scale before and after treatment.
    RESULTS AND CONCLUSION: There was no obvious adverse effect in the 27 spinocerebellar ataxia patients during, before or after mesenchymal stem cells treatment. The effect of autologous bone marrow mesenchymal stem cells in six patents was not significant; for the other 21 patients treated with allogeneic umbilical cord mesenchymal stem cells transfusion, the subjective symptoms of the patients were improved, and the International Cooperative Ataxia Rating Scale scores were decreased significantly at 3 months after treatment when compared with those before treatment (P < 0.05). The results suggest that umbilical cord mesenchymal stem cells treatment is safe and able to ameliorate the clinical symptoms and improve life quality of spinocerebellar ataxia patients to some extent.

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    Bone marrow mesenchymal stem cells from diabetic versus normal rats for treatment of myocardial infarction
    Yang Zhong-lu, Wang Hui-shan
    2013, 17 (27):  5026-5032.  doi: 10.3969/j.issn.2095-4344.2013.27.013
    Abstract ( 378 )   PDF (2195KB) ( 732 )   Save

    BACKGROUND: Current studies have shown that bone marrow mesenchymal stem cells from normal or young people usually serve as a source of transplanted cells in stem cell transplantation treatment of myocardial infarction.
    OBJECTIVE: To compare the therapeutic effects of bone marrow mesenchymal stem cells from diabetic and normal rats on myocardial infarction.
    METHODS: Under sterile conditions, bone marrow mesenchymal stem cells from normal and diabetic rats were harvested. Then, rat models of myocardial infarction were established and randomly divided into three groups: 100 μL cell suspension containing 105-106 bone marrow mesenchymal stem cells from F2 normal or diabetic rats was injected into myocardial infarction lesions, and 100 μL Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum was injected serving as blank control. After 1 month, hematoxylin-eosin staining for myocardial infarction lesions was performed for histomorphological observation. Bcl-2 expression was detected by immunohistochemistry method.
    RESULTS AND CONCLUSION: Based on cell morphology observation and flow cytometry identification, high-purity bone marrow mesenchymal stem cells could be obtained using rat femoral bone marrow adherent culture. Cell growth curve showed that normal rat bone marrow mesenchymal stem cells grew faster than those from diabetic rats. At 1 month after transplantation, histomorphological improvement was seen in the infarcted area after transplantation of normal rat bone marrow mesenchymal stem cells as compared with the other two groups. In addition, the Bcl-2 expression in the infarcted area was higher in the normal rat cell group than the the other two groups. These findings indicate that bone marrow mesenchymal stem cells from normal rats grow faster than those from diabetic rats, and the cells from normal rats have better therapeutic effects on myocardial infarction.

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    Efficacy and safety of neural stem cell transplantation for the treatment of Parkinson’s disease
    Zhang Xiao-ying
    2013, 17 (27):  5033-5040.  doi: 10.3969/j.issn.2095-4344.2013.27.014
    Abstract ( 1010 )   PDF (793KB) ( 1072 )   Save

    BACKGROUND: At present, the clinical application of amplified neural stem cells in the treatment of nervous system diseases is still in exploratory stage.
    OBJECTIVE: To investigate efficacy and safety of neural stem cell transplantation for the treatment of Parkinson’s disease.
    METHODS: The embryonic brain tissues aged 12 weeks were obtained for proliferation in vitro, then the brain tissues (5×106-2×107) were prepared into the 4-6 mL suspension. The suspension was injected into 30 cases of Parkinson’s disease through lumbar puncture and carotid injection, once a week, continuously for 3 weeks. Unified Parkinson’s disease rating scale, Hoehn-Yahr scale and Schwab & England Activities of Daily Living Scale scoring were performed prior to and after transplantation. Blood and urine routine, liver function, renal function and adverse reactions were detected and evaluated prior to and after transplantation.
    RESUTLS AND CONCLUSION: The 12-week-old embryos cultured neural stem cells could be stably amplified in vitro with the capacities of differentiation into neurons, astrocytes and oligodendrocytes. The follow-up results showed that neural stem cell transplantation could effectively control the pathological process of Parkinson’s disease, restore the damaged brain function, and improve the patient’s neurological function without significant complications. It is feasible and effective to use the in vitro long-term amplified human neural stem cell transplantation in the treatment of Parkinson’s disease.

