Effects of cytomegalovirus on the adhesion of bone marrow stromal cells

AIM:To investigate the effect of cytomegalovirus (CMV) on the expression of ICAM-1 and VCAM-1 in bone marrow stromal cells and on the adhesion of bone marrow stromal cells to hematopoietic cells.METHODS:Flow cytometry was used to detect the expression of adhesion molecules ICAM-1 and VCAM-1 in bone marrow stromal cells, MTT method was used to perform the adhesion assay of bone marrow stromal cells to normal hematopoietic cells. RESULTS:Bone marrow stromal cells could be infected by the CMV used in this experiment; CMV below the dose of 100TCID₅₀ could not destroy bone marrow stromal cells apparently; The expression of ICAM-1 increased at the early stage(18 h) of CMV infection, the expression of ICAM-1 decreased at the late stage (120 h) of CMV infection. Inactived CMV could also increase the expression of ICAM-1 as alive CMV; The adhesion rate of bone marrow stromal cells to hematopoietic cells increased at the early stage of CMV infection. CMV had no significant effects on the expression of VCAM-1 in bone marrow stromal cells. CONCLUSION:The adhesion capacity of bone marrow stromal cells to hematopoietic cells increased at the early stage of CMV infection, while the adhesion capacity of bone marrow stromal cells to hematopoietic cells decreased at the late stage of CMV infection.     

Study on changes of neurological function and neuron apoptosis after intravenous administration of bone marrow stromal stem cells for treating permanent focal cerebral ischemia in rats

AIM: To explore the survivorship and the mechanism of the intravenous administration of bone marrow stromal stem cells (BMSCs) for treating permanent focal cerebral ischemia in rats. METHODS: After purified, proliferated, and marked with BrdU, the BMSCs were injected intravenously into rats 1 d after focal cerebral ischemia.Modified neurological severity score (mNSS) was evaluated before and following 1, 7, 14 and 28 d after middle cerebral artery occlusion (MCAO). Rats were executed at 1, 7, 14 and 28 d after MCAO. Brain sections were stained with hematoxylin and eosin (HE) for determining the infarct volume. Slides were stained by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and immunostaining for cleaved caspase-3 method for apoptosis detection and mechanism exploration in situ.RESULTS: mNSS in BMSCs-transplanted group at 14th day and 28th day of MCAO was significantly lower than that in control group(P<0.05). TUNEL-positive cells in the hippocampus and thalamus area of BMSCs-transplanted rats were significantly fewer than those in control rats at 14th day and 28th day of MCAO(P<0.05). Double immunostaining showed that small grafted BMSCs and small endogenous neural cell apoptosis depended on the capase-3 in hippocampus.CONCLUSION: The intravenous administration of BMSCs promotes the recovery of neurological function of rats with focal cerebral ischemia. The therapeutic effect of BMSCs on rats with focal cerebral ischemia may be derived from the reduction of apoptosis and the mobility and migration of endogenous neural stem cells in the ischemic boundary zone.     

Protective effects of bone marrow stromal cells on ischemia/reperfusion hippocampal slices

AIM:To study the protective effect of bone marrow stromal cells (MSCs) on ischemia /reperfusion hippocampal slices.METHODS:Ischemia/reperfusion models of hippocampal slices from newborn rats were established. MSCs obtained from adult bone marrow were cultured, isolated and purified. Cell death was assessed using propidium iodide fluorescence. And brain-derived neurotrophic factor (BDNF) expression in MSCs was determined by immunocytochemistry and Western blot.RESULTS:Maximal dead cells appeared in hippocampal slices 3 to 7 days after reperfusion. When the slices were co-cultured with MSCs, only a few cells were dead. The protective effect of MSCs on the slices was diminished significantly when anti-BDNF antibody was added to the medium. The protein of BDNF was faintly expressed in MSCs under normal conditions. When MSCs were co-cultured with ischemia /reperfusion hippocampal slices, the expression of BDNF in MSCs was increased gradually especially when co-cultured for 3 to 7 days. However, MSCs co-cultured with normal hippocampal slices expressed BDNF at a lower level at any times of co-culture.CONCLUSIONS:In an in vitro model of simulated ischemia, MSCs reduce cell death. Ischemia/reperfusion hippocampal slices co-cultured with MSCs promote the expression of BDNF in MSCs, which in turn protect the ischemic neurons.     

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.     

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.     

