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 explore effects of zinc on the contents of cycl in D2, cycl in-dependent kinase 4 (CDK4), and their DNA and total cellular protein in human umbil ical cord blood-drived mesenchymal stem cells (hUCBMSCs). Methods hUCBMSCs were isolated and cultured by density gradient centrifugation adherence method in vitro. At the serial subcultivation, the hUCBMSCs were randomly divided into 7 groups. In control group, hUCBMSCs were cultured with DMEM medium (containing 15%FBS). In treatment groups, hUCBMSCs were cultured with DMEM medium (containing 15%FBS plusZnSO4?7H2O). The final concentrations of zinc were 0.5, 1.5, 2.5, 3.5, 4.5, and 5.5 mg/L, respectively. The cellular surface antigens of CD29, CD34, CD44, and CD45 at the 3rd generation of hUCBMSCs were detected by flow cytometry. MTT assay was used to detect cell activity of the 3rd generation of hUCBMSCs. The optimum concentration of zinc was selected by the results of MTT as experimental group. The cell growth curves of experimental group and control group were drown by counting cell. The cell surface antigen, reproductive cycle, and DNA content were detected by flow cytometry motheds. The contents of cycl in D2 and CDK4 were detected by Western blot method. Results The positive expression rates of CD29 and CD44 were more than 70% in hUCBMSCs. The cell activity of 2.5 mg/L treatment group was superior to other treatment groups, as experimental group. At 7, 14, and 28 days, the contents of DNA, total cellular protein, cycl in D2, and CDK4 of hUCBMSCs were significantly higher in experimental group than those in control group (P lt; 0.01). The percentage of hUCBMSCs at S stage and prol iferation index in experimental group were also significantly higher than those in control group (P lt; 0.01). Conclusion Zinc (0.5-4.5 mg/L) has the promoting effect on the hUCBMSCs activity, and 2.5 mg/L is the optimal concentration. Zinc (2.5 mg/L) can accelerate the prol iferation and DNA reproduction of hUCBMSCs and increase the contents of cycl in D2 , CDK4, and cellular total protein.
Objective To monitor the stem cell migration into the bone defect following an injection of the labeled mesenchymal stem cells (MSCs) by the enha nced green fluorescent protein (EGFP)technology and to provide insights into an application of MSCs for the fracture healing. Methods Isolated MSCs from the rabbit femur marrow were culture-expanded and were labeled by the transfection with the recombinant retrovirus containing the EGFP gene. Then, some labeled MSCs were cultured under the osteogenic differentiation condition and the phenotype was examined. After the fracture of their bilateral ulna, 18 rabbits were divide d into two groups. The labeled MSCs were injected into the aural vein at 1×107 cells／kg in the experimental group and the unmarked MSCs were injected in the control group 24 hours before surgery, and 1 and 24 hours after surgery, res pectively. Necropsies were performed 2 days after surgery in the two groups. The sections from the left defects were observed under the fluorescence microscope and the others were analyzed by the bright-field microscopy after the HE staining. Results The EGFP did not affect the MSCs viability. After the labeled cells were incubated in the osteogenic medium alkaline phosphatase, the calcium nodule s were observed. All the rabbits survived. The tissue of haematoma was observed in the bone defects and the fluorescent cells were found in the experimental gr oup, but no fluorescent cells existed in the control group. Conclusion The EG FP labeled MSCs can undergo osteogenic differentiation in vitro and can mig rate into bone defects after their being injected into the peripheral vein.