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    Myeliod sarcoma in cardio-phrenic angle after allogeneic peripheral blood stem cell transplantation
    He Yi, Li Xu-dong, Wang Dong-ning, Hu Yuan, Wang Wen-wen, Liu Jia-jun, Lin Dong-jun, Huang Ren-wei
    2013, 17 (27):  5041-5047.  doi: 10.3969/j.issn.2095-4344.2013.27.015
    Abstract ( 370 )   PDF (1962KB) ( 450 )   Save

    BACKGROUND: Allogeneic hematopoietic stem cell transplantation is the main method to cure leukemia, but the patients receiving allogeneic hematopoietic stem cell transplantation still have to face the risk of recurrence. Myeloid sarcoma is a rare extramedullary relapse mode with worse clinical outcomes, so it is necessary to understand the characteristics of myeloid sarcoma and relative treatment methods.
    OBJECTIVE: To analyze the clinical characteristics and treatment methods of myeliod sarcomas in cardio-phrenic angle after allogeneic peripheral blood stem cell transplantation.
    METHODS: One case was diagnosed as single myeloid sacoma in the cardio-phrenic angle after allogeneic peripheral blood stem cell transplantation. The patient underwent surgical resection of the mass, chemotherapy and radiotherapy. The clinical effect, complications and survival situation were observed.
    RESULTS AND CONCLUSION: The patient suffered from bacteremia, fungal pneumonia and even life-threatening sepsis shock during two courses chemotherapies. Then, the patient received radiotherapy for mediastinum and the myeloid sacoma never relapsed. However, the patient suffered central nervous system leukemia after free of the disease for 25 months. Myeliod sarcoma after allogeneic hematopoietic stem cell transplantation is rare and its manifestation is changeful. The diagnosis mainly depends on histopathology and immunohistochemistry. Surgery, chemotherapy, radiotherapy, second transplantation and molecular targeted
    drugs are the choices of treatment strategy. However, the optimal treatment strategy for individual patients remains to be determined.

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    In vitro culture of human embryonic striatum-derived neural stem cells
    Fan Ming-chao, Wang Qiao-ling, Liu Ke, Zhang Xin, Guan Yun-qian, Sun Peng
    2013, 17 (27):  5048-5056.  doi: 10.3969/j.issn.2095-4344.2013.27.016
    Abstract ( 320 )   PDF (494KB) ( 513 )   Save

    BACKGROUND: Neural stem cells are always derived from animals, and unsuitable for human transplantation treatment.
    OBJECTIVE: To explore the in vitro culture methods of human embryonic striatum-derived neural stem cells, and to observe the biological characteristics.  
    METHODS: The human embryonic striatum were separated from the embryo at a gestational age of 8-16 weeks that received induction of labor with water bag, and then the embryonic striatum was in vitro cultured in the serum-free Dulbecco’s modified Eagle’s medium. The cells were passaged after neurospheres formation, and then the cells were induced to differentiation with the Dulbecco’s modified Eagle’s medium/F12 containing 10% fetal bovine serum.
    RESULTS AND CONCLUSION: The in vitro cultured human embryonic striatum-derived neural stem cells grew rapidly and could express nestin. Colony formation assay showed the cell clone formation rate was 6.0%-7.0%. 5-Bromodeoxyuridine incorporation assay showed the cell proliferation rate was 37.9%. Immunofluorescence staining showed that the cells after induction and differentiation could express Tuj-1, glial fibrillary acidic protein and nestin, but not express myelin basic protein. The results indicate that human embryonic striatum-derived neural stem cells cultured in the serum-free medium can maintain their biological characteristics and have self-renewal capacity, and the cells can differentiate into the neurons and astrocytes induced by the fetal bovine serum.