Strontium ranelate promotes differentiation of bone marrow mesenchymal stem cells to osteoblasts by increasing expression of bone morphogenetic protein 2

AIM: To explore whether strontium ranelate (Sr) promotes differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to osteoblasts by increasing the expression of bone morphogenetic protein 2 (BMP-2). METHODS: Rat BMSCs were isolated, purified and cultured, then were induced to differentiate into osteoblasts. The cells were treated with different concentrations of Sr or noggin (an inhibitor of BMP-2) according to the experimental purposes. The activity of alkaline phosphatase (ALP) was measured by colorimetry. Mineralized nodules were measured by alizarin red staining. The expression of BMP-2 was detected by Western blotting. RESULTS: Treatment with Sr at concentrations of 0.1 mmol/L to 7 mmol/L for 7 d obviously increased the activity of ALP,and Sr at concentration of 3 mmol/L produced the maximum effect. Exposure of the cells to Sr at concentration of 3 mmol/L for 21 d significantly increased mineralized nodules. Exposure of the cells to Sr at concentrations of 0.1 mmol/L to 7 mmol/L for 7 d markedly increased the expression of BMP-2. Preconditioning with noggin at concentration of 100 μg/L for 2 h not only inhibited Sr-induced expression of BMP-2, but also antagonized the increase in the activity of ALP and mineralization induced by Sr in BMSCs. CONCLUSION: Up-regulation of the expression of BMP-2 may be one of the mechanisms by which Sr promotes differentiation of rat BMSCs to osteoblasts.     

Salvianolic acid B reduces apoptosis of bone marrow stem cells induced by hydrogen peroxide

AIM: To investigate the effect of salvianolic acid B (Sal B) on apoptosis of rat bone mesenchymal stem cells(BMSCs) induced by hydrogen peroxide(H₂O₂). METHODS: BMSCs were incubated with Sal B at the concentration of 1, 10 or 100 μmol/L while treated with lethal concentration of H₂O₂ (500 μmol/L). The effect of Sal B at different concentrations on the viability of BMSCs was detected by MTT. Flow cytometry were used to determine the protective role of Sal B in apoptosis of BMSCs. The changes of chromatin distribution in BMSCs were observed by Hoechst 33342 staining. The expression of p-ERK1/2 was detected by Western blotting. RESULTS: Sal B protected the BMSCs against H₂O₂ as the cell viability was increased from (53.60±4.21)% to (85.33±9.08)% or (75.78±6.28)% in a dose-dependent manner. After exposed to H₂O₂, about 50%-65% BMSCs displayed apoptotic morphology. Treatment with Sal B at the concentrations of 10 and 100 μmol/L reduced the cytotoxic effect of H₂O₂ on BMSCs to about 32% and 47%, respectively. The results of flow cytometric analysis confirmed the cytoprotective effect of Sal B. This protective effect was concomitant with significant reduction of ROS generation. Moreover, H₂O₂ time-dependently induced a pronounced increase in ERK1/2 phosphorylation,which was effectively inhibited by Sal B.CONCLUSION: Sal B protects BMSCs against H₂O₂-induced apoptosis. Sal B may exert its protective effect on BMSCs by triggering intracellular anti-apoptosis mechanism as well as reducing the oxidative stress.     

Difference of immune response to transplantation of BMSCs and non-BMSCs-derived cells in animals

AIM: To analyze the characteristics of immune response in the animals after transplantation of bone marrow stromal cells (BMSCs) or non-BMSCs-derived cells.METHODS: Rat BMSCs and Buffalo rat liver (BRL) cells were transplanted into SD rats and New Zealand rabbits by intravenous and subcutaneous injections. The differences of immune response in the animals received these two types of cells were evaluated by comparative analysis of the lymphocytes and monocytes in peripheral blood as well as the histopathological characteristics of the subcutaneous transplantation sites.RESULTS: The increase intensity of the lymphocyte number in BMSCs group was lower than that in BRL group, regardless of allograft or xenograft. The immune reaction of the animals to BMSCs transplantation was a little slower. It was obviously dissimilar that the increase intensity of monocyte number in BMSCs group was higher than that in BRL group. Pathological cutaneous sections showed that neither obvious necrosis nor inflammatory cells was observed in the vicinity of BMSCs transplantation sites.CONCLUSION: The intensity of immune response induced by BMSCs is lower than that by BRL cells. The anti-rejection ability of BMSCs in cell transplantation is better than that of BRL cells.     

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.     