Objective To investigate the effect of the synthetic bone morphogenetic protein 2 (BMP-2)derived peptide on the osteogenic induction in the marrow mesenchymal stem cells (MSCs）and to evaluate the osteoinductivity and dosedependence of the BMP-2 derived peptide in vitro. Methods MSCs of 4-week old Wistar rats were separated and cultured. In the 3rd passage, the conditional culture medium was changed, in which the BMP-2-derived peptide in the following doses was added: 300,200, 100, 50, and 0 μg/ml, respectively (Groups A-E). The activity of alkaline phosphatase (ALP）and the amount of calciumdeposition were meassured at 5，10，15 and 20 days during the culture with the conditional culture medium. The real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was performed to measure the mRNA expressions of collagen type Ⅰ, osteopontin (OPN), and osteocalcin（OCN）and to measure the osteoinductivity of the BMP-2-derived peptide in the different concentrations.Results Under the inverted phase contrast microscope, MSCs cultured in the conditional culture medium for 3-4 days were changed in shape, from long fusiform to short fusiform or polygon. As the concentration of the BMP-2-derived peptide increased, the time for MSCs to change into the osteoblasts decreased. There was a significantly greater level of the ALP activity and amount of the calcium deposition in Groups A and B than in the other groups（Plt;0.05）. However,there was no significant difference between Group A and Group B （Pgt;0.05）. Theresult of FQPCR showed that after MSCs were cultured in the different doses of theconditional culture medium for 14 days, the mRNA expressions of collagen type Ⅰ, OPN andOCN were at higher levels. An increasing order in the level of the cycle threshold (Ct) was found in the following groups: Agt;Bgt;Cgt;D. Almost no expression was found in Group E. The Ct levels were significantly greater in Groups A and B thanin Groups C and D（Plt;0.05）. However, there was no significant difference between Group A and Group B （Pgt;0.05）.ConclusionThe BMP-2-derived peptide can greatly promote differentiation of MSCs into the osteoblasts, the promotion of osteogenesis has a dosedependent pattern, and the best inducing dosage is 200 μg/ml.
Objective To provide theoretical evidence for clinical application of the epidermal stem cells after an investigation on changes of the epidermal stem cells during the survival process after the fullthickness skin autograft. Methods On the backs of 42 Wistar rats, orthotopic transplantation models (1.5 cm×1.5 cm) of the fullthickness skin autograft were made. According to the time of the specimen taking, at 1, 3, 5, 7, 14, 21 and 30 days after operation, the rats were randomly divided in 7 groups (Groups 1-7). Specimens taken in each group before operation were used as controls. At each time point, the gross observation was made on the transplanted skin flaps, from which the skin tissues were harvested at each time point before and after operation. The routine pathological and the immunohistochemical examinations were performed on the specimens, which were stained by HE and were observed for immunohistochemical changes and the changes in the cells positive for integrinβ-1 and p63. Results All the fullthickness skin autografts survived 3 days after operation except the skin autograft in 1 rat in both Group 5 and Group 6, which was infected around the transplanted skin flap. In Groups 1-4, cell edema, inflammatory cell infiltration, and increased fibrocytes were observed. In Groups 5-7, the maturity degree of the epithelial cells became higher and higher, and the fibrocyte proportion was lowered. In each group the cell positivity rate for integrin β1 was lower than the cell positivity rate for p63. The positive cells were arranged in disorder, distributed into the layers of the epidermis and gradually concentrated in the basal layer of the epidermis and the bulge of the folliculus pili. The positive cells were also found in the other layers of the epidermis.The positive cells were gradually decreased in number, and reached the lowest level in Group 2. There was a significant difference in the above variables in Groups 1,2,3,5,6 and 7 between before and after operations (P＜0.05). Conclusion During the survival process of the fullthickness skin autograft, the proportion of theepidermal stem cells is gradually decreased at first; Then, the proportion isgradually increased, even beyond the normal level; finally, the proportion is decreased again. The distribution of the epidermal stem cells appear in disorder, almost distributed in the layers of the epidermis; finally, the almost normal distribution can be found.
ObjectiveTo summarize the isolation procedures, molecular characterization, and differentiation and vascularization capacity of adipose-derived stem cells (ADSCs), in order to discuss the potential value of ADSCs for the repairment and regeneration of adipose tissues. MethodsRelated literatures about ADSCs were retrieved to summarize the potential value of ADSCs for the repairment and regeneration of adipose tissues. ResultsAs mesenchymal stem cells, ADSCs was rich in human adipose tissues. ADSCs possessed the potential to differentiate toward a variety of cell lineages, such as adipogenic, chondrogenic, osteogenic, cardiomyogenic, myogenic, and angiogenic. Besides, its capacity of adipogenic differentiation could maintain several passages. The most importantly, ADSCs could secrete significant amounts of angiogenesis-related cytokines, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), which increased the angiogenesis of adipose tissue. ConclusionsADSCs play a key role in adipose tissue engineering, autologous adipose tissue grafting, and soft tissue wound repairing, which have important application prospect for breast reconstruction.
