ObjectiveTo comprehensively analyze the recent advancements in the field of mesenchymal stem cells (MSCs) derived exosomes (MSCs-exosomes) in tissue repair. MethodsThe literature about MSCs-exosomes in tissue repair was reviewed and analyzed. ResultsExosomes are biologically active microvesicles released from MSCs which are loaded with functional proteins, RNA, and microRNA. Exosomes can inhibit apoptosis, stimulate proliferation, alter cell phenotype in tissue repair of several diseases through cell-to-cell communication. ConclusionMSCs-exosomes is a novel source for the treatment of tissue repair. Further research of MSCs-exosomes biofunction, paracellular transport, and treatment mechanism will help the transform to clinical application.
Objective To observe the effects of cryopreservation and resuscitation on the biological characteristics of mesenchymal stem cells (MSCs) derived f rom human umbilical cord blood. Methods MSCs were isolated and cultured f rom human umbilical cord blood in vitro. The cells were passaged , and the third generation of MSCs were cryopreserved in－196 ℃ liquid nitrogen for 4 weeks with cryopreservation medium , which contained 10 % dimethyl sulfoxide (DMSO) and 90 % fetal calf serum ( FCS) . The morphology , proliferation and differentiation of MSCs were investigated and compared with those of MSCs before cryopreservation. Results There was no significant difference of morphology between pre-cryopreserved MSCs and the ones af ter resuscitation. It was observed that all MSCs were spindle-shaped and showed adherence growth characteristic before and af ter cryopreservation. The cell growth curves of MSCs were also similar before and af ter cryopreservation. Even though the curve of resuscitated MSCs descended a little as compared with that of pre-cryopreserved MSCs , there was no significant difference ( Pgt; 0. 05) . After 2-week adipocytic differentiation induction , fat drops could be found in the kytoplasm of MSCs and they were red when stained with oil-red O staining , which suggested that MSCs could be induced and differentiated into adipocytes. Af ter 4-week osteoblastic differentiation induction , MSCs could be induced and differentiated into osteoblasts , and calcium node showed black when stained with Von Kossa staining. There were no significant changes of the differentiating ability of MSCs into adipocyte and osteoblast before and after cryopreservation. Conclusion MSCs derived from human umbilical cord blood maintains their biological characteristics af ter cryopreservation and resuscitation.
Objective To determine if mesenchymal stem cells ( MSCs) could be reconstructed as a vehicle for angiopoietin-1 ( Ang1) gene therapy in lung injury. Methods MSCs were obtained from adult male inbred mice and cultured to passage four. The cells were identified by fluorescence-activated cell sorting ( FACS) analysis and cell differentiation detection. Lentiviral vectors contained GFP and Ang1 gene were conducted in 293T cells through three plasmids co-transfection method. Then MSCs were transduced with Ang1 gene efficiently through lentiviral vectors. The mRNA expression of Ang1 in MSCs was detected by RT-PCR before and after transfection. Also fluorescence from MSCs was detected by fluorescence microscope every day after transfection. Two hours after LPS inhalation, mice were infused via jugular veinwith normal saline ( NS group) , lentiviral vector carrying Ang1 ( Ang1 group) , lentiviral vector carrying GFP ( MSCs group) , and lentiviral vector carrying Ang1 /GFP ( MSCs-Ang1 group) , respectively. Kaplan-Meier survival analysis was performed to compare the effects of MSCs-Ang1 on survival. And ectogenic MSCs origined lung cells were investigated in receipt mice. Results After passaged and purification,MSCs were confirmed to have the potential of differentiation. The lentiviral vectors carrying Ang1 and GFP were also identified. After transfection, the mRNA expression of Ang1 in MSCs was enhanced. Through the fluorescence microscope,MSCs get the most green fluorescence expression five days after the transfection when MOI was 20. Kaplan-Meier survival analysis showed that MSCs-Ang1 infusion had improved survival rates of lung injury rats compared with the control, but it did not reach statistical significance ( P = 0. 066) . Cells expressing GFP in lung tissues can be observed after MSCs were transplanted in vivo. Conclusions MSCs expressing Ang1 high can be constructed through lentiviral vector transfer, and MSCs-origined cells can be detected in receipt lungs after transplantation. So MSCs may serve as a vehicle for gene therapy in lung injury.
