Objective To explore the in vitrodifferentiation of the rat mesenchymal stem cells (MSCs ) into the skeletal muscle cells induced by the myoblast differentiation factor (MyoD) and 5-azacytidine. Methods The MSCs were taken from the rat bone marrow and the suspension of MSCs was made and cultured in the homeothermia incubator which contained 5% CO2at 37℃. The cells were observed under the inverted phase contrast microscope daily. The cells spreading all the bottom of the culture bottle were defined as onepassage. The differentiation of the 3rd passage of MSCs was induced by the combination of 5-azacytidine, MyoD, transforming growth factor β1, and the insulin like growth factor 1. Nine days after the induction, the induced MSCs were collected, which were analyzed with the MTT chromatometry, theflow cytometry, and the immunohistochemistry. Results The primarily cultured MSCs grew as a colony on the walls of the culture bottle; after the culture for 5-7 days, the cells were shaped like the fibroblasts, the big flat polygonal cells, the medium sized polygonal cells, and the small triangle cells; after the culture for 12 days, the cells were found to be fused, spreadingall over the bottle bottom, but MSCs were unchanged too much in shape. After the induction by 5-azacytidine, some of the cells died, and the cells grew slowly. However, after the culture for 7 days, the cells grew remarkably, the cell volume increased gradually in a form of ellipse, fusiform or irregularity. After theculture for 14 days, the proliferated fusiform cells began to increase in a great amount. After the culture for 18-22 days, the myotubes increased in number and volume, with the nucleus increased in number, and the newly formed myotubes and the fusiform myoblst grew parallelly and separately. The immunohistochemistry for MSCs revealed that CD44 was positive in reaction, with the cytoplasm ina form of brown granules. And the nucleus had an obvious border，and CD34 was negative. The induced MSCs were found to be positive for desmin and specific myoglobulin of the skeletal muscle. The flow cytometry showed that most of the MSCs and the induced MSCs were in the stages of G0/G1，accounting for 79.4％ and 62.9％，respectively; however, the cells in the stages of G2/S accounted for 20.6％ and 36.1％. The growth curve was drawn based on MTT，which showed that MSCs weregreater in the growth speed than the induced MSCs. The two kinds of cells did not reach the platform stage，having a tendency to continuously proliferate.ConclusionIn vitro,the rat MSCs can be differentiated into the skeletal muscle cells with an induction by MyoD and 5-azacytidine, with a positive reaction for the desmin and the myoglobulin of the skeletal muscle. After the induction, the proliferation stage of MSCs can be increased, with a higher degree of the differentiation into the skeletal muscle.
Objective To observe the influence of human umbilical cord mesenchymal stem cells (hUCMSC) transplanted into the tail vein of diabetic rats on apoptosis of retinal neurons and the retinal expression level of glial fibrillary acidic protein (GFAP). Methods Seventy clean male Sprague-Dawley rats were randomly divided into the normal control group (group A), diabetes mellitus (DM) only group (group B), DM + balanced salt solution (BSS) group (group C), DM + hUCMSC group (group D), with 10 rats in each group. DM rats were induced by intraperitoneal injection of streptozotocin. Apoptosis of retinal cells was assayed by dUTP nick end labeling. Immunohistochemistry and Western blot was performed to detect the retinal expressions of GFAP in rats. Results Compared with group A, large numbers of apoptotic cells could be found in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) of group B and group C, however the apoptotic cells in group D were significantly reduced than group B and C. The expression of GFAP was mainly located in the retinal GCL and retinal nerve fibre layer (RNFL) in group A, throughout the inner plexiform layer (IPL) in group B and C, only distributed in RNFL and GCL in group D. It was obvious that the expression of GFAP in group B and C was higher than group A. Compared with group B and C, the expression of GFAP in group D was significantly reduced. The difference of GFAP expression among the 4 groups was significant (F=79.635, P<0.05). Conclusion hUCMSC could inhibit the apoptosis of retinal cells and activation of glial cells in early DM rats.
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 review the advance in the experimental studies and evaluate the potential therapeutic application of the mesenchymal stem cells(MSCs). Methods The related articles published in China and theother countries during the recent years were extensively reviewed and analyzed. Results The MSCs were widely used in the cell-transplantation therapy and the tissue engineering because of their pluripotency of differentiation into various kinds of cells. They were also frequently used in the gene therapy because they could stably express the transfected objective genes. Because of their immunomodulatory function, the MSCs could also be used in the immunotherapy. Conclusion The MSCs are the stem cells, which have characteristics of renewing themselves, having multipotency, and being easy to undergo amplification in vitro.The MSCs are ideal target cells for the cell therapy, tissue engineering, gene therapy, and immunotherapy.
