芪参益气滴丸联合间充质干细胞移植改善小鼠心功能与巨噬细胞表型的相关性研究

目的:基于心肌梗死后巨噬细胞在损伤心肌修复过程中的主要效应及炎症影响干细胞的存活,探讨芪参益气滴丸是否可以影响巨噬细胞表型从而达到提高骨髓间充质干细胞的存活率、加强心肌细胞保护作用。方法:本研究采用eGFP+/-雄性实验小鼠,将小鼠随机分为PBS组(MI+PBS)、干细胞移植芪参益气滴丸组(MI+MSCs+QSYQ)和干细胞组(MI+MSCs),每组20只实验小鼠。心肌梗死模型造模前2周干细胞移植芪参益气滴丸组开始予芪参益气滴丸溶液灌胃(3.9 mg/mL,无菌水溶解),而其他两组则予等量无菌水灌胃作为对照。将实验前期分离培养所获得小鼠骨髓间充质干细胞计数后PBS重悬备用,采取开胸结扎心脏前降支建立小鼠心肌梗死模型,建立心肌梗死模型后运用30G进样针抽取20μL干细胞悬液,在心肌小鼠模型梗死区取4个点均匀注射,每个点注射量约5μL。空白组则予抽取等量的PBS,操作方法一致,术后撤除呼吸机,缝合术口。造模前、干细胞移植后2周、3周行超声心动图检测,评估心功能及左室重构情况,观察3周后处死实验小鼠,留取心脏标本予HE染色计算心肌梗死面积,并最终于荧光显微镜下观察巨噬细胞表型特征。结果:干细胞移植3周后心脏超声评估结果显示MI+MSCs+QSYQ组的LVIDd、LVIDs明显小于另外两组,而心脏FS和EF值均明显升高(P<0.05),心肌HE染色结果提示与单纯注射PBS相比较,移植MSCs能够明显减少梗死面积,且明芪参益气滴丸联合MSCs能够进一步减少心肌梗死面积。免疫荧光双标方提示间质充质干细胞移植3周后3组实验小鼠心肌梗死区均无M1型巨噬细胞亚群滞留,而存在大量M2型巨噬细胞浸润,并且MI+MSCs+QSYQ组的M2型巨噬细胞数量最多。结论:心肌梗死后骨髓间充质干细胞移植具有促进小鼠巨噬细胞向M2型表型转化的作用,而芪参益气滴丸联合骨髓间充质干细胞移植能够进一步增加巨噬细胞向M2型表型转化及炎症消退、心肌的修复效应。

Mesenchymal stromal cell-based therapy: Regulatory and translational aspects in gastroenterology

The past decade has witnessed an outstanding scientific production focused towards the possible clinical applications of mesenchymal stromal cells(MSCs) in autoimmune and chronic inflammatory diseases. This raised the need of novel standards to adequately address quality, efficacy and safety issues of this advanced therapy. The development of a streamlined regulation is currently hampered by the complexity of analyzing dynamic biological entities rather than chemicals. Although numerous pieces of evidence show efficacy in reducing intestinal inflammation, some inconsistencies between the mechanisms of action of rodent vs human MSCs suggest caution before assigning translational value to preclinical studies. Preliminary evidence from clinical trials showed efficacy of MSCs in the treatment of fistulizing Crohn's disease(CD), and preparations of heterologous MSCs for CD treatment are currently tested in ongoing clinical trials. However, safety issues, especially in longterm treatment, still require solid clinical data. In this regard, standardized guidelines for appropriate dosing and methods of infusion could enhance the likelihood to predict more accurately the number of responders and the duration of remission periods. In addition, elucidating MSC mechanisms of action could lead to novel and more reliable formulations such as those derived from the MSCs themselves(e.g., supernatants).

Mesenchymal stromal cells' role in tumor microenvironment:involvement of signaling pathways

Mesenchymal stromal cells(MSCs) are adult multipotent stem cells residing as pericytes in various tissues and organs where they can differentiate into specialized cells to replace dying cells and damaged tissues. These cells are commonly found at injury sites and in tumors that are known to behave like "wounds that do not heal." In this article, we discuss the mechanisms of MSCs in migrating, homing, and repairing injured tissues. We also review a number of reports showing that tumor microenvironment triggers plasticity mechanisms in MSCs to induce malignant neoplastic tissue formation, maintenance, and chemoresistance, as well as tumor growth. The antitumor properties and therapeutic potential of MSCs are also discussed.