Objective To explore the effects of bone marrow mesenchymal stem cells ( BMMSCs) on pulmonary fibroblasts of patients with nonspecific interstitial pneumonitis ( NSIP) , and investigate the therapeutic mechanism of BMMSCs for interstitial pulmonary fibrosis. Methods Human BMMSCs, human pulmonry fibroblasts ( HPFs) from NSIP patients, and normal HPFs were primary cultured in vitro. Then HPFs fromNSIP patients were co-cultured with BMMSCs or normal HPFs using Transwell co-culture system. After 24 hours, levels of transforming growth factor β1 (TGF-β1) and interferon inducible protein 10 ( IP-10) in culture supernatants were detected by ELISA method. Meanwhile, interleukin-6 ( IL-6) , IL-8, and monocyte chemotactic protein-1 ( MCP-1) in co-culture supernatants were detected by liquid chip. After co-cultured for 48 hours, total protein of HPFs was extracted and the expression level of alpha smooth muscle actin ( α-SMA) secreted by HPFs were detected by Western blot.Results HPFs from NSIP patients secreted higher level of IL-6, IL-8, and MCP-1 than normal HPFs, and secreted high level of α-SMA. In the Transwell co-culture system, human BMMSCs significantly reduced the levels of IL-6, IL-8, and MCP-1 secreted from HPFs of NSIP patients, and reduced the high expression of α-SMA in HPFs of NSIP patients. Conclusion Human BMMSCs can significantly reduce the secretion of IL-6, IL-8, MCP-1, and the expression of α-SMA in HPFs from NSIP patients.
Objective To explore the migration and differentiation of bone marrow mesenchymal stem cells（MSCs） in lung . Methods MSCs were harvested from a male Wister rat. Sixty female Wister rats were randomly divided into four groups. The pulmonary fibrosis model was established by intratracheal instillation of bleomycin in group A-D. Immediately and 7 days after bleomycin administration respectively，the rats in group B and C received infusion with 5-bromodeoxynridine （BrdU） labeled MSCs via tail vein. And the rats in group D were infused MSCs without BrdU labeling serving as a negative control. The sry gene of Y chromosome was detected by polymerase chain reaction （PCR）. Double immunofluorescence staining was used to detected BrdU and surfactant associated protein-C （SP-C） expression in lung tissue，fresh bone marrow，and the 5th generation MSCs. Reverse transcriptipon-PCR was used to detect the expressions of SP-C mRNA and AQP-5 mRNA. Results The sry gene was detected in bleomycin induced lung injury tissues of the rats after MSCs infusion immediately and on the 7th day The MSCs in lung tissue could transformed into cells with ACEⅡ morphological features and molecular phenotype. The transformation rate was higher in the rats received MSCs infusion immediately than the rats received on 7th day. The 5th generation MSCs and fresh bone marrow expressed SP-C mRNA，without AQP-5 mRNA and SP-C expression. Conclusions Exogenous MSCs can be transplanted into injured lung tissues and transform into AECⅡ，especially in early stage of lung injury. The differentiation potential of MSCs can be activated in injury micro-environment.
Objective Bone marrow mesenchymal stem cells （MSCs） have been suggested to play an important role in the treatment of a variety of pulmonary diseases. The present study was aimed at evaluating the therapeutical effect of MSCs transplantation on emphysematous rats，and explore its influence in local and systemic inflammation. Methods Emphysema rat model was established by cigarette smoking. MSCs were transfected with lentivirus vector carrying green fluorecent protein （GFP） and the transfected MSCs in lung of smoke rats were detected by imaging system for small animals. Thirty-six SD rats were randomly divided into a control group，an emphysema group，and a MSCs transplantation group. The total and differential cell counts in bronchoalveolar lavage fluid （BALF） were measured. TNF-α and IL-1β levels in BALF and serum were measured by ELISA. Malonaldehyde （MDA） level in lung tissue was detected by chromatometry. Emphysema changes were evaluated by mean linear intercept （MLI） of lung under light microscope by HE staining. Results The transfected MSC in different lung lobes were found to be alive at four weeks after intrapulmonary delivery. Compared with the emphysema group，the total cell count in BALF，TNF-α and IL-1β levels in BALF and serum，MDA level in lung tissue and MLI were significantly reduced intheMSCs transplantation group（Plt;0.01）. Conclusions Transplantation of MSCs can mediate down-regulation of TNF-α and IL-1β in BALF and serum，attenuate inflammation，oxidative stress and emphysema change of lung，suggesting that MSCs have significant therapeutic effects on emphysema.
Objective To isolate，culture and expand bone marrow mesenchymal stem cells （MSCs） in vitro，induce MSCs to differentiate directionally towards chondrocytes，and provide experimental basis for clinical application of MSCs and construction of tissue engineering tracheal cartilage. Methods Cultured MSCs were isolated from bone marrow of Sprague-Dawley rats，purified using adherence separation，and identified by flow cytometry analysis. Transforming growth factor β1 （TGF-β1）and insulin-like growth factor 1 （IGF-1） were used as main induction factors to induce MSCs to differentiate directionally towards chondrocytes. The expression of collagen typeⅡwas evaluated by immunocytochemical staining 21 days after induction. Light microscope and electron microscope were used to observe tiny and ultrastructural changes of the cells before and after induction. Results The expression of collagen typeⅡwas positive by immunocytochemical staining 21 days after induction. MSCs were fusiform before induction under light microscope and electron microscope. After induction，the cells became larger，polygon，star-shaped or triangular. Transmission electron microscope showed that the cells had abundant organelles，larger nuclei and more nucleoli after induction. Conclusion Abundant organelles，larger nuclei and more nucleoli are the ultrastructure changes of chondrocytes differentiated from MSCs，indicating that the cells are active in differentiation and metabolism.
Abstract： Objective To study the difference of gene expression profile of bone marrow mesenchymal stem cells （MSCs） cultured in vitro from coronary heart disease patient with or without diabetes mellitus by Affymetrix Gene array. Methods One male patient at age of 53 years with coronary heart disease and diabetes mellitus was included in this study with the diagnosis of coronary heart disease and type 2 diabetes mellitus. Another male patient at age of 51 years with coronary heart disease without diabetes mellitus was also included in this study with the diagnosis of coronary heart disease. MSCs of the two patients were isolated and purified by the methods of density gradient centrifugation with lymphocyte separation medium for human and adherent filtration. The MSCs expression profile of cytokines and signal transduction genes were examined by Affymetrix gene array. Results There were 27 functional protein genes expression in the patient with coronary heart disease and diabetes mellitus relating to cell apoptosis, cytokine, and signal transduction. Among them, the expression of 13 functional genes, including TNFRSF10B, TNFRSF21, NGF, CAV2, ITGA8, TNS1, ITGA2, AKT3, MBP, MAP2, INHBA, FST, PLA2G5, increased significantly in the patient with coronary heart disease and diabetes mellitus. However, the expression level of 14 genes, including EPR1, BIRC5, HELLS, BCL2, HGF, CASP1, SEPP1, ITGA9, MAP2K6, RUNX3, TGFBR2, RUNX2, CTNNB1, CDC42, decreased significantly. Conclusion The gene expression profile of bone marrow MSCs from coronary heart disease patient with diabetes mellitus is significantly different from the patient with coronary heart disease patient without diabetes mellitus.
