Effect of mesenchymal stem cells infusion on hematopoietic recostitution after peripheral blood stem cell transplantation in mice

AIM: To study the effect of mesenchymal stem cells (MSCs) infusion on hematopoietic recovery after peripheral blood stem cell transplantation in mice. METHODS: BALB/c mice conditioned by high dose chemotherapy/radiotherapy were infused with10⁶ peripheral blood mononuclear cells (PBMC) mobilized by granulocyte colony-stimulating factor (PBSCT group),10⁴ MSCs culture-expanded in vitro and10⁶ PBMC(experimental group1),10⁶ MSCs and10⁶ PBMC(experimental gruop 2). Survival rate within 4 weeks, white blood cell count, bone marrow nucleated cells (BMNC), granulocyte-macrophage colony forming unit(GM-CFU) and fibroblast colony forming unit (F-CFU) were examined. RESULTS: Survival rate, BMNC, GM-CFU, F-CFU were significantly higher in experimental group 2 than that in PBSCT group (P<0.05), WBC recovery was faster (P<0.01) and F-CFU level was higher in experimental group1than that in PBSCT group (P<0.05). CONCLUSION: Mesenchymal stem cells infusion enhanced hematopoietic reconstitution after peripheral blood stem cell transplantation.     

Effect of transplantation of hRAMP1 gene-modified bone marrow MSCs on postangioplasty neointima formation in rabbits

AIM: To explore the effect of transplantation of human receptor activity-modifying protein 1 ( hRAMP1 ) gene-modified bone marrow mesenchymal stem cells (MSCs) on neointima formation after carotid balloon angioplasty in carotid atherosclerosis rabbits. METHODS: MSCs were collected through density gradient centrifugation and adherent culture. MSCs were transfected with adenovirus vector carrying hRAMP1 gene to generate hRAMP1 gene-modified MSCs (hRAMP1-MSCs). All animals with carotid atherosclerosis and balloon angioplasty were randomly divided into hRAMP-MSCs group, MSCs group and control group. After the model was established, MSCs transfected with pAd2-EGFP-hRAMP1 or pAd2-EGFP and PBS were injected to the ear vein,respectively. The injured carotid arteries were harvested to detect the homing of MSCs,reendothelialization and neointima thickness 7 d, 14 d and 28 d after cell transplantation. The plasma samples were collected for detecting vascular endothelial growth factor (VEGF) by ELISA. The expression of endothelial nitric oxide synthase (eNOS) in injured carotid arteries was measured by Western blotting. RESULTS: The expression of CD31 and EGFP was observed in the neointima at different time points in hRAMP1-MSCs group and MSCs group. Compared to control group, the reendothelialization of carotid significantly increased in both hRAMP1-MSCs group and MSCs group at different time points (P<0.05), and that in hRAMP1-MSCs group showed better than that in MSCs group (P<0.05). The area of neointima and the rate of restenosis were lower in hRAMP1-MSCs group and MSCs group than those in control group, and those in hRAMP1-MSCs group were significantly lower than those in MSCs group. The plasma level of VEGF and the expression of eNOS in the injured carotid arteries were significantly higher in both hRAMP1-MSCs group and MSCs group than those in control group at different time points (P<0.05), and those in hRAMP1-MSCs group were better than those in MSCs group (P<0.05). In the injured carotid arteries, the expression level of proliferating cell nuclear antigen (PCNA) in hRAMP1-MSCs group was the lowest,with the middle level in MSCs group and the highest level in control group. CONCLUSION: The hRAMP1 gene-modified MSCs are better in promoting reendothelialization and attenuating neointima than natural MSCs. The recombinant hRAMP1 adenovirus vectors dont affect the differentiation potential of MSCs into endothelial cells.These findings indicate that the modified stem cells have the potency of more effective reendothelialization to decrease restenosis after angioplasty.     

