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.     

Differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro via cardiotrophin 1

AIM: To investigate the effects of cardiotrophin 1 (CT-1) on differentiation of swine bone marrow mesenchymal stem cells (MSCs) into cardiomyocyte-like cells in vitro.METHODS: MSCs were isolated and proliferated from Tibet miniswine. Adipogenic and osteogenic potentials were identified. MSCs were divided into 4 groups for induction: untreated group, 5-azacytidine (5-Aza) group,CT-1 group and 5-Aza combined with CT-1 group. After induction for 4 weeks, the expression of cardiac cell markers including α-actin and cardiac troponin-T (cTnT) was estimated by immunofluorescence staining. Finally, the rates of red fluorescence positive-staining cells were calculated. RESULTS: The expression of α-actin in the 4 groups by red fluorescence staining was as follows: the differentiation rate of cardiomyocyte-like cells in combination group was 29.90%±4.76%, significantly higher than that in 5-Aza group (17.73%±2.34%, P<0.01), CT-1 group (6.63%±0.55%, P<0.01) and untreated group (1.62%±0.09%, P<0.01). The differentiation rate in 5-Aza group was significantly higher than that in CT-1 group (P<0.01) and untreated group (P<0.05). The differentiation rate in CT-1 group was significantly higher than that in untreated group (P<0.01). The expression of cTnT in the 4 groups was as follows: the differentiation rate of cardiomyocyte-like cells in combination group was 36.50%±4.09%, significantly higher than that in 5-Aza group (14.37%±1.65%, P<0.01), CT-1 group (7.50%±0.61%, P<0.01) and untreated group (1.12%±0.23%, P<0.01). The differentiation rate in 5-Aza group was significantly higher than that in CT-1 group (P<0.01) and untreated group (P<0.01). The differentiation rate in CT-1 group was significantly higher than that in untreated group (P<0.01).CONCLUSION: Appropriate concentrations of 5-Aza (10 μmol/L) and CT-1 (0.1 μg/L) induce swine bone marrow MSCs to differentiate into cardiomyocyte-like cells in vitro. CT-1 combined with 5-Aza significantly increases the differentiation rate.     

Differentiation of rat bone marrow mesenchymal stem cells expressing hepatocyte growth factor into hepatocyte-like cells

AIM:To observe the hepatic differentiated efficiency of rat bone marrow mesenchymal stem cells (rMSCs) expressing hepatocyte growth factor (HGF) in three-dimensional microenvironment formed by hanging drop. METHODS:rMSCs were isolated and cultured in vitro, and flow cytometry was used to detect the expression of CD44, CD90, CD34 and CD45. Recombinant retrovirus carrying cDNA of human HGF (pLNCX2-hHGF) was constructed and infected with rMSCs (hHGF-rMSCs). hHGF expression in hHGF-rMSCs was detected by RT-PCR, immunofluorescence staining and ELISA. hHGF-rMSCs were cultured in hanging drop for 21 days. The expression of albumin (ALB),cytokeratin-18 (AFP) and alpha fetoprotein (CK-18) were detected by RT-qPCR and immunofluorescence staining in the 7th, 14th and 21st day, respectively. The secretion of albumin in cultured supernatant was measured by ELISA. RESULTS:CD44 and CD90 were highly expressed in the third generation of rMSCs, but CD34 and CD45 were hardly expressed. The expression of hHGF at mRNA and protein level were all detectable in the hHGF-rMSCs, and the secretion in the cultured supernatant was about (123.71±8.81) μg/L in a period of 21 days. In the hHGF-rMSCs, ALB, AFP and CK-18 were highly expressed at mRNA level from the 7th to the 21st day, and there were significant differences compared with rMSCs at the same time point (P<0.01). The results of immunofluorescence staining showed that the protein expression of AFP was negative on day 7 and 14, and positive on day 21; while the protein expression of ALB and CK-18 was positive on day 7 and lasted until day 21. ALB was positively observed in the culture supernatant of hHGF-rMSCs from 7th to 21st day measured by ELISA, and there was significant difference between the hHGF-rMSCs and rMSCs (P<0.01). CONCLUSION:hHGF transduced-rMSCs can be induced to differentiate into hepatocyte-like cells after cultured in hanging drop which provides a three-dimensional microenvironment. All these results might help to provide new seed cells for cell therapy of clinical liver diseases and in vitro bioartificial liver.     

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.     

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.     

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.     

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.     

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.     

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.     

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).     

Mechanisms of catalpol-induced osteogenic differentiation in SD rat bone marrow mesenchymal stem cells

AIM: To investigate the mechanisms for catalpol-induced osteogenic differentiation of SD rat bone marrow mesenchymal stem cells (BMSCs) in vitro. METHODS: The cells were divided into control group, osteoinduction group and catalpol group. The activity of alkaline phosphatase (ALP) was measured at 7 d, 14 d and 21 d after catapol treatment, meanwhile ALP positive cell numbers and calcium nodes were counted at 14 d and 21 d after catapol treatment,respectively. The mRNA expression of Runx2, osteocalcin, Wnt3a, β-catenin, Wnt5a and Wnt11 was detected at 7 d, 14 d and 21 d after catapol treatment by real-time PCR. RESULTS: Catalpol at 2.0 mg/L increased ALP activity and ALP positive cell numbers significantly(P<0.05), meanwhile, it also increased calcium nodes numbers in cultured BMSCs (P<0.05). Compared with control group, catalpol increased the mRNA expression of Runx2 significantly at 14 d, but not at the 7 d and 21 d. Catapol also promoted the mRNA expression of osteocalcin significantly from 7 d to 21 d. Compared with control group, the mRNA expression of Wnt3a and β-catenin in catalpol group was increased at 14 d and 21 d. In addition, the mRNA expression of Wnt5a and Wnt11 in catalpol group was higher than that in control group at 14 d, but that was decreased at 21 d. CONCLUSION: Catalpol induces differentiation of BMSCs into osteoblast by increasing the mRNA expression of Runx2, and promotes the differentiation and mature of these osteoblasts by increasing ALP secretion, osteocalcin mRNA expression and calcium deposition. The activation of Wnt signaling pathway may be involved in this pro-osteogenic differentiation process.     

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.     

Biological characteristics of chronic myelogenous leukemia bone marrow derived mesenchymal stem cells

AIM: To study the biology characteristics of mesenthymal stem cells (MSCs) derived from chronic myelogenous leukemia(CML) and normal adult bone marrow.METHODS: Mononuclear cells from chronic myelogenous leukemia (n=19) and normal adult (n=8) bone marrow were obtained, cultured in expanded medium with low serum concentration.Cell morphology, cell cycle, immunophenotype and in vitro differentiation capacity were investigated.The differentiations of osteocytes and adipocytes were detected by von Kossa staining and Oil-red O staining.The chimeric oncogene BCR/ABL and Ph chromosome, two hall marks of CML, were detected in CML derived MSCs, normal adult MSCs, CML derived hematopoietic cells and K562 cells.RESULTS: CML and normal adult derived MSCs showed similar characteristics in cell morphology, phenotype and growth pattern.A typital fibrablast like morphology was observed.Under suitable conditions, CML and normal adult MSCs had the similar ability to differentiate into adipocytes and osteoblasts in vitro.Moreover, CML and normal adult MSCs did not express BCR/ABL gene products and Ph chromosome was not observed.CONCLUSIONS: We isolated and cultured a population of cells with characteristics of multipotent stem cells from CML bone marrow.There were similar biologic characteristics and differentiation ability between normal adult and CML bone marrow-derived MSCs.     

Effect of bone marrow mesenchymal stem cells on engraftment of hematopoietic stem/progenitor cells in sensitized mice

AIM: To evaluate the effects of bone marrow-derived mesenchymal stem cells (MSCs) on engraftment of hematopoietic stem/progenitor cells in sensitized mice. METHODS: Mouse bone marrow-derived MSCs were cultured by adherent culture method. MSCs combined with or without hematopoietic stem/progenitor cells were implanted into the sensitized mouse model, which was established by allogeneic splenocyte transfusion, and were divided into 6 groups: MSC intervention groups, including sensitized mice with MSCs on day 11, sensitized mice with MSCs on day 0 and sensitized-mice with MSCs both on day 11 and day 0; control groups, including sensitized mice without MSC intervention, non-sensitized mice without MSC intervention and non-sensitized mice without MSCs or transplantation of hematopoietic stem/progenitor cells. The survivors were assessed after transplantation and hematopoietic recovery was monitored weekly including hematological change, immune function reconstruction, bone marrow cell recovery, chimera analysis and graft-versus-host disease development. RESULTS: Compared with different control groups, MSC intervention did not prolong the survival rates of the sensitized model mice after lethal irradiation. CONCLUSION: Under the experimental conditions, MSC combined with C57BL/6 bone marrow hematopoietic stem/progenitor cells fail to promote the growth of engraftment in C57BL/6 allogeneic splenocyte-sensitized BALB/c mice in vivo.     

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.     

