Electrophysiological characteristics of the membrane current of cardiomyocytes-like cells derived from rat bone marrow mesenchymal stem cells

AIM: To study the electrophysiological characteristics of the membrane currents of cardiomyocytes-like cells derived from rat bone marrow mesenchymal stem cells (MSCs).METHODS: MSCs were induced,cultured and identified according to the reference.At the fourth week after treatment with 5-azacytidine(5-aza),cardiomyocytes-like cells were detected for the membrane current with the whole-cell patch-clamp technique and compared with the undifferentiated MSCs.RESULTS: The undifferentiated MSCs only expressed potassium currents.MSCs were stained positive for troponin T after treatment with 5-aza,and two kinds of inward currents and three kinds of outward currents were expressed.They respectively were the fast inward sodium current (I_Na),the L-type calcium current (I_Ca),the transient outward potassium current (I_to),the ultra-rapid delayed rectifier potassium current (I_kur) and the slow delayed rectifier potassium current (I_ks).Compared with the undifferentiated MSCs,the potassium currents of cardiomyocytes-like cells derived from MSCs were mainly made up of I_kur and I_ks.CONCLUSION: After treatment with 5-azacytidine,MSCs are differentiated into cardiomyocytes-like cells,which express the current of I_Na,I_Ca,I_to,I_kur and I_ks.     

Study on the biological characteristics of rat bone marrow mesenchymal stem cells

AIM: To investigate the basic biological characteristics of adult rat bone marrow mesenchymal stem cells(rBMMSCs), and compare to that of human BMMSCs (hBMMSCs). METHODS: rBMMSC and hBMMSCs were separated from bone marrow with the difference of adherence and Ficoll-Paque reagent, and expanded in culture medium in vitro, respectively. The proliferation and growth characteristics of the primary and different passage culture of rBMMSCs and hBMMSCs were analysed. The neural differentiation capacity of rBMMSCs with passages were observed. To detect the surface antigens of rBMMSCs, the labeled cells were analysed on a FACScan flow cytometer. The karyotype of rBMMSCs were detected by blocking cellular fission with colchicines. RESULTS: rBMMSCs and hBMMSCs have a strong self-renewal capacity. Approximately (4-8)×10¹² and (3-4)×10¹² cells were obtained after passage 15 in vitro, respectively. The ability of proliferation, CFU-Fs, and neural differentiation of rBMMSCs and hBMMSCs were decreased gradually with passages, but the ability of proliferation and CFU-Fs of rBMMSCs were higher than that of hBMMSCs at different passage. FACScan result showed rBMMSCs were uniformly positive for CD29 and CD44, and negative for CD11b, CD45, CD61, CD71, CD80, CD86,MHCⅠ and MHCⅡ. rBMMSCs had an normal karyotype, which had an average of 37.0±4.0 to 40.5±2.5 chromosomes. CONCLUSION: Adult rBMMSCs have strong self-renewal and neural differentiation capacity, and have an normal karyotype. So rBMMSCs can be used as the seed cells for tissue engineering.     

Differentiation of bone marrow mesenchymal stem cells into chondrocytes

AIM: To investigate the differentiation of human bone marrow mesenchymal stem cells (MSC) into chondrocytes in vitro and determine factors involving in the differentiation process. METHODS: MSC were separated from iliac bone marrow with lymphocyte separating medium using density centrifugation. Cells were cultured and expanded in medium until reaching required number. MSC was induced to differentiate into chondrocytes by adopting high cell density, supplying growth factor and using micromass culture. Cells were observed by HE staining. Matrix of cartilage was detected by alcian blue and toludine blue and cartilage specific collagen II was detected by immunohistochemistry. RESULTS: The structure of the micromass assumed that of cellular cartilage, alcian blue staining were uniformly positive and toludine blue detected diffuse metachromasia substance, cells uniformly expressed collagen Ⅱ. CONCLUSION: High cell density, growth factor and appropriate culture conditions are critical to induce differentiation of MSC into chondrocytes.     