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    Astragalus injection strengthens biological viability of rat neural stem cells
    Zhang Li1, Luo Xiu-cheng, Yang Shi-zhao, Zhang Jun-feng, Yang Ji-ping, Zhao Zhao-hua, Zhang Hui
    2013, 17 (27):  5057-5062.  doi: 10.3969/j.issn.2095-4344.2013.27.017
    Abstract ( 311 )   PDF (2108KB) ( 587 )   Save

    BACKGROUND: Neuroscience and brain science researches have paid attention to the effect of astragalus membranaceus in the treatment of neurologic impairment disease and neural regeneration. Studying astragalus membranaceus effects on neural stem cells are becoming a new research direction.
    OBJECTIVE: To explore the effects of astragalus injection on biological viability of rat neural stem cells.
    METHODS: Neural stem cells of Wistar rats were separated and cultured. Immunofluorescence staining was applied to identify the neural stem cells. The purified cells were gained by the second subcultivation in vitro, and then the cells were randomly divided into control group and astragalus injection groups with various concentrations (50, 200, 400 g/L) to culture for 6, 12 and 24 hours. The activity of cells was tested by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, and then the immunohistochemistry was applied to detect the expressions of neuron-specific enolase and glial fibrillary acidic protein in the 50 g/L astragalus injection group after induced for 7 days.
    RESULTS AND CONCLUSION: The viability of neural stem cells increased significantly after intervention with different concentrations of astragalus injection for 6 hours as compared with the control group (P < 0.05). However, there was no difference in the cell viability after treated with different concentrations of astragalus injection for 24 hours (P > 0.05). Compared with the control group, the cells in the 50 g/L astragalus group differentiated rapidly, and the number of positive cells for neuron-specific enolase was increased significantly (P < 0.05). The neural stem cells proliferation was hastened, and its differentiation was promoted by the interference of astragalus injection.

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    Bone marrow stem cells co-transfected with transforming growth factor beta 3 and bone morphogenetic protein 2
    Wang Ti-jun, Wang Chang-yao, Xia Chang-suo, Sui Ai-hua, Wang Ying-zhen
    2013, 17 (27):  5063-5069.  doi: 10.3969/j.issn.2095-4344.2013.27.018
    Abstract ( 288 )   PDF (2324KB) ( 523 )   Save

    BACKGROUND: Bone morphogenetic protein 2 and transforming growth factor β are important factors in bone regeneration, increasing the expressions of bone morphogenetic protein 2 and transforming growth factor β can promote the osteogenic differentiation of bone marrow mesenchymal stem cells.
    OBJECTIVE: To construct the lentivirus vector carrying bone morphogenetic protein 2 and transforming growth factor β3, and to observe the expression of lentivirus vector in bone marrow mesenchymal stem cells. 
    METHODS: The recombinant lentiviral vectors carrying transforming growth factor β3, bone morphogenetic protein 2 and green fluorescent protein were constructed with recombinant lentiviral technology, and then the recombinant lentiviral vectors were used to transfect the passage 3 rabbit bone marrow mesenchymal stem cells in vitro cultured (transfection group). The bone marrow mesenchymal stem cells transfected with single gene lentivirals (single gene transfection group) carrying transforming growth factor β3 and bone morphogenetic protein 2 or single lentivirals were as control (control group). At 1 week after trasfection, the total RNA and protein were extracted from each group for detection.
    RESULTS AND CONCLUSION: The green fluorescence bone marrow mesenchymal stem cells transfected with transforming growth factor β3 and bone morphogenetic protein 2 gene for 3 days could be observed under fluorescence microscope, and the transfection efficiency was over 90%. Reverse transcription-PCR and Western blot results showed the mRNA and protein expressions of transforming growth factor β3 and bone morphogenetic protein 2 in the transfection group were higher than those in the single gene transfection group and the control group. The results indicate that lentivirus can successfully transfect transforming growth factor β3 and bone morphogenetic protein 2 into the bone marrow msenchymal stem cells and achieve its high expression, and these two genes have the synergistic effect of promoting expression.

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    Autologous bone marrow mesenchymal stem cells and related chemokines in
    fracture microenvironment
    Zhang Xian-yu, Wang Wei, Chen Wei, Chen Jian-mei, Xu Hao
    2013, 17 (27):  5070-5079.  doi: 10.3969/j.issn.2095-4344.2013.27.019
    Abstract ( 319 )   PDF (3138KB) ( 659 )   Save

    BACKGROUND: The oriented migration of bone marrow mesenchymal stem cells may depend on the interaction between local chemotactic factors and cell surface receptors. However, which chemotactic factors may mediate the oriented migration of bone marrow mesenchymal stem cells towards the fracture site remains unclear.
    OBJECTIVE: To tag autologous bone marrow mesenchymal stem cells, evaluate its role in bone healing, and detect the highly expressed factors associated with migration of bone marrow mesenchymal stem cells in the microenvironment.
    METHODS: The fluorescence/chimeric C57BL/6 mouse models were established, then left shankbone fracture models were also produced. The percentages of green fluorescent protein positive cells to all cells at the fracture site and the percentage of osteoblasts differentiated from bone marrow mesenchymal stem cells to all the osteoblasts were detected at different time points. The role of bone marrow mesenchymal stem cells in the fracture repairing was evaluated. The levels of chemotactic factors protein expression at the fracture site in different time points were detected with immunohistochemistry technology.
    RESULTS AND CONCLUSION: The percentage of green fluorescent protein positive cells to all cells at the fracture site was (3.011±0.911)%, (9.031±0.145)%, (12.064±0.145)% at 1, 5, 14 days postoperatively; and osteoblasts differentiated from bone marrow mesenchymal stem cells accounted for 50% of all the osteoblasts. After fracture, the stromal cell derived factor-1, colony stimulating factor, hepatocyte growth factor, monocyte chemoattractant protein-1, and matrix metalloproteinases-9 were expressed to varying degrees in the microenvironment, while the expression of granulocyte colony-stimulating factor was negative. The expression of stromal cell derived factor-1 in the fracture microenvironment was the highest, mainly due to the migration of bone marrow mesenchymal stem cells. Experimental findings indicate that, autologous bone marrow mesenchymal stem cells participate in and play an important role in bone healing. The stromal cell derived factor-1 plays an important role in promoting bone marrow mesenchymal stem cells migration and promoting bone healing.

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    Gene expression and calcium ion concentration variation in bone marrow mesenchymal stem cells differentiating into neuron-like cells
    Pan Guo-qiang, Qi Feng-ping, Xu Hong, Wang Xiang-li
    2013, 17 (27):  5080-5086.  doi: 10.3969/j.issn.2095-4344.2013.27.020
    Abstract ( 304 )   PDF (950KB) ( 535 )   Save

