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04 June 2014, Volume 18 Issue 23 Previous Issue    Next Issue

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A Notch signaling pathway inhibitor affects chondrogenesis of human umbilical cord mesenchymal stem cells Zhang Jia-wen, Luo Er-mei, Wu Yan-hui, Fu Shu-ying, Chen Dan-chun, Yu Li, Tang Ming-qiao 2014, 18 (23):  3609-3615.  doi: 10.3969/j.issn.2095-4344.2014.23.001 Abstract ( 295 )   PDF (836KB) ( 654 )   Save BACKGROUND: Notch singling pathway is very important for cell proliferation and differentiation, but its role is still unknown during chondrogenesis of human umbilical cord mesenchymal stem cells. OBJECTIVE: To investigate the effect of N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)- phenylglycinet-butyl ester (DAPT) on inducing human umbilical cord mesenchymal stem cell differentiation into chondrocytes. METHODS: Human umbilical cord mesenchymal stem cells were isolated from human umbilical cord, then were induced to differentiate into chondrocytes. There were four experimental groups: non-induced group, high-glucose Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum and 1% double antibody; induced group, induced medium containing 6.25 mg/L insulin, 6.25 mg/L transferrin, 10 μg/L transforming growth factor beta 1, 0.1 μmol/L dexamethasone, 50 mg/L vitamin C, 5% fetal bovine serum and 1% double antibody; dimethyl sulfoxide group, induced medium containing 0.1% dimethyl sulfoxide; DAPT group, induced medium containing 5 μmol/L DAPT. RESULTS AND CONCLUSION: After chondrogenic induction, the morphology of human umbilical cord mesenchymal stem cells became polygon and positive for toluidine blue and immunofluorescence staining; the expression of Jag-1, PS-1, Notch-1 and Hes-1 decreased significantly (P < 0.01). After the addition of DAPT, compared with the induced group, the relative gene expression of Jag-1, PS-1 and Hes-1 decreased markedly (P < 0.01), the relative gene expression of Notch-1 decreased obviously as well (P < 0.05), and the contents of proteoglycan and collagen type II proteins decreased significantly (P < 0.01). At the same time, the relative gene expression of proteoglycan decreased obviously (P < 0.05), and the relative gene expression of collagen type II decreased in part. Notch signaling pathway exists in human umbilical cord mesenchymal stem cells, once chondrogenesis begins, the signaling strength will decline rapidly. DAPT may prevent human umbilical cord mesenchymal stem cells from differentiating into chondrocytes by Jag-1-Notch-1-Hes-1 pathway. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Cdc42 takes a role in the chemotaxis of umbilical cord mesenchymal stem cells to inflammatory cytokines Liu Su-rui, Li Jun-xia, Yang Xiao-ya, Li Zhu, Gao Yu-hua, Xu Sheng-ru, Wang Geng-yin 2014, 18 (23):  3616-3621.  doi: 10.3969/j.issn.2095-4344.2014.23.002 Abstract ( 322 )   PDF (826KB) ( 426 )   Save BACKGROUND: The homing ability of mesenchymal stem cells is closely associated with the effects of cell transplantation. Clarifying the mechanism of chemotaxis and migration will contribute to enhance the clinical application of mesenchymal stem cells. OBJECTIVE: To investigate the effect of Cdc42 in the homing of human umbilical cord mesenchymal stem cells. METHODS: First, mesenchymal stem cells were isolated from human umbilical cord, and co-cultured with tumor necrosis factor α, interleukin-1β, and transforming growth factor β. Western blot assay was used to test the level of Cdc42. Besides, Cdc42 siRNA was synthesized by chemical method to transfect the cells, and cell migration and adhesion were measured by Transwell and Matrigel separately. Meanwhile, the activity of signal molecule, extracellular regulated protein kinase 1/2, was evaluated by western blot. RESULTS AND CONCLUSION: The results indicated that the inflammation factors induced the highly expression of Cdc42 in human umbilical cord mesenchymal stem cells, almost double level to controls. siRNA notably inhibited the migration and adhesion of human umbilical cord mesenchymal stem cells through Cdc42 down-regulation, and the extracellular regulated protein kinase 1/2 and phosphorylation form were also decreased simultaneously. In a word, we speculate Cdc42 plays a role in the chemotaxis of human umbilical cord mesenchymal stem cells in vitro. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Expression of Cbfα1/p56 subtype in bone marrow mesenchymal stem cells from rat mandible Zhou Jian-hua, Xu Yan-bin, Qiu Jian-zhong, Chen Zheng-gang, Jiang Hong-bing, Wang Li-li 2014, 18 (23):  3622-3626.  doi: 10.3969/j.issn.2095-4344.2014.23.003 Abstract ( 234 )   PDF (1868KB) ( 464 )   Save BACKGROUND: Unlike the ilium derived from the paraxial mesoderm, the mandible from cranial neural crest has a unique mechanism. Core binding factor α1 (Cbfα1) is a key transcription factor for skeletogenic process. However, the role of Cbfα1/p56 subtype in mandible tissue is yet not clear. OBJECTIVE: To research the expression of Cbfα1/p56 subtype in bone marrow mesenchymal cells from rat mandible in vitro. METHODS: Bone marrow mesenchymal stem cells from rat mandible and ilium were in vitro isolated and purified by primary culture. The characteristics of bone marrow mesenchymal cells were compared through the methods of enzyme linked immunosorbent assay and real-time PCR, including growth curve, alkaline phosphatase activity and relative mRNA expression of Cbfα1 subtypes. RESULTS AND CONCLUSION: Bone marrow mesenchymal cells from rat mandible and ilium were successfully obtained. Bone marrow mesenchymal cells from the mandible proliferated more rapidly, alkaline phosphatase activity of which was higher than iliac cells. The relative mRNA expression of Cbfα1/p56 subtype in bone marrow mesenchymal cells from the mandible was more than that in iliac cells at 6 days of culture (P < 0.05), while the expression of Cbfα1/p57 in each time showed no statistical significance (P > 0.05). The results showed that Cbfα1/p56 is very significant in the early osteogenic differentiation of bone marrow mesenchymal cells from the mandible. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Expression of vascular endothelial growth factor in bone marrow mesenchymal stem cells under hypoxic conditions Guo Hui, Zhang Yu-juan, Wei Xiao-guang, Xu Biao, Chen Yong-zhen 2014, 18 (23):  3627-3632.  