Objective To investigate the myogenic differentiation of mesenchymal stem cells (MSCs) after being transplanted into the local muscle tissues. Methods The serious muscleinjured model was established by the way of radiation injury, incising, and freezing injury in 36 mouses. Purified MSCs derived from bone marrow of male mouse and MSCs induced by5-azacytidine(5-Aza-CR) were transplanted into the local of normal muscle tissues and injured muscle tissues of femal mouse. The quantity of MSCs and the myogenic differentiation of implanted MSCs were detected by the method of double labeling, which included fluorescence in situ DNA hybridization (FISH) and immuno-histochemistry on the 1st, 3rd, 6th, 9th, 12th, and 15th day after transplantation. Results The quantity of implanted MSCs decreased as timepassed. MSCs’ differentiation into myoblasts and positive expression of desmin were observed on the 15th day in purified MSCs group and on the 6th day in induced MSCs groups. Conclusion MSCs could differentiate into myoblasts after being implanted into the local of muscle tissues. The differentiationoccurs earlier in the induced MSCs group than that in purified MSCs group.
【Abstract】 Objective To isolate and culture human amniotic fluid-derived mesenchymal stem cells (HAFMSCs),to investigate a better cryopreservation protocol of HAFMSCs and to observe the biocharacteristics and the multi-potential of HAFMSCs after cryopreservation for the further fundamental researches and cl inical appl ications. Methods HAFMSCswere isolated from the amniotic fluid of pregnant women during the second trimester by the improved two-step method.HAFMSCs were cryopreserved with different cryopreservation protocols (containing different contents of FBS and DMSO atcryoprotectant) in l iquid nitrogen for 12 weeks. The biocharacteristics of the HAFMSCs after cryopreservation were analyzed. The growth characteristics were observed by MTT method and the growth curves were drawn. The surface antigens of HAFMSCs were detected using flow cytometry, including CD29, CD34, CD44, CD45, CD73, and CD90. The adi pogenic and osteogenic differentiation abil ities of HAFMSCs were observed. The mRNA levels of Oct-4 and Nanog of the HAFMSCs were compared between before and after cryopreservations. Results At 12 weeks after cryopreservation, different protocols had different effects on the cell viabil ity; the better formula of cryoprotectant was 50% DMEM, 40% FBS, and 10% DMSO. After cryopreservation, the cells proliferated rapidly and the growth curves showed “S” shape, which was the same as the cells before cryopreservation. Phenotype showed that HAFMSCs were positive for the surface markers CD29, CD44, CD73, and CD90, and negative for CD34 and CD45. After 21 days of adi pogenic differentiation, the l ipid droplets were observed by oil red O staining. After 21 days of osteogenic differentiation, the calcium mineralizations were verified by von Kossa staining. There was no significant difference (P gt; 0.05) in the mRNA levels of Oct-4 and Nanog between before and after cryopreservations. Conclusion HAFMSCs have rapid proliferation and multi-potential in vitro. The cells have high viabil ities and no changes of the biocharacteristics and differentiation potential ities after cryopreservation for 12 weeks. Cryoprotectant containing 50% DMEM, 40% FBS, and 10% DMSO is a better cryopreservation protocol.
Objective To review the research appl ication and advance of synovium-derived mesenchymal stem cells (SMSCs) in tissue engineering. Methods The recent related l iterature was reviewed, concerning isolation method, characteristics of SMSCs, and its appl ication in tissue engineering. Results SMSCs are multi potent cell population with characteristics of easy isolation and high prol iferation, which have been appl icated in the cartilage, tendon, l igament, and bone tissue engineering. Conclusion SMSCs is a new member of mesenchymal stem cells family. It appears to be promising seedcells for tissue engineering, but further research is needed.
Objective To establish a method of constructing skin-equivalents (SE) by the hair follicle stem cells (HFSC) and the fibroblasts. Methods The K19 immunostainning was employed to localize the HFSC in the human scalp from the cosmetic surgery. The isolated HFSC through the enzyme digestion were seeded on the dermal equivalent (DE) formed by polymerization of the fibroblasts and collagen. After being cultured between the air-liquid interface for 14 days, SE were harvested and used for an evaluation. Results HFSC were located mainly in the outer root sheath in the hair follicle. Based on DE, the growing HFSC could build a fullydeveloped and multilayered epidermis with the basal membrane formedb etween the epidermis and the dermis. The fibroblasts were active and spread evenly in the collagen matrix. Conclusion The hair follicle stem cells located in the outer root sheath can be successfully used to construct skin-equivalents in vitro and have a promising clinical use in the treatment.