Distribution of mesenchymal stem cells in mdx mice after transplantation

AIM: To investigate the distribution of mesenchymal stem cells in mdx mice after transplantation. METHODS: P5 mesenchymal stem cells (MSCs) of SD rats labeled with ［3H］－TdR were injected intravenously into the mdx mice preconditioned with 7 Gy γ ray. The mice were killed at 24 h, 48 h, 2 weeks, 1 month, 2 months, 4 months after transplantation of MSCs. Blood, lung, liver, bone marrow, heart, and skeletal muscle were collected, then the irradiated quantity was detected to calculate tissue specific localization account using scintillascope. RESULTS: Specific localization account in lung was the highest at 24 h. At 48 h liver was the highest. After transplantation the account of bone marrow increased and at 2 weeks reached the highest, then decreased as time going but was still higher than that of other organs. The account of skeletal muscle and heart also increased. CONCLUSION: At early time after transplantation, the MSCs labeled by ［3H］－TdR mainly distribute in lung and liver, then homing to bone marrow increasingly and the account is the highest at 2 weeks. MSCs migrate to injured organs, such as skeletal muscle and heart. The migration suggests that MSCs can settle down in muscles and provide evidence for MSCs to differentiate into myocytes.     

Effects of bone mesenchymal stem cell transplantation on silicosis fibrosis in rats

AIM: To study the effects of exogenous bone mesenchymal stem cell (BMSC) transplantation on silicosis fibrosis in rats, and to explore the dose-effect relationship. METHODS: BMSCs were isolated and cultured from male 5-week-old SD rats in vitro. Fifty healthy female SD rats were randomly divided into 5 groups: control group, silicosis model group, BMSCs treatment A group (1×10⁹ cells/L), BMSCs treatment B group (3×10⁹ cells/L) and BMSCs treatment C group (5×10⁹ cells/L). The silicosis model was made by one-time infusion of silica dust suspension using the non-exposed tracheal intubation, and different doses of BMSCs were given for intervention therapy. All the rats were sacrificed on the 21st day after the model was established. The morphological changes of the lung tissues were observed by HE staining and Masson staining. The localization and distribution of tumor necrosis factor α (TNF-α) and transforming growth factor β (TGF-β) were determined by the method of immunohistochemistry. The protein levels of TNF-α, TGF-β, collagen type I and collagen type III were detected by Western blotting. The sex-determining region (SRY) protein was searched by an immunofluorescence method to confirm the homing of BMSCs. RESULTS: Compared with control group, the silicosis model group had significant alveolitis changes, silicon nodule formation, collagen deposition and other pathological characteristics. Compared with silicosis model group, the pathological changes in BMSCs treatment A group were improved. The conditions of BMSCs treatment B group were also improved significantly. However,the pathological changes in BMSCs treatment C group were increased obviously. The protein levels of TNF-α, TGF-β, collagen type I and collagen type III in the lung tissues ranked as follows: BMSCs treatment C group > silicosis model group > BMSCs treatment A group > BMSCs treatment B group > control group. The difference between BMSCs treatment C group and silicosis model group was not statistically significant, and the differences between the other groups were statistically significant. The SRY-positive cells were observed in BMSCs treatment B group, but no significant expression in the heart, liver, spleen and kidney tissues was observed. CONCLUSION: The exogenous BMSC transplantation antagonizes the development of silicosis fibrosis in rats, which has dose-effect relationship.     

Effects of meglumine cycle adenylate phosphate combined with transplantation of mesenchymal stem cells on heart functions in rat model of heart failure

AIM: To investigate the therapeutic effects of recombinant meglumine cycle adenylate phosphate (MCA) and bone marrow mesenchymal stem cells (BMSCs) on the enhancement of the cell survival and improvement of the cardiac functions in the rat model of adriamycin-induced cardiomyopathic heart failure. METHODS: BMSCs were isolated and expanded using the pre-plating method. Doxorubicin was used by intraperitoneal injection into the Wistar rats to establish the model of cardiomyopathic heart failure. The model animals randomly received the injection of PBS, MCA, BMSCs or MCA+BMSCs respectively, and normal controls were without any treatment. Four weeks after injection, the cardiac functions were determined by echocardiography and multichannel physiological recorder. The levels of brain natriuretic peptide (BNP) were measured by ELISA. The positive rate of BrdU-labeled BMSCs in the myocardium was analyzed by the method of immunohistochemistry. The expression of myocardium-specific protein, GATA-4, connexin 43(Cx43) and cardiac troponin 1(cTNI), was detected by Western blotting. