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08 May 2018, Volume 22 Issue 13 Previous Issue    Next Issue

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Long non-coding RNA H19 facilitates bone marrow mesenchymal stem cell survival and vascularization in hypoxic-ischemic conditions in vitro Hou Jing-ying, Wang Lei, Long Hui-bao, Wu Hao, Wu Quan-hua, Zhong Ting-ting, Zhou Chang-qing, Guo Tian-zhu, Chen Xu-xiang, Wang Tong 2018, 22 (13):  1969-1975.  doi: 10.3969/j.issn.2095-4344.0430 Abstract ( 279 )   PDF (1477KB) ( 231 )   Save BACKGROUND: Our previous study demonstrated that bone marrow mesenchymal stem cells (MSCs) presented with a low survival rate and newly formed vascular-like structures was sparsely distributed in the local infarct tissues after cell transplantation, which certainly impaired the therapeutic efficacy. Long non-coding RNA-H19 (lncRNA-H19) has been confirmed to be associated with MSCs differentiation and mediate vascularization.                   OBJECTIVE: To observe the influence of lncRNA-H19 on the survival and vascularization potential of MSCs in vitro and to explore the possible mechanism. METHODS: MSCs were obtained and cultured in vitro. Cells were divided into five groups: MSCs+H19, MSCs+H19 negative control (MSCs+H19 NC), MSCs+si-H19, MSCs+si-H19 negative control (MSCs+si-H19 NC) and MSCs groups. MSCs+H19 and MSCs+H19 NC groups were transfected with lncRNA-H19 and lncRNA-H19 scramble RNA respectively, while MSCs+siH19 and MSCs+si-H19 NC groups were transfected with lncRNA-H19 siRNA and lncRNA-H19 siRNA scramble respectively. Cells were cultured under hypoxic-ischemic condition (serum-free medium, 1% O2) for 24 hours. Then, cell proliferation and apoptosis were evaluated using MTS and TUNEL, respectively. Cell supernatant from each experimental group was further co-cultured with human umbilical vein endothelial cells to induce vascularization. The expression of vascular endothelial growth factor A (VEGFA) was thereafter detected using western blot assay RESULTS AND CONCLUSION: Compared with MSCs+H19 NC and MSCs groups, MSCs+H19 group presented with significantly higher proliferation rate, lower apoptosis percentage and a larger number of vascular branches on matrigel (P < 0.01). There was a significantly higher expression of VEGFA in the MSCs+H19 group than MSCs+H19 NC and MSCs groups. Compared with the MSCs and MSCs+si-H19 NC groups, MSCs+H19 group presented with significantly lower proliferation rate, higher apoptosis percentage and a less number of vascular branches on matrigel (P < 0.01). In addition, VEGFA expression was distinctly downregulated in the MSCs+si-H19 group in comparison with the MSCs+si-H19 NC and MSCs groups. These findings indicate that lncRNA-H19 effectively promotes MSCs survival and vascularization under hypoxic-ischemic condition in vitro, and this effect may be associated with the upregulation of VEGFA. 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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Atorvastatin effects on proliferation and apoptosis of rat bone marrow-derived endothelial progenitor cells in vitro Zhang Ri-lin, Chen Shu-ling, Li Shang-hai, Ning Yi-ming, Li Qing-jun, Ye Xiao-min, Liang Wei-jun 2018, 22 (13):  1976-1980.  doi: 10.3969/j.issn.2095-4344.0491 Abstract ( 331 )   PDF (1385KB) ( 173 )   Save BACKGROUND: Atorvastatin has a cardiovascular protective effect that significantly improves endothelial function and promotes the mobilization, migration, and differentiation of endothelial progenitor cells. However, the screening of atorvastatin concentration for in vitro cell culture is not well documented. OBJECTIVE: To investigate the effects of different concentrations of atorvastatin on rat bone marrow-derived EPCs growth characteristics. METHODS: Bone marrow mononuclear cells from Sprague-Dawley rats were induced in selective culture fluid to culture EPCs. Immunofluorescence staining was used to identify cell surface markers. Harvested EPCs were divided into control group and atorvastatin groups with four different concentrations (0.01, 0.1, 1, and 10 μmol/L) for culture. The growth and proliferation of EPCs were observed under light microscope and MTT assay. Flow cytometry was used to detect apoptosis in EPCs. Nitric oxide and endothelial nitric oxide synthase levels in the culture fluid were measured by nitrate reductase method. RESULTS AND CONCLUSION: The number of cells tended to increase in the control and atorvastatin groups, and it was highest in the 1 μmol/L atorvastatin group. The cell number in the 10 μmol/L atorvastatin group began to decrease at 7 days of culture. Among the five groups, the apoptotic rate of cells was lowest in the 1 μmol/L atorvastatin group and highest in the 10 μmol/L atorvastatin group. The levels of nitric oxide and endothelial nitric oxide synthase were significantly higher in the 0.01, 0.1 and 1.0 μmol/L atorvastatin groups compared with the control group (P < 0.01), but lower in the 10 μmol/L atorvastatin group compare with the other groups (P < 0.01). Overall, atorvastatin can promote the proliferation of endothelial progenitor cells and reduce apoptosis by increasing the production of endothelial nitric oxide synthase and nitric oxide, and 1 μmol/L atorvastatin is most suitable for the EPCs culture. 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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Gelatin fibrous scaffolds promote fibrogenic differentiation of human dental pulp stem cells Jiang Li-ming, Xia Shang, Song Ge, Chen Xu 2018, 22 (13):  1981-1986.  doi: 10.3969/j.issn.2095-4344.0495 Abstract ( 354 )   PDF (7036KB) ( 152 )   Save BACKGROUND: A tooth can be led to lose viability, split easily and miss immune defensive response by pulpitis and pulp necrosis. Determining how to achieve dental pulp regeneration has become a research focus in dentistry. The physicochemical properties and biocompatibility of scaffold materials are crucial for proliferation and differentiation of stem cells. OBJECTIVE: To study whether a gelatin scaffold can induce dental pulp stem cells to differentiate into fibroblasts. METHODS: Gelatin scaffolds at different concentrations were prepared by electrospinning method. The surface morphology and physical properties of gelatin scaffolds were tested by using scanning electron microscope and tensile tests. The human dental pulp stem cells (hDPSCs) were seeded on the scaffolds and the cell proliferation and fibrogenic differentiation were tested using MTT and RT-PCR. RESULTS AND CONCLUSION: The fiber diameter of the 7.5% gelatin scaffold was (2.02±0.36) μm, and it was increased to (3.15±0.52) μm after cross-linking. In the 15% gelatin scaffold, fiber bonding was detected and strengthened until the emergence of flat structures after cross-linking. Both 7.5% and 15% gelatin scaffolds could promote the adhesion and growth of hDPSCs. On day 7, the cell number on the 7.5% gelatin scaffold was significantly higher than that on the 15% gelatin scaffold (P < 0.05). The levels of Collagen I, α-SMA, Periostin and Fibronectin were also higher in the 7.5% gelatin scaffold than in the 15% gelatin scaffold (P < 0.05). In conclusion, 7.5% gelatin scaffold is more beneficial to the proliferation and fibrogenic differentiation of hDPSCs. 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 SP7/Osterix and alkaline phosphatase in bone marrow mesenchymal stem cells with Cbfal/RUNX2 gene silencing regulated by the water extracts from Bushen Huoxue Decoction Cheng Ying-xiong, Luo Yi-wen, Wang Bin, Wu Zhi-fang, Shen Wei, Luo Hui, Sun Shi-dong, Huang Wen-qiang 2018, 22 (13):  1987-1992.  doi: 10.3969/j.issn.2095-4344.0493 Abstract ( 450 )   PDF (1650KB) ( 156 )   Save BACKGROUND: Bushen Huoxue Decoction (BSHXD) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs) in vitro. Exploring the molecular mechanisms involved is of clinical benefits. OBJECTIVE: To discuss the changes in the expression of SP7/Osterix and alkaline phosphatase (ALP) in BMSCs with Cbfal/RUNX2 gene silencing regulated by the water extracts from BSHXD. METHODS: BMSCs were isolated and cultured by the bone marrow adherent method, and BMSCs at passage 3 were used in the assay. BMSCs were transfected with nothing (blank control group), Cbfal/RUNX2 gene silencing lentivirus (silencing group), and negative viral vector (negative control group), respectively. Then, the cells were cultured in 100 mg/L BSHXD water extract, and 3 days later, the protein and mRNA expression of RUNX2 and Osterix was detected by western blot and qPCR, respectively. Activity of ALP in the BMSCs was also detected in each group. RESULTS AND CONCLUSION: The transfection efficiency of Cbfal/RUNX2 gene silencing lentivirus was about 90%. The protein and mRNA expressions of RUNX2 and Osterix were significantly decreased in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus as compared with the other two groups, and so was the ALP activity (P < 0.01). After treated with the water extracts from BSHXD, the expression of RUNX2 and Osterix as well as the ALP activity in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus increased significantly (P < 0.01). To conclude, the water extract from the BXHXD can up-regulate the expression of RUNX2 and Osterix and the activity of ALP, thus promoting BMSCs osteogenic 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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Bone marrow mesenchymal stem cells on Matrigel: growing and changing Zhang Bin-bin, Gao Quan-wen, Li Bing, Huang Sha, Yao Bin 2018, 22 (13):  1993-1998.  doi: 10.3969/j.issn.2095-4344.0427 Abstract ( 433 )   PDF (7727KB) ( 233 )   Save BACKGROUND: Matrigel as an extracellular matrix complex can facilitate cell proliferation, differentiation and collagen secretion in a cell culture system. OBJECTIVE: To establish a three-dimensional culture model of Matrigel combined with bone marrow mesenchymal stem cells (BMSCs) and to observe the morphology, proliferation and survival of BMSCs in the Matrigel three-dimensional culture model. METHODS: BMSCs were isolated and cultured by the whole bone marrow adherent method, followed by osteogenic and adipogenic induction and identification. The growth curve of passage 3 BMSCs was determined through the CCK-8 experiment. Passage 2 BMSCs were combined with Matrigel, and the morphology and proliferation of BMSCs were observed by hematoxylin-eosin staining under a phase contrast microscope. The viability of the cells was evaluated by the Live/Dead staining. RESULTS AND CONCLUSION: (1) BMSCs cultured by the whole bone marrow adherent method had the ability to differentiate into osteoblasts and adipocytes. (2) BMSCs exhibited an “S”-shaped growth curve, which was consistent with the growth characteristics of normal cells. (3) Under the phase contrast microscope, BMSCs cultured by the Matrigel model were extended and interconnected in a three-dimensional network growth state with good proliferation. The Matrigel gradually became soft and collapsed over time (7days) and a few cells were still in a network growth after 14 days. Hematoxylin-eosin staining results showed that the cell cytoplasm was larger at 4 days and the cells became thinned and mutually cross-linked at 7 days. (4) The active percentage of BMSCs in the Matrigel model was 92.57%, 95.54% and 97.37% at 1, 4, 7 days of culture, respectively. To conclude, BMSCs cultured on the Matrigel has good proliferation and high viability.  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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Bone marrow mesenchymal stem cells improve learning ability of the aging rat Liu Yang, Wang Fei-qing, Liu Yan-qing, Li Hong-ri, Zhang Bo, Li Yan-ju 2018, 22 (13):  1999-2004.  doi: 10.3969/j.issn.2095-4344.0506 Abstract ( 300 )   PDF (1396KB) ( 135 )   Save BACKGROUND: At present, studies have shown that bone marrow mesenchymal stem cells (BMSCs) have self-renewal ability, which can be used as ideal seed cells for repairing tissue and organ damages caused by aging and lesions. OBJECTIVE: To study the changes in the levels of oxidation, inflammatory factors and neurotrophic factors (BDNF) in the brain of aging rats undergoing BMSCs transplantation, and to analyze the mechanism underlying the repair of learning and memory ability in the aging rats. METHODS: A total of 30 clean Sprague-Dawley rats were randomly divided into control group, model group and BMSCs group, 10 rats in each group. Aging models were made in the rats by 3-month subcutaneous injection of D-galactose. After modeling, BMSCs treatment was performed via tail vein injection in the BMSCs group. The injection was performed once a week, for 8 continuous weeks. Morris water maze was used to detect the learning and memory abilities of the rats in each group after the final injection of BMSCs. Superoxide dismutase activity in the brain tissue of rats was detected by xanthine oxidase method. Level of malondialdehyde in the rat brain tissue was detected by thiobarbituric acid method. Total antioxidant capacity of the brain tissue was detected by Fe3+ reduction method. Real-time PCR and western blot assay were used to detect the expression of brain-derived neurotrophic factor mRNA and protein in the brain tissue of the aging rat, respectively. RESULTS AND CONCLUSION: Compared with the model group, the BMSCs group exhibited significantly higher activity of superoxide dismutase, stronger total antioxidant capacity, and higher levels of brain-derived neurotrophic factor mRNA and protein (P < 0.05), but the lower malondialdehyde level in the brain (P < 0.05). Compared with the model group, there was less time and higher frequency for passing through the platform in the BMSCs group (P < 0.05). Our findings further indicate that BMSCs can improve the abilities of learning and memory in aging rats, and the underlying mechanism is likely to improve antioxidant capacity and to regulate the level of brain-derived neurotrophic 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 Figures and Tables | References | Related Articles | Metrics

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Platelet-rich plasma combined with naringin induces osteogenic differentiation of human bone marrow mesenchymal stem cells in vitro Nong Ju-an, Li Xiao-feng, Fang De-peng, Zhan Long, Yang Yuan 2018, 22 (13):  2005-2010.  doi: 10.3969/j.issn.2095-4344.0511 Abstract ( 389 )   PDF (4881KB) ( 161 )   Save BACKGROUND: Platelet-rich plasma (PRP) and naringin can both promote proliferation and induce osteogenic differentiation of mesenchymal stem cells. However, their combined use is rarely reported. OBJECTIVE: To observe the effect of PRP combined with naringin on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. METHODS: BMSCs at passage 3 were divided into four groups: (1) blank control group, cells were cultured in α-MEM; (2) PRP group, cells were cultured in α-MEM containing PRP; (3) naringin group, cells were cultured in α-MEM containing naringin; and (4) combined group, cells were cultured in α-MEM containing PRP and naringin. The contents of used PRP and naringin were 12.5% and 50 μg/L respectively. Cell proliferation was detected by MTT assay. Expression of related genes in hBMSCs was detected by RT-PCR. Alkaline phosphatase staining, collagen type I immunohistochemical staining, and alizarin red staining were used to analyze the osteogenic differentiation of hBMSCs. RESULTS AND CONCLUSION: The proliferation of hBMSCs was increased in each group, especially in the combined group. Cells in all the groups except the blank control group were positive for alkaline phosphatase staining, collagen type I immunohistochemical staining, and alizarin red staining, and the positive effect was more obvious in the combined group. However, negative or weakly positive response was found in the blank control group. At 7 and 14 days, the expression of alkaline phosphatase and collagen type I was significantly higher in the PRP, naringin and combined groups than the blank control group (P < 0.05); at 14 days, the expression of alkaline phosphatase and collagen type I was significantly higher in the combined group than the PRP and naringin groups (P < 0.05). To conclude, PRP combined with naringin can promote the proliferation of hBMSCs and induce the osteogenic differentiation of hBMSCs. Moreover, there is a synergistic effect between PRP and naringin.  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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Influence of Wnt/beta-catenin signal transduction pathway on the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells Peng Qin, Yin Yan-feng, Guan Zheng, Lv Sha, Su Wen-jun, Shan Hai-yan, Zhang Lei 2018, 22 (13):  2011-2019.  doi: 10.3969/j.issn.2095-4344.0510 Abstract ( 425 )   PDF (8034KB) ( 160 )   Save BACKGROUND: Umbilical cord mesenchymal stem cells can be induced to differentiate into hepatocyte-like cells in vitro and in vivo. However, the exact mechanism is still unknown. Existing studies have shown that the Wnt/β-catenin signaling pathway is closely related to this process. OBJECTIVE: To explore the effect of Wnt/β-catenin signaling pathway on the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells and its potential molecular mechanism. METHODS: Human umbilical cord mesenchymal stem cells were extracted from the neonatal umbilical cord by tissue adherent method. After being cultured and purified, the umbilical cord mesenchymal stem cells at passages 4-6 were divided into four groups: control group (DMEM culture group), hepatocyte-like differentiation group, activator Wnt3a group (adding 20 μg/L Wnt3a, an activator of Wnt/β-catenin signaling pathway, under the differentiation condition), and inhibitor Dkk-1 group (adding 20 μg/L Dkk-1, an inhibitor of Wnt/β-catenin signaling pathway, under the differentiation condition). Induced cells were collected respectively on days 7, 14, 21, 28. Their mRNA and protein expressions of α-fetoprotein (AFP), albumin (ALB), hepatocyte nuclear factor 4α (HNF4α) and Cytokeratin-19 (CK-19) in the cells were detected by real-time quantitative PCR and western blot respectively. Meanwhile, Periodic Acid-Schiff staining, low-density lipoprotein uptake test and indocyanine green absorption test were applied to detect the function of hepatocyte-like cells. RESULTS AND CONCLUSION: Compared with the control group, expressions of AFP and HNF4α mRNA and protein as well as ALB mRNA were significantly up-regulated in the hepatocyte-like differentiation group, activator Wnt3a group and inhibitor Dkk-1 group (P < 0.05). Whereas, there was a decrease in the CK-19 expression at mRNA and protein levels (P < 0.01) in these three groups. Compared with the hepatocyte-like differentiation group, the mRNA and protein expressions of AFP and HNF4α, and the mRNA expression of ALB were significantly down-regulated in the activator Wnt3a group (P < 0.05). Compared with hepatocyte-like differentiation group and activator Wnt3a group, the inhibitor Dkk-1 group had higher expression of AFP, HNF4α mRNA and their proteins as well as the mRNA expression of ALB (P < 0.05). Findings from the Periodic Acid-Schiff staining, low-density lipoprotein uptake test and indocyanine green absorption test showed more positive cells in the inhibitor Dkk-1 group than in the hepatocyte-like differentiation group and least positive cells in the activator Wnt3a group. Overall, these findings suggest that the inhibition of Wnt/β-catenin signaling pathway promotes the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells; conversely, the cell differentiation can be inhibited via the Wnt/β-catenin 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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A comparative study on serum-free and serum culture methods of human umbilical cord mecenchymal stem cells Zhang Xue-juan, Liu Gao-mi-yang, Liu Ju-fen, Song Yi-jia, Lin Qing-keng, Bai Ying-ying, Pan Xing-hua 2018, 22 (13):  2020-2026.  doi: 10.3969/j.issn.2095-4344.0496 Abstract ( 503 )   PDF (4697KB) ( 211 )   Save BACKGROUND: Studies have shown increasing risks and problems in the serum culture system, such as immune rejection, batch differences and virus risk. In addition, with the discovery and application of exosomes, the serum-free culture system is becoming an increasing concern. OBJECTIVE: To compare the similarities and differences between the serum-free culture system and the traditional serum culture system, which lays the foundation for the clinical transformation of human umbilical cord mesenchymal stem cells (hUCMSCs) and provides experimental data. METHODS: Umbilical cord was collected from term infants of cesarean section under aseptic condition, and hUCMSCs were isolated and cultured by explant tissue technique. hUCMSCs was cultured with 10% fetal bovine serum (FBS) and 15% serum substitutes (AGS) from the original generation. Then an inverted microscope was used to observe cell morphological changes. Flow cytometry was used to detect cell surface markers. Cell counting kit-8 was used to detect cell proliferation. Induced differentiation experiment was used to detect cell differentiation potential. Western Blot was used to detect the protein levels of oct4, nanog and sox2. RESULTS AND CONCLUSION: Under the inverted microscope, hUCMSCs cultured with AGS showed more uniform vortex-like growth, and those cultured with FBS gradually appeared with cell differentiation or aging with the increase of cell generations. hUCMSCs cultured by both methods expressed CD73,CD90 and CD105 but lowly expressed CD34 and CD45, and there was no significant difference between the two culture methods. FBS method was superior to AGS method in proliferation ability. Results from the induced differentiation experiments showed that hUCMSCs cultured by both methods had adipogenic, osteogenic and chondrogenic abilities, and there was no significant difference between the two culture methods. hUCMSC cultured by both methods expressed oct4 and nanog but showed no significant difference in level, while the expression of sox2 was significantly higher in the hUCMSCs cultured by AGS than by FBS (P < 0.05). To conclude, the hUCMSCs cultured with AGS are in accordance with the international standards of mesenchymal stem cells. The AGS method as an alternative to the FBS method can become a preferred method for hUCMSCs culture. 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 umbilical cord blood mesenchymal stem cells differentiate into neuron-like cells: in vitro induction by mouse nerve growth factor Chen Jun, Yang Zi-jin 2018, 22 (13):  2027-2032.  doi: 10.3969/j.issn.2095-4344.0492 Abstract ( 389 )   PDF (5095KB) ( 238 )   Save BACKGROUND: Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) are a kind of adult stem cells in the human umbilical cord blood, which have the potential to differentiate into neuron-like cells and can be used for the treatment of a variety of nervous system diseases. How to effectively induce hUCB-MSCs differentiation into neuron-like cells is always a hotspot in the stem cell research, which is of high scientific research value. OBJECTIVE: To explore the induction effect of mouse nerve growth factor (mNGF) on the differentiation of human umbilical cord blood-derived mesenchymal stem cells into neuron-like cells in vitro.  METHODS: The donated hUCB-MSCs were resuscitated and the cell morphology after culture was observed to draw a cell growth curve. Passage 5 cells were cultured in the culture medium containing 0 (blank control), 50, 100, 150, 200 μg/L mNGF, and the cell morphology was observed and recorded daily under inverted phase contrast microscope. Immunocytochemistry detection was used to examine the expression of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) at 7 days of induction, and then the NSE and GFAP positive expression was calculated by Image-Pro Plus 6.0 software.  RESULTS AND CONCLUSION: The viability of resuscitated hUCB-MSCs was up to over 90%. The cell morphology was in long shuttle shape and spindle shape with unequal size, and the cells presented with “S”-shaped growth curve and entered the logarithmic growth phase at 3-6 days. Typical neuron-like changes were observed after induction by mNGF; however, there was no change in the cell morphology in the control group. Immunocytochemical staining showed that the induced cells were positive for both NSE and GFAP, and the highest positive rates of NSE and GFAP were observed after induction by 100 μg/L mNGF (P < 0.05). In the control group, there was no positive expression of NSE and GFAP. To conclude, mNGF can induce the in vitro differentiation of hUCB-MSCs into neuron-like cells, and 100 μg/L mNGF can achieve the best induction 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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Isolation and identification of human adipose-derived stem cells and its exosomes Li Hong-chao, Jin Yin-peng, Wang Xi, Li Li, Wang Xiao-jin, Zhou Rong, Chen Cheng-wei, Fu Qing-chun, Cheng Ming-liang 2018, 22 (13):  2033-2038.  doi: 10.3969/j.issn.2095-4344.0494 Abstract ( 645 )   PDF (3933KB) ( 320 )   Save BACKGROUND: Currently, mesenchymal stem cells have been widely explored and applied in scientific research field, and many studies suggest that the underlying mechanism of mesenchymal stem cells mainly relies on its exosomes. OBJECTIVE: To isolate and identify human adipose-derived stem cells and its exosomes, and to identify their biological characteristics. METHODS: Human adipose tissue was digested with collagenase I, and adipose-derived stem cells were isolated and purified. Immunophenotype, osteogenic and adipogenic abilities of adipose-derived stem cells were identified. Exosomes were isolated by using ultrafiltration method. Morphology of exosomes was observed by Nanosight and electron microscope. Expression of proteins in exosomes was detected by antibody array method. RESULTS AND CONCLUSION: Adipose-derived stem cells exhibited long spindle-like or fibroblast-like appearance, expressed CD73, CD44, CD90, CD105 and had the potential to differentiate into many tissues, including bone and adipose tissues. The exosomes had the similar size, with the diameter of 30-150 nm. They possessed many proteins including FLOT1, ICAM, ALIX, CD81, CD63, ANXA5, TSG101, and so on. Findings from the present study indicate the successful isolation of exosomes from human adipose-derived 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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Monosialotetrahexosyl ganglioside at an optimal concentration: inducing neuron-like differentiation of human umbilical cord mesenchymal stem cells Zhang Peng, Zhao Zong-mao, Li Jian-hua, Liu Hui, Liu Yong-jun, Li Min-jie, Chen Ming-wei, Shen Lun,He Lei 2018, 22 (13):  2039-2044.  doi: 10.3969/j.issn.2095-4344.0490 Abstract ( 330 )   PDF (3532KB) ( 176 )   Save BACKGROUND: Studies have confirmed that monosialotetrahexosyl anglioside can induce human umbilical cord mesenchymal stem cells to differentiate into neuron-like cells, but little is reported on its optimal concentration. OBJECTIVE: To explore the optimal concentration of monosialotetrahexosyl ganglioside that induces human umbilical cord mesenchymal stem cells to differentiate into neuron-like cells in vitro. METHODS: Human umbilical cord mesenchymal stem cells were isolated by using collagenase digestion method, and after expansion, passage 3 cells were randomly allocated into five groups. When 70%-80% of cells were confluent, 50, 100, 150 and 200 mg/L monosialotetrahexosyl ganglioside induction solutions were added in corresponding experimental groups, while cells in the blank control group were cultured in the same volume of L-DMEM medium. Cell morphology was observed under inverted phase contrast microscope. Expression levels of microtubule-associated protein 2, neurofilament protein and glial fibrillary acidic protein were measured by using immunohistochemistry at 6 hours after induction. RESULTS AND CONCLUSION: Human umbilical cord mesenchymal stem cells were isolated successfully and sub-cultured stably. These cells could express surface markers of mesenchymal stem cells. Monosialotetrahexosyl ganglioside at the optimal concentration of 150 mg/L was confirmed to induce the neuron-like differentiation of human umbilical cord mesenchymal stem cells, and differentiated cells could express microtubule-associated protein 2 and neurofilament protein as neuron-specific markers.  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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Immunogenicity of insulin producing cells differentiating from human umbilical cord mesenchymal stem cells Li Lan-lan, Li Ning, Yang Xiao-fei, Chen Tao, Ou Gang-wei, Li Fu-rong 2018, 22 (13):  2045-2050.  doi: 10.3969/j.issn.2095-4344.0488 Abstract ( 317 )   PDF (4170KB) ( 301 )   Save BACKGROUND: Human umbilical cord mesenchymal stem cells (hUC-MSCs) have low immunogenicity and it is unclear whether insulin producing cells (IPCs) that differentiate from hUC-MSCs have low immunogenicity. OBJECTIVE: To investigate the immunogenicity of IPCs differentiating from hUC-MSCs in vitro and after IPCs transplantation into the host. METHODS: (1) The hUC-MSCs were induced to differentiate into IPCs according to the modified scheme. Flow cytometry assay was used to detect the immunophenotype and apoptotic rate of IPCs in a cytotoxicity test. (2) Cell counting kit-8 was used to detect the proliferative capacity of human peripheral blood mononuclear cells in the one-way mixed lymphocyte assay. (3) The IPCs were then transplanted into   the abdominal cavity and left renal capsule of mice, and then the infiltration of immune cells was detected by flow cytometry and immunohistochemistry. RESULTS AND CONCLUSION: The IPCs highly expressed HLA-ABC and lowly expressed HLA-DR, CD40 and CD80. The apoptosis rate of IPCs increased with the increase of pre-sensitized splenocytes in the cytotoxicity test. In the one-way mixed lymphocyte assay, IPCs inhibited the proliferation of human peripheral blood mononuclear cells when the target ratio was 10:1 and 50:1. After IPCs transplantation, the number of lymphocytes was increased in the transplanted site. In summary, our results show that IPCs that differentiate from hUC-MSCs maintain low immunogenicity in vitro, but have some immunogenicity after transplantation into the host due to microenvironment changes. 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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MicroRNA-520a-3p induces apoptosis in lung cancer stem cells via modulation of MAP3K2 Yan Jun, Zhong Zhi-hong, Shi Hua-qiu 2018, 22 (13):  2051-2056.  doi: 10.3969/j.issn.2095-4344.0504 Abstract ( 268 )   PDF (1279KB) ( 167 )   Save BACKGROUND: Studies have confirmed that microRNA (miR)-520a-3p has a regulatory role in lung cancer stem cells, but the specific function and mechanism of action are still unknown. OBJECTIVE: To investigate the influence of miR-520a-3p on the apoptosis of lung cancer stem cells, and to explore the underlying mechanism.  METHODS: The magnetic activated cell sorting method was utilized to separate the CD133+ lung cancer stem cells from the lung cancer A549 cell line. The expression of miR-520a-3p in the lung cancer stem cells was determined by the real-time PCR assay. Liposome transfection assay was used to up-regulate the miR-520a-3p expression level in the lung cancer stem cells, and flow cytometry assay was applied to detect the influence of miR-520a-3p expression on the apoptosis of the lung cancer stem cells. Moreover, the modulation of MAP3K2 gene by the miR-520a-3p was analyzed by the dual-luciferase reporter gene assay, and the influence of miR-520a-3p expression on the protein expression of MAP3K2, Bcl-2 and Caspase-3 was analyzed by western blot assay.  RESULTS AND CONCLUSION: The real-time PCR showed that the expression level of miR-520a-3p in CD133+ lung cancer stem cells was significantly lower than that in the CD133- lung cancer cells (P < 0.05). Overexpression of miR-520a-3p significantly increased the apoptotic rate of CD133+ lung cancer stem cells (P < 0.05). The dual-luciferase reporter gene assay results suggested that inhibition of miRNA-520a-3p could increase the luciferase activity of the reporter plasmids containing the 3’-untranslated region (3’-UTR) of MAP3K2 gene (P < 0.05), and overexpression of miR-520a-3p could decrease the luciferase activity of the reporter plasmids containing the 3’-UTR of MAP3K2 gene (P < 0.05). Moreover, miR-520a-3p overexpression also decreased the protein levels of MAP3K2 and Bcl-2 in the CD133+ lung cancer stem cells, P < 0.05), and increased the Caspase-3 protein level (P < 0.05). To conclude, in the lung cancer stem cells, miR-520a-3p was in a low-expressed status. miR-520a-3p could inhibit the expression of MAP3K2 gene, thereby inducing the cell apoptosis. 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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Role of ABCG2 gene in proliferation and invasion of colorectal cancer stem cells Sun Feng 2018, 22 (13):  2057-2062.  doi: 10.3969/j.issn.2095-4344.0503 Abstract ( 391 )   PDF (4742KB) ( 214 )   Save BACKGROUND: ABCG2 transporter can mediate multidrug resistance. ABCG2 overexpression can enhance the tolerance of stem cells to chemotherapeutic drugs. However, the mechanism of ABCG2 gene in the proliferation and invasion of colorectal cancer stem cells has not been confirmed. OBJECTIVE: To investigate the role of ABCG2 gene in the proliferation and invasion of colorectal cancer stem cells. METHODS: Colorectal cancer cell lines HCT116 were routinely isolated and cultured, and were randomly divided into four groups. CD133 positive cells were isolated by immunomagnetic beads method, and were transfected with ABCG2-siRNA and ABCG2 overexpression plasmids to construct colorectal cancer stem cell models with ABCG2 low expression and overexpression, and were set as low expression group and overexpression groups, respectively. The remaining HCT116 cells were set as normal and empty plasmid transfected control groups. The proliferation and invasion of colorectal cancer stem cells in different models were determined by MTT assay and Transwell assay, respectively. Expressions of matrix metalloproteinase 9 (MMP-9) mRNA and protein were detected by enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), respectively. RESULTS AND CONCLUSION: (1) Flow cytometry results showed that: CD133 positive cells in colorectal cancer cell line HCT116 accounted for 2.4%, while increased to 94.51% of the cell line after being sorted by immunomagnetic beads, suggesting that isolated and cultured cells were colorectal cancer stem cells. (2) MTT assy showed no significant difference between the four groups of cells prior to the cell transfection (P > 0.05). However, MTT value in the low expression group was significantly lower than that in the overexpression group after cell transfection (P < 0.05). (3) Results from the Transwell assay showed that the cells in the low expression groups did not have the ability of migration; on the contrary, in the overexpression group, the number of cells crossing the basement membrane was relatively increased and the ability of cell migration and invasion was enhanced significantly (P < 0.05). (4) The MMP-9 protein level was lower in the low expression group than in the overexpression group (P < 0.05). Similar results were yielded in the PCR detection. To conclude, down-regulation of ABCG2 protein expression can inhibit colorectal cancer stem cell proliferation and invasion. ABCG2 gene can change the viability of colorectal cancer stem cells by regulating the MMP-9 expression. 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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Y-27632 promotes differentiation of human embryonic stem cells into neuron-like cells He Da-yan, Yang Yu-hong 2018, 22 (13):  2063-2067.  doi: 10.3969/j.issn.2095-4344.0753 Abstract ( 493 )   PDF (3737KB) ( 191 )   Save BACKGROUND: Y-27632 is a Rho-associated coil-formed protein kinase inhibitor that can regulate the self-renewal of stem cells, promote clonal formation and cell survival, and regulate and protect neuronal cell growth and development. How to improve the differentiation efficiency of embryonic stem cells into neuron-like cells is highly important for nerve injury repair and nerve regeneration. OBJECTIVE: To investigate the effect of Y-27632 on the differentiation efficiency and function of human embryonic stem cells into neuron-like cells. METHODS: After resuscitation, human embryonic stem cells at passage 16 were subjected to morphological observation and staining identification. The embryoid bodies were prepared by suspension culture, and after 8 days of incubation, the cells were cultured in Sato medium containing different concentrations of Y-27632 (0, 5, 10, 20, 40 μmol/L) for 10 days and identification by staining. After induction for 18 days, the differentiation efficiency and neurite outgrowth were identified by immunofluorescence staining. RESULTS AND CONCLUSION: The human embryonic stem cells co-expressed Oct4 and SSEA-3 stem cell specific markers. After suspension culture for 8 days and further adherent culture for 10 days, the cells could be differentiated into neuron-like cells with neurogenic morphology and expressing Tuj-1. Y-27632, especially at a concentration of 10 μmol/L, not only promoted cell proliferation (a significant increase in adherent cells an Tuj-1 positive cells), but also facilitated cell differentiation into neurons. Immunofluorescence staining findings showed that 10 μmol/L Y-27632 significantly increased the number of Tuj-1 positive cells and neurites and the length of neurites after 18 days of differentiation. These results indicate that Y-27632 not only promotes the differentiation of human embryonic stem cells into neuron-like cells, but also accelerates neurite outgrowth. 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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Coculture with mesenchymal stem cells facilitates the proliferation of hematopoietic stem cells under different coculture modes Wang Shu-yue, Lin Fan-li, Qian Yi, Chen Xiao-qing, Liu Yang, Li Shu-tan, Cheng Yan, Xiong Hao, Huang Chun-lan 2018, 22 (13):  2068-2074.  doi: 10.3969/j.issn.2095-4344.0508 Abstract ( 398 )   PDF (6415KB) ( 319 )   Save BACKGROUND: Although a large number of related studies have been carried out, there is still a lack of practical methods to amplify hematopoietic stem cells (HSCs) in vitro. Mesenchymal stem cells (MSCs) secrete a variety of cytokines that promote the HSCs proliferation and inhibit their differentiation. These cytokines play an important role in maintaining the hematopoietic microenvironment and regulating HSCs function. OBJECTIVE: To investigate the effect of bone marrow MSCs on the proliferation of HSCs in vitro under different coculture modes. METHODS: Mesenchymal stem cells from the bone marrow of C57BL/6 mice were cultured in vitro using the whole bone marrow adherent culture. CD117+ cells (HSCs) were sorted from passage 3 cells by using miniMACS magnetic beads sorting. Then, CD117+ cells were co-cultured with MSCs under different coculture models, including single culture of HSCs (control group), Transwell coculture (upper chamber, HSCs; lower chamber, MSCs) and two-dimensional contact coculture (coculturing HSCs and MSCs in 24-well plates). The morphology of HSCs was observed under phase contrast microscope and fluorescence microscope, and the number of active cells of HSCs was counted at 1, 3, 5, and 7 days after coculture. RESULTS AND CONCLUSION: During the coculture of 1-7 days, the number of HSCs in the two groups was increased with culture time (P < 0.05). After 3 days of coculture, HSCs in each group was grown into the logarithmic growth phase, and morphological changes in some HSCs were detected at 5 days of coculture. At 7 days of coculture, the viabilities of HSCs in different culture models were ranked as follows: single culture model < Transwell coculture model < two-dimensional contact coculture model (P < 0.05). These findings suggest that MSCs can effectively promote the proliferation of HSCs in vitro, and the promotion effect is increased under contact coculture conditions. 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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Bone marrow mesenchymal stem cells combined with acellular dermal matrix for repair of urethral injury Fu Jin-shan, Zhou Zhi-yan, Xu Si-yi 2018, 22 (13):  2075-2080.  doi: 10.3969/j.issn.2095-4344.0486 Abstract ( 361 )   PDF (5171KB) ( 272 )   Save BACKGROUND: In recent years, the development of tissue engineering provides more choices for the repair of urethral injury. OBJECTIVE: To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) combined with acellular dermal matrix in the repair of urethral injuries. METHODS: Passage 3 BMSCs from New Zealand white rabbits were inoculated on the acellular dermal matrix to construct tissue-engineered urethra grafts. Thirty-six New Zealand rabbits were randomized into three groups (n=12 per group). Experimental group was implanted with BMSCs-acellular dermal matrix complex at urethral injury. Control group was implanted with acellular dermal matrix material at urethral injury. Normal group had neither injury nor treatment. At postoperative 4, 8 and 12 weeks, the repaired urethral tissue was subjected to hematoxylin-eosin staining. At postoperative 12 weeks, immunohistochemical staining and urodynamic study were performed. RESULTS AND CONCLUSION: At postoperative 4 weeks, thin-layer epithelial regeneration was visible in the urethra defect area of the experimental group, and the continuity was better. The urethra mucosa of the control group was discontinuous. At postoperative 8-12 weeks, the urethral epithelial layer in the experimental group became thickened, exhibiting a good continuity with the normal urethral epithelium, thickened mucosa, and smooth and continuous urethral mucosa; the regenerated urethral mucosa of the control group exhibited good continuity, but less regenerated epithelial layers. At postoperative 12 weeks, immunohistochemical results showed the repaired urethra in the experimental group was positive for uroplakin IIIa, CK AE1/AE3, and α-smooth muscle actin. The maximum urethral pressure in the experimental group showed no significant difference before and after operation, while the postoperative pressure in the control group showed a significant increase (P < 0.05). Overall findings indicate that the combination of BMSCs and acellular dermal matrix has better efficacy than the acellular dermal matrix alone. 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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Hydroxyapatite/beta-tricalcium phosphate scaffolds combined with adipose-derived stem cells for the treatment of spinal defects in rabbits Wang Teng-fei, Song Xing-hua, Maimaitiaili Abulikemu, Chen Jiang-tao, Tao Ying, Yang Yong 2018, 22 (13):  2081-2086.  doi: 10.3969/j.issn.2095-4344.0505 Abstract ( 440 )   PDF (5159KB) ( 295 )   Save BACKGROUND: Repair of bone defects is not only a clinical problem, but also a hot topic in the field of orthopedics. Although autologous bone grafting is considered as the “gold standard” for bone repair, its use is limited due to the limited source of autogenous bone, bone infections and pains that are easy to occur in the donor region. Allograft bones are always associated with immune rejection, slow healing, and infection. Therefore, it is imminent to develop new materials for bone repair. OBJECTIVE: To explore the effect of rabbit adipose-derived stem cells (rADSCs) as seed cells and hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) composite as a carrier on the repair of rabbit vertebral defects. METHODS: Thirty-eight 3-month-old New Zealand white rabbits were selected, and two of them were used to culture rADSCs in vitro. Passage 3 rADSCc were inoculated on HA/β-TCP scaffolds and then cultured in vitro for 2 weeks. A 5 mm×5 mm×3 mm bone defect was prepared at the anterior edge of L4/5 vertebral body in the remaining 36 rabbits. These model rabbits were then randomized into cell-scaffold composite group, scaffold group and control group with no intervention, with 12 rabbits in each group. rADSCs/HA/β-TCP composite and HA/β-TCP scaffold were implanted into the cell-scaffold and HA/β-TCP groups, respectively. Anteroposterior and lateral DR of the spine and Lane-Sandhu X-ray were performed at 4, 8, 12 postoperative weeks. All rabbits were sacrificed at 12 postoperative weeks and specimens were collected for gross and histopathological observations. RESULTS AND CONCLUSION: Under the gross observation, bone defects in the cell-scaffold group were essentially replaced by new bone tissues, which was significantly better than that in the scaffold group and control group. At 12 postoperative weeks, the material implanted was basically absorbed in the cell-scaffold group, partially absorbed in the scaffold group and poorly absorbed in the control group in which there was a clear boundary with the surrounding tissues and patchy calcified shadows were visible. X-ray results showed that the repair effect in the cell-scaffold group was better than that in the scaffold group and control group (P < 0.05). Histopathological findings showed the marked absorption of the implant in the cell-scaffold group, partial residual in the scaffold group with some fibrous calluses and osteoid tissues, and a large amount of fiber tissues and a small amount of calluses in the control group. Overall, the rADSCs/HA/β-TCA has a good ability to repair bone defects. 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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Estrogen regulates osteogenic differentiation of human periodontal ligament stem cells via Wnt/beta-catenin signaling pathway Mao Jie, Zhou Yi-fei, Wu Xiao-ling, Yu Jing-hong, Dang Hai-xia, Xu Xiao-mei 2018, 22 (13):  2087-2092.  doi: 10.3969/j.issn.2095-4344.0498 Abstract ( 456 )   PDF (6328KB) ( 343 )   Save BACKGROUND: Estrogen can promote the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs), but the molecular mechanism is unclear. OBJECTIVE: To study the regulatory effect of estrogen on the osteogenic differentiation of hPDLSCs via Wnt/β-catenin signaling pathway. METHODS: The hPDLSCs were isolated and purified by digestion method combined with limited dilution clone method. Three experimental groups were set as follows: osteogenic induction only (control group); 1×10-7 mol/L estrogen with osteogenic induction (estrogen group); and 100 μg/L Wnt3a protein with osteogenic induction (Wnt3a group). Alkaline phosphatase activity was detected at 1, 3, 5, 7 days of osteogenic induction. Western blot was used to detect the expression of Wnt/β-catenin signaling pathway related proteins β-catenin, P-GSK-3β, GSK-3β, CyclinD1 and osteoblast-related proteins Runx2 and OCN after 7 days of osteogenic induction. RESULTS AND CONCLUSION: The activity of ALP in all groups increased with time. The expression level of ALP in the estrogen group and Wnt3a group was higher than that in the control group at 1, 3, 5 and 7 days of induction (P < 0.05), while there was no significant difference between the former two groups (P > 0.05). The western blot results showed that the expression levels of β-catenin, P-GSK-3β, CyclinD1, Runx2 and OCN in the estrogen group and Wnt3a group were higher than those in the control group (P < 0.05), while the expression of GSK-3β was lower than that in the control group (P < 0.05). But there were no differences in the expression of Wnt/β-catenin signaling pathway related proteins and mid-late osteogenic markers between estrogen group and Wnt3a group (P > 0.05). To conclude, estrogen can enhance the osteogenic differentiation of hPDLSCs, and the underlying mechanism is likely to activate the Wnt/β-catenin signaling pathway in activated hPDLSCs exposed to estrogen. 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 and multi-differentiation potential of chondrogenic stem cells from rat lumbar endplates Wang Xiao, Xu Hong-guang, Xiao Liang, Liu Chen, Jin Zhong-xing 2018, 22 (13):  2093-2097.  