Congestive heart failure is a complication of myocardial infarction threatening human health. Although the pharmacotherapy is effective, it is still a worldwide challenge to thoroughly repair the injured myocardium induced by myocardial infarction. It has been demonstrated that mesenchymal stem cells (MSCs) can repair infarcted myocardium. Much evidence shows that MSCs can generate new myocardial cells in both human and animals' hearts. This review aims at discussing the therapeutic progress of the congestive heart failure treated with MSCs.
Objective To explore the method that can inducethe mesenchymal stem cells (MSCs) to differentiate into the neuronlike cells in vitro．Methods The neuron-like cells were isolated froman SD rat (age, 3 months; weight, 200 g). They underwent a primary culture; theinduced liquid supernatant was collected, and was identified by the cell immunohistochemistry. The C3H1OT1/2 cells were cultured, as an MSCs model, and they were induced into differentiation by β-mercaptoethanol (Group A) and by the liquid supernatant of the neuron-like primary cells (Group B), respectively. The cells were cultured without any induction were used as a control (Group C). Immunohistochemistrywas used to identify the type of the cells． Results The result of the immunochemistry showed that the cells undergoing the primary culture expressed the neurofilament protein (NF) and the neuronspecific enolase (NSE), and they were neuron-like cells. β-mercaptoethanol could induce the C3H1OT1/2 cells toexpress NF and NSE at 2 h, and the expression intensity increased at 5 h. The liquid supernatant of the primarily-cultured neuron-like cells could induce theC3H1OT1/2 cells to express NF and NSE at 1 d, but the expression intensity induced by the liquid supernatant was weaker than that induced by β-mercaptoethanol. The positivity rate and the intensity expression of NSE were higher than those of NF. Conclusion MSCs can differentiate into the neuron-like cells by β-mercaptoethanol and the microenvironment humoral factor, which can pave the way for a further study of the differentiation of MSCs and the effectof the differentiation on the brain trauma repair.
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 study the culture and purification of the fetal mouse liver mesenchymal stem cells(MSCs) in vitro and to investigate their differentiation potential and the composite ability with true bone ceramic(TBC). Methods The single cell suspension of MSCs was primarily cultured and passaged, which was prepared from the fetal mouse liver; the flow cytometry was applied to detectCD29, CD34, CD44 and CD45. The osteogenic differentiation was induced in chemical inducing system; the osteogenic induction potency was tested. The purified fetal mouse liver MSCs were compounded with TBC covered with collagen type Ⅰ in vitro and the cell attachment and proliferation to the TBC were observed. Results The primary MSCs of fetal mouse liver were easy to culture in vitro. They proliferated well and were easy to subcultured. The proliferation ability of primary and passaged MSCs was similar. Flow cytometric analysis showed the positive results for CD29, CD44 and the negative results for CD34, CD45. After 7 days of induction, the MSCs expressed collagen type I and alkaline phosphatase(ALP) highly. After 14 days of induction, the fixed quantity of ALP increased significantly. After 28 days of induction, calcium accumulation was observed by Von Kossa’s staining. Many liver MSCs attached to the surface of TBC. Conclusion The MSCs of the fetalmouse liver can be obtained, subcultured and purified easily. After culturing in chemical inducing system, the MSCs of fetal mouse liver can be successfully induced to osteoblast-like cells, attach to the surface of TBC and proliferate well.
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.
Objective To fabricate a novel gelatinchondroitin sulfate-sodium hyaluronate tri-copolymer scaffold and to confirm the feasibility of serving as ascaffold for cartilage tissue engineering. Methods Different scaffolds was prepared with gelatin-chondroitin sulfatesodium hyaluronate tri-copolymer by varying the freezing temperatures (-20℃,-80℃ and liquid nitrogen). Pore size, porosity, inter pores and density were observed with light microscopy and scanning electron microscopy (SEM). The load-stiffness curves were compared between different scaffolds and normal cartilage. The number of MSCs attaching to different scaffolds and the function of cells were also detected with MTT colorimetric microassay. Results The pore size was 300±45, 230±30 and 45±10 μm; the porosity was 81%, 79% and 56%; the density was 9.41±0.25, 11.50±0.36 and 29.50±0.61 μg/mm3 respectively in different scaffolds fabricated at -20℃,-80℃ and liquid nitrogen; the latter two scaffolds had nearly the same mechanical property with normal cartilage; the cell adhesion rates were 85.0%, 87.5% and 56.3% respectively in different scaffolds and the scaffolds can mildly promote the proliferation of MSCs. Conclusion Gelatin-chondroitin sulfatesodium hyaluronate tricopolymer scaffold fabricated at -80℃ had proper pore size, porosity and mechanical property. It is a novel potential scaffold for cartilage tissue engineering.