doi: 10.3969/j.issn.2095-4344.2014.23.004 Abstract ( 362 )   PDF (1972KB) ( 481 )   Save BACKGROUND: Whether transplanted bone marrow mesenchymal stem cells under hypoxic conditions can survive is crucial for the successful cell transplantation. Therefore, studies on the growth of bone marrow mesenchymal stem cells under hypoxic conditions in vitro can provide experimental evidence for in vivo cell transplantation. OBJECTIVE: To observe the expression of vascular endothelial growth factor in bone marrow mesenchymal stem cells under hypoxia. METHODS: Rat bone marrow mesenchymal stem cells were obtained and cultured, and observed under light microscopy. Passage 3 cells were cultured under normoxia (21% O2) and hypoxia (3% O2), respectively, for 72 hours. Then cell counting kit-8 assay and flow cytometry were employed to detect cell proliferation in the two groups. Western blot assay was adopted to detect the expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in the two groups. RESULTS AND CONCLUSION: (1)Rat bone marrow mesenchymal stem cells were obtained and cultured successfully, which were fusiform cells and had uniform shape under the light microscope. (2)The results of cell counting kit-8 assay showed that the number of cells in the hypoxic group was higher than that in the normoxic group at each time point, and cell viability increased significantly at hours 36 and 48 (P < 0.05). (3)The results of flow cytometry demonstrated that the proportion of cells in S phase and cell proliferation index in the hypoxic group were significantly increased, compared with the normoxic group (P < 0.05). (4)Western blot results showed that there was a small amount of the expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in the normoxic group, but the expression of these two proteins in the hypoxic group was increased in a time-dependent manner (P < 0.05). These findings suggest that hypoxia can induce proliferation of rat bone marrow mesenchymal stem cells cultured in vitro, and also raise hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a time-dependent manner. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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LIM mineralization protein expression in LIM mineralization protein-1 gene-transfected bone marrow mesenchymal stem cells Jin Xia-sheng, Wang Zi-jiang, Xiang Chuan 2014, 18 (23):  3633-3638.  doi: 10.3969/j.issn.2095-4344.2014.23.005 Abstract ( 314 )   PDF (2023KB) ( 690 )   Save BACKGROUND: In vitro experiments suggest that LIM mineralization protein-1 (LMP-1) gene can increase the expression of LMP-1 protein in osteoporotic bone marrow mesenchymal stem cells. OBJECTIVE: To investigate the LMP-1 expression in osteoporotic bone marrow mesenchymal stem cells transfected with RV-LMP-1-GFP in vitro. METHODS: Twelve SD female rats were selected and subjected to bilateral ovariectomy for establishment of osteoporosis models. After 2 months of feeding, bilateral femurs, tibiae, and humeri of rats were taken to isolate and culture bone marrow mesenchymal stem cells. Passage 3 cells were taken and randomly divided into ovariectomized group and LMP-1 transfection group. Another six rats only underwent removal of the same amount of fat tissue around the ovary, and passage 3 cells which were harvested as those in the former two groups served as sham group. RT-PCR and western blot assay were performed to determine the expression of LMP-1 protein and mRNA. RESULTS AND CONCLUSION: Under an inverted fluorescence microscope, transfected bone marrow mesenchymal stem cells from osteoporotic rats showed green fluorescent expression. The three groups all could express LMP-1 at the protein and mRNA levels. The expression levels of LMP-1 protein and mRNA in the LMP-1 transfection group were significantly higher than those in the ovariectomized group and sham group (P < 0.05), but there was no statistical difference between the latter two groups (P > 0.05). The successful expression of the RV-LMP-1-GFP gene in osteoporotic SD rat bone marrow mesenchymal stem cells was realized at the protein and mRNA levels; moreover, the expression of LMP-1 was increased dramatically. These findings indicate that LMP-1 gene is successfully transferred into osteoporotic SD rat bone marrow mesenchymal stem cells, and significantly elevates the expression of LMP-1 mRNA and protein. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Separation, culture and identification of rabbit bone marrow mesenchymal stem cells by iliac puncture: operation details and techniques Zhang Cong, Liu Hong-mei, Li Qing-wei, Chen Guo-wu, Liang Xiao, Meng Chun-yang 2014, 18 (23):  3639-3644.  doi: 10.3969/j.issn.2095-4344.2014.23.006 Abstract ( 389 )   PDF (827KB) ( 837 )   Save BACKGROUND: Bone marrow mesenchymal stem cells are considered as commonly used seed cells to construct tissue-engineered for repair of bone and cartilage defects. It is of great significance for cytology and tissue engineering experiments to study the common problems existing in the basic operation and how to avoid these problems in a timely manner. OBJECTIVE: To summarize the common problems existing in the process of operation in order to provide reliable methods about separation, culture and identification of bone marrow mesenchymal stem cells for beginners and researchers. These can reduce or avoid some errors and problems during operation. METHODS: Sixteen New Zealand white rabbits were selected as experiment objects, and bone marrow mesenchymal stem cells were separated from rabbits by iliac puncture, purified and augmented by using density gradient centrifugation combined with adherent culture method. Then cell morphology was observed by inverted phase contrast microscope, growth curve detected by MTT method and cell phenotype identified by flow cytometry. RESULTS AND CONCLUSION: We encountered some problems in the process of separation and culture, when we operated the first five rabbits. After carefully summarizing and analysis of the reasons, the operation was successfully completed on the rest 11 rabbits. Bacteria pollution and cell aging were not found in the process of cell culture. What is more, the cells at passage 3 appeared with high-expression of CD29, and CD44, but low expression of CD14 and CD34. The cell growth curve showed that the proliferation activity of cells at passages 3 and 5 was higher than that at passage 10. Although the technology of separation, culture and identification of bone marrow mesenchymal stem cells is mature, the failure will be happen if we do not pay attention to the details of operation. By strictly carrying out normal operations, we can get high purity of bone marrow mesenchymal stem cells, which lays a good foundation for cell and animal experiments in the future. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Treatment of spinal cord injury by transfection of double gene recombinant adenovirus vector into rat bone marrow mesecnhymal stem cells Xu Yan-mei, Sun Fei, Hui Chun-ying, Wang Wei 2014, 18 (23):  3645-3652.  doi: 10.3969/j.issn.2095-4344.2014.23.007 Abstract ( 246 )   PDF (1276KB) ( 487 )   Save BACKGROUND: Brain-derived neurotrophic factor (BDNF) has widespread effects on dopaminergic neurons, cholinergic neurons and other neurons, which can promote more differentiation of stem cells into neuron-like cells. Hypoxia inducible factor-1α (HIF1α) can improve tissue and cell viability in the ischemic environment and maintain the local microenvironment of hemangioblasts, which has a more far-ranging physiological role than genes. OBJECTIVE: By constructing double gene recombinant adenovirus vector with BDNF and three points mutant HIF1α to complete the transfection of adenovirus vector into rats mesenchymal stem cells and then study the promoting nerve regeneration and angiogenesis effect of these two genes to spinal cord injury in vitro in constant oxygen conditions. METHODS: (1)We finished the site-directed mutagenesis of 402, 564 and 803 amino acids in human HIF1α gene CDS region by PCR method and we finished recombination of mutation posterior HIF1α gene and BDNF into adenovirus pAdEasy-1 system. Packaging