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 study the effect of adenovirus bone morphogenetic protein 2 gene(Ad-BMP-2) transfer inducing mesenchymal stem cells (MSCs) compounded with fibrin gel on repair of rabbit cartilage defect. Methods ①BMP-2 and collagen type Ⅱ in MSCs transferred by Ad-BMP-2 were examined by RT-PCR, aniline dyeing and immunohistochemical analysis in vitro. ②MSCs were cultured in fibrin gel for 9 days, and were examined with electron microscope. ③Fortytwo rabbits suffering from cartilage defect were divided into 3 groups:the defects were treated with Ad-BMP-2 transfer inducing MSCs compounded with fibrin in group A, with MSCs compounded with fibringel in group B and with no implants in group C as control. HE and aniline dyeing, immunohistochemical analysis and biomechanics study were carried out in the 4th, 8thand 12th weeks. Results ①The positive results were observed for BMP-2 and collagen type Ⅱ with RT-PCR on the 3rd day and 5th day respectively, being statisticallysignificant difference when compared with control group(P＜0.05). ②Ad-BMP-2 transfer inducing MSCs cultured in fibrin gel were positively stained by aniline dyeing and immunohistochemstry. ③The therapy effect of group A was better than that of the other two groups in histology, biochemistry and biomechanics, and the biomechanic and histological features of repaired cartilage were similar to those of the natural cartilage. Conclusion Ad-BMP-2 can induce the expressionof collagen type Ⅱ and mucopolysaccharide in MSCs by secreting BMP-2, and can reconstruct articular cartilage defects better when compounded with fibrin gel.
ObjectiveTo comprehensively analyze the recent advancements in the field of mesenchymal stem cells (MSCs) aging,and summary its achievements and its difficulty at the present.
MethodsThe literature about MSCs aging was reviewed and analyzed.
ResultsInducible telomerase reactivation of MSCs is successful to extend the life span of senescent cells,but it also has potential safety hazard.The age range presented in the research of age-related cell senescence is inconsistent,resulting in different outcomes.Many ways to improve cell in vitro culture conditions will help delay aging.Recent research indicates that oxidative stress theory is seemed to not completely explain cell aging.
ConclusionFurther research of MSCs aging mechanism will help the tissue engineering transform to clinical application.
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 investigate the effects of the recombinanthuman bone morphogenetic protein 2 (rhBMP-2) and/or the osteogenic agents on proliferation and expression of the osteoblast phenotype differentiation of the SD rat mesenchymal stem cells（MSCs）. Methods The rat MSCs were cultured in vitro and were randomly divided into the experimental groups(Groups A-I) and the control group. In the experimental group, MSCs were induced by rhBMP2 in different doses (10, 50, 100 and 200 μg/L) in Groups BE, the osteogenic agent alone (Group A) and by the combined use of rhBMP-2 ［in different doses (10,50, 100 and 200 μg/L)］ and the osteogenic agent in Groups F-I. The MTT colorimetric assay was used to evaluate the proliferation, and the activities of alkaline phosphatase (ALP) and osteocalcin (OC) were observed at 3, 6, 9, 12 days, respectively. Results The inverted phase contrast microscopy showed that MSCs by primary culture for 12 hours were adhibited, with a fusiform shape at 48 hours. At 4 days they were polygonal or atractoid, and were spread gyrately or radiately at 6 days. At 10 days, they were spread at the bottom of the bottle.The statistical analysis showed that the expression of the osteoblast phenotype differentiation of MSCs could be induced in the experimental groups. The proliferation of MSCs could be enhanced in a dosedependent manner in GroupsB-E. The expression of the osteoblast phenotype differentiation, which was tested by the activities of ALP and OC, was significantly higher in Groups F-I than in Groups A-E. Conclusion The combined use of rhBMP-2 and the osteogenic agents can enhance the MSC proliferation and induce an expressionand maintenance of the osteoblast phenotype differentiation of the rat MSCs.
Abstract: Objective To study the integration of transplanted cells and host cells by detection of the cellcell junction after transplantation of the myocardiumlike cell derived from the canine umbilical cord blood. Methods The mesenchymal stem cell(MSCs) was transfected by Laz-Z after harvest, culture, induced by 5-azacytidine(5-aza). Thirty-six adult hybrid dogs were randomly divided into cell transplantation group and control group. The canine of myocardium infarction was established. 107 MSCs were transplanted into dogs with acute myocardium infarction by coronary artery infusion and local injection in cell transplantation group and physiologic saline was used in the control group. The specimens were harvested and detected by immunofluorescence for 2, 4 and 8 weeks respectively. Results The umbilical cord blood MSCs were fusiform or spindleshaped. They presented clonal and knittinglike growth.The MSCs could differentiate into myocardium-like cell by the induction of 5-aza and express α-actin, desmin, connexin43.The transplanted cells could survive more than 8 weeks after transplantation. Cadherin and connexin 43 were found in the position of cellcell junction of transplanted cells group and between transplanted cells and host cells. Cadherin and connexin 43 were found in the hose cells of the control group. Conclusion The umbilical cord blood MSCs is able to differentiate into myocardiumlike cell in vitro and form cellcell junction in vivo to communicate with surrounding cells.
Objective?To review the recent progress of the researches in construction of tissue engineered osteochondral composites, and to discuss the challenges in construction of tissue engineered osteochondral composites.?Methods?The recent literature on the construction of tissue engineered osteochondral composites was extensively reviewed and analyzed.?Results?The studies on the construction of tissue engineered osteochondral composites are relatively more in vivo, the current focus is that different tissues derived mesenchymal stem cells are widely used to be seed cells; single-phase scaffold has been limited, studies on biphase scaffold and triphase scaffold are new trends; the design and performance of bioreactor need to be further optimized in the future.?Conclusion?The construction of tissue engineered osteochondral composites will be a promising method for the treatment of cartilage defects.
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 observe the effects of the bone marrow mesenchymal stem cells (BMSCs) on the expression of neurotrophic factor protein gene in the retinal detachment (RD) rabbits.
Methods
60 healthy rabbits were randomly divided into control group (group A), retinal detachment with PBS group (group B), retinal detachment with BMSCs group (group C), 20 rabbits in each group. RD model were established for rabbits in group B and C. 10 μl PBS was injected into the subretinal space of rabbits in group B, while 10 μl CM-Dil labeled BMSC PBS was injected into subretinal space of rabbits in group C. The rabbits in the group A received no treatment. At 1, 2 and 4 weeks after modeling, the mRNA expression of basic fibroblast growth factor (bFGF), brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) were measured by real-time quantitative PCR.
Results
At 1, 2 and 4 weeks after modeling, the mRNA expression of bFGF, BDNF, CNTF on retinal tissue were increased significantly in group C as compared with group A and B (P < 0.01). At 1 week after modeling, the mRNA expression of bFGF and CNTF on retinal tissue were increased significantly in group B as compared with group A, the mRNA expression of BDNF on retinal tissue in group B was similar with group C. At 2 and 4 weeks after modeling, the mRNA expression of bFGF, BDNF, CNTF were decreased in group B as compared with group A.
Conclusion
Subretinal transplantation of BMSC can increase the mRNA expression of bFGF, BDNF and CNTF on retinal tissue in RD rabbits.
Mesenchymal stem cells (MSCs) are considered as an ideal treatment for multiple diseases including ocular disease. Recent studies have demonstrated that MSCs-derived exosomes have similar functions with MSCs. Exosomes are nanovesicles surrounded by a phospholipid layer that shuttle active cargo between different cells. They are capable of passing the biological barrier and have potentials to be utilized as natural carrier for the ocular drug delivery.