Proliferation and differentiation of bone marrow mesenchymal stem cells in process of bone loss in ovariectomized rats

AIM: To study the function of proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) for bone loss in the pathogenesis of osteoporosis (OP) in ovariectomized rats. METHODS: Animal model of OP was established by ovariectomy (OVX,bilateral ovarian resection) in 10-week-old healthy female Sprague-Dawley (SD) rats.BMSCs were isolated, cultured and purified by the combination of density gradient centrifugation, adhesion separation and limited dilution method, and cultured in vitro to the 3rd~4th passage in all experiments. The BMSCs phenotype appraisal was studied by flow cytometry. Colony-forming assay was applied to detect the BMSCs proliferation ability. The MTT method was used to analyze the growth curves of BMSCs. After adipogenic induction (ADI), lipid drops were observed by oil red O staining to compare the adipogenic potential between the 2 kinds of BMSCs. After osteogenic induction (OSI), calcium nodules were observed by alizarin red staining (ARS). The mRNA expression levels of BMSCs osteogenesis-related proteins, for instance, Runx2, osteocalcin (OCN) and osteopontin (OPN) were measured by RT-PCR. RESULTS: Compared with sham group, the colony-forming ability of BMSCs in OVX group became decreased, the proliferation capacity was declined, the osteogenic potential was decreased, and the adipogenic potential was increased(P<0.05). CONCLUSION: In ovariectomized OP rats, the proliferation and osteogenesis of BMSCs decrease, and the adipogenesis of BMSCs increases, which may cause rapid bone loss and play an important role in the pathogenesis of OP.     

High expression of PDX-1 in bone marrow mesenchymal stem cells mediated by superfect

AIM: To construct a eukaryotic expression vector containing pancreatic duodenal homebox-1 (PDX-1) and to elevate the expression efficiency of exogenous gene in rat bone marrow mesenchymal stem cells (MSCs). METHODS: Recombinant vector containing PDX-1 was constructed. Flow cytometry was used to identify the cell cycle of bone marrow mesenchymal stem cells (MSCs) cultured in vitro. Recombinant vector containing PDX-1 was transfected into bone marrow MSCs using superfect in medium. After being selected by G418, RT-PCR and Western blotting were used to investigate the expression of PDX-1 in MSCs. RESULTS: Restricted enzyme analysis and sequencing showed that PDX-1 gene segment was consistent with that in GenBank. Flow cytometry showed that there were about 85.9% cells at the cell cycle of G0/G1. The whole cells transfected emitted green fluorescence under flow cytometry. The efficiency of transfection was above 40%. RT-PCR and Western blotting demonstrated that there was expression of PDX-1 in transfected bone marrow MSCs. CONCLUSION: Recombinant vector containing PDX-1 was constructed successfully. Superfect mediated expression of exogenous gene in bone marrow MSCs in a high efficiency, and bone marrow MSCs containing exogenous gene are an ideal cells for gene therapy.     

Meglumine cyclic adenylate induces differentiation of bone marrow mesenchymal stem cells into cardiomyocytes in vitro

AIM: To study the effect of meglumine cyclic adenylate (MCA) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes in vitro. METHODS: The whole bone marrow adherent culture method was used to isolate, culture and amplify the BMSCs. The surface markers of BMSCs were determined by flow cytometry analysis. MCA at concentrations of 10^-2 mol/L, 10^-3 mol/L, 10^-4 mol/L, 10^-5 mol/L, 10^-6 mol/L and 10^-7 mol/L was added to the culture medium containing the second generation of BMSCs.5-Azacytidine(5-Aza) was used as a positive control. The cell viability was measured by MTT method.The cAMP content in BMSCs was detected by ELISA. The mRNA expression of GATA-4, Cx43 and β-MHC in MCA group and MCA+H89 (a PKA inhibitor) group was measured by SYBR-RT-PCR. The differentiation effects of MCA and 5-Aza were compared by flow cytometry. RESULTS: Most of the BMSCs expressed CD44 and CD71, and did not express CD45. MCA inhibited the viability of BMSCs in a time-and dose-dependent manner, and MCA atthe concentration of 10^-2 mol/L showed particularly remarkable effect. MCA significantly increased intracellular cAMP level in BMSCs in a concentration-dependent manner. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group were significantly higher than that in blank group (P<0.05), and the highest effect was under the condition of MCA induction at the concentration of 10^-3 mol/L for 3 days. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group was higher than that in 5-Aza group and H89+MCA group (both P<0.05). Differentiation rate in MCA group was slightly higher than that in 5-Aza group (20.24%±1.02% vs 18.39%±0.58%, P<0.05). CONCLUSION: MCA stimulates BMSCs to increase intracellular cAMP production and inhibits the viability of BMSCs, thus promoting the mRNA expression of GATA-4, β-MHC and Cx43 through the cAMP/PKA signaling pathway.