Selection and amplification of the liver stem cell subset from rat bone marrow cells with a medium containing cholestatic serum in vitro

AIM: To explore the feasibility of direct separat and selective enlargement of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro. METHODS: Bone marrow cells of rats were cultured with selective media containing 2%, 5%, 7% and 10% cholestatic rat serum, respectively. The BDLSC were then induced to proliferate with the addition of hepatocyte growth factor (HGF) on the firth day. BDLSC were characterized using immunocytochemistry and RT-PCR for lineage markers, glycogen staining and urea synthetic assay for functions 2 weeks later. RESULTS: Bone marrow cells were unble to form colony in the presence of 2% cholestatic serum and apopotosis appeared gradually in 7% or 10% cholestatic serum. The BDLSC survived in the medium containing 5% cholestatic serum while the other types of cells did not. The survival cells proliferated with a high speed during the second week and then formed hepatocyte-like colony-forming units (H-CFU). Cells in the H-CFU expressed the characteristic proteins of fetal hepatocytes. Furthermore, they had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: The selective micro-environment effectively selected BDLSC from the bone marrow cell, and will be a new way to provide an abundant source of donor hepatocytes for clinical cell therapy.     

Adhesion of bone marrow stromal cell in children with acute lymphoblastic leukemia

AIM and METHOD: The adhesion behavior of bone marrow stromal cells(BMSC) in children with acute lymphoblastic leukemia(ALL) to bone marrow mononuclear cells(BMMC) and Raji cells were investigated by MTT method. The expression of adhesion molecules ICAM-1 and VCAM-1 were detected by flow cytometry. RESULTS: The adhesion ability of BMSC in acute period of ALL to BMMC was lower than that of control group. The adhesion ability of BMSC in long term remission period of ALL to Raji cells higher than that of control group. The expression of ICAM-1 on the surface of BMSC in acute period of ALL is much lower than that of control group. CONCLUSIONS: The adhesion of BMSC to BMMC or tumor cells in children with ALL was abnormal. The abnormal adhesion behavior might be partially due to the changed expression of adhesion molecules on BMSC.     

Adult rat and human bone marrow mesenchymal stem cells differentiate into neurons with Musk's polypeptide

AIM: To investigate the differentiation from adult rat and human bone marrow mesenchymal stem cells (BMMSCs) into neuron with musk polypeptide (Mu-P).METHODS: Adult rat and human BMMSCs were induced with Mu-P.Neuron-specific enolase (NSE),neurofilament (NF),Nestin,glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry.RESULTS: Simple methods with Mu-P induced adult rat and human BMMSCs exhibiting a neuronal phenotype,expressing Nestin at 3 hours to 5 hours,and expressing NE and NF at 5 hours to 7 days.But the neuron-like cells didn't express the glial astrocyte marker GFAP.CONCLUSION: Adult rat and human BMMSCs can be induced to differentiate into neurons with Mu-P.     

Lentivirus mediated CCN1 gene on growth and migration of rat bone marrow mesenchymal stem cells

AIM：To investigate the role of cysteine-rich 61 (Cyr61/CNN1) in proliferation and migration of bone marrow mesenchymal stem cells (BMSCs). METHODS：The lentiviral vector carrying CCN1 (Lenti-GFP-CCN1) was constructed and then transfected into the rat BMSCs. The cells were divided into non-transfection group, transfection group (transfected with Lenti-GFP-CCN1) and negative control group (Lenti-GFP). The fluorescence intensity of the transfected BMSCs was observed under inverted fluorescence microscope. The effects of CCN1 on the proliferation and migration of BMSCs were detected by MTT assay and scratch wound healing assay. RESULTS：The proliferation of BMSCs transfected with Lenti-GFP CCN1 had no significant difference compared with negative control group and control group. The width/thickness ratio of migrated BMSCs in wound healing was significantly higher in Lenti-GFP-CCN1 group than that in negative control group and control group (P<0.05). CONCLUSION：Exogenous CCN1 promotes the migration of BMSCs.     

Remaining residual bone marrow cells through bone marrow transplantation from female to male in mice

AIM:To investigate the remaining residual bone marrow cells after bone marrow transplantation (BMT) from female to male in mice by detecting the male Y chromosome from the blood cells. METHODS:Bone marrow cells from either male or female C57BL/6 mice were injected via tail vein to the corresponding male or female mice at 1×107 per animal 6 h after irradiation exposure to different doses of ［137Cs］. The survival rate of BMT was calculated after 14 d. The numbers of leukocytes in peripheral blood were measured and Y chromosome levels were also assayed in the male recipent mice. RESULTS:With radiation doses of 1 000 rad and 950 rad, the hematopoietic function of the female recipient mice quickly restored, but the male recipient mice had only 48% survival rate. With the radiation dose of 900 rad, the male recipient mice all survived and their hematopoietic function quickly restored. The peripheral leukocyte counts returned to normal 13 d after BMT. The Y chromosome genes in the peripheral blood cells were detected in 5 weeks after BMT in the male recipient mice, suggesting that the bone marrow cells in the male mice were completely destroyed by radiation, and the bone marrow cells from female mice completely replaced those in the male mice. CONCLUSION: After irradiation at a dose of 900 rad, the male mice can be used as BMT recipients without endogenous bone marrow cells. This study warrants male recipient mouse model in BMT for further investigation on the function of bone marrow cell-specific genes after global gene manipulations of the animals.     