Microparticles derived from bone marrow mesenchymal stem cells promote angiogenesis in rat myocardial infarction model

AIM: To observe the effects of microparticles derived from bone marrow mesenchymal stem cells (MSC-MPs) on angiogenesis and cardiac function in a rat myocardial infarction model. METHODS: MSCs were obtained from Sprague-Dawley rats. MSCs were treated under serum-free condition in hypoxia for 72 h, and the microparticles were isolated from the supernatants. The phenotypic profile of MSC-MPs was determined by bead-based flow cytometry and the morphology was observed under a transmission electron microscope. The rat myocardial infarction model was established. The cardiac function was evaluated by echocardiography after the intramyocardial injection of MSC-MPs. The myocardial infarct size was observed by Masson staining. The blood vessel density in the peri-infarcted area was measured using immunohistochemical staining for von Willebrand factor and α-smooth muscle actin. The expression of vascular endothelial growth factor (VEGF) was analyzed by real-time PCR. RESULTS: Apoptotic MSCs released a large quantity of microparticles which were phenotypically similar to the parent MSCs and 100~1 000 nm in diameter. The cardiac functions of myocardial infarction rat model were improved at 7 d and 28 d after intramyocardial injection of MSC-MPs compared with control group. The myocardial infarct size was reduced and angiogenesis was promoted significantly in the infarcted heart injected with MSC-MPs 28 d after treatment. MSC-MPs treatment also increased the expression level of VEGF within 7 d.CONCLUSION: MSC-MPs protect cardiac tissue from ischemic injury and improve cardiac function by promoting angiogenesis after myocardial infarction.     

Biological characteristics of long passaging rat mesenchymal stem cells

AIM:To investigate multi-potential of rat bone marrow mesenchymal stem cells (rBMMSC) and mutation inclination, the rBMMSC were long passaged in vitro. METHODS:Cellular cycles of different passages were assayed by FACSan flow cytometry and karyotypes of passage 6, passage 25 and passage 45 were compared by G-binding analysis. RESULTS:The early passages and long-term passages all showed strong proliferation; passage 6, passage 25 and passage 45 all showed normal karyotype. CONCLUSION:Long-term culture and passage of rBMMSC still remains strong proliferation. With this capability, the mutation inclination is not enhanced.     

Differentiation of bone marrow mesenchymal stem cells in dilated cardiomyopathy

AIM: To investigate the feasibility of differentiation of bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes and vascular endothelial cells in dilated cardiomyopathy (DCM). METHODS: 20 rabbits were randomly divided into two groups. MSCs were isolated from bone marrow and cultured. Adriamycin was applied to the two groups to create rabbit DCM models. At 3 weeks after the creation of DCM models, the experiment group animals received intramyocardial injection of autologous MSCs. 4 weeks after transplantation, the implanted sites were examined to identify the labelled cells and to investigate its differentiation through immunofluorescence. RESULTS: At 4 weeks after the MSCs transplantation, the implanted cells were found in the experiment group and some differentiated into cardiomyocytes and vascular endothelial cells, which was not founded in the control group. CONCLUSION: Autologous bone marrow mesenchymal stem cells can differentiate into cardiomyocytes and vascular endothelial cells in dilated cardiomyopathy.     

Ultrastructure and function of human bone marrow mesenchymal stem cells

AIM: To study the cytoskeleton of mesenchymal stem cells (MSCs), the ultrastructure and function relationship by using atomic force microscope (AFM). METHODS: The ultrastructures and morphological feature of MSCs cultured for 1 d and 5 d were studied by AFM. RESULTS: The special structures that possess peculiar morphological characteristic of MSCs such as cytoskeleton, pseudopod, microfilament etc were identified by AFM, and these special structures are difficult to observe under electronic microscopy or other conventional optical microscopy. CONCLUSIONS: AFM is a powerful tool to study ultrastructures, morphological features, and cytoskeleton of stem cells in near physiological conditions. Its application prospect in cellular biology is extensive. The special cytoskeleton and other structures of MSCs observed above may represent the structural base of multi-differentiation potential of MSCs.     