    BACKGROUND: Monosialotetrahexosylganglioside plays an important role in a variety of physiological processes, such as the nerve cell growth and development, differentiation, regeneration and intracellular and extracellular information transmission. 
    OBJECTIVE: To investigate the effects of monosialotetrahexosylganglioside on the changes of gene expression and calcium ion concentration in the course of inducing the mesenchymal stem cells into neuron-like cells with Woodbury’s method.
    METHODS: The mesenchymal stem cells from Sprague Dawley rats were cultured after isolated and purified, After 5 passages in culture, the cell integrated into a dense monolayer, and treated with 50 mmol/L
    monosialotetrahexosylganglioside for 24 hours as the monosialotetrahexosylganglioside group; then the mesenchymal stem cells were induced into neuron-like cells with the methods of Woodbury after pre-cultured for 24 hours, and set the control group. The protein and mRNA expression levels of growth-associated protein 43, neuron-specific enolase, neurofilament and nestin were detected by immunocytochemistry and real-time PCR, respectively. The fluorescence intensity of intracellular free calcium ion before and after inducing was detected by laser scan confocal microscope.
    RESULTS AND CONCLUSION: After induction, the expression levels of growth-associated protein 43, neuron-specific enolase, neurofilament and nestin of the mesenchymal stem cells in the monosialotetrahexosylganglioside group were higher than those in the control group (P < 0.05), demonstrating that monosialotetrahexosylganglioside could promote the differentiation of mesenchymal stem cells into neuron-like cells. The fluorescence intensity in mesenchymal stem cells was increased gradually in two groups after the medium was replaced by the induction medium, attained its peak value at 100 seconds and then decreased gradually, but the fluorescence intensity was still higher than that before the induction at 20 minutes. The fluorescence intensity of intracellular free Ca2+ was increased significantly in the monosialotetrahexosylganglioside group when compared with the control group (P < 0.05), suggesting that monosialotetrahexosylganglioside could increase the concentration of intracellular free Ca2+, and intracellular free Ca2+ may be useful in the course of induction. The changes of protein expression levels of growth-associated protein 43, neuron-specific enolase, neurofilament and nestin were not significant after induction, indicating that Woodbury classic induced programme could regulate the post-transcriptive protein level.

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    Safety and clinical application of induced pluripotent stem cells
    Zhang Yang, Li Xiu-lan
    2013, 17 (27):  5087-5093.  doi: 10.3969/j.issn.2095-4344.2013.27.021
    Abstract ( 371 )   PDF (637KB) ( 800 )   Save

    BACKGROUND: Induced pluripotent stem cells are obtained from somatic cells by reprogramming method. The safety of induced pluripotent stem cells has attracted much attention because of their huge and potential value in clinical application.
    OBJECTIVE: To review the current studies addressing the safety and clinical application of induced pluripotent stem cells.
    METHODS: The PubMed database between 2006 and 2012 was retrieved by the first author to search the correlative documents concerning the safety and clinical application of induced pluripotent stem cells. Totally 203 papers were primarily gotten. Finally, 47 papers were included.
    RESULTS AND CONCLUSION: At present, the main methods to enhance the safety of induced pluripotent stem cells include avoiding usage of c-Myc gene, another mediate way replacing the retrovirus, direct leading of reprogramming factor protein, safer donor cells, micromolecule compound and other in-transgenosis ways. Induced pluripotent stem cells have extensive clinic treatment prospects, and can be used for the build of disease-specific induced pluripotent stem cells line.

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    Thinking and actuality of induced pluripotent stem cells to treat spinal cord injury
    Liu Wen-jun, Zhang Hui, Yin Zong-sheng
    2013, 17 (27):  5094-5100.  doi: 10.3969/j.issn.2095-4344.2013.27.022
    Abstract ( 324 )   PDF (684KB) ( 599 )   Save

    BACKGROUND: Currently, therapies for spinal cord injury include steroid pulse therapy, surgical decompression and stem cell therapy. A lot of work has been focused on stem cell therapy for spinal cord injury which has become a hot spot.
    OBJECTIVE: To summarize the research status and progress in induced pluripotent stem cells for treatment of spinal cord injury.
    METHODS: A computer-based online search of PubMed (1980-01/2011-11) was performed for articles in English using the keywords of “spine injury, induced pluripotent stem cells, IPS”
    RESULTS AND CONCLUSION: A total of 35 articles were included in result analysis, in which, stem cell therapy for treatment of spinal cord injury is agreed or supported. Induced pluripotent stem cells are isolated directly from the body, which solves ethical and moral issues in the transplantation of stem cells, meanwhile avoids allograft rejection and also provides a large source of cells. Stem cell therapy for spinal cord injury is widely used in animal experiments but few in clinical application. However, stem cell therapy has a good effect in the animal experiments, and shows a higher safety. The existing problems of induced pluripotent stem cells to treat spinal cord injury mainly include immature differentiating method of induced pluripotent stem cells and more complications. Therefore, induced differentiation and security of induced pluripotent stem cells for treatment of spinal cord injury will become a research key.

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