viral and titration determination were also finished. (2)Four kinds of virus fluids and blank group were selected for subsequent experiments. We observed transfection efficiency after transfection of virus into bone marrow mesenchymal stem cells and detected the expressions of BDNF and HIF1α mRNA and protein in transfection cells. (3) The protein expression of vascular endothelial growth factor, downstream formation vascular gene of HIF1α, in cells in all groups was detected by western blot assay. RESULTS AND CONCLUSION: (1)The site-directed mutagenesis of 402, 564 and 803 amino acids to alanine in coding sequence region was successful. The construction of four kinds of adenoviral recombinants was successful and identification of packaging was completed. (2)The level of BDNF gene mRNA and protein expression in experimental and positive control 1 groups was significantly higher than that in the other groups (P < 0.05). The level of HIF1α mRNA and protein expression in experimental and positive control 2 groups was significantly higher than that of the other groups (P < 0.05). The level of vascular endothelial growth factor protein was also increased in the experimental and positive control 2 groups, which was significantly different from other groups (P < 0.05). These findings indicate that the BDNF and HIF1α proteins are largely and efficiently expressed in constant oxygen conditions after single vector double gene adenovirus system transfection into mesenchymal stem cells and high-efficiency expression of vascular endothelial growth factor protein is promoted so that this is a new direction for treatment of spinal cord injury by gene therapy combined with cell transplantation. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Bone marrow mesenchymal stem cells protect myocardial function in acute myocardial infarction through a paracrine mechanism Zou Song-ping, Wang Yu, Li Chun-yu, Fu Xue-dan, Liu Yan-li 2014, 18 (23):  3653-3659.  doi: 10.3969/j.issn.2095-4344.2014.23.008 Abstract ( 250 )   PDF (455KB) ( 558 )   Save BACKGROUND: Adult cardiomyocytes show no regenerative ability, and cell therapy for myocardial regeneration and repair may improve myocardial ischemic injury function. OBJECTIVE: To confirm the effect and reveal the mechanism of bone marrow mesenchymal stem cells (BMSCs) on acute myocardial infarction (AMI). METHODS: BMSCs were isolated, cultured from bone marrow of Sprague-Dawley rats using density gradient centrifugation. AMI models were produced in 20 rats by ligating the left anterior descending (LAD) coronary artery, and randomly divided into model group and BMSCs group. In the BMSCs group, cells were subsequently injected with a sterile microinjection via the tail vein. RESULTS AND CONCLUSION: Six months postoperatively, the cardiac function was improved, the vessel density was increased, the percentage of apoptotic cells was decreased in the BMSCs group than that in the model group; the expression levels of inflammatory factors, including vascular endothelial growth factor, von Willebrand factor, transforming growth factor 3β, and interleukin-1β mRNA were significantly improved in the BMSCs group than that in the model group. These results showed that BMSCs can protect the myocardium from AMI by regulating the secretion of inflammatory cytokines and angiogenic factors. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Pasteurization affects the physicochemical properties and biological function of hemoglobin? Li Feng-juan, Feng Jie, Li Shen, Yang Cheng-min 2014, 18 (23):  3660-3663.  doi: 10.3969/j.issn.2095-4344.2014.23.009 Abstract ( 314 )   PDF (633KB) ( 967 )   Save BACKGROUND: Pasteurization is a perfect method for albumin virus inactivation, which may not be required for virus inactivation validation. However, there are no systematical reports concerning virus inactivation of hemoglobin blood substitutes. OBJECTIVE: To explore the effects of pasteurization on the physicochemical properties and biological function of hemoglobin blood substitutes. METHODS: Appropriate cord blood samples were taken followed by centrifugation, washing blood, rupture of membranes, stabilizer treatment. In the control group, the samples were placed in 55 ℃ water bath, and when the temperature of hemoglobin solution reached (55±1) ℃, a heat treatment began and lasted for 2 hours. In the pasteurization group, the samples were placed in 60 ℃ water bath, and when the temperature of hemoglobin solution reached (60±1) ℃, a heat treatment began and lasted for 10 hours. The heating process was under continues nitrogen protection. Then, the hemoglobin solution was placed in ice bath and cooled to below 4 ℃ followed by low-speed centrifugation and filtration via microporous membrane, purification and viral inactivation thereby to obtain cord blood hemoglobin. RESULTS AND CONCLUSION: The products in the pasteurization group were all red clear liquid. There was no significant difference between the two groups in the yield, methemoglobin concentration, and oxygen-carrying capacity. The purification of the two groups was more than 98%. Two kinds of purification methods had no effects on the oxygen-carrying capacity of hemoglobin. Therefore, pasteurization method can replace thermosensitive purification method of 55 ℃, 2 hours. The pasteurization method will not only ensure the physicochemical and biological properties of hemoglobin, but also achieve the purpose of virus inactivation. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Therapeutic effect of erythropoietin gene-modified bone marrow mesenchymal stem cell transplantation on rat cerebral infarction Li Jin-yan, Wang Liang 2014, 18 (23):  3664-3669.  doi: 10.3969/j.issn.2095-4344.2014.23.010 Abstract ( 349 )   PDF (831KB) ( 427 )   Save BACKGROUND: Previous studies have shown that erythropoietin can protect neurons and promote nerve regeneration. OBJECTIVE: To explore the therapeutic effect of erythropoietin gene-modified bone marrow mesenchymal stem cell transplantation via caudal vein on rat cerebral infarction. METHODS: Western blot assay was used to identify the expression of exogenous erythropoietin in bone marrow mesenchymal stem cells. A model of middle cerebral artery occlusion was established in Wistar rats using thread method. And then, model rats were randomly divided into model group (PBS injection via the caudal vein), transplantation group (transplantation of bone marrow mesenchymal stem cell suspension), erythropoietin group (transplantation of erythropoietin-transfected bone marrow mesenchymal stem cell suspension). Neurologic function was assessed at 3 days, 1, 2, 3, 4 weeks after cell transplantation. Four weeks after transplantation, the rats were decapitated after anesthesia to take brain tissues for RT-PCR detection of Bcl-2/Bax gene expression. Cell apoptosis was measured by TUNEL. Hematoxylin-eosin staining and fluorescence microscopy were employed to observe the survival and distribution of PKH26-labeled bone marrow mesenchymal stem cells. RESULTS AND CONCLUSION: Western blot results showed that erythropoietin-transfected bone marrow mesenchymal stem cells could express the erythropoietin in vitro. At 1, 2, 3, 4 weeks after transplantation, the neurological defect scores in the transplantation group and erythropoietin group were significantly lower than those in the model group (P < 0.05, P < 0.01). The expression of bcl-2 gene in the infarct region was significantly higher in the erythropoietin group than the transplantation and model groups (P < 0.05), but the expression of bax was significantly decreased (P < 0.05). In the erythropoietin group, the number of apoptotic cells was reduced, and the number of PKH26 positive cells was increased as compared with the other two groups (P < 0.05). These findings indicate that the transplantation of erythropoietin-modified bone marrow mesenchymal stem cells via caudal vein can significantly improve the neurological function in the rats with cerebral infarction.   中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Umbilical cord mesenchymal stem cell transplantation for treatment of diabetic lower limb vascular disease Li Xiao-ling, Zhu Lv-yun, Song Guang-yao, Jia Li-jing, Yang Shao-ling, Hu Li-ye 2014, 18 (23):  3670-3675.  doi: 10.3969/j.issn.2095-4344.2014.23.011 Abstract ( 595 )   PDF (793KB) ( 670 )   Save BACKGROUND: Compared with bone marrow and autologous peripheral blood stem cells, umbilical cord mesenchymal stem cells are characterized as more primitive, more powerful amplification and lower immunogenicity, no ethical problems, which are more important to the elderly patients with diabetes mellitus. OBJECTIVE: To evaluate the efficacy and safety of umbilical cord mesenchymal stem cells transplantation in the treatment of the elderly patients with diabetic lower limb vascular disease. METHODS: Fifty-six elderly patients with diabetic lower limb vascular disease were randomly divided into observation group and control group. The control group was treated with conventional therapy, while the observation group was treated with umbilical cord mesenchymal stem cells transplantation. RESULTS AND CONCLUSION: Observation group showed a higher efficiency than the control group, with significant difference (P < 0.05). After treatment, foot skin temperature, transcutaneous oxygen pressure, and ankle brachial index were all improved in both two groups, and the ankle brachial index showed a better value in the observation group (P < 0.05). There were no significant adverse reactions in the two groups. Umbilical cord mesenchymal stem cells transplantation is a simple, safe and effective therapy for the elderly patients with diabetic lower limb vascular disease, with better short-term curative effect. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Therapeutic effects of mesenchymal stem cell transfusion on different damaged organs in graft-versus-host disease Lu Ying, Zhang Xiang-zhong, Liu Xiang-fu, Li Fang, Lin Dong-jun 2014, 18 (23):  3676-3681.  doi: 10.3969/j.issn.2095-4344.2014.23.012 Abstract ( 451 )   PDF (650KB) ( 526 )   Save BACKGROUND: Because of their immunological properties, bone marrow mesenchymal stem cells transfusion is developed as a new treatment for refractory graft-versus-host disease. OBJECTIVE: To analyze the safety and curative effect of bone marrow mesenchymal stem cells transfusion on treating different organ damages in graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. METHODS: Eight patients with malignant hematologic disease were included in this study. The patients developed severe steroid-resistant graft-versus-host disease after allogeneic hematopoietic stem cell transplantation and received transfusion of mesenchymal stme cell (1×106/kg) together with the primary therapy of immunosuppressive agent. RESULTS AND CONCLUSION: For the totally eight patients, six got response (two cases of complete remission, and four cases of partial remission) and two showed no remission. Four of five cutaneous damages were ameliorated and one showed no effect. For three cases of oral graft-versus-host disease, two acquired complete remission and one showed partial remission. Two cases of liver graft-versus-host disease and two cases of astro-intestinal graft-versus-host disease obtained complete remission. No response was displayed to three cases of ocular graft-versus-host disease, one case of bronchiolitis obliterans, and one case of urinary graft-versus-host disease. In the median follow-up of 28 months (7-62 months), three patients developed posttransplant lymphoproliferative disorders within 3 months after mesenchymal stem cells transfusion. Administration of mesenchymal stem cells is safe for treatment of severe graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Mesenchymal stem cells transfusion may be a promising therapy for refractory cutaneous , astro-intestinal, liver and oral graft-versus-host disease but not for pulmonary, ocular and urinary graft-versus-host disease. Whether mesenchymal stem cells transfusion is associated with posttransplant lymphoproliferative disorders needs more case data. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Biological characteristics and dopaminergic neural-like cell differentiation potential of human amniotic membrane-derived mesenchymal stem cells Zhou Wen-ran, Li Xin, Wang Wen-bo, Xie Yan-xia, Tang Na, Yan Ying 2014, 18 (23):  3682-3690.  doi: 10.3969/j.issn.2095-4344.2014.23.013 Abstract ( 384 )   PDF (3389KB) ( 917 )   Save BACKGROUND: Human amniotic membrane-derived mesenchymal stem cells (AMSCs) are considered to be one kind of adult stem cells that can be easily obtained in large quantities without using an invasive method. Because of their low immunogenicity, anti-inflammatory properties, multipotency of differentiation and without ethical issue, human amniotic membrane-derived mesenchymal stem cells have been proposed as a good candidate to be used in cell therapy and regenerative medicine. However, the biological properties and the differentiation capacity of human amniotic membrane-derived mesenchymal stem cells are still poorly characterized. OBJECTIVE: To establish a practical method for isolation and purification of human amniotic membrane-derived mesenchymal stem cells, and to study the biological characteristics and dopaminergic neural-like cell differentiation potential of the human amniotic membrane-derived mesenchymal stem cells. METHODS: Human amniotic membrane-derived mesenchymal stem cells were disassociated and isolated from the amniotic membrane by trypsin and collagenase based enzymic digestion, and purified by percoll mediated density gradient centrifugation. Expressions of surface antigens and transcription factors of the human amniotic membrane-derived mesenchymal stem cells were determined by flow cytometry and western blot assays. Based on the osteogenic and adipogenic induction, the multipotent differentiation capability of human amniotic membrane-derived mesenchymal stem cells was determined. Induction of neural cell differentiation of human amniotic membrane-derived mesenchymal stem cells was conducted in Neurabasal conditioning medium with ATRA supplement. Neural cell associated bio-markers were determined by immunofluoresence staining and confocal microscope. RESULTS AND CONCLUSION: In this study, we performed a practical method to isolate and purify human amniotic membrane-derived mesenchymal stem cells and amniotic epithelial cells simultaneously, with high cells yield. We demonstrated a group of constitutive