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28 March 2017, Volume 21 Issue 9 Previous Issue    Next Issue

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Effect of berberine on the proliferation and apoptosis of lung cancer stem cells and the possible mechanism Sun Yan-zhen, Li Zhen, Yuan Zheng 2017, 21 (9):  1313-1318.  doi: 10.3969/j.issn.2095-4344.2017.09.001 Abstract ( 407 )   PDF (807KB) ( 738 )   Save BACKGROUND: Previous studies have demonstrated that berberine represses multiple tumors and tumor stem cells, but the effect of berberine on lung cancer stem cells (LCSCs) remains unclear. OBJECTIVE: To explore the effect of berberine on the proliferation and apoptosis of LCSCs and the possible mechanism. METHODS: CD133+ LCSCs were separated from A549 cells by immunomagnetic beads. The effects of different  concentrations (0, 2.5, 5, 10, 20, 40 mg/L) of berberine on the proliferation and apoptosis of LCSCs were determined by MTT and flow cytometry analysis, respectively. In order to further affirm the effect of berberine on the proliferation and apoptosis of LCSCs, the expression levels of Ki67, Bax and Bcl-2 protein were detected by western blot. In addition, to investigate the potential mechanism by which berberine exerts regulatory effects on LCSCs, the expression levels of Hedgehog signaling pathway-associated proteins (PTCH1, SHH, Gli-1 and SMO) were determined. RESULTS AND CONCLUSION: After magnetic cell sorting, the content of the CD133+ fraction was enriched up to 84.13%. MTT and flow cytometry assays showed that berberine inhibited proliferation and promoted apoptosis of LCSCs in a concentration-dependent manner. Western blot analysis showed that the expression levels of Ki67, Bcl-2, PTCH1, SHH, Gli-1 and SMO proteins of LCSCs cultured in the medium with 20 mg/L berberine were dramatically decreased compared to the control, while the expression level of Bax protein was markedly increased compared to the control. These findings suggest that berberine may inhibit proliferation and promote apoptosis for LCSCs through the Hedgehog signaling 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 Figures and Tables | References | Related Articles | Metrics

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Expression of stem cell surface marker CD133 in hepatocellular carcinoma and its positive subset proliferation Zhang Bao-qi 2017, 21 (9):  1319-1323.  doi: 10.3969/j.issn.2095-4344.2017.09.002 Abstract ( 435 )   PDF (936KB) ( 407 )   Save   BACKGROUND: CD133 has been used as a marker to separate and study different tumor stem cells by using its protein specific features. OBJECTIVE: To investigate the expression of stem cell surface marker CD133 in hepatocellular carcinoma and the in vitro proliferation of its positive subsets. METHODS: The human hepatoma MHCC97-H cell lines were cultured and sorted, CD133+ and CD133- MHCC97-H cells were obtained, and the expression of CD133 and cell migration and invasion were detected. After 7 days of culture, cell proliferation ability and Notch expression were detected. After 14 days of culture, cell cloning efficiency was detected. CD133+ and CD133- MHCC97-H cells were implanted into nude mice subcutaneously, and 4 weeks later, tumor formation in mice was detected. RESULTS AND CONCLUSION:(1) CD133 expression and cell clone formation: The expression of CD133 and cell cloning efficiency of CD133+ MHCC97-H cells were significantly higher than those of CD133- MHCC97-H cells (P < 0.05). (2) Cell proliferation: After 3-7 days of culture, the absorbance value of CD133+ MHCC97-H cells was significantly higher than that of CD133- MHCC97-H cells (P < 0.05). (3) Cell migration and invasion: The number of CD133+ MHCC97-H cells passing through the cell membrane was significantly more than that of CD133- MHCC97-H cells (P < 0.05). (4) The Notch protein expression of CD133+ MHCC97-H cells was significantly higher than that of CD133- MHCC97-H cells (P < 0.05). (6) Tumor formation test: The tumor size of CD133 - MHCC97-H cells was larger than that of CD133- MHCC97-H cells (P < 0.05). To conclude, CD133+ hepatocellular carcinoma cell subsets with strong proliferation ability have some characteristics of cancer stem cells, and strong invasion, metastasis and tumorigenic abilities.   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 Figures and Tables | References | Related Articles | Metrics

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A preliminary establishment of the “single-cell-transplant-umorigenic-test”model for identifying liver cancer stem cells Jia Wei, Cen Yan-hui, Bao Juan, Yang Rui, He Guo-zhen, Wu Xiao-jun, Li Yi-yi 2017, 21 (9):  1324-1328.  doi: 10.3969/j.issn.2095-4344.2017.09.003 Abstract ( 407 )   PDF (3224KB) ( 433 )   Save BACKGROUND: There is no effective method to identify liver cancer stem cells until now, which has become one of the major challenges in this field. OBJECTIVE: To explore a more effective and suitable way for the identification of liver cancer stem cells in hepatocellular carcinoma cell lines. METHODS: Firstly, the single cell separation technique was used to obtain single cells, which were seeded into 96-well plates one by one. Secondly, cell sublines derived from single cell colonies (tumorigenic colonies) were selected and obtained. At last, the cells from these clones were transplanted into the forelimb armpits of nude mice to observe the tumorigenic ability. RESULTS AND CONCLUSION: The number of holes of single cells obtained was 371 by the single cell separation technique, and the success rate was 96.4%. According to the growth of cell clone, tumorigenic colonies were selected to be transplanted to the forelimb armpits of nude mice, and the tumor formation rate of the colonies was 100%. The identification model of “single-cell-transplant-tumorigenic-test” for liver cancer stem cells is confirmed to be preliminarily established, which lays the technology and methodological basis for the follow-up research.   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 Figures and Tables | References | Related Articles | Metrics

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Distribution and morphological characteristics of CD44+/SOX2+ cancer stem cells in colorectal carcinoma Yuan Hang, Liu Bin, Tie Ya-teng, Sun Ya-min, Xin Qing, Su Qin-jun, Li Shuang-ming 2017, 21 (9):  1329-1333.  doi: 10.3969/j.issn.2095-4344.2017.09.004 Abstract ( 437 )   PDF (3701KB) ( 354 )   Save   BACKGROUND: CD44 is commonly used as a marker of colorectal cancer stem cells. In recent years, it is found that transcription factor SOX2 is a potential marker of colorectal cancer stem cells. OBJECTIVE: To observe the number, distribution and morphological characteristics of CD44+/SOX2+ cells in colorectal cancer, and to explore whether CD44+/SOX2+ is a marker of colorectal cancer stem cells. METHODS: Immunehistochemical double staining method was used to observe the number and distribution of CD44+/SOX2+ cells in 10 cases of normal intestinal mucosa, 20 cases of colorectal adenoma and 88 cases of cancer tissue specimens. Hematoxylin-eosin staining was used to observe the morphological characteristics of CD44+/SOX2+ cells in colorectal cancer tissues. RESULTS AND CONCLUSION: Immunehistochemical double staining showed that: there were no CD44+/SOX2+ cells in normal intestinal mucosa, a very small amount of CD44+/SOX2+ cells in colorectal adenoma, and a small amount of CD44+/SOX2+ cells in colorectal carcinoma. The CD44+/SOX2+ cells in highly and moderately differentiated adenocarcinoma were scattered or distributed focally in the basal part of crypt or along the co-walled gland. In poorly differentiated adenocarcinoma, they were scattered or distributed focally in other cancer cells. Hematoxylin-eosin staining showed that the CD44+/SOX2+ cells were small, round or oval structure , with less cytoplasm, large nuclear, obvious nucleolus, and the mitotic phenomenon was rarely seen. There was no correlation between the expression of CD44+/SOX2+ and age, gender, location, depth of invasion in colorectal cancer patients; there was a negative correlation with differentiation and a positive correlation with TNM staging, distant metastasis. To conclude, CD44+/SOX2+ cells are in line with the characteristics of tumor stem cells in terms of distribution and morphology character, and CD44+/SOX2+ may be a marker of colorectal cancer 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 Figures and Tables | References | Related Articles | Metrics

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Ectopic osteogenesis of bone marrow stromal stem cells under bone morphogenetic protein 2/vascular endothelial growth factor 165 co-transfections Zhou Hang-yu, Xia De-lin, Gan Sheng-yuan, Shao Xue-lei 2017, 21 (9):  1334-1339.  doi: 10.3969/j.issn.2095-4344.2017.09.005 Abstract ( 423 )   PDF (4295KB) ( 449 )   Save   BACKGROUND: Double gene transfection using bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor 165 (VEGF165) for bone marrow stromal stem cells (BMSCs) to induce osteogenesis provides experimental basis for the study on tissue engineering bone. OBJECTIVE: To investigate the ability of BMP2 and VEGF165 double gene modified rat BMSCs to induce osteogenesis. METHODS: BMSCs were isolated from the femur and tibia of four 4-week-old Sprague-Dawley rats by whole bone marrow adherent culture. Passage 3 BMSCs were randomized into five groups: non-transfection group, empty plasmid group, BMP2 transfection group, VEGF165 transfection group, BMP2 and BMP2/VEGF165 transfection group (co-transfection group). Then, western blot assay was used to detect expression of BMP2 and VEGF165 at 48 hours after transfection, and the activity of alkaline phosphatase was detected in each group at 7 days after transfection. RESULTS AND CONCLUSION: Highly expressed BMP2 in BMP2 and co-transfection groups and highly expressed VEGF165 in VEGF165 and co-transfection groups were found after transfection. The expression of BMP2 or VEGF165 in the co-transfection group was significantly higher than that in the BMP2 or VEGF165 transfection group after transfection, respectively (P < 0.05). Western blot results showed that the activity of alkaline phosphatase was ranked as: co-transfection group > BMP2 transfection group > VEGF165 transfection group > empty plasmid group and non-transfection group. There was a significant difference in the activity of alkaline phosphatase between co-transfection group and any of single gene transfection groups (P < 0.05). To conclude, BMP2/VEGF165 co-transfection promotes the ectopic osteogenesis of BMSCs.   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 Figures and Tables | References | Related Articles | Metrics

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Comparison of lentiviral vector and adenoviral vector mediated gene transfer into rabbit bone marrow mesenchymal stem cells Li Shi-peng, Li Qiang, Shi Zheng-song, Cai Wei-liang, Ding Yin-kuan, Tao Xuan 2017, 21 (9):  1340-1345.  doi: 10.3969/j.issn.2095-4344.2017.09.006 Abstract ( 477 )   PDF (4392KB) ( 407 )   Save BACKGROUND: Both lentiviral vector and adenoviral vector are considered as good vectors for gene mediation, and their differences in transferring bone morphogenetic protein 2 (BMP-2) into rabbit bone marrow mesenchymal stem cells (BMSCs) are unclear. OBJECTIVE: To compare the duration, efficiency and the deviation of exogenous gene expression after rabbit BMSCs transfection using lentiviral vector and adenoviral vector which are used to mediate enhanced green fluorescent proteins (EGFP) and BMP-2. METHODS: Rabbit BMSCs at passage 5 were exposed to Ad-EGFP-BMP-2 (group A) or Lenti-EGFP-BMP-2 (group B) with multiplicity of infection of 100, as transfection groups. And in control group (group C), the same quality of culture medium was required equivalent to the groups A and B. The expression of EGFP was observed by inverted fluorescence microscope at various time intervals. And the expression of exogenous gene BMP 2 in cells was detected and analyzed by immunohistochemical staining at 72 hours after transfection as well as by western blot at 72 hours, 1, 3 weeks after transfection. RESULTS AND CONCLUSION: The intense green fluorescence emerged under the microscope at 24-48 hours after transfection in group A, which was stronger than group B, reached the peak at 72 hours, and then decreased at 1 week until disappearance at 3 weeks. No EGFP expression was detected in group C. High expression of BMP-2 was found in group A but was dramatically downregulated after 1 week. Group B showed the high expression of EGFP/BMP-2 persisted for a longer period after transfected that even lasted for 3 weeks. Overall, the lentiviral vector and adenoviral vector can efficiently transfect rabbit BMSCs and stably express the target gene of EGFP/BMP-2. Under the same MOI, compared to the adenoviral vector, transfection of lentiviral vector to rabbit BMSCs is more effectively and expression of EGFP/BMP-2 can be persistent in a longer term.   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 Figures and Tables | References | Related Articles | Metrics

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Effect of interleukin-12 gene modified bone marrow mesenchymal stem cells on the growth of ovarian cancer cells Zhang Xia, Du Zhi-xiang, Wang Na, Meng Ya-li 2017, 21 (9):  1346-1351.  doi: 10.3969/j.issn.2095-4344.2017.09.007 Abstract ( 475 )   PDF (1873KB) ( 383 )   Save BACKGROUND: Bone marrow mesenchymal stem cells are characterized by wide sources and low immunogenicity. Especially, these cells are easy to import and express foreign genes, and thus have obvious superiorities as anti-tumor gene therapy vectors. OBJECTIVE: To investigate the effects of interleukin-12 gene modified bone marrow mesenchymal stem cells on the proliferation, cell cycle and apoptosis of ovarian cancer cells. METHODS: Interleukin-12 recombinant adenovirus vector was used to transfect bone marrow mesenchymal stem cells. Then, the expression of interleukin-12 mRNA and protien in transfected bone marrow mesenchymal stem cells was detected by RT-PCR and western blot assay, respectively. The level of interleukin-12 in cell supernatant was determined by ELISA. The SKOV3 cells were co-cultured with the supernatant of bone marrow mesenchymal stem cells transfected with (transfection group) or without (control group) interleukin-12 recombinant adenovirus vector. The proliferation of SKOV3 cells was determined by MTT assay. Flow cytometry was used to detect the cell cycle and apoptosis of SKOV3 cells. RESULTS AND CONCLUSION: RT-PCR and western blot results showed that interleukin-12 mRNA and protein were expressed in transfected bone marrow mesenchymal stem cells, but not found in empty vector group and blank control group. ELISA results showed that the content of interleukin-12 in the supernatant of bone marrow mesenchymal stem cells was (68.78±12.35) μg/L in the interleukin-12 transfection group after 48 hours culture, and no interleukin-12 expression was detected in the empty vector group and the blank control group. Interleukin-12-transfected bone marrow mesenchymal stem cells significantly inhibited the proliferation of SKOV3 cells, and the proliferation inhibition rate was increased with time (P < 0.05). The proportion of G1-phased SKOV3 cells was higher in the transfection group than in the control group (P < 0.05), and the percentage of G2-phased SKOV3 cells was lower in the transfection group than in the control group (P < 0.05). The apoptosis rate of SKOV3 cells in the transfection group was higher than that in the control group (P < 0.05). Our experimental findings indicate that bone marrow mesenchymal stem cells transfected with interleukin-12 recombinant adenovirus vector can express interleukin-12, inhibit the proliferation and induce apoptosis of ovarian cancer 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 Figures and Tables | References | Related Articles | Metrics

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Vascular endothelial growth factor transfection induces human bone marrow mesenchymal stem cells differentiating into endothelial-like cells under hypoxia Hu Ji-hong, Jia Jia, Lu Juan, Wang Qiu-ping, Zhao Jing-miao, Jin Li-mei, Li Jin-juan 2017, 21 (9):  1352-1356.  doi: 10.3969/j.issn.2095-4344.2017.09.008 Abstract ( 395 )   PDF (4901KB) ( 441 )   Save BACKGROUND: It has been found that vascular endothelial growth factor can induce the differentiation of bone marrow mesenchymal stem cells into endothelial cells, but can the vascular endothelial growth factor gene promote the differentiation of bone marrow mesenchymal stem cells into vascular endothelial cells in the damaged organ under the hypoxic environment? OBJECTIVE: To observe whether human bone marrow mesenchymal stem cells induced by vascular endothelial growth factor could differentiate into vascular endothelial cells under hypoxia. METHODS: The third passage of human bone marrow mesenchymal stem cells were cultured in vitro. Cells in the control group were cultured with conventional culture medium, while those in experimental group were cultured with adenovirus vector containing vascular endothelial growth factor in 5% O2. After 2 weeks of culture, morphological observation and surface-related molecular detection were performed. The levels of vascular endothelial growth factor and endothelial nitric oxide synthase were detected by ELISA. The expression of endothelin and prostacyclin was detected by RT-PCR and western blot assay. RESULTS AND CONCLUSION: (1) The number of cells in the control group was more than that in the experimental group. The cells in the control group were crowded and arranged irregularly, showing a fiber-like growth, while those in the experimental group were mostly triangular or polygonal, exhibiting a colony-like growth. (2) CD31 was negative in the control group, while CD105 was positive and the positive rate was 99.7%, indicating that the cells still showed the phenotype of bone marrow mesenchymal stem cells. The positive rate of CD31 was significantly increased to 30.33% in the experimental group and the positive rate of CD105 expression was decreased to 58.11%, indicating a typical phenotype of endothelial cells. (3) Compared with the control group, the expression of endothelin, vascular endothelial growth factor and endothelial nitric oxide synthase increased significantly in the experimental group (P < 0.05), and the expression of prostacyclin decreased significantly (P < 0.05). All these findings indicate that vascular endothelial growth factor can promote the differentiation of human bone marrow mesenchymal stem cells into vascular endothelial cells under hypoxia.   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 Figures and Tables | References | Related Articles | Metrics

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Isolation, identification and biological characteristics of rat bone marrow mesenchymal stem cells Cao Hua, Gao Jian-hua, Liu Xiao-long, Lin Si-si 2017, 21 (9):  1357-1361.  doi: 10.3969/j.issn.2095-4344.2017.09.009 Abstract ( 467 )   PDF (3190KB) ( 370 )   Save BACKGROUND: How to effectively harvest bone marrow mesenchymal stem cells with homogeneity is crucial for cartilage tissue engineering research. OBJECTIVE: To isolate and culture rat bone marrow mesenchymal stem cells and to observe biological characteristics of the cells. METHODS: Bone marrow mesenchymal stem cells were isolated from the femur and tibia of 30 Sprague-Dawley rats using the whole bone marrow adhesion method. Cell surface markers were identified using flow cytometry, cell proliferation ability was observed through cell counting, and cell survival rate was determined by MTT assay. RESULTS AND CONCLUSION: The harvested cells were mostly round and oval, which partially showed a triangular shape. Cell fusion appeared after 12-15 days of inoculation. The proliferation of passaged cells was accelerated. Two hours after passage, the cells evenly covered the bottom of the culture dish. With the increasing of cell generations, the cell proliferation ability was gradually decreased. The survival rates of cells at passages 1-5 were  over 95%, significantly higher than those at passages 6 and 7 (P < 0.05). The harvested cells were negative for CD34, but positive for CD44. To conclude, the whole bone marrow adhesion method is effective to isolate rat bone marrow mesenchymal stem cells with stable growth and strong proliferation ability. Passage 5 cells can be selected as seed cells for cartilage 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 Figures and Tables | References | Related Articles | Metrics

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Effect of the imprinting loss of insulin like growth factor 2 gene during the differentiation from mouse embryonic stem cells to islet-like cells in vitro Liu Feng, Peng Yu-huan, Tang Jia-zhen, Jang Shan 2017, 21 (9):  1362-1367.  doi: 10.3969/j.issn.2095-4344.2017.09.010 Abstract ( 593 )   PDF (4601KB) ( 384 )   Save BACKGROUND: Insulin like growth factor 2 (IGF2) is an important embryonic mitosis growth promoting factor, which plays a critical role in the process of maintaining normal cell growth. The gene expression of IGF2 is under epigenetic regulation in the way of genomic imprinting. Imprinting loss of IGF2 is likely to be associated with the abnormal development of the individual and tumorigenesis. OBJECTIVE: To investigate the effect of imprinting loss of IGF2 gene on the differentiation of mouse embryonic stem cells into islet-like cells. METHODS: Two kinds of mouse embryonic stem cells (SF1-G and SF1-1) were induced to differentiate into islet-like cells in vitro. The expression of Insulin gene was detected by real-time PCR and cell immunofluorescence. The expression of IGF2 was detected by the polymerase chain reaction-restriction fragment length polymorphism in the cells before and after induced differentiation. The level of insulin released at terminal differentiation stage was tested by insulin release assay in vitro. RESULTS AND CONCLUSION: (1) There was no change in the imprinting state of the two kinds of mouse embryonic stem cells with normal and imprinted IGF-2 gene before and after differentiation. (2) In the induced cells, the expression level of insulin in the SF1-1 group was higher than that in the SF1-G group, although there was no significant difference in the insulin release between the two kinds of cells. (3)The imprinting loss of IGF-2 gene may be related to the up-regulation of insulin mRNA expression in terminal cells during induced differentiation.    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 Figures and Tables | References | Related Articles | Metrics

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Adenovirus transfection of MafA induces human umbilical cord mesenchymal stem cells to express pancreatic cell-specific genes Wang Hong-wu, Ni Ping, Lai Xiu-lan, Feng Xue-yong, Lin Li-min, Ma Lian 2017, 21 (9):  1368-1372.  doi: 10.3969/j.issn.2095-4344.2017.09.011 Abstract ( 405 )   PDF (3613KB) ( 328 )   Save BACKGROUND: Human umbilical cord mesenchymal stem cells (HUMSCs) can differentiate into insulin-producing cells after induced by chemical drugs or co-culture methods, but insulin secretion is extremely low. Therefore, to induce mature pancreatic beta cell differentiation from stem cells by adenovirus transfection of specific genes involved in the development of pancreas is a research hotspot in recent years. OBJECTIVE: To study the differentiation potential of HUMSCs into insulin-producing cells after transfection with musculoaponeurotic fibrosarcoma oncogene homologue A (MafA). METHODS: Ad-MafA-EGFP was transferred into passage 3 HUMSCs. After 7 days of induction, changes of cell morphology were observed by inverted phase contrast microscope. Expression of pancreatic cell-specific genes (glucagon, PDX1, Nkx2.2) was detected by PCR technique. RESULTS AND CONCLUSION: After Ad-MafA-EGFP transfection, no significant morphological changes were observed in the HUMSCs under inverted phase contrast microscope. It was confirmed by fluorescence microscope that Ad-MafA-EGFP was transferred into the HUMSCs. After induction, the expression of human pancreatic precursor cell-related genes, including glucagon, PDX1 and Nkx2.2, was increased as detected by PCR. To conclude, these findings could provide experimental evidence for further differentiation and maturation of pancreatic β cells from HUMSCs.   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 Figures and Tables | References | Related Articles | Metrics