Wnt signaling pathway is involved in catalpol-induced proliferation of rat bone marrow mesenchymal stem cells

AIM：To investigate the changes of Wnt signaling pathway in catalpol-induced proliferation of rat bone marrow mesenchymal stem cells (BMSCs). METHODS：The BMSCs were isolated from SD rats, purified by differential time adherent method and divided into control group and catalpol (1.0 mg/L) group. Flow cytometry was used to detect the proliferation index of BMSCs. The mRNA levels of Wnt3a, Wnt5a, Wnt11 and β-catenin was evaluated by real-time PCR. In addition, the protein expression level of β-catenin was determined by Western blotting. RESULTS：Prolife-ration index was increased from 8.90%±0.46% to 17.93%±1.68% after treatment with catalpol (P<0.01). Compared with control group, the mRNA expression of Wnt5a, Wnt11 and β-catenin was all increased with catalpol treatment. No difference of Wnt3a mRNA expression between control group and catalpol group was observed. Meanwhile, the protein expression of β-catenin was increased in catalpol group compared with control group. CONCLUSION：Catalpol promotes BMSCs going into the cell cycle. Classical and non-classical Wnt signaling pathways are activated in this process.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

Expression of recombinant myocardin and its effect on human bone marrow mesenchymal stem cell reprogramming into vascular smooth muscle cells

AIM: To optimize the expression and purification of recombinant myocardin, and to explore the effect of recombinant myocardin on the transdifferentiation of human bone marrow mesenchymal stem cells (hBMSCs) into vascular smooth muscle cells (vSMCs). METHODS: The prokaryotic expression vector for myocardin was constructed. The best ways to induce the expression of myocardin recombinant protein and to optimize the isolation and purification methods were explored. Nano-transducers were applied to encapsulate myocardin to form nano-protein complexes. Nano-myocardin was transducted into hBMSCs and the protein transduction efficiency was measured. After transduction of the protein was successful, smooth muscle myosin heavy chain, a specific marker of vSMCs, was detected, and the transformation of hBMSCs into vSMCs was also observed. RESULTS: Compared with the traditional methods, the expression of myocardin recombinant protein was higher by high-concentration induction method, and the new elution method was able to obtain more myocardin recombinant protein. The nano-myocardin complexes were successfully transduced into the hBMSCs and induced the transformation of hBMSCs into vSMCs. CONCLUSION: The recombinant myocardin was efficiently transduced into the cells and reprogrammed hBMSCs into vSMCs.     

Effects of azathioprine on proliferation,cell cycles and apoptosis of me-senchymal stem cells from rat bone marrow in vitro

AIM: To investigate the effects of azathioprine (AZA) on the proliferation, cell cycle and apoptosis of mesenchymal stem cells (MSCs) from the bone marrow of Sprague-Dawley rats in vitro. METHODS: MSCs were cultured in low-glucose DMEM containing 10% FBS,and treated with AZA at concentrations of 50 mg/L, 100 mg/L, 200 mg/L and 300 mg/L for 24 h, 48 h and 72 h. The effects of AZA on the growth curve and proliferation of MSCs were tested by cell counter under microscope. The apoptosis and cell cycle was determined by flow cytometry. RESULTS: Pure MSCs were gained by 3 times of passages. No significant effect of AZA at concentration of less than 100 mg/L on the proliferation, cell cycle and apoptosis of MSCs was observed (P>0.05). Under the condition of more than 200 mg/L for 72 h, AZA inhibited the growth of MSCs.The inhibitory rate was more than 66%, and the rate of apoptosis was increased (P<0.05). However, at the concentration of 300 mg/L for 72 h, AZA decreased the apoptotic rate and the necrotic rate of MSCs was obviously increased (P<0.05). Using AZA at concentration of more than 200 mg/L, as the action time prolonged, the MSCs in G₀/G₁ phase were increased, and those in S phase were decreased (P<0.05). CONCLUSION: At some concentrations, AZA significantly affects the proliferation, apoptosis and cell cycle of MSCs. Large dose of AZA may cause MSCs to death.