expressions of neural antigens and embryonic associated transcription factor proteins (OCT-4, SOX-2 and KLF4) in fresh isolated human amniotic membrane-derived mesenchymal stem cells as well as in human amniotic membrane-derived mesenchymal stem cells after in vitro passage, which suggested that the human amniotic membrane-derived mesenchymal stem cells not only possessed intrinsic tendency to neural cell differentiation, but also maintained their stem cell characteristics after in vitro passage. We stimulated the human amniotic membrane-derived mesenchymal stem cells in the neurobasal-A and B27 based conditioning medium to induce neural cell differentiation. The induced human amniotic membrane-derived mesenchymal stem cells displayed an up-regulation of expression in panel of neural and dopaminergic associate molecules (β-tubulin III, neuron-specific nuclear protein, tyrosine hydroxylase, glial fibrillary acidic protein, myelin basic protein and nestin) by flow cytometry and immunofluorescence staining, which demonstrated the multipotent differentiation capability and dopaminergic neuron-like differentiation potential of the human amniotic membrane-derived mesenchymal stem cells. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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In vitro culture and differentiationof goat neural stem cells Xia Xu-ke1, 2, Zhang Wei3, Yu Jie-feng1, 4, Zhang Ying1 2014, 18 (23):  3691-3695.  doi: 10.3969/j.issn.2095-4344.2014.23.014 Abstract ( 435 )   PDF (2169KB) ( 391 )   Save BACKGROUND: Different types of nerve regulatory factors and glial cells have been reported to exert different roles in the differentiation and maturation of neural stem cells, but culturing neural stem cells in large animals is relatively rarely reported. OBJECTIVE: To explore the way for culturing goat neural stem cells and to detect the outcome after in vitro differentiation. METHODS: The neural stem cell was separated and cultured from the newborn goat cerebral cortex and the anti-nestin immunocytochemical staining was performed for cell identification. At the same time, anti-S100 active Schwann cells were gotten from the sciatic nerve. Then in vitro differentiation was preformed and the outcome was detected by the immunocytochemical stain of anti-glial fibrillary acidic protein, anti-microtubule-associated protein 2 and anti-S100. Cells without primary antibodies served as controls. Gray values were calculated and compared. RESULTS AND CONCLUSION: The Schwann cells were cultured successfully, which were active to the anti-nestin immunocytochemical staining and anti-S100 staining. After differentiation, the products were active to anti-glial fibrillary acidic protein and anti-microtubule-associated protein 2 immunocytochemical stain, but negative to the anti-S100. And significant difference was found in gray values. The goat neural stem cells and Schwann cells were successfully cultured and identified. After the differentiation, the astrocytes and neurons were detected, but the Schwann cells were not found. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Efficacy and oncological safety of bone marrow mesenchymal stem cell transplantation for colitis in inflammatory bowel disease models in mice He Xiao-wen, Chen Ze-xian, Zhang Long-juan, He Xiao-sheng, Lian Lei, Ke Jia, Lin Xu-tao, Chen Xi, Wu Xiao-jian, Lan Ping 2014, 18 (23):  3696-3701.  doi: 10.3969/j.issn.2095-4344.2014.23.015 Abstract ( 303 )   PDF (2188KB) ( 505 )   Save BACKGROUND: Transfusion of bone marrow mesenchymal stem cells may become a novel and effective biological therapy for inflammatory bowel disease in clinical practice. Nevertheless, the oncological safety of the treatment is worrisome, and is a key to determine whether mesenchymal stem cells can be widely used in treatment of inflammatory bowel disease, and deserves further investigation. OBJECTIVE: To evaluate the therapeutic effect of bone marrow mesenchymal stem cell transfusion against inflammatory bowel disease in mouse models, and to clarify the effects of mesenchymal stem cells on tumorigenesis of inflammatory bowel disease. METHODS: Mouse model of colitis was established using Balb/c (H-2d) mice exposed to dextran sulfate sodium. Syngeneic bone marrow mesenchymal stem cells were transfused into mouse model through caudal vein. The therapeutic effect of mesenchymal stem cells was compared and observed, and pathological remission of colitis was evaluated. Mouse model of colitis-driven colon carcinogenesis was established using Balb/c (H-2d) mice exposed to dextran sulfate sodium and azoxymethane. Tumor formation within the murine colon was compared and observed after transfusion of mesenchymal stem cells. RESULTS AND CONCLUSION: In models of dextran sulfate sodium-induced colitis, weight loss and fecal occult blood were lessened in the bone marrow mesenchymal stem cell group compared with the phosphate buffered saline group. Histological damage score of colitis was less in the bone marrow mesenchymal stem cell group: mucosal structure of distal colon was almost intact under microscope, and there was small area of epithelial defects and cryptal defects. Inflammatory cell infiltration, proliferation of capillary and small vessels could be observed in mucosa and submucosa. Homing and colonization of mesenchymal stem cells in submucosa of inflamed colon could also be observed by in vivo tracing. In the dextran sulfate sodium/azoxymethane model of colitis-driven colon carcinogenesis, the number of intestinal tumors and tumor load were obviously less in the bone marrow mesenchymal stem cell group than in the control group. Results indicated that transfusion of bone marrow mesenchymal stem cells can apparently improve colitis lesions of mice with inflammatory bowel disease and inhibit carcinogenesis of colitis, which may provide theoretical support for the biological safety of mesenchymal stem cells transplantation for inflammatory bowel disease. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Osteogenetic ability of mouse spermatogonial stem cells cultured in vitro Hu Hong-mei, Li Wei, Sun Xin-ming, Jiang Fen, Yang Gan-jun 2014, 18 (23):  3702-3706.  doi: 10.3969/j.issn.2095-4344.2014.23.016 Abstract ( 291 )   PDF (747KB) ( 448 )   Save BACKGROUND: Spermatogonial stem cells are a kind of adult stem cells, which have self-renewal and differentiation potential, and can be differentiated into specific cells in vitro, suggesting that the spermatogonial stem cells may be possibly differentiated into osteoblasts. But the related research has not been reported. OBJECTIVE: To observe the biological characterization and osteogenic process of mouse spermatogonial stem cells cultured in vitro. METHODS: Spermatogonial stem cells were obtained from the testicle of mice aged 15-20 days, and were cultured on the feeder layer from bone marrow stroma cells in vitro. When cultured for 3 days, the cells were cultured in the conditioned medium (experimental group) and basic medium (control group). The cells proliferation capability and osteogenic property were examined by phase-contrast microscope, alkaline phosphatase activity and type I collagen immunofluorescence staining. RESULTS AND CONCLUSION: Spermatogonial stem cells proliferated faster in the experiment group than in the control group. Cells grew rapidly in colony-like shape in the conditioned medium at 3-6 days, the three-dimensional feeling enhanced, cell mass and clusters continued to increase in size, the extracellular matrix was increased in number and the cytoplasmic bridge was not obvious. After culture for 15 days, cells in the two groups were positive for alkaline phosphatase staining that the cytoplasmic membrane was dyed black. Under the fluorescent microscope, green fluorescence was visible in the experimental group, suggesting the cells in the experimental group was positive for type I collagen, but negative in the control group, which is similar with the biological characteristics of osteoblasts. These findings indicate that spermatogonial stem cells possess the osteogenic capability under induction conditions, which are expected to provide seed cells for bone tissue engineering. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Phytohaemagglutinin stimulates the proliferation of peripheral blood mononuclear cells and expression of secretory cytokines Wang Ding, Song Bing, Zhong Xuan, Sun Xiao-fang, Fan Yong 2014, 18 (23):  3707-3714.  doi: 10.3969/j.issn.2095-4344.2014.23.017 Abstract ( 396 )   PDF (1010KB) ( 811 )   Save BACKGROUND: Phytohemagglutinin (PHA) can stimulate the peripheral blood mononuclear cells (PBMCs) into cell cycle, and cause their immune activation, which is a common immune proliferation model. However, the role of non-PBMC ingredient of peripheral blood is unclear, as well as the expression of endothelial cells related cytokines. OBJECTIVE: To study the effect of whole blood culture and PBMCs alone culture with PHA on the PBMC proliferation and apoptosis, expression of inflammatory cytokine and endothelial cell secreted cytokine markers. METHODS: Morphological changes of PBMCs separated from normal karyotype human peripheral blood individually cultured with or without PHA were observed. The PBMCs were collected by whole blood culture or PBMC separated culture. mRNA was extracted for the fluorescence quantitative RT-PCR, which was applied to detect the cell proliferation, apoptosis, and expression of inflammatory cytokine and endothelial cell secreted cytokines. The statistic analysis was used for the significance explication. RESULTS AND CONCLUSION: PBMCs alone cultured ere different from those undergoing whole blood culture. The PHA could up-regulate the gene expression of Ki67, proliferating cell nuclear antigen, Caspase 3, interferon-γ, tumor necrosis factor-β and interleukin-6, but down-regulate Protein C. This indicted that PHA could promote the proliferation and apoptosis of PBMCs and up-regulate the expression of inflammatory cytokines, but down-regulate the expression of endothelial cells secreted coagulation cytokines. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Immortalization and screening of cardiac progenitor cells in mice Zhu Gao-hui, He Tong-chuan, Xiong Feng, Zhu Min1, Chen Yuan 2014, 18 (23):  3715-3722.  doi: 10.3969/j.issn.2095-4344.2014.23.018 Abstract ( 425 )   PDF (2457KB) ( 478 )   Save BACKGROUND: Transplantation of exogenous stem cells for functional cardiac cell death or apoptosis easily induced complications after transplantation. Therefore, cardiac progenitor cells of heart itself have been ideal seed cells. OBJECTIVE: To establish stable cell lines of cardiac progenitor cells from mouse heart, and to provide ideal cell models for studying proliferation and differentiation factors affecting cardiac progenitor cells during adult myocardial cells were damaged. METHODS: (1) Myocardiocytes were isolated from embryonic 15.5 days mice. (2) The cultured myocardiocytes were immortalized using retrovirus SSR69. Immortalized monoclonal myocardial cells were obtained using antibiotic selection and infinite dilution. (3) Monoclonal cell line with the property of cardiac progenitor cells was screened out. RESULTS AND CONCLUSION: (1) Myocardiocytes were successfully isolated and cultured. Partial cells showed obvious beating. (2) Myocardiocytes infected with retrovirus SSR69 were cloned and got 76 clones, then were named as CP15-#, (3) Screening the first 20 clones, the reasonable clones with the characteristics of cardiac progenitor cells were obtained according to myocardial cell marker genes. The results suggested that immortalized cardiac progenitor cells were established mediated by reversible SV40 T antigen. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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In vitro culture and neuron-like differentiation of human dental pulp stem cells Fang Cheng-zhi, Yang Yu-jia, Yao Yue, He Xue-hua 2014, 18 (23):  3723-3726.  doi: 10.3969/j.issn.2095-4344.2014.23.019 Abstract ( 424 )   PDF (1322KB) ( 534 )   Save BACKGROUND: The discovery and concept of pulp tissue-derived stem cells is beneficial to the understanding of tooth development and regeneration and repair mechanisms from the cellular level. OBJECTIVE: To understand the induced differentiation capacity and induced conditions in vitro of human dental pulp stem cells into neuron-like cells. METHODS: Pulp tissue was separated from human healthy third molars. Single cell suspensions were prepared and seeded into 6-well plates containing alpha-modified minimum essential medium supplemented with 15% fetal bovine serum. Subconfluent cultures (first passage) of colony forming cells were induced with butylhydroxy anisole, forskolin, β-mercaptoethanol, basic fibroblast growth factor. RESULTS AND CONCLUSION: Immunofluorescence and reverse transcription-PCR assay showed that human dental pulp stem cells positively expressed stro-1, Col-I, dentin sialoprotein after 2 weeks of induction. Nestin and neuron-specific enolase were strongly expressed, but the gingival fibroblasts were negatively expressed. It indicates that adult stem cells in human dental pulp have a high neuron-like cell differentiation potential under a certain inductive condition. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Rabbit bone marrow mesenchymal stem cells transfected with recombinant adenovirus vectors carrying basic fibroblast growth factor: variation of cell phenotypes Cai Tao-yi, Chen Xiong-sheng, Jia Lian-shun, Sun Yan-qing, Lin Bin, Chen Chang-qing 2014, 18 (23):  3727-3721.  doi: 10.3969/j.issn.2095-4344.2014.23.020 Abstract ( 370 )   PDF (1796KB) ( 538 )   Save BACKGROUND: Exogenous basic fibroblast growth factor (bFGF) plays an important role in the ligament tissue healing process, and the use of transgenic methods to transfect exogenous genes into cells can promote the secretion of bFGF. OBJECTIVE: To observe phenotypic changes and the bFGF protein expression after bFGF recombinant adenovirus was used to transfect rabbit bone marrow mesenchymal stem cells (BMSCs). METHODS: Passage 2 BMSCs were divided into three groups: Ad.bFGF-eGFP group, Ad.eGFP group and control group. Under a phase contrast microscope we observed the changes in cell morphology. The expression of bFGF protein in BMSCs was determined by enzyme-linked immunosorbent assay (ELISA). The proliferative curve was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). RESULTS AND CONCLUSION: The transfected cells showed a uniform phenotype of fibroblasts. MTT colorimetric assay revealed that more proliferative activity of transfected BMSCs was shown in the Ad.bFGF-eGFP group than in the Ad.eGFP group and control group. ELISA results showed that expression of bFGF protein was higher in the Ad.bFGF-eGFP group than in the Ad.eGFP group and control group (P < 0.05). BFGF recombinant adenovirus can induce the differentiation of BMSCs into fibroblasts, increase proliferative ability and promote the expression of bFGF protein. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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The expression of long non-coding RNA AK089560 in mesenchymal stem cells undergoing osteogenic and adipogenic differentiation Zuo Chang-qing, Lu Han-yun, Zhong Yue-chun, Wang Zong-gui, Dai Zhong, Liu Yu-yu, Wu Tie 2014, 18 (23):  3732-3738.  doi: 10.3969/j.issn.2095-4344.2014.23.021 Abstract ( 447 )   PDF (1756KB) ( 658 )   Save BACKGROUND: Recent studies have found that stem cell pluripotency and differentiation is regulated by many long non-coding RNAs (LncRNAs). The expression and effect of LncRNA AK089560 during differentiation of stem cells is unclear. OBJECTIVE: To investigate the expression of LncRNA AK089560 in mesenchymal stem cells C3H10T1/2 undergoing osteogenic and adipogenic differentiation. METHODS: Osteogenic differentiation of mesenchymal stem cells C3H10T1/2 was induced by recombinant human bone morphogenetic protein-2 and evaluated using alkaline phosphatase staining. The adipogenic differentiation of mesenchymal stem cells C3H10T1/2 was induced by three factors (dexamethasone, indomethacin and insulin) and evaluated by oil red O staining. The dynamical expression of LncRNA AK089560 was detected by qRT-PCR assay. The AK089560 secondary structure was predicted using RNAfold software. The relationship between AK089560 and neighboring protein-coding genes was analyzed using UCSC genome browser and visualized by fancyGENE online software. RESULTS AND CONCLUSION: Over 70% of C3H10T1/2 cells were positive for alkaline phosphatase after osteogenic induction and more than 80% of the cells positive for oil red O staining after adipogenic induction.  qRT-PCR results showed that the expression of LncRNA AK089560 at days 2, 4, 6 of both osteogenic and adipogenic differentiation was significantly decreased compared with the control group (P < 0.05). Bioinformatics analysis showed that there was a stem-loop structure for AK089560 and sense overlap relationship between AK089560 and protein-encoding gene Sema3a. These findings indicate that LncRNA AK089560 expression is reduced during osteogenic differentiation and adipogenic differentiation, showing that AK089560 may be involved in regulating the multi-directional differentiation of stem cells. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Culture and biological characteristics of neural stem cells from caveolin-1 knockout embryonic mice Liu Bai-yan, Yu Yue, Yi Jian, Chen Xue-mei, Cai Guang-xian 2014, 18 (23):  3739-3744.  doi: 10.3969/j.issn.2095-4344.2014.23.022 Abstract ( 317 )   PDF (2110KB) ( 399 )   Save BACKGROUND: Caveolin-1 is expressed in mammalian brain and involved in the normal development of the brain, which can affect the proliferation of neural stem cells in the brain. OBJECTIVE: To acquire neural stem cells from caveolin-1 knockout embryonic mice in vitro and study their biological characteristics. METHODS: The whole brain was separated from C57BL/6 mice and caveolin-1 knockout C57BL/6 mice respectively at encyesis 14-16 days. Single cell suspension was obtained by enzyme digestion, and cultured in the conditioned medium of neural stem cells. Following 7 days of primary culture, the cells were induced in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10% fetal bovine serum for 7 days.  RESULTS AND CONCLUSION: The major cells of the cell suspensions from the fetal mouse brain were dead at 1 day after culture, and some single cells floated in the medium and their transmittance were better, and then they gradually formed multicellular balls after 3 days. A small amount of cells were adhered at the bottom of culture plate after passage, and a great amount of cell balls appeared after 7 days. The proliferation rate of neural stem cells from caveolin-1 knockout mice was higher than that from normal mice. The cell balls were nestin-positive and their differentiated cells was positive for neurofilament 200, glial fibrillary acidic protein or O4, respectively. All of the cells from normal mouse brain were positive for caveolin-1, but the cells from caveolin-1 knockout mice were negative for caveolin-1 by immunocytochemistry. Moreover, the speed of cell ball formation and the number of cell balls in neural stem cells from caveolin-1 knockout mice were better than those from normal mice. Caveolin-1 negative neural stem cells were cultured successfully from caveolin-1 knockout mouse brain, and the results show that caveolin-1 can promote the proliferation of neural stem cells and inhibit their differentiation in vitro. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Transforming growth factor beta 3 induced odontoblast-like differentiation of stem cells from human exfoliated deciduous teeth Zhou Hui, Lin Jin-mei, Ren Fei, Liu Jian-ping, Zhang Jin-cai, Xu Ping-ping, Yang Qin,Chen Xiao-chun 2014, 18 (23):  3745-3750.  doi: 10.3969/j.issn.2095-4344.2014.23.023 Abstract ( 253 )   PDF (647KB) ( 598 )   Save BACKGROUND: Studies have reported that the superfamily of transforming growth factors exert a role in the mineralization of various stem cells, but the combination effects of transforming growth factor β3 and heparin on proliferation and mineralization ability of stem cells from human exfoliated deciduous teeth remains to be studied. OBJECTIVE: To explore the effect of transforming growth factor β3 on odontoblast-like differentiation of stem cells from human exfoliated deciduous teeth. METHODS: Human deciduous teeth were collected using enzyme digestion. The 3rd passage dental pulp stem cells were incubated with 25 μg/L recombinant human transforming growth factor β3, 10 U/mL heparin or their combination. The dentin sialophosphoprotein mRNA and dentinsialoprotein expressions were detected by Q-PCR and western blot assay. Alkaline phosphatase activity was determined using alkaline phosphatase kit. RESULTS AND CONCLUSION: Stem cells from human exfoliated deciduous teeth grew well after induction. The activity of alkaline phosphatase in the combination group was significantly higher than that in the transforming growth factor β3, heparin and control groups (P < 0.01). After combination induction, the cells were strongly positive for alizarin red staining. Results from α-PCR and western blot assay showed that the expressions of dentin sialophosphoprotein were both remarkably increased at mRNA and protein levels. In summary, stem cells from human exfoliated deciduous teeth can differentiate into odontoblast-like cells under the induction of transforming growth factor β3 plus heparin. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Umbilical cord-derived mesenchymal stem cell culture: dyeing and tracer technique Huang Xia, Pan Xing-hua, Pang Rong-qing, Ruan Guang-ping, Cai Xue-min 2014, 18 (23):  3751-3755.  