Effect of inhibiting release of exosomes on biological characteristics of bone marrow mesenchymal stem cells

AIM:To investigate the effect of inhibiting the release of exosomes on the biological characteristics of bone marrow mesenchymal stem cells (BM-MSCs) and the underlying mechanisms. METHODS:The exosome releasing-deficient mouse model was constructed by knockout of Rab27a using TALEN technique. The BM-MSCs were isolated and cultured. The exosomes were extracted from the culture medium using total exosome isolation kit and quantified by nanoparticle tracking analysis (NTA). The size and morphology of the exosomes were observed under transmission electron microscope. To evaluate the proliferation ability of BM-MSCs, the BM-MSCs were labeled with 5-ethynyl-2'-deoxyuridine (EdU) and the expression level of proliferating cell nuclear antigen (PCNA) was determined by Western blot. Moreover, hypoxia tolerance of BM-MSCs in vitro was evaluated via TUNEL staining and MTS assay. RESULTS:The count of exosomes released by BM-MSCs isolated from Rab27a knockout mice was significantly reduced. Inhibition of exosome release resulted in decreases in the viability of the BM-MSCs and their resistance to hypoxia. CONCLUSION:Inhibition of exosome release from the BM-MSCs results in significantly decreased proliferation ability and resistance to hypoxia.     

Biological characteristics of bone marrow mesenchymal stem cells from GFP transgenic mice transfected with human β-nerve growth factor gene

AIM:To investigate the changes of biological characteristics of GFP transgenic mouse bone marrow mesenchymal stem cells (MSCs), which were transfected with human β-nerve growth factor (β-NGF) gene in a recombinant eukaryotic expression vector. METHODS:MSCs obtained from GFP transgenic mice were isolated, cultured and purified by the whole bone marrow adherence methods. Human β-NGF gene was transfected into the MSCs by a recombinant eukaryotic expression vector. The β-NGF expression in the MSCs was detected by the method of immunocytochemistry. Hippocampal neurons from neonatal mice were cultured with culture supernatant of the MSCs transfected with pcDNA3-β-NGF and the biological characteristics of the MSCs were investigated 3 d after culture under inverted phase-contrast microscope. RESULTS:The β-NGF positive rate of MSCs in pcDNA3-β-NGF transfection group ［(37.12±2.14)%］ was significantly higher than that in MSCs control group ［(2.36±0.62)%］ and blank control group ［(1.43±0.76)%］.The neurite length of neonatal mouse hippocampal neurons cultured with culture supernatant from pcDNA3-β-NGF-transfected MSCs ［(31±3)μm］ was significantly longer than that in negative control group ［(23±4)μm］, suggesting that MSCs transfected with β-NGF gene maintained better biological characteristics. CONCLUSION: The constructed recombinant eukaryotic expression vector of human β-NGF gene can be transfected into MSCs efficiently and NGF can be effectively expressed in MSCs. MSCs transfected with β-NGF gene are capable of stable expression and secretion of β-NGF, and maintenance of better biological characteristics.     

Study on Cx43 communication function of cardiomyocyte-like cells derived from rat bone marrow mesenchymal stem cells in vitro

AIM: To approach the gap junction distribution and communication function of cardiomyocyte-like cells derived from rat bone marrow mesenchymal stem cells (MSCs) in vitro.METHODS: MSCs were isolated by extraction of bone marrow specimens,gradient centrifugation and the adherence of culture plates.MSCs were culture in vitro,treated with 5-azacytidine and incubated for 24 h.The induced MSCs,which had been incubated for 2,3 and 4 weeks,were divided into group Ⅰ,Ⅱ and Ⅲ.In addition,the normal cardiomyocyte cells were used as control group.The distribution of connexin 43(Cx43) and the mean fluorescence redistribution rate were detected in every group with the laser scanning confocal microscope.RESULTS: Cx43 protein grain density in induced MSCs was increased with the lasting of incubation by quantitive analysis of Cx43 distribution.After 4 weeks,the Cx43 protein density in induced MSCs was nonsignificant deviation with control group (63.87±12.43,64.87±12.15,P>0.05).The diversify tendency of the mean fluorescence redistribution rate was approximation with the result of Cx43 in every group.The results showed that groupⅠ was 19.59%±6.08%,groupⅡ was 37.17%±3.84%,groupⅢ was 46.82%±2.69%,and control group was 49.71%±5.53%.CONCLUSION: MSCs can be differentiated to cardiomyocyte-like cells,which have been induced and incubated for 4 weeks in vitro.The communicational function of those MSCs is similar to the normal cardiomyocyte cells.     