Culture and Identification of Human Amniotic Mesenchymal Stem Cells

Objective To establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs). Methods hAMSCs were isolated from human amniotic membrane by trypsin-collagenase digestion, and cultured in Dulbecco's modified Eagle's medinm/F12 medium supplemented with 10% fetal bovine serum. Phenotypic characteristics of these cells were analyzed by means of immunocytochemistry and flow cytometry. Results The cells successfully isolated from human amniotic membrane expressed representative mesenchymal cell surface markers CD44, CD90, and vimentin, but not CD45. Conclusions This study establishes a potential method for isolation of hAMSCs from human amnion, in vitro culture, and identification. The isolated cells show phenotypic characteristics of mesenchymal stem cells.

Intravenous injection of mesenchymal stem cells is effective in treating liver fibrosis

AIM: To compare the influence of different transplant sites in bone marrow mesenchymal stem cell (MSC)-based therapy for liver fibrosis. METHODS: MSCs isolated from Sprague Dawley (SD) rats were induced into hepatocyte-like cells. Liver fibrosis in SD rats was induced with carbon tetrachloride. Following hepatocyte induction in vitro, 4',6-diamidino- 2-phenylindole (DAPI)-labeled MSCs were transplanted by intravenous, intrahepatic, and intraperitoneal injection. Histopathological staining, immunohistochemistry, and biochemical analysis were used to compare the morphological and functional liver regeneration among different MSC injection modalities. The expression differences of interleukins, growth factor, extracellular matrix, matrix metalloproteinases, and tissue inhibitor of metalloproteinase were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR) andenzyme linked immunosorbent assay (ELISA). RESULTS: Four days after exposure to hepatocyte differentiation medium, MSCs that did not express hepatocyte markers could express α-fetoprotein, albumin, and cytokeratin 18. The results of histopathological staining, immunohistochemistry, and biochemical analysis indicated that intravenous injection is more effective at rescuing liver failure than other injection modalities. DAPI-labeled cells were found around liver lobules in all three injection site groups, but the intravenous group had the highest number of cells. PCR and ELISA analysis indicated that interleukin-10 (IL-10) was highest in the intravenous group, whereas il1β, il6, tnfα and tgfβ, which can be regulated by IL10 and are promoters of liver fibrosis, were significantly lower than in the other groups. CONCLUSION: MSC administration is able to protect against liver fibrosis. Intravenous injection is the most favorable treatment modality through promotion of IL10 expression.

Comparison of human amniotic fluid-derived and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells： Characterization and myocardial differentiation capacity

Objective To compare the characterization and myocardial differentiation capacity of arnniotic fluid-derived mesenchymal stromal cells （AF MSCs） and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells （WJ MSCs）. Methods The human AF MSCs were cultured from amniotic fluid samples obtained by amniocentesis. The umbilical cord WJ MSCs were obtained from Wharton＇s Jelly of umbilical cords of infants delivered full-term by normal labor. The morphology, growth curves, and analyses by flow cytometry of cell surface markers were compared between the two types of cells. Myocardial genes （GATA-4, c-TnT, a-actin, and Cx43） were detected by real-time PCR and the corresponding protein expressions were detected by Western blot analysis after myocardial induced in AF MSCs and WJ MSCs. Results Our findings revealed AF MSCs and WJ MSCs shared similar morphological characteristics of the fibroblastoid shape. The AF MSCs were easily obtained than the WJ MSCs and had a shorter time to reach adherence of 2.7 ± 1.6 days to WJ MSCs of 6.5 ± 1.8 days. The growth curves by MTT cytotoxic assay showed the AF MSCs had a similar proliferative capacity at passage 5 and passage 10. However, the proliferative capacities ofWJ MSCs were decreased at 5 passage relative to 10 passage. Both AF stem cells and WJ stem cells had the characteristics of mesenchymal stromal cells with some characteristics of embryonic stem cells. They express CD29 and CD105, but not CD34. They were positive for Class I major histocompatibility （MHC I） antigens （HLA-ABC）, and were negative, or mildly positive, for MHC Class II （HLA-DR） antigen. Oct-4 was positive in all the two cells types. Both AF MSCs and WJ MSCs could differentiate along myocardium. The differentiation capacities were detected by the expression of GATA-4, c-TnT, a-actin, Cx43 after myocardial induction. Conclusions Both AF MSCs and WJ MSCs have the potential clinical application for myogenesis in cardiac regenerative therapy.

