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.     

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.     

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.     

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.     

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.     

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.     

In vivo use of alginate scaffold with bone marrow mesenchymal stem cells in acute hepatic failure rats

AIM: To evaluate the use of alginate scaffold with bone marrow mesenchymal stem cells (BMSCs) in a rat acute hepatic failure model. METHODS: BMSCs were labeled with chloro- methylbenzamido dialkylcarbocyanine (CM-DiI), cultured in alginate scaffold and transplanted into the liver of acute hepatic failure rats with 70% liver resection. Four weeks later, the scaffold-cell complexes were taken out, and their abilities to secret albumin (ALB) and store glycogen were examined. The survival rate and liver functions of the rats were also examined. RESULTS: CM-DiI was an effective cell tracer. The BMSCs in alginate scaffold secreted ALB and stored glycogen in acute hepatic failure rats. The survival rate and liver functions of the rats treated with scaffold-cell complexes were better than those of the rats treated with alginate alone. CONCLUSION: Alginate scaffold with BMSCs can be used as artificial hepatic tissues in acute hepatic failure rats.     

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.     

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.     

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.     

Aged bone marrow stem cells can be rejuvenated by cell fusion with young bone marrow stem cells

AIM: To investigate the function of aged bone marrow mesenchymal stem cells (BMSCs) fused with young BMSCs in mice. METHODS: The cell fusion model, which was made by C57BL/6 mouse BMSCs labeled with PKH26 membrane red fluorescence (young cells, age of 2-3 months, Y) and (old cells, age of 18-24 months, O), and young and old BMSCs of green fluorescent protein (GFP) transgenic C57BL/6 mouse, was established by the induction of polyethylene glycol 1500 (PEG 1500). The cell fusion rate and cell surface markers were detected by flow cytometry. The morphology and nuclear characteristics of the fused cells were observed under fluorescence microscopy. In this study, the age dependent changes in BMSCs proliferation and differentiation potential in Y group, O group, and another three fusion groups (Y-Y group, Y-O group, O-O group) were examined. The proliferation potentials in 5 groups were compared by counting cell numbers at days 2, 4, 6, and 8. The osteogenic and adipogenic differentiation potentials of the cells in 5 groups were determined by using standard differentiation procedures. RESULTS: The fusion rate of 30.45%±4.13% was obtained by PEG 1500 induction. No significant difference of the fusion rates in Y-Y, Y-O and O-O groups was observed. Fused BMSCs coincided with the common BMSCs were reactive to the BMSCs lineage-specific CD44, Sca-1 surface markers and negative for the hematopoietic stem cells (HSCs) lineage-specific surface markers such as CD34, CD117, CD31, and CD45. The percentage of increasing cell numbers in Y-O group was significantly higher than that in O-O group at days 2, 4, 6, and 8. The positive rate of the area stained with Alizarin red, which represents osteogenic differentiation potential of BMSCs, was significantly higher in Y-O group than that in O-O group ［(25.46%±1.52%) vs (13.85%±1.69%), P<0.01］. In Y-O group, the higher rate of the positive area stained with oil red O, which represents adipogenic differentiation potential of BMSCs, was observed as compared to that in O-O group ［(12.99%±2.61%) vs (6.03%±1.71%), P<0.05］. CONCLUSION: Aged bone marrow stem cells can be rejuvenated by cell fusion with young bone marrow stem cells, particularly the proliferation and differentiation potentials.     

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.     

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).     

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.     

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 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.     

Lithium chloride promotes neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy

AIM: To study the influence of lithium chloride (LiCl) on the neuronal differentiation of rat bone marrow mesenchymal stem cells (MSCs), and to explore whether autophagy was involved in this process. METHODS: MSCs were isolated and cultured in vitro. The cells were divided into LiCl group and control group. MSCs were treated with β-mercaptoethanol as an inducer for triggering the cells to differentiate into neurons. The expression of neuronal markers-neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2), and autophagic marker-microtubule-associated protein 1 light chain 3 (LC3) were measured by immunofluorescence method and Western blot. An autophagy activator rapamycin and autophagy inhibitor 3-methyladenine (3-MA) were applied to modulate the autophagy in the LiCl treated-cells. The protein expression of NSE and MAP-2 were determined by Western blot. RESULTS: After induction, the expression of NSE and MAP-2 were increased. The percentage of NSE-and MAP-2-positive cells and the expression of NSE and MAP-2 in the LiCl group were greater than those in control group (P<0.05). After induction, the number of LC3-positive dots and the expression of LC3-Ⅱ in LiCl group were greater than those in control group (P<0.05). The expression of NSE and MAP-2 increased when the autophagy was modulated by rapamycin in LiCl treated-cells, and on the contrary, the expression of NSE and MAP-2 were inhibited as autophagy was modulated by 3-MA. CONCLUSION: Lithium chloride may promote the neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy.     

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.