Mechanism of telomere mainteance in human bone marrow mesenchymal stem cells

AIM： To study the telomere maintenance mechanism in mesenchymal stem calls (MSCs).〖WT5"HZ〗 METHODS：MSCs were isolated from healthy human bone marrow by their adherence to plastic and then were checked with CD14-FITC,CD45-FITC,CD44-FITC,HLA-DR-FITC,CD34-PE,CD29-PE and CD166-PE.Telomere length and ECTR DNA in MSCs were detected by Southern blotting.The localization of TRF1 and promyelocytic leukemia (PML) in MSCs were detected with immunofluorescence staining.TRAP protocol was performed to detect the telomerase activity in MSCs and MSCs-derived adipocytes.Western blotting and TRAP protocol were applied to measure telomerase activity of MSCs,which were synchronized by serum starvation and aphidicolin treatment.〖WT5"HZ〗RESULTS：The telomere in length seemed shorter and relatively more homogeneous in MSCs and HeLa cells than that in WI-38-2RA cells.TRF1 did not concide with PML nuclear body in MSCs and HeLa cells while it exclusively did in WI-38-2RA cells.ECTR DNA was negative in MSCs and HeLa cells but positive in WI-38-2RA cells.Telomerase was negative in MSCs but it was positive in MSCs-derived adipocytes detected by TRAP.Moreover,a cell cycle-dependent expression profile of telomerase was found in MSCs when they were synchronized by serum starvation and aphidicolin treatment.Untreated MSCs expressed very low level of telomerase probed by Western blotting with 2C4 mAb,but the telomerase level had significantly increased when these cells were trapped in S phase.〖WT5"HZ〗CONCLUSION：The telomere of MSCs is maintained by telomerase pathway instead of alternative lengthing of telomere(ALT) and the level of telomerase expression is associated with cell cycle stage.     

Gene transfer into different passages of bone marrow mesenchymal stem cells

AIM: To investigate the efficiency and stability of adenovirus-medicated gene transfer into different passages of bone marrow mesenchymal stem cells (BMSCs). METHODS: BMSCs were obtained from bone marrow of SD rats and cultured. Then passage 3 (P3) and P8 BMSCs were transfected with Ad-CMV-GFP, respectively. The transfection ratio was evaluated by flow cytometry. At the same time coxsackie and Ad receptor (CAR) of different passages of BMSCs was estimated by RT-PCR and Western blotting. RESULTS: The green fluorescence was observed 24 h after transfection, while the strength of fluorescence increased with time and the peak was at 7 days. It was seen that the transfection ratio was over 80% and there was no difference between P3 and P8 BMSCs (P>0.05). Flow cytometry analysis by different gates showed the transfection ratio was high in BMSCs in the period of productive metabolism. The mRNA expression of CAR in P3, P6 and P8 was similar, and the same change was observed in the protein expression of CAR in P3 and P8 BMSCs. CONCLUSION: Ad-CMV-GFP is transferred to BMSC effectively and sustained about 28 days. It is suspected that BMSCs in mitotic phase are easy to be transferred by Ad-CMV-GFP and different passages of BMSCs from P3 to P8 BMSCs can be as high-effectively gene vehicle.     

Age-related changes of bone marrow mesenchymal stem cells in senile osteoporosis

AIM：To investigate the different functions of bone marrow mesenchymal stem cells (BMMSCs) in age-related osteoporosis. METHODS：The senescence-accelerated mice (SAMP6) were used in the experiment. The BMMSCs were isolated from femora and tibiae by flushing. Flow cytometric analysis was performed with MSCs-related monoclonal antibodies. The expression of differentiation genes was tested by real-time RT-PCR. RESULTS：In the progress of age-related osteoporosis, BMMSCs exhibited a decrease in osteogenesis and an increase in adipogenesis. Transforming growth factor β(TGF-β) signaling was significantly changed along with aging in SAMP6 mice. CONCLUSION：The functional changes of BMMSCs may play an important role in senile osteoporosis. The alteration of TGF-β-related gene expression may be the molecular mechanism of dysfunction in BMMSCs.     

Rapid proliferation of bone marrow mesenchymal stem cells enhanced by xanthosine

AIM: To explore the effect of xanthosine (Xs) on proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: Xs was directly added to the culture system and the effects of Xs on proliferation and differentiation of BMSCs were observed. RESULTS: In the presence of Xs, the growth rate of the 10th passage cells was almost similar to that of the 4th passage cells and no downtrend was observed. However, in control group (without Xs), the growth rate of the 10th passage cells was obviously declined. The BMSCs promoted by Xs still kept up a vigorous capability of differentiation into hepatocytes. CONCLUSION: As an inhibitor of asymmetric cell kinetics, Xs can promote the conversion of BMSCs from asymmetric cell kinetics to symmetric cell kinetics, and keeps synchronously the ability of differentiation from BMSCs into hepatocytes as well. It is helpful for enhancing the proliferation efficiency of BMSCs in vitro, and will have extensive application of clinical practice.     

Notch signaling in bone marrow mesenchymal stem cells induced by lipopolysaccharide

AIM: To investigate the roles of Notch signaling in lipopolysaccharide (LPS)-induced proliferation and secretion of interleukin-6 (IL-6) and chemokine CXCL1 in bone marrow mesenchymal stem cells (BMSCs).METHODS: BMSCs were isolated by whole bone marrow culture. The expression levels of Notch signaling pathway receptors and ligands in the BMSCs treated with LPS were measured by qPCR and Western blot. The proliferation of BMSCs was analyzed by MTT assay and viable cell counting. The secretion levels of IL-6 and CXCL1 induced by LPS were measured by ELISA.RESULTS: Treatment with LPS at 1 mg/L effectively induced the proliferation of BMSCs and the secretion of IL-6. Obvious expression of Notch receptors and ligands in the BMSCs was observed, and LPS had little effect on the mRNA and protein levels of Notch receptors and ligands, but LPS increased the protein levels of Hes1 and Hey1, the target genes of Notch signaling. LPS at 1 mg/L increased the proliferation of BMSCs, whereas DAPT (Notch signal inhibitor) reduced the basal and LPS-induced proliferation of BMSCs (P<0.01). LPS treatment robustly increased the secretion of IL-6 and CXCL1 as assessed by ELISA. However, inhibition of Notch signaling almost completely abolished LPS-induced secretion of IL-6 and CXCL1 (P<0.05).CONCLUSION: Inhibition of Notch signaling reduced not only the proliferation of BMSCs but also IL-6 and CXCL1 secretion induced by LPS.     

Ectopic hTERT gene expression in human bone marrow mesenchymal stem cells

AIM: To investigate the effect of transfection of hTERT gene into human mesenchymal stem cells (MSCs) on their telomerase activity and life-span.METHODS: Human MSCs were transfected with a pEGFP-hTERT plasmid by liposome-mediated transfection. Then the hTERT mRNA expression in MSCs was detected by RT-PCR. The activity of telomerase in transfected MSCs was detected by PCR and ELISA. The telomerase-positive MSCs was cultured in vitro and induced into neuron-like cells with EGF and bFGF. Neuron-specific markers (NF-M, MAP₂) were detected by RT-PCR.RESULTS: hTERT fragment was identified in the hTERT-transfeced cells but not in the untransfected human bone marrow MSCs. The untransfected human MSCs remained telomerase-negative but the hTERT-transfected cells showed robust telomerase activity. The telomerase-negative MSCs entered a nondividing state and senesced after about 20 to 25 passages. In test group, however, telomerase-positive MSCs to date had undergone 35 passages. RT-PCR analysis showed that telomerase-positive MSCs expressed neuron-specific markers, such as NF-M or MAP₂ after induced with EGF and bFGF in vitro. CONCLUSION: Ectopic expression of the hTERT gene in human MSCs reconstitutes telomerase activity. The transfection of hTERT gene into human MSCs extends their replicative life span and maintains their multipotent differentiation capacity.     