Objective To study the influence of three different ways of myogenic induction on Ca2+ regulation of mesenchymal stem cells (MSCs) derived from umbilical cord blood. Methods From January 2007 to April 2010, three different ways of myogenic induction including the adoptions of 5azacytidine, extraction of myocardium, and myocardial differentiation medium were used to induce MSCs derived from the umbilical cord blood of dogs in Xinhua Hospital of Shanghai Jiaotong University. Confocal laser scanning microscope was used to detect cells induced by the three abovementioned methods, cardiomyocytes and Ca2+ combined with Fluo3/AM inside the MSCs. For each group of cells, 2 to 5 visual fields were chosen, and 30 visual fields were recorded for each kind of cells. The mean fluorescence intensity of ten images shot in one minute was used to reflect the concentration of free intracellular Ca2+. Furthermore, the change of the concentration was continuously monitored by optical density(OD) value. Results After induction, the Ca2+ concentration inside the MSCs was significantly higher than that inside the cardiomyocytes (F=59.400, P=0.000). There was a statistical difference among the intracellular Ca2+ concentration induced respectively by 5azacytidine, extraction of myocardium, and myocardial differentiation medium (F=18.988, P=0.000). No significant difference existed between the intracellular Ca2+ concentration induced by 5-azacytidine and extraction of myocardium (OD value: 1 076.88±44.65 vs. 1 040.90±37.48, P=0.186), while the intracellular Ca2+ concentration induced by 5azacytidine was significantly higher than that induced by myocardial differentiation medium (OD value: 1 076.88±44.65 vs. 973.91±46.49, P=0.001), and the intracellular Ca2+ concentration induced by extraction of myocardium was significantly higher than that induced by myocardial differentiation medium (OD value: 1 040.90±37.48 vs. 973.91±46.49, P=0.001). The concentration of intracellular Ca2+ induced by all the three different methods fluctuated spontaneously, which was quite similar with the cardiomyocytes, but the frequency and the scope of the fluctuation were quite different. Ca2+ was released instantly by KCl stimulation in the two groups of MSCs pretreated by 5-aza and extraction of myocardium. Though MSCs pretreated by myocardial differentiation medium had response to KCl stimulation, Ca2+ could not be released in this group. On the contrary, the duration of Ca2+ release was prolonged. Conclusion Ca2+ regulation system of MSCs derived from umbilical cord blood can be influenced by these myogenic inductions. However, the reason and effect of the differences need to be elucidated by further investigation.
Objective To observe the morphology and growing status of mesenchymal stem cells(MSCs) of human bone marrow in vitro, in order to confirm that MSCs of human bone marrow are ideal seed cells and provide basic theory for further MSCs research. Methods The methods of density gradient centrifugation with lymphocyte separation medium for human and adherent filtration were used to isolate and purify MSCs of human bone marrow. We observed the cellular growth status and morphology of the primary MSCs and the surface antigens of second passage MSCs were tested. Results The primary culture cells fused into monolayer after 14-16 d. The passage cells kept the same morphological characteristics of primary culture cells. Ultrastructure of the second passage MSCs showed that the shape of nuclei was irregular, there were multiple nucleoli in some of the nuclei, and morphological differentiation of intracytoplasm organelles was immature. The growth curve of the first, fifth and tenth passage cells showed a logarithmic growth at day 3, a peak growth at day 5, and no clones occurred after tenth passage. Cloning efficiency of first passage, fifth passage and tenth passage was respectively 25.83%±2.93%, 14.67%±1.63% and 4.67%±0.52%. Test of MSCs phenotypic characteristics showed a high homogeneity among the cells and surface antigen profiles were positive for CD29, CD44 and negative for CD34, CD45. Conclusion The methods of density gradient centrifugation with lymphocyte separation medium for human and adherent filtration are simple, economic and efficient to isolate and purify MSCs from human bone marrow. With a high proliferating ability in vitro, MSCs from human bone marrow are ideal seed cells for tissue engineers.