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Tree shrew umbilical cord mesenchymal stem cells: isolation, cultivation and osteogenic and adipogenic differentiation Ruan Guang-ping, Zhu Lu, Liu Ju-fen, Li Zi-an, Wang Jin-xiang, Pang Rong-qing, Pan Xing-hua 2017, 21 (9):  1373-1377.  doi: 10.3969/j.issn.2095-4344.2017.09.012 Abstract ( 390 )   PDF (3356KB) ( 389 )   Save BACKGROUND: Studies have shown that umbilical cord mesenchymal stem cells are ideal seed cells for tissue engineering research. OBJECTIVE: To isolate, culture and identify tree shrew umbilical cord mesenchymal stem cells, in order to establish a standardized tree shrew umbilical cord mesenchymal stem cell lines. METHODS: Caesarean-isolated tree shrew umbilical cord samples were used to isolate and culture umbilical cord mesenchymal stem cells using tissue explant adherent method. Flow cytometry assay was used to detect cell surface markers. Osteogenic and adipogenic induction media were used to induce umbilical cord mesenchymal stem cells to differentiate into osteoblasts and adipocytes. RESULTS AND CONCLUSION: The cultured umbilical cord mesenchymal stem cells expressed CD90 and CD105 with the positive rate of 99.9% and 99.8% respectively. Hematopoietic stem cell marker CD34 expression rate was 0.0% and the endothelial cell marker CD31 expression rate was 0.7%, in line with the characteristics of umbilical cord mesenchymal stem cell surface markers. Calcium nodules by alizarin red staining and lipid droplets by oil red O staining were observed in the induced cells. These experimental findings indicated that umbilical cord mesenchymal stem cells from tree shrews capable of osteogenic and adipogenic differentiation were successfully isolated and cultured.   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 Figures and Tables | References | Related Articles | Metrics

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Therapeutic effect of bone marrow mesenchymal stem cells on multiple organ dysfunction syndrome He Fa-lian, Liu Gao-mi-yang, Liu Ju-fen, Li Zi-an, Wang Qiang, Pan Xing-hua 2017, 21 (9):  1378-1383.  doi: 10.3969/j.issn.2095-4344.2017.09.013 Abstract ( 273 )   PDF (5864KB) ( 411 )   Save BACKGROUND: Clinical treatment of multiple organ dysfunction syndrome has been a big problem today, while the treatment of bone marrow mesenchymal stem cells may provide a new research direction. OBJECTIVE: To investigate the therapeutic effects of bone marrow mesenchymal stem cells transplanted into multiple organ dysfunction syndrome models. METHODS: According to body weight of rats, endotoxin in the 1 mg/kg dose was injected into the femoral vein to build the multiple organ dysfunction syndrome models in rats, and the surviving rats were randomly divided into treatment and control groups. Then the rats in the treatment group were injected intravenously third-generation bone marrow mesenchymal stem cells (5×106), while rats in the control group injected intravenously with an equal volume of saline. After 5 days of treatment, the blood routine examination, blood biochemistry and coagulation tests were conducted, and then the liver, kidney, heart, and spleen of rats were captured to observed pathological changes through the hematoxylin-eosin staining. RESULTS AND CONCLUSION: One week after cell transplantation, liver function and biochemical indicators were improved significantly in the treatment group compared with the control group (P < 0.05), and the white blood cell count in the treatment group was closer to normal levels (P < 0.05). Hematoxylin-eosin staining results showed that in the multiple organ dysfunction syndrome rats appeared to have different degrees of inflammatory lesions and necrosis in the heart, liver, spleen, and kidneys, while inflammatory infiltration and necrotic lesions decreased significantly in the treatment group compared with the control group. To conclude, bone marrow mesenchymal stem cells exert remarkable therapeutic effects on multiple organ dysfunction syndrome.   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 Figures and Tables | References | Related Articles | Metrics

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Chondroitinase with adipose-derived mesenchymal stem cell transplantation: a therapeutic strategy for retinal degeneration in rats Chu Miao, Ju Chao-juan, Zhao Li-na, Cui Zeng-min, Song Geng 2017, 21 (9):  1384-1389.  doi: 10.3969/j.issn.2095-4344.2017.09.014 Abstract ( 339 )   PDF (4305KB) ( 333 )   Save BACKGROUND: Studies have found that chondroitin sulfate proteoglycans degradation with chondroitinase can promote the migration of Müller cells on the retina, but whether it could promote the migration of adipose-derived mesenchymal stem cells in retinal degeneration rats is unclear. OBJECTIVE: To investigate the effect of chondroitin sulfate proteoglycans degradation with chondroitinase on adipose-derived mesenchymal stem cell treatment for retinal degeneration in rats. METHODS: Human adipose-derived mesenchymal stem cells were isolated and cultured. A retinal degeneration rat model was established followed by administration of adipose-derived mesenchymal stem cells+chondroitinase into the subretinal space. The migration of adipose-derived mesenchymal stem cells and retinal cell apoptosis in rats after transplantation were observed. RESULTS AND CONCLUSION: The human adipose-derived mesenchymal stem cells could be successfully cultured. The labeling rate of BrdU to the human adipose-derived mesenchymal stem cells was more than 90.0%. At 7 days after modeling, the outer nuclear layer of the retina was collapsed, and a large amount of photoreceptor cells were dissected. The outer nuclear layer was attached to the Bruch’s membrane, and the retina was arched. The central retina and peripheral retina were damaged. In normal rats, the retinal layers were clear, and the photoreceptor cells arranged regularly; and the retinal pigment epithelium was complete. The migration rate of adipose-derived mesenchymal stem cells in adipose-derived mesenchymal stem cells+chondroitinase group was higher than that in adipose-derived mesenchymal stem cells group (P < 0.05). There was no significant difference in the apoptotic rate of retinal cells between the two groups (P > 0.05). These experimental findings show that chondroitin sulfate proteoglycans degradation with chondroitinase can enhance the migration ability of human adipose-derived mesenchymal stem cells on the retina.   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 Figures and Tables | References | Related Articles | Metrics