Biological characteristics and induced differentiation of bone marrow-derived mesenchymal stem cells from sensitized mice in vitro

AIM: To evaluate the biological characteristics and differentiating potentials of bone marrow-derived mesenchymal stem cells (MSCs) from sensitized mice by allogeneic splenocyte transfusion in vitro. METHODS: Adherent culture method was applied for culturing the bone marrow-derived MSCs from sensitized mice. The cell morphology was examined and the surface marker profiles were analyzed by flow cytometry. The differentiating potentials of the MSCs into osteogenic, adipogenic and myogenic lineages were explored. The bone marrow-derived MSCs from the normal mice were collected and served as controls. RESULTS: Both the bone marrow-derived MSCs from sensitized and normal mice were exhibited a homogeneous distinctive morphology and were positive for the surface markers CD29, CD105, CD44 and Sca-1, negative in CD 34 and CD11b. The abilities of both MSCs to differentiate into osteogenic, adipogenic and myogenic pathways in the same condition were also observed. CONCLUSION: There is no difference in the biological characteristics and induced differentiating potentials between the sensitized mouse bone marrow-derived MSCs by allogeneic splenocytes transfusion and the MSCs from normal mice.     

Human bone marrow mesenchymal stem cells differentiate toward endothelial lineage in vitro

AIM：To investigate the cytological basis and differentiating conditions of human bone marrow mesenchymal stem cells(hMSCs) differentiated into cells of the endothelial lineage in vitro.METHODS：hMSCs were isolated by density gradient centrifugation and fractionated on a 1 073 g/L Percoll.The combination of VEGF165 and various matrix proteins including fibronectin (FN) and typeⅠ collagen (Col) was used to induce hMSCs in vitro.Cells were characterized by immunohistochemistry,cytochemistry,FACS and ultrastructure to identify and detect the differentiated population and markers.RESULTS：hMSCs was positive for KDR.PAS reaction was positive and ultrastructure of hMSCs showed glycogen-pool in ectoplasm.Glycogen reducing or disappear suggested that stem cells have occurred differentiation.Induction of hMSCs resulted in the increase of KDR,β1 integrin and CD34.CONCLUSION：hMSCs were induced to a transit population (TP) that differentiated toward the endothelial progenitor cells (EPC),but not a really EPC.hMSCs pedigree diagram of differentiation was hMSCs→TP→EPC→endothelial cells (ECs).     

Trichostatin A promotes mouse bone marrow mesenchymal stem cells to differentiate into insulin-secreting cells

AIM：To investigate whether trichostatin A (TSA), a new revulsant,can induce mouse mesenchymal stem cells to differentiate into insulin-secreting cells and to explore the appropriate concentration of TSA. METHODS：The mesenchymal stem cell line from C57BL/6 mice was cultured in vitro and divided into 5 groups before treated with different concentrations of TSA, (group A: DMSO; group B~E: treated with 25 nmol/L, 50 nmol/L, 100 nmol/L and 200 nmol/L of TSA, respectively). After exposed to different cultured media for 10 d during the 2 stages, the cells were detected by the following methods: the insulin-secreting cells in each group were identified by dithizone staining and the results were calculated with immunohistochemical half quantitative analysis. The insulin secreted by insulin-secreting cells in each group was identified by immunofluorescence, and the mean fluorescence intensity of insulin was compared. The content of insulin in each group was quantified by ELISA. The appropriate concentration of TSA was determined according to the above results. RESULTS：TSA treatment for 10 d promoted the mouse bone marrow mesenchymal stem cells to differentiate into insulin-secreting cells which produced insulin. The immunohistochemistry and immunofluorescence imaging analysis of insulin-secreting cells showed that the insulin staining positive area, positive ratio, total density of insulin expression and mean fluorescence intensity of insulin in group B were significantly higher than those in the other TSA-treated groups. When the concentrations of TSA gradually increased, the content of insulin reduced accordingly. The content of insulin in group B was significantly higher than that in the other TSA-treated groups. CONCLUSION：TSA treatment for 10 d promotes bone marrow mesenchymal stem cells from C57BL/6 mice to differentiate into insulin-secreting cells and the appropriate concentration of TSA is 25 nmol/L.