doi: 10.3969/j.issn.2095-4344.2014.23.024 Abstract ( 302 )   PDF (614KB) ( 409 )   Save BACKGROUND: The culture of umbilical cord-derived mesenchymal stem cells is extremely important for studies on umbilical cord mesenchymal stem cells. Optimization of cell culture technology is crucial for clinical application of mesenchymal stem cells and even cell therapy. Meanwhile, the labeling and tracer technique of umbilical cord-derived mesenchymal stem cells is a hotspot in stem cell transplantation. OBJECTIVE: To review the research and development of the cell markers and tracer methods of umbilical cord-derived mesenchymal stem cells. METHODS: A computer-based search of VIP, CNKI, Medline, Highwire and Foreign Journals Integration System databases was performed for articles concerning culture and labeling of umbilical cord-derived mesenchymal stem cells published from January 2001 to October 2013. The keywords were “stem cells, mesenchymal stem cells, umbilical cord-derived mesenchymalstem cells, cell culture, labeling methods” in Chinese and English, respectively. Finally, 35 articles were included in result analysis. RESULTS AND CONCLUSION: Umbilical cord-derived mesenchymal stem cells have not yet been widely used, mainly because of the immature isolation, culture and staining techniques of umbilical cord-derived mesenchymal stem cells. These techniques are worthy of further optimization studies. Although in recent years, cell markers and tracer technology of umbilical cord-derived mesenchymal stem cells have made great progress, there are still many problems need to be solved. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Inflamed dental pulp stem cells: initial research and future development Zhao Hua-xiang, Zhao Shan-mei, Xin Xin, Zhang Bo, Ma Ning-hu, Li Mu-jia, Zhang Meng-qi, Li Ang 2014, 18 (23):  3756-3761.  doi: 10.3969/j.issn.2095-4344.2014.23.025 Abstract ( 412 )   PDF (647KB) ( 877 )   Save BACKGROUND: Inflamed dental pulp stem cells are a new kind of dental pulp stem cells, and there is no systematic review on the cells by now. OBJECTIVE: To systematically review the research progress in inflamed dental pulp stem cells. METHODS: A computer-based online search in PubMed, Web of Science, CNKI, WanFang and VIP databases was performed for related articles published from the establishment of the databases to February 2014. The keywords were “(pulptis or inflam* dental pulp* or human dental pulp with irreversible pulpitis) and stem cell*” in English and Chinese, respectively. Hand searching was also done to obtain further information or papers about the studies. The results were qualitatively analyzed to comprehensively summarize the progress in the research of inflamed dental pulp stem cells. RESULTS AND CONCLUSION: Totally 11 papers were involved in result analysis that comprehensively review the research progress in inflamed dental pulp stem cells at the following aspects: the research of history, material origin, cell culture, cell-surface markers, proliferation ability, multi-directional differentiation potential, animal models and clinical use. Researches of inflamed dental pulp stem cells are still in the initial stage, and cultivating conditions and the establishment of animal models are still in the exploratory phase. Controversies still exist in the capacity of proliferation and multi-directional differentiation of the inflamed dental pulp stem cells. And fewer studies have been done in the characteristics of immunity, subpopulation and clinical use of the inflamed dental pulp stem cells. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Thyroid cancer stem cells and thyroid cancer treatment: theory and applications Sai Heng, Bi Li-fu, Wu Yan 2014, 18 (23):  3762-3767.  doi: 10.3969/j.issn.2095-4344.2014.23.026 Abstract ( 347 )   PDF (580KB) ( 378 )   Save BACKGROUND: Thyroid cancer stem cells are one of the reasons for tumor resistance that promotes tumor development. The research of thyroid cancer stem cells has provides a new clinic means for the diagnosis and treatment of thyroid cancer. OBJECTIVE: To overview the discovery, identification, and correlation of thyroid cancer stem cells with thyroid cancer. METHODS: A computer-based online search of PubMed database and Wan Fang database between 1995-01/2014-01 was performed to search related articles with the key words of “thyroid cancer, cancer stem cell, stem cell, cancer suppressor gene” in English and Chinese, respectively. Literatures related to thyroid cancer stem cells were selected; in the same field, the articles published lately in authoritative journals were preferred. RESULTS AND CONCLUSION: A total of 561 literatures were primarily selected, and 57 documents were involved for result analysis according to inclusion criteria. Cancer stem cells have become a focus in the study of carcinogenesis. Stem cells also exist in the thyroid gland and its tumor. At present, there are several ways to isolate and identify thyroid cancer stem cells. The tumor thyroid stem cells are closely related to the occurrence, transfer and treatment of tumors. But whether we can cure thyroid cancer through restraining or eliminating thyroid cancer stem cells is still unknown that needs further studies. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics

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Haploidentical hematopoietic stem cell transplantation in children with severe aplastic anemia Lu Jing-yuan, Lu Quan-yi, Lin Jin-zong, Hu Jia-sheng, Hong Xiu-li, Chen Ya-mei 2014, 18 (23):  3768-3772.  doi: 10.3969/j.issn.2095-4344.014.23.027 Abstract ( 374 )   PDF (567KB) ( 718 )   Save BACKGROUND: The main therapy of severe aplastic anemia in children is immunosuppressive therapy or stem cell transplantation, but the latter one is restricted due to few donor sources. Haploidentical hematopoietic stem cell transplantation is commonly used in leukemia, but it is still rarely reported in the treatment of aplastic anemia. OBJECTIVE: To investigate the effect of haploidentical hematopoietic stem cell transplantation combined with placenta-derived mesenchymal stem cell transplantation for children with severe aplastic anemia. METHODS: A 7-year-old girl who had been confirmed as having severe aplastic anemia for 1.5 years received a cotransplantation of haploidentical hematopoietic stem cells combined with placenta-derived mesenchymal stem cells on July 9th, 2012. The donor was her mother. The preconditioning regimen consisted of fludarabine, cyclophosphamide, and anti-thymocyte globulin. RESULTS AND CONCLUSION: Time of neutrophil recovery (> 0.5×109/L) was +9 days, and hematopoietic reconstruction was complete at +12 days. The short tandem repeat analysis showed 100% donor’s genotype at +100 days. Immunosuppressive drugs were stopped at +8 months, and no acute or chronic graft-versus-host disease occurred. With a follow-up of 18 months, she was in the disease-free survival period. Our findings suggest that the cotransplantation of allogeneic haploidentical hematopoietic stem cells and placenta-derived mesenchymal stem cells is a new effective approach for children with severe aplastic anemia, which is worth exploring in the future. 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： Figures and Tables | References | Related Articles | Metrics