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The overexpression of miR-378 promotes the therapeutic effects of bone marrow mesenchymal stem cell transplantation on myocardial infarction Zhang Xiu-min, Yu Bo, Li Xue-yuan 2017, 21 (9):  1390-1396.  doi: 10.3969/j.issn.2095-4344.2017.09.015 Abstract ( 389 )   PDF (4949KB) ( 380 )   Save BACKGROUND: Recently, miR-378 has been shown to modulate the anti-hypoxia capacity of bone marrow mesenchymal stem cells (BMSCs) and reduce cell apoptosis under hypoxic conditions. OBJECTIVE: To investigate the benefits of miR-378-upregulated BMSC transplantation in a rat model of acute myocardial infarction. METHODS:Primary rat BMSCs were cultured in vitro. Until passage 3, the cells were infected with the lentivirus carrying synthetic miR-378 gene fragments. A rat model for acute myocardial infarction was constructed by ligating the left anterior descending artery. Thereafter, the animals were randomly assigned to three groups: control group (n=10), BMSCnull group (n=16) and BMSCmiR-378 group (n=16). In the latter two groups, 50 μL of normal saline containing 1×107 empty virus-transfected or miR-378-transfected BMSCs was injected into the region of myocardial infarction, respectively. Only 50 μL of normal saline was injected in the control group. Twenty-four hours later, the apoptosis of transplanted BMSCs was evaluated with TUNEL, and expression level of vascular endothelial growth factor and transforming growth factor-β was detected using western blot assay. Four weeks after treatment, the left ventricular function of rats was assessed by echocardiography, and then histological and molecular biology analyses were performed. RESULTS AND CONCLUSION: At 24 hours postoperatively, there were less apoptotic BMSCs and higher expression levels of vascular endothelial growth factor and transforming growth factor-β in the BMSCmiR-378 group than in the BMSCnull group (n=6, P < 0.001). Four weeks later, there were more transplanted BMSCs and BMSCs-derived cardiomyocytes in the BMSCmiR-378 group than the BMSCnull group (n=10, P < 0.001). Moreover, increased new vessel density (P < 0.001), decreased infarcted area (P < 0.001), preserved left ventricular ejection fraction (P < 0.05), reduced left ventricular end-diastolic volume (P < 0.05) were found in the BMSCmiR-378 group, compared with the other two groups. The above parameters were better in the BMSCnull group than the control group (P < 0.05). Overall, the upregulation of miR-378 could enhance the capability of BMSCs against hypoxia, and consequently promote myocardial repair after implantation, providing a new strategy for cell therapy of myocardial 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 Figures and Tables | References | Related Articles | Metrics

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Effects of basic fibroblast growth factor modified skeletal muscle satellite cell transplantation on neovascularization following acute myocardial infarction Xu Zhi-feng, Li Jing-lai, Han Zhen, Feng Gang, Ren Ming-ming 2017, 21 (9):  1397-1401.  doi: 10.3969/j.issn.2095-4344.2017.09.016 Abstract ( 318 )   PDF (4178KB) ( 327 )   Save BACKGROUND: Previous studies have found that skeletal muscle satellite cell transplantation can induce angiogenesis in myocardial infarction area, reduce infarct size and improve cardiac function. But the overall effect is not satisfactory. OBJECTIVE: To observe the survival of basic fibroblast growth factor (bFGF) gene modified skeletal muscle satellite cells in acute myocardial infarction and to observe the expression of bFGF gene and the effect of cell transplantation on angiogenesis in myocardial infarction area. METHODS: Eighteen New Zealand white rabbits were divided into three groups by random: skeletal muscle satellite cell group (control group), bFGF gene enhanced skeletal muscle satellite cell group (experimental group) and blank control group. The left anterior descending branch of the coronary artery of the rabbits was ligated so as to establish an animal model of acute myocardial infarction in the former two groups. After labeled by DAPI before transplantation, the skeletal muscle satellite cells, bFGF gene modified skeletal muscle satellite cells and the equivalent amount of DMEM/F12 were injected into the local infarct myocardium correspondingly. Samples were taken 4 weeks after transplantation. Then, the survival of skeletal muscle satellite cells and the expression of bFGF gene were observed under light microscope and fluorescence microscope, and the neovascularization in the myocardial infarction area was examined by immunohistochemical staining. RESULTS AND CONCLUSION: No DAPI-labeled cells were visible in the blank control group, but in the other two groups, a large amount of DAPI-labeled skeletal muscle satellite cells were seen in the infarction area. Enhanced green fluorescent protein was highly expressed in the experimental group. Microvessel density in the infarction area was highest in the experimental group followed by the control and blank control groups (P < 0.05). These findings indicate that bFGF gene modified skeletal muscle satellite cells can survive and promote neovascularization in the acute myocardial infarction area.   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 Figures and Tables | References | Related Articles | Metrics

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Immunological impact of autologous peripheral blood stem cell infusion on lung adenocarcinoma Wang Mei-jiang, Qi Lin 2017, 21 (9):  1402-1407.  doi: 10.3969/j.issn.2095-4344.2017.09.017 Abstract ( 366 )   PDF (4435KB) ( 529 )   Save BACKGROUND: Although clinical methods for autologous peripheral blood stem cell separation and identification are relatively more, there is no unified standard. Moreover, little is reported on immune effects on lung adenocarcinoma during autologous peripheral blood stem cell transplantation. OBJECTIVE: To study the effect of autologous peripheral blood stem cell transplantation on immunological function of lung adenocarcinoma rats by exploring the separation and identification methods of autologous peripheral blood stem cells. METHODS: The mononuclear cells were isolated from the peripheral blood of Wistar rats by density gradient centrifugation method and then identified. Eighteen Wistar rats were randomly divided into routine treatment group and cell transplantation group, with nine rats in each group. Animal models of lung adenocarcinoma were established in the rats, and underwent no treatment in the routine treatment group but injection of autologous peripheral blood stem cell suspension (0.2 mL, 2.5×106/L) via the tail vein in the cell transplantation group. Immune indicators were detected in two groups after treatment. Three rats from each group were anesthetized and scarified at 4, 10, 14 days after treatment, respectively. Afterwards, we detected the effects of autologous peripheral blood stem cell transplantation on immunological function of lung adenocarcinoma rats. RESULTS AND CONCLUSION: Newly isolated mononuclear cells from rat peripheral blood showed a round or oval shape and were relatively small in size. After 5 days of culture, autologous peripheral blood stem cells grew rapidly, and the round cells began to spread to both ends and became spindle-shaped, thereby forming small colonies. Autologous peripheral blood stem cells induced in vitro experienced three stages successively: cell arrest, logarithmic and plateau phases. Flow cytometry showed that sorted cells were identified as autologous peripheral blood stem cells by expressing CD90, CD133 and Flk-1. After 4, 10 and 14 days culture, the expression of CD133 increased first and then descended, and Flk-1 showed an increasing trend. Compared with the routine treatment group, the expression of tumor necrosis factor-α induced protein 6 in the cell transplantation group was significantly higher (P < 0.05), while the expression of nuclear factor κB was significantly lower (P < 0.05). In conclusion, the isolation and identification methods of autologous peripheral blood stem cells are simple and can be used to secrete a large number of nutritional factors in the treatment of lung adenocarcinoma, which can enhance body immunity in the treatment.   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 Figures and Tables | References | Related Articles | Metrics

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Repairing articular cartilage defect of the knee joint by synovial mesenchymal stem cells and chondrocytes Hu Xin, Yin Xiao-peng, Gong Zhong-cheng, Shao Bo, Ling Bin, Liu Hui, Rekemu Abasi, Wang Bing, Hu Lu-lu, Wang Yue, Lin Zhao-quan 2017, 21 (9):  1408-1413.  doi: 10.3969/j.issn.2095-4344.2017.09.018 Abstract ( 356 )   PDF (8218KB) ( 344 )   Save BACKGROUND: As is known to all, it is difficult to repair cartilage defect of the knee joint. Fortunately, it is reported that a chitosan scaffold with the mixture of synovial mesenchymal stem cells and chondrocytes enables the formation of cartilage matrix. OBJECTIVE: To observe the chondrogenic differentiation of synovial mesenchymal stem cells co-cultured with chondrocytes on a chitosan/type I collagen composite scaffold after being transplanted into an animal model. METHODS: The synovial mesenchymal stem cells and chondrocytes harvested from the knee joint of Sprague-Dawley rats were co-cultured at a ratio of 1:2 on the chitosan/type I collagen composite scaffolds and transplanted into the defect site of the knee joint in rat models of cartilage defect of the knee joint. RESULTS AND CONCLUSION: After 4, 8, 12 weeks, the hematoxylin-eosin staining, the toluidine blue staining of aggrecan, and safranine O staining showed that there were increasing chondrocytes in the rat knee joint. The immunohistochemical staining of type II collagen showed the positive expression of type II collagen in the cartilage tissues and extracellular matrix of the knee joint. To conclude, the combination of two kinds of cells cultured in the chitosan/type II collagen composite scaffold can form cartilage-like tissues in vivo, and improve the quality of cartilage repair.   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 Figures and Tables | References | Related Articles | Metrics

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Orthotopic transplantation of human amniotic mesenchymal stem cells for treatment of cerebral infarction in rats Wang Yu-ying, Su Xu, Liu Bo, Liu Juan, Wan Xue 2017, 21 (9):  1414-1419.  doi: 10.3969/j.issn.2095-4344.2017.09.019 Abstract ( 312 )   PDF (6806KB) ( 421 )   Save BACKGROUND: Preliminary experimental study found that the human amniotic mesenchymal stem cells (hAMSCs) transplantation can improve nerve injury symptoms of rats with cerebral infarction. OBJECTIVE: To observe the survival, colonization and differentiation of hAMSCs in the infarct area of cerebral infarction rats. METHODS: Sixty Sprague-Dawley rats were randomly assigned into hAMSCs transplantation, model or sham operation groups (n=20/group). Animal models of middle cerebral artery occlusion were produced in the model and transplantation groups by Zea-Longa method. One day after modeling, rats in the hAMSCs transplantation group were given in situ transplantation of 10 μL of hAMSCs (2×106) into the damaged striatum and cortex, while those in the model and sham operation group were given the same volume of PBS. Within 1 week after transplantation, rat neurological defects were assessed and changes in their body mass were continuously monitored. Two weeks after transplantation, TTC staining was used to observe cerebral infarct size, hematoxylin-eosin staining was used for pathological observation of brain tissues, and immunofluorescent staining was used to detect expression of neuron-specific nuclear protein.  RESULTS AND CONCLUSION: With time, weight loss was increased while neurologic deficit scores were gradually reduced in the hAMSCs and model groups. Compared with the model group, the weight loss and neurologic deficit scores were lower in the hAMSCs group,; however, there was a significant difference in the neurologic deficit scores but not in the weight loss between the two groups. Additionally, the hAMSCs significantly reduced infarct size, attenuated pathologic injury, and decreased the number of inflammatory cells. Immunofluorescence staining showed that the hAMSCs were observed at 1 week after transplantation under inverted luorescence microscope, and gradually differentiated into nerve cells at 2 weeks after transplantation. In conclusion, transplanted hAMSCs may migrate to and survive in the cerebral infarct region, and differentiate into nerve cells in situ in 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 Figures and Tables | References | Related Articles | Metrics

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Human amniotic epithelial cells: isolation, identification and multi-directional differentiation Li Ping, Wang Jiu-cun, Lu Yao, Xu Hui-li 2017, 21 (9):  1420-1425.  doi: 10.3969/j.issn.2095-4344.2017.09.020 Abstract ( 404 )   PDF (4677KB) ( 439 )   Save BACKGROUND: Human placenta is a stable source for human amniotic epithelial cells, which is becoming a cell source in the regenerative medicine that attracts widespread attentions. OBJECTIVE: To establish the method of isolation, culture, and adipogenic, chondric and osteogenic differentiation of human amniotic epithelial cells. METHODS: Trypsin-EDTA digestion was used to isolate human amniotic epithelial cells from human amnion tissue, which were then cultured and identified in vitro. The growth curve of the cells was observed in 12 days. Passage 1 human amniotic epithelial cells were induced to differentiate into adipocytes, chondrocytes and osteoblasts, and conventional cultured cells were used as controls. After 16 days induction, oil red O, Masson and alkaline phosphates staining methods were carried out, and adipogenic transcription factor, type II collagen, osteopontin, alkaline phosphatase mRNA expressions were detected using real-time fluorescene quantitative PCR. RESULTS AND CONCLUSION: Human amniotic epithelial cells were successfully obtained from human amnion tissue. Immunofluorescence data showed the expression of epithelial cell surface marker CK19. Passage 1 cells had a strong ability to divide and proliferate. Compared with passage 1 ones, passage 2 cells showed a slight decrease in proliferation ability, and the proliferation ability of passage 3 cells was the worst. Red lipid droplets, brilliant blue cartilage matrix and reddish brown calcium nodes were detected by oil red O, Masson and alkaline phosphates staining after adipogenic, chondrogenic and osteogenic differentiation, respectively. With the time prolonged, the expressions of adipogenic transcription factor, type II collagen, osteopontin and alkaline phosphatase mRNA were increased. These results demonstrated that human amniotic epithelial cells could be isolated from human amniotic membrane by enzyme digestion method, and these amniotic epithelial cells could be induced to differentiate into differentiate into adipocytes, chondrocytes and osteoblasts.   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 Figures and Tables | References | Related Articles | Metrics

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Effect of pilose antler polypeptides on apoptosis and membrane stability of mitochondria in cardiac stem cells Huang Xiao-wei, Xu Yan, Liu Yue-xin, Li Zhi-meng, Qu Xiao-bo 2017, 21 (9):  1426-1431.  doi: 10.3969/j.issn.2095-4344.2017.09.021 Abstract ( 358 )   PDF (5375KB) ( 319 )   Save BACKGROUND: Cardiac stem cells can differentiate into cardiomyocytes in vitro under induction of pilose antler polypeptides, which provides a new therapeutic idea for myocardial injury. OBJECTIVE: To explore the effect of pilose antler polypeptides on the apoptosis rate and membrane stability of mitochondria in cardiac stem cells. METHODS: We chose healthy male Wistar rats born 2 days to extract cardiac stem cells. The culture dish was used as the experimental unit, and extracted cells were divided into the following four groups (n=12). Blank control group: The same amount of buffer was added for induction; 5-azacytidine group: induced with 5-azacytidine (3 μmol/L); pilose antler polypeptides group: induced with pilose antler polypeptides (800 mg/L); combined group: induced with pilose antler polypeptides (800 mg/L) and 5-azacytidine (3 μmol/L). After 48 hours induction, apoptosis rate of cardiac stem cells in each group was detected with flow cytometry. The membrane potential of mitochondria in cardiac stem cells was detected with immunofluorescence. The expression level of Nkx 2.5, GATA4, ATF-2, and MEF-2C in cardiac stem cells was detected using western blot assay. RESULTS AND CONCLUSION: There were small, round and bright cells after 2 weeks culture, and cell colonies of cardiac stem cells formed. The apoptosis rate of cardiac stem cells in the 5-azacytidine group increased significantly (P < 0.05) and its membrane potential decreased significantly compared with the blank control group (P < 0.05). The apoptosis rate of cardiac stem cells in the pilose antler polypeptides group and combined group decreased significantly compared with the 5-azacytidine group (P < 0.05, P < 0.01), and their membrane potential increased significantly (P < 0.05, P < 0.01). The results of western blot showed that the expression level of Nkx2.5 increased significantly in the pilose antler polypeptides group and combined group compared with the 5-azacytidine group (P < 0.05), while the expression levels of GATA4 and ATF-2 in each group were low and there were no significant differences among groups (P > 0.05). The expression level of MEF-2C in each group was at a middle level, and there were no significant differences among groups (P > 0.05). To conclude, our experimental findings indicate that pilose antler polypeptides could decrease the apoptosis rate and improve membrane stability of mitochondria in cardiac stem cells. The mechanism may be related to the increased expression of Nkx 2.5, but whether the mechanism is related to GATA4, ATF-2 and MEF-2C needs to be further studied.   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 Figures and Tables | References | Related Articles | Metrics

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In vitro EdU labeling of peripheral blood mononuclear cells in rabbits Zhao Ming-lei, Zhen Dong-qin, Huang Jian-fa, Li Wei-hua, Wang Wen-cong, Li Zhi-quan, Zhang He-ning,Xian Bi-kun, Peng Yu-ting, Zhou Min-yi, Huang Bing 2017, 21 (9):  1432-1438.  doi: 10.3969/j.issn.2095-4344.2017.09.022 Abstract ( 453 )   PDF (4804KB) ( 393 )   Save BACKGROUND: The proliferation of peripheral blood stem cells among peripheral blood mononuclear cells (PBMCs) in vitro remains unclear. There is no optimal marker for tracing PBMCs transplanted in vivo. OBJECTIVE: To observe the degree of PBMC proliferation in stem cell medium by EdU labeling and to explore the feasibility of EdU-labeled peripheral blood stem cells. METHODS: New Zealand rabbit PBMCs were isolated and cultured for 1 to 5 days in stem cell medium supplemented with EdU. The cells were observed and counted at 0, 1, 2, 3, 4 and 5 days in culture. The cells were harvested at each time point and stained with EdU fluorescent reagents. Then, confocal microscopy and flow cytometry were used to detect EdU-labeled cells. RESULTS AND CONCLUSION: (1) Freshly isolated rabbit PBMCs were rounded and showed clear outline. After 1 day culture, most of the cells were suspended in the medium, spherical or round. There were also a few cell clusters and adherent cells scattered in a triangle or polygon shape; after 2 days culture, more cell debris were observed, and most cells were round; when cultured for 3-5 days, increased cell debris, smaller cell mass and decreased cell density significantly were observed. (2) With the prolongation of culture time, the cell count decreased gradually. (3) When cultured for 1 day, EdU labeled cells in red were scattered. The number of cells marked with EdU red label increased significantly at day 2 and remained unchanged after 3 days of culture. At 5 days of culture, the number of red cells markedly decreased; the highest positive rate of EdU-labeled cells was (2.38±0.10)% at 2 days after culture. To conclude, these results showed that the proportion of proliferating cells in rabbit PBMCs was very low. EdU is capable of labeling proliferative cells among PBMCs.   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 Figures and Tables | References | Related Articles | Metrics

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Effects of Kangguzengsheng Capsules-containing serum on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells Song Yong-zhou, Tong Jiu-hui, Liu Hui-ling, Ma Wei, Li Ming 2017, 21 (9):  1439-1443.  doi: 10.3969/j.issn.2095-4344.2017.09.023 Abstract ( 404 )   PDF (1052KB) ( 391 )   Save BACKGROUND: Our previous studies have found that the Kangguzengsheng Capsules can promote fracture healing. OBJECTIVE: To explore the influence of Kangguzengsheng Capsules-containing serum on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. METHODS: Forty Sprague-Dawley rats were randomly divided into four groups. The capsule powder was resolved into 1 mL natural saline and intragastrically administered into rats according to 1.16 g/100 g, 3.48 g/100 g, 10.44 g/100 g in low, medium and high dose groups. Rats in control group were given equal volume of natural saline. After consecutive administration for 12 days, blood samples from the abdominal aorta were collected and serum samples were isolated and preserved until use. Bone marrow mesenchymal stem cells were isolated and purified by the whole marrow adhesion method, and identified by flow cytometry. Harvested cells were divided into four groups and cultured in the osteogenic culture medium containing different kinds of serum samples as described above. MTT method was adopted to test the cell proliferation at 24, 48 and 72 hours of culture. Alkaline phosphatase activity in cells was detected at 7 and 14 days of culture. Alkaline phosphatase staining was performed at 7 days of culture, and alizarin red staining performed at 14 days of culture. RESULTS AND CONCLUSION: Compared with the blank control group, bone marrow mesenchymal stem cells showed no significant changes in the proliferative ability and alkaline phosphatase activity in the low dose group, but these two indices were significantly increased in the high and medium dose groups at 48 and 72 hours of culture or at 7 and 14 days of culture, respectively (P < 0.05). Cells positive for alkaline phosphatase and alizarin red staining were observed in the low, medium and high dose groups, and the cell staining was most remarkable in the high dose group. To conclude, Kangguzengsheng Capsules-containing serum can promote the proliferation and osteogenic differentiation of bone marrow 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 Figures and Tables | References | Related Articles | Metrics

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Runx2 over-expression effect on expression of osteogenesis related genes in osteogenic differentiation of human umbilical cord blood mesenchymal stem cells Zhou Da-kai, Li Hui-ning, Ma Shan-shan, Cheng Tian 2017, 21 (9):  1444-1449.  doi: 10.3969/j.issn.2095-4344.2017.09.024 Abstract ( 440 )   PDF (1074KB) ( 408 )   Save BACKGROUND: Runx2 plays a central role in osteogenic differentiation, which is of important significance in new bone formation. OBJECTIVE: To observe the effect of Runx2 over-expression on osteogenic differentiation of human umbilical cord blood mesenchymal stem cells. METHODS: Runx2 was generated by RT-PCR and the recombinant adenovirus (pAd-Runx2) was constructed. The viral titer was determined by air dilution method. After being transfected into human umbilical cord blood mesenchymal stem cells, the green fluorescence was observed under fluorescence microscope. Real-time fluorescent quantitative PCR and western blot were used to detect mRNA expression changes of osteogenesis related genes, Runx2, OCN, BMP-2, ALP, in human umbilical cord blood mesenchymal stem cells at 1, 3, 7 and 14 days after transfection. RESULTS AND CONCLUSION: The recombinant adenovirus was successfully constructed and the titer was 1.7×1010 pfu/L. After infection by pAd-Runx2, human umbilical cord blood mesenchymal stem cells expressed green fluorescence protein clearly under the fluorescence microscope. Cells in the transfected group differentiated into osteoblast-like cells, and those in the control group stayed the same as pre-infected. The expression of Runx2, OCN, BMP-2 and ALP in the transfected group increased over time to some extent, but these changes were not detected in the control group. These findings indicate that Runx2 over-expression can promote the osteogenic differentiation of human umbilical cord blood 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 Figures and Tables | References | Related Articles | Metrics

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Allogeneic hematopoietic stem cell transplantation for intermediate cytogenetic risk acute myeloid leukemia Zhang Wen-hui, Chi Kai-kai, Chen Yu-qing, Yang Jing, Zhu Zun-min, Sun Kai, Zhang Yin 2017, 21 (9):  1450-1455.  doi: 10.3969/j.issn.2095-4344.2017.09.025 Abstract ( 557 )   PDF (1077KB) ( 385 )   Save BACKGROUND: Previous studies have shown that HLA-identical sibling allogeneic peripheral blood hematopoietic stem celltransplantation (allo-HSCT) provides higher disease-free and overall survival rates for patients with intermediate cytogenetic risk acute myeloid leukemia (AML) in complete remission (CR). But prognosis factors have not been fully defined. OBJECTIVE: To evaluate the outcome of patients with intermediate cytogenetic risk AML undergoing HLA-matched allo-HSCT in CR, and to analyze the prognostic factors. METHODS: Fifty cases of intermediate cytogenetic risk AML in CR receiving HLA-matched allo-HSCT from January 2009 to January 2015 were retrospectively analyzed. Primary outcome measures of the study included overall survival (OS), relapse rate and non-relapse mortality. RESULTS AND CONCLUSION: The 4-year OS of the study population reached to 64%, and the relapse rate and NRM reached to 18% and 20% respectively. Incidence of acute graft-versus-host disease was 26%. Different prognosis was observed between female donor/male recipient (FDMR) combination transplant and control (4-year OS: 50% vs. 71.9%, P=0.041), between patients requiring more than one course of induction chemotherapy to achieve CR and control (4-year OS: 40% vs. 70%, P=0.038), between older age (≥ 40 years) and control (4-year OS: 44.4% vs. 68.3%, P=0.056). The 4-year OS for matched sibling donor and matched unrelated donor was 63.2% and 66.7% (P=0.427), respectively. Further analysis revealed significantly high non-relapse mortality in FDMR combination transplant (P=0.024) and older age (≥ 40 years; P=0.043). Multivariate analysis revealed three negative prognostic factors: FDMR combination (P=0.031, RR=1.38, 95% CI: 1.03-1.95), requiring more than one course of induction chemotherapy to achieve CR (P=0.016, RR=1.46, 95% CI: 1.10-1.98) and older age (≥ 40 years; P=0.024, RR=1.63, 95% CI: 1.32-2.12). To conclude, HLA-matched allo-HSCT is a choice for the intermediate cytogenetic risk AML case in CR. FDMR combination, requiring more than one course of induction chemotherapy to achieve CR and older age (≥ 40 years) are confirmed as risk factors of poor prognosis for HLA-matched allo-HSCT patients with intermediate cytogenetic risk AML in CR. To these cases, the donor-recipient sex combination is more important than the donor type in donor selection.   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 Figures and Tables | References | Related Articles | Metrics

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CD4+CD25+ regulatory T cells and graft-versus-host disease after hematopoietic stem cell transplantation Guo Zhi, Tong Chun, Chen Hui-ren 2017, 21 (9):  1456-1462.  doi: 10.3969/j.issn.2095-4344.2017.09.026 Abstract ( 360 )   PDF (1092KB) ( 282 )   Save BACKGROUND: Autoreactive T cells are a group of specialized cells that exert a peripheral immunosuppressive effect through certain mechanisms ensuring self-tolerance within the adaptive immune system. The discovery of the latest surface markers for natural CD4+CD25+ regulatory T cells has re-emphasized the concept of peripheral regulation or suppression of T cells. Several groups have begun to investigate the role of regulatory T cells in animal models of allogeneic hematopoietic stem cell transplantation. OBJECTIVE: To review the recent results regarding protection from graft-versus-host disease by adoptively transferred CD4+ CD25+ regulatory T cells in mice and to discuss the latest findings from clinical studies on hematopoietic stem cell transplantation. METHODS: We retrieved CNKI, PubMed and Medline databases for articles concerning CD4+CD25+ regulatory T cells and graft-versus-host disease published from 2000 to 2015. According to inclusion and exclusion criteria, 46 papers were included in result analysis. RESULTS AND CONCLUSION: CD4+CD25+ regulatory T cells expressing the transcriptional repressor FOXP3 mediate immunoregulatory functions and are critical for the prevention of autoimmune diseases. As peripheral tolerance induction is a prerequisite for successful allogeneic hematopoietic stem cell transplantation, the role of CD4+CD25+ regulatory T cells in transplantation models and clinical trials is now under investigation in many laboratories. CD4+CD25+ regulatory T cells play an important role in the development of graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. CD4+CD25+ regulatory T cells not only effectively prevent and treat graft-versus-host disease but also retain graft-versus-leukemia 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 Figures and Tables | References | Related Articles | Metrics

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Gene therapy with induced pluripotent stem cells: a hope for beta thalassemia? Li Ling-li, Zhang Feng-bo, Li Qi, Ma Yan-lin 2017, 21 (9):  1463-1469.  doi: 10.3969/j.issn.2095-4344.2017.09.027 Abstract ( 672 )   PDF (1047KB) ( 474 )   Save BACKGROUND: Beta thalassaemia is a monogenic disease, which lacks effective clinical treatments. Hematopoietic stem cell transplantation currently is the only radical treatment for beta thalassaemia, but the limits of suitable donor and costs minimize its clinical application. Given the technology of reprogramming using somatic cells is well established, gene therapy using induced pluripotent stem cells has become the new direction of beta thalassaemia treatment. OBJECTIVE: To put forward the advantages of CRISPR/Cas9 technology in gene therapy of beta thalassaemia in the future by summarizing the mechanisms of three kinds of gene editing technologies and the preliminary experimental results in animal models. METHODS: In order to search relevant articles about beta thalassaemia, the first author retrieved PubMed database and CNKI (from 1989 to 2015) using the key words of “beta thalassemia, genetic therapy, genome editing, homologous recombination, iPSCs” in English and Chinese, respectively. After eliminating literatures which were irrelevant to research purpose or containing a similar content, 67 articles were chosen for further analysis. RESULTS AND CONCLUSION: Gene editing technology has made considerable progress and three kinds of directed gene editing technologies have been developed, including ZFNs, TALENs, CRISPR/Cas technology. By targeting induced pluripotent stem cells from thalassemi patients, these three kinds of gene editing technologies have been expected to correct pathogenic genes of thalassemia. The CRISPR/Cas system is more simple, rapid, safe and efficient than the others. The CRISPR/Cas9 system is expected to repair β-globin genes in the induced pluripotent stem cells, germ cells, fertilized eggs and embryos from beta thalassaemia patients, laying the foundation for future clinical application.   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 Figures and Tables | References | Related Articles | Metrics

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Effect and mechanism of umbilical cord blood stem cells in the treatment of stroke Liu Jie, Zhang Guo-hua 2017, 21 (9):  1470-1476.  doi: 10.3969/j.issn.2095-4344.2017.09.02 Abstract ( 331 )   PDF (1032KB) ( 374 )   Save BACKGROUND: Studies have confirmed that umbilical cord blood stem cells can improve the neurological function after stroke, but the mechanism of umbilical cord blood stem cells in the treatment of stroke is unclear.  OBJECTIVE: To review the effect and mechanism of umbilical cord blood stem cells in the treatment of stroke. METHODS: The first author searched PubMed and CNKI databases for relevant articles published from January 2005 to December 2015 using key words of “umbilical cord blood stem cells, stroke” in English and Chinese, respectively. RESULTS AND CONCLUSION: With the further research on the neural differentiation of umbilical cord blood stem cells, umbilical cord blood stem cells have become a hot research topic in the field of neural system disease therapy. At present, some experimental studies on cell transplantation for stroke have achieved ideal curative effects. However, the mechanism of umbilical cord blood stem cells in the treatment of stroke is not clear. Known therapeutic mechanisms include the recombination of blood vessels, nerve loops and other damage tissue structures, secretion of various nutritional factors to reduce endogenous apoptosis, promotion of endogenous blood vessels and nerve regeneration, etc. But the following problems need to be further studied, including whether the transplanted cells can be fully filled to replace necrotic nerve cells and promote recovery of nerve function; how these survived stem cells establish the neural connections; how these transplanted cells reduce the apoptosis and necrosis of the host cells to restore the cell function.   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 Figures and Tables | References | Related Articles | Metrics