Effects of insulin-like growth factor-1 on the properties of mesenchymal stem cells in vitro

Objective:To explore the effects of insulin-like growth factor-1(IGF-1) on migration,proliferation and differentiation of mesenchymal stem cells(MSCs).Methods:MSCs were obtained from Sprague-Dawley rats by a combination of gradient centrifugation and cell culture techniques and treated with IGF-1 at concentrations of 5-20 ng/ml.Proliferation of MSCs was determined as the mean doubling time.Expression of CXC chemokine receptor 4(CXCR4) and migration property were determined by flow cytometry and transwell migration essay,respectively.mRNA expression of GATA-4 and collagen II was determined by reverse transcription-polymerase chain reaction(RT-PCR).Results:The mean doubling time of MSC proliferation was decreased,and the expression of CXCR4 on MSCs and migration of MSCs were increased by IGF-1,all in a dose-dependent manner,while the optimal concentration of IGF-1 on proliferation and migration was different.IGF-1 did not affect the expression of GATA-4 or collagen II mRNA.Conclusions:IGF-1 dose-dependently stimulated the proliferation of MSCs,upregulated the expression of CXCR4,and accelerated migration.There was no apparent differentiation of MSCs to cardiomyocytes or chondrocytes after culturing with IGF-1 alone.

Effects of mesenchymal stem cells transfected with human hepatocyte growth factor gene on healing of burn wounds

Objective： To explore the effects of bone marrow-derived mesenchymal stem cells （BMSCs）transfected with adenoviral vector carrying hepatocyte growth factor （HGF, Ad-HGF） on burn wound healing.Methods： BMSCs from male Wistar rats were separated and purified with Percoll separating medium by density gradient centrifugation and cultured with DMEM containing 20% fetal bovine serum （FBS）. Then BMSCs were transfected with Ad-HGF at the optimal gene transduction efficiency of 100 multiplicity of infection （MOI）. The efficiency of transfection and the expression of HGF in the suspension were detected by flow cytometry and enzyme linked immunosorbent assay （ELISA） respectively. Thirtytwo female rats were subjected to 90℃ water for 12 seconds to induce a partial thickness skin burn. The animals were randomly divided into mesenchymal stem cells （MSCs） treatment group （Group A）, Ad-HGF treatment group （Group B）,Ad-HGF-modified MSCs treatment group （Group C） and saline control group （Group D）. On days 3, 5, 7, 14 and 21 postburn, HE and Sirius red stain were performed to observe the burn wound healing and collagen content. The content of hydroxyproline in wounds was also detected.Transplanted cells and the expression of（sex-determining region Y） SRY gene were detected by in situ hybridization and polymerase chain reaction （PCR）, while the expression of HGF in wound tissues was detected by ELISA.Results： The result of flow cytometry showed that the transfection efficiency was 86.41% at 100 MOI. Compared with the control group, the content of HGF in the supernatant after transfection increased time-dependently and peaked at 48 h, showing significant differences at 24 h, 48 h,72 h and 96 h （P＜0.01 ）. Results of HE stain revealed that the range of re-epidermidalization in Group C was significantly larger than that in other groups in the first week. Three weeks postburn, the epidermis was significantly thicker in Group C than in other groups and the nails of dermis inserted into the derma of burn wounds. Sirius red stain showed that the content of collagen Ⅰ in Group C was much less compared with that in other groups 21 days postburn. In situ hybridization revealed an expression of SRY gene in burned female rats, consistent with the finding of PCR. Immunohistochemistry demonstrated the largest increase of HGF expression in Group C, whose contents of hydroxyproline,however, decreased on day 7 postburn. Compared with other groups, the content of HGF in the wounds of Group C increased obviously on day 14 after transfection （P＜0.05） and there was no significant difference among Groups A, B and D.Conclusion： Our study suggests that transplantation of MSCs modified with Ad-HGF has positive effects on the healing of burn wounds probably through differentiation and release of relevant cytokines.

Mesenchymal stem cells as therapeutic agents and in gene delivery for the treatment of glioma

Mesenchymal stem cells(MSCs) are plastic-adherent cells with a characteristic surface phenotype and properties of self-renewal, differentiation, and high proliferative potential. The characteristics of MSCs and their tumortropic capability make them an ideal tool for use in cell-based therapies for cancer, including glioma. These cells can function either through a bystander effect or as a delivery system for genes and drugs. MSCs have been demonstrated to inhibit the growth of glioma and to improve survival following transplantation into the brain. We briefly review the current data regarding the use of MSCs in the treatment of glioma and discuss the potential strategies for development of a more specific and effective therapy.