doi: 10.3969/j.issn.2095-4344.0485 Abstract ( 432 )   PDF (3914KB) ( 125 )   Save BACKGROUND: ClonePix, a cell cloning and screening system, can quickly and efficiently screen cell clones undergoing suspension culture with low melting point agarose, which can be used to provide a sufficient number of seed cells for biological therapies.  OBJECTIVE: To isolate and identify chondrogenic stem cells from the rat lumbar endplate. METHODS: The lumbar endplates of 10 Sprague-Dawley rats with an age of 4 weeks were digested using trypsin and type II collagenase to isolate primary chondrogenic stem cells, followed by suspension culture with low melting point agarose. Then, the cell clones were selected by ClonePix and expanded in vitro for morphological observation. The multidirectional differential potential of cell clones was identified through osteogenesis, adipogenesis and chondrogensis tests, and the monoclonal formation ability was determined. RESULTS AND CONCLUSION: The cell clones could be successfully separated by using the agarose culture system, which were spindle-shaped after in vitro expansion. The osteogenesis, adipogenesis and chondrogensis capacities of the cells were identified using alizarin red, oil red O and safranin staining, respectively. Single cells of the clone group were inoculated into 10 cm culture dishes, and after 12 days of in vitro expansion culture, colonies of cells were observed with the naked eye. With the increase of cell seeding density, the number of cell colonies decreased. When the cells were inoculated into 10 cm culture dishes at a density of 100 cells, the colony forming ability was strongest, and more than 20 cell colonies could be formed. These findings indicate that chondrogenic stem cells that are isolated by the agarose gel culture system have multidirectional differentiation potential and high proliferation ability.  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 ginsenoside Rg3 on mouse neural stem cell differentiation in vitro Zhang Feng-lan, Yang Lu-jun, Hu Wei-yan, Zhou Jiao-yue, Ma Sheng-xia, Xiao Zhi-cheng 2018, 22 (13):  2098-2103.  doi: 10.3969/j.issn.2095-4344.0751 Abstract ( 430 )   PDF (5525KB) ( 262 )   Save BACKGROUND: Application of neural stem cells (NSCs) is of great current interest in neuroscience, but NSCs origin is very limited. And they always differentiate into a large percentage of glial cells and small percentage of neurons in natural differentiation process, so researchers should take effective measures to promote NSCs differentiation into certain offsprings. Previous studies have shown that ginseng saponin ingredients, such as Rb1 and Rg1, have certain influence on NSCs differentiation, but it is unclear whether Rg3 plays a role on NSCs differentiation. OBJECTIVE: To preliminarily investigate the effect of ginsenoside Rg3 on mouse NSCs differentiation into neurons and astrocytes in vitro. METHODS: The fetal cortices of embryonic 14 days (E14) C57BL/6 mice were isolated for culturing primary NSCs. Then passaged NSCs were identified by their purity with NSCs specific antibodies, Nestin and Sox2, by immunofluorescence staining. NSCs were induced for 3 days in the differentiation medium containing ginsenoside Rg3 of different concentrations (blank control, 50 and 250 nmol/L). After that, immunofluorescence staining was used to identify differentiated neurons with neuronal specific antibody, Tuj1, and differentiated astrocytes with astrocyte specific antibody, GFAP. Then, we calculated and statistically analyzed Tuj1+/DAPI and GFAP+/DAPI percentages in the three different groups. Besides, real-time PCR assay was used to test Tuj1 and GFAP mRNA expression in the three groups after 3 days of differentiation.  RESULTS AND CONCLUSION: Primary and passaged NSCs were successfully cultured and almost of cells were positive for both Nestin and Sox2, so these high-purity NSCs could be used in the following experiments. Immunofluorescence staining and statistical analysis results showed that compared with the blank control and 250 nmol/L groups, 50 nmol/L group had an obviously increased neuronal percentage after 3 days differentiation (P < 0.01), while the blank control and 250 nmol/L groups had no significant difference (P > 0.05); compared with the blank control group, 50 and 250 nmol/L groups had significantly increased astrocyte percentages (P < 0.05), whereas there was no obvious difference between 50 and 250 nmol/L groups (P > 0.05). The results of real-time PCR assay were similar with the above immunofluorescence results. In conclusion, 50 nmol/L ginsenoside Rg3 can enhance mouse NSCs differentiation into neurons and astrocytes, while 250 nmol/L ginsenoside Rg3 can only promote mouse NSCs differentiation into astrocytes. 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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Screening and evaluation of Smo-siRNA targeted to inhibition of Molt-4 cell proliferation Zhu Hua-min, Wang Xu, Xu Yan, Yang Li-jian, Chen Shao-hua, Wu Xiu-li, Li Yang-qiu 2018, 22 (13):  2104-2108.  doi: 10.3969/j.issn.2095-4344.0483 Abstract ( 344 )   PDF (3920KB) ( 162 )   Save BACKGROUND: Studies have shown that the occurrence and development of T lymphocytic leukemia is related to the abnormality of Hedgehog pathway. The Smo gene is a key gene in this signaling pathway and controls the transmission of Hedgehog signaling into the cell membrane. OBJECTIVE: To design and screen a highly efficient and specific Smo-siRNA which is able to downregulate the Smo gene expression in Molt-4 cells, thereby inhibiting the Molt-4 cells proliferation and inducing apoptosis. METHODS: (1) Smo-siRNAs numbered 1, 2 or 3, and the scrambled non-siRNA control (SC) were obtained by chemosynthesis. Untreated and sc-treated cells were used as controls. (2) Smo expression levels in Molt-4 cells were analyzed using qRT-PCR at 24, 48, 72 hours after siRNAs delivered by NuclefectorTM. Cell proliferation in vitro was assayed by the cell counting kit-8. The morphology and percentage of apoptotic cells were revealed by Hoechst33258 staining and flow cytometry, respectively. RESULTS AND CONCLUSION: (1) Smo-siRNAs were successfully transferred into Molt-4 cells, and exhibited best silencing results. After transfection with Smo-siRNA1, the mRNA level of Smo was significantly reduced (P < 0.05), and the lowest level was at 48 hours after transfection. (2) Cell proliferation of Molt-4 cells was significantly inhibited by Smo-siRNA at 24 hours after transfection. (3) Hoechst staining results showed morphological changes of Molt-4 were in accordance with those of apoptotic cells. (4) The apoptotic rate was significantly increased in the Smo-siRNA group compared with the control group (P < 0.05). Findings from this study showed that suppression of Smo by RNA interference could effectively inhibit proliferation and induce apoptosis in Molt-4 cells, indicating that Smo-siRNA as gene targeted therapy or synergistic treatment has therapeutic potential in T-cell malignancies. 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 adiponectin in the treatment of nephrotic syndrome rats undergoing adipose stem cell transplantation Liu Qian, Zhang Yu-jing, Liu Jing, Wang Jing, Xie Jin 2018, 22 (13):  2109-2113.  doi: 10.3969/j.issn.2095-4344.0519 Abstract ( 336 )   PDF (1185KB) ( 115 )   Save BACKGROUND: Adiponectin can regulate glucose and lipid metabolism in the body and protect body tissues and organs. Therefore, we combine it with stem cell transplantation for the treatment of nephrotic syndrome. OBJECTIVE: To investigate the therapeutic effect of adiponectin combined with adipose stem cell transplantation in the rats with nephrotic syndrome.  METHODS: Rat adipose stem cells were resuscitated and cultured in vitro, and labeled using CD-Dil prior to the transplantation. Eighty Sprague-Dawley rats were randomly assigned into a normal control group, a model group, an adipose stem cell transplantation group, and a combined group (adiponectin and adipose stem cell transplantation), with 20 rats in each group. Rats in the normal control group received no treatment. The remaining rats in the latter three groups were given a single intravenous injection of adriamycin (6 mg/kg) to prepare animal models of nephrotic syndrome, followed by a tail vein injection of normal saline, 2×106/L adipose stem cell suspension, and 1 μg/kg adiponectin plus 2×106/L adipose stem cell suspension, respectively. Administration of adiponectin in the combined group was given once a day, for 3 consecutive days. RESULTS AND CONCLUSION: (1) Compared with the normal control group, higher levels of 24-hour urinary protein, blood cholesterol and urea nitrogen as well as lower serum albumin level were observed in the remaining three groups at 28 days after modeling. Compared with the model group, the levels of 24-hour urinary protein, blood cholesterol and urea nitrogen in the adipose stem cell transplantation group were decreased, while the level of serum albumin was increased. Compared with the adipose stem cell transplantation group, the levels of 24-hour urinary protein, blood cholesterol and urea nitrogen in the combined group were decreased, while the level of serum albumin was increased. (2) At 28 days after modeling, severest renal damage appeared in the model group, while the damage was reduced in the adipose stem cell transplantation group and considerably relieved in the combined group accompanied by improved inflammation and edema. (3) The number of CM-Dil positive cells in renal tissues was highest in the combined group followed by the adipose stem cell transplantation group, but no positive cells were detected in the normal control and model groups. Our experimental findings reveal that adipose-derived stem cell transplantation has a certain therapeutic effect on nephrotic syndrome, and adiponectin can enhance this therapeutic effect, by improving renal function and effectively inhibiting the pathological changes of renal tissues. 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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Superparamagnetic iron oxide labeling influences in vitro differentiation of induced pluripotent stem cells Xie Qing-song, Shan Yu-dong, Fu Xiao-jun, Xu Xin-long, Hua Jie, Wen Lu-tong 2018, 22 (13):  2114-2119.  doi: 10.3969/j.issn.2095-4344.0487 Abstract ( 360 )   PDF (2044KB) ( 163 )   Save BACKGROUND: Superparamagnetic iron oxide (SPIO) labeling technology is a classic noninvasive tracing method, which has been widely used in the stem cell transplantation. Induced pluripotent stem cells (iPSCs) are currently one of the most promising seed cells for cell transplantation. Whether SPIO labeling can also be used to noninvasively trace induced pluripotent stem cells is rarely reported, and concern has been raised about whether SPIO markedly impacts the differentiation of iPSCs. OBJECTIVE: To investigate the effects of SPIO labeling on the differentiation of iPSCs in vitro.  METHODS: Rat fibroblasts were isolated and cultured. Efficient recombinant vector and plasmids that were packaged by virus and contained target genes (Oct4, Sox2, Klf4 and c-Myc) were transfected into 293T cells for virus packaging and production. The packaging lentiviral vectors that contained target genes infected rat fibroblasts to obtain iPSCs. SPIO-labeled (experimental) or unlabeled (control) iPSCs were subjected to neural induction and differentiation. Prussian blue staining and transmission electron microscope observation were performed for SPIO-labeled iPSCs. Immunohistochemical method was used to detect neuron-specific enolase expression after induced differentiation. Flow cytometry was used to detect the proportion of neurons and glial cells differentiated from iPSCs.  RESULTS AND CONCLUSION: There were dense iron particles in the cytoplasm of SPIO-labeled iPSCs shown by Prussian staining and under transmission electron microscope. Differentiated iPSCs were positive for neuron-specific enolase. In addition, the proportion of neurons and glial cells showed no difference between the experimental and control groups. To conclude, SPIO labeling has no obvious effect on the capacity of iPSCs differentiating into neurons. Reasonable application of this new cell labeling technique will promote the development of seed cells in regenerative medicine. 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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Rolling adhesion of HL-60 cells treated with all-trans retinoic acid on E-selectin Xiao Jing, Li Qu-huan, Yang Bi-shan, Fang Ying, Wu Jian-hua 2018, 22 (13):  2120-2125.  doi: 10.3969/j.issn.2095-4344.0499 Abstract ( 392 )   PDF (1370KB) ( 113 )   Save BACKGROUND: All-trans retinoic acid (ATRA) is an ideal therapy for acute promyelocytic leukemia, which can induce promyelocytes to differentiate to mature granulocytes. However, differentiation syndrome is still a high risk for the patients undergoing ATRA therapy. Occurrence of this complication is closely related with cellular morphology change and expression and function of cellular adhesion molecules, especially selectin and integrin families. OBJECTIVE: To reveal rolling adhesion behavior and mechanical mechanism of ATRA treated HL-60 cells on the substrate coated with E-selectin under different fluid shear forces. METHODS: Using the equipment of parallel plate flow chamber, untreated and ATRA treated HL-60 cells were driven to roll on E-selectin-coated substrate. The mean rolling velocity and mean stop time were calculated. Here, the HL-60 cells were incubated in the medium containing 1×10-6 mol/L ATRA for 0, 48, 72, 96 hours. The substrates were captured with 40 μg/L E-selectin overnight and the shear stresses were set to 0.02, 0.04, 0.06 Pa. RESULTS AND CONCLUSION: The velocity of untreated/treated HL-60 cells decreased firstly and then increased with monotonously increasing shear stress. On the contrary, the mean stop time and factional stop time increased firstly and then decreased. Therefore, we deduced that the flow enhanced rolling adhesion was regulated by the catch bond for the HL-60 cells rolling on E-selectin under flow. On the other side, rolling velocities decreased under the same shear stress even if treated with or without ATRA, and the mean stop time and factional stop time increased inversely, which further illustrate the rolling velocity is mainly regulated by stop time.  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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Dental pulp stem cells in tissue engineering: application and development Shao Miao-miao, Liu Zhong-xi, Xu Nuo, Liu Qing-hua, Wang Dong, He Jian-ya, Li Xiao-jie 2018, 22 (13):  2126-2132.  doi: 10.3969/j.issn.2095-4344.0429 Abstract ( 547 )   PDF (1264KB) ( 280 )   Save BACKGROUND: As a special source of stem cells, dental pulp stem cells (DPSCs) make much progress in the development of tissue engineering field due to their high proliferation and self-renewal ability. In the certain conditions DPSCs can be induced to differentiate into a variety of specialized tissue cells, providing a new way for tissue engineering development. OBJECTIVE: To review the main progress in the DPSCs biological characteristics, original source, isolation method, and its related application in tissue engineering research. METHODS: “Dental pulp stem cell, differentiation, regenerative medicine, tissue engineering” in English and Chinese were termed as the keywords to search relevant articles about DPSCs and tissue engineering published from 2005 to 2017 in PubMed, Medline, WanFang, and CNKI databases. After removal of repetitive or irrelevant articles, 66 articles were finally reviewed. RESULTS AND CONCLUSION: With the development of tissue engineering and regenerative medicine, the effective combination of DPSCs and tissue engineering scaffolds have be further achieved. Recent studies on DPSCs focus on the properties of DPSCs differentiating into odontoblasts and osteocytes/osteoblasts and on the potential of nerve repair, vascular remodeling, corneal reconstruction and chondrogenic 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