Labeling of cynomolgus monkey bone marrow-derived mesenchymal stem cells for cell tracking by multimodality imaging

Recently,transplantation of allogeneic and autologous cells has been used for regenerative medicine.A critical issue is monitoring migration and homing of transplanted cells,as well as engraftment efficiency and functional capability in vivo.Monitoring of superparamagnetic iron oxide(SPIO) particles by magnetic resonance imaging(MRI) has been used in animal models and clinical settings to track labeled cells.A major limitation of MRI is that the signals do not show biological characteristics of transplanted cells in vivo.Bone marrow mesenchymal stem cells(MSCs) have been extensively investigated for their various therapeutic properties,and exhibit the potential to differentiate into cells of diverse lineages.In this study,cynomolgus monkey MSCs(cMSCs) were labeled with Molday ION Rhodamine-BTM(MIRB),a new SPIO agent,to investigate and characterize the biophysical and MRI properties of labeled cMSCs in vitro and in vivo.The results indicate that MIRB is biocompatible and useful for cMSCs labeling and cell tracking by multimodality imaging.Our method is helpful for detection of transplanted stem cells in vivo,which is required for understanding mechanisms of cell therapy.

Effect of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro

Purpose： To investigate the influence of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro. Methods： Primary osteoblasts, bone marrow-derived mesenchymal stem cells （BMSCs, cultured in osteoinductive medium） and RAW264.7 cells cultured in osteoclast inductive medium were all subjected to a 1000μstrain （μs） at 1 Hz cyclic mechanical stretch for 30 min （twice a day）. Results： After mechanical stimulation, the alkaline phosphatase （ALP） activity, osteocalcin protein level of the osteoblasts and BMSCs were all enhanced, and the mRNA levels of ALP and collagen type I increased. Additionally, extracellular-deposited calcium of both osteoblasts and BMSCs increased. At the same time, the activity of secreted tartrate-resistant acid phosphatase, the number of tartrate-resistant acid phosphatase-positive multinucleated cells, matrix metalloproteinase-9 protein levels of RAW264.7 cells and the extracellular calcium solvency all decreased. Conclusion： The results demonstrated that 1000 μs cyclic mechanical loading enhanced osteoblasts activity, promoted osteoblastic differentiation of BMSCs and restrained osteoclastogenesis of RAW264.7 cells in vitro.

Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor

Objective:Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focusing on combining gene transfection with tissue engineering techniques.The aim of this study is to investigate the effect of connective tissue growth factor(CTGF) on the proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells(MSCs).Methods:A CTGF-expressing plasmid(pCTGF) was constructed and transfected into MSCs.Then expressions of bone morphogenesis-related genes,proliferation rate,alkaline phosphatase activity,and mineralization were examined to evaluate the osteogenic potential of the CTGF gene-modified MSCs.Results:Overexpression of CTGF was confirmed in pCTGF-MSCs.pCTGF transfection significantly enhanced the proliferation rates of pCTGF-MSCs(P<0.05).CTGF induced a 7.5-fold increase in cell migration over control(P<0.05).pCTGF transfection enhanced the expression of bone matrix proteins,such as bone sialo-protein,osteocalcin,and collagen type I in MSCs.The levels of alkaline phosphatase(ALP) activities of pCTGF-MSCs at the 1st and 2nd weeks were 4.0-and 3.0-fold higher than those of MSCs cultured in OS-medium,significantly higher than those of mock-MSCs and normal control MSCs(P<0.05).Overexpression of CTGF in MSCs enhanced the capability to form mineralized nodules.Conclusion:Overexpression of CTGF could improve the osteogenic differentiation ability of MSCs,and the CTGF gene-modified MSCs are potential as novel cell resources of bone tissue engineering.

Identification of neuron-related genes for cell therapy of neurological disorders by network analysis

Bone mesenchymal stem cells（BMSCs） differentiated into neurons have been widely proposed for use in cell therapy of many neurological disorders. It is therefore important to understand the molecular mechanisms underlying this differentiation. We screened differentially expressed genes between immature neural tissues and untreated BMSCs to identify the genes responsible for neuronal differentiation from BMSCs. GSE68243 gene microarray data of rat BMSCs and GSE18860 gene microarray data of rat neurons were received from the Gene Expression Omnibus database. Transcriptome Analysis Console software showed that 1248 genes were up-regulated and 1273 were down-regulated in neurons compared with BMSCs. Gene Ontology functional enrichment, protein-protein interaction networks, functional modules, and hub genes were analyzed using DAVID, STRING 10, BiN GO tool, and Network Analyzer software, revealing that nine hub genes, Nrcam, Sema3 a, Mapk8, Dlg4, Slit1, Creb1, Ntrk2, Cntn2, and Pax6, may play a pivotal role in neuronal differentiation from BMSCs. Seven genes, Dcx, Nrcam, Sema3 a, Cntn2, Slit1, Ephb1, and Pax6, were shown to be hub nodes within the neuronal development network, while six genes, Fgf2, Tgfβ1, Vegfa, Serpine1, Il6, and Stat1, appeared to play an important role in suppressing neuronal differentiation. However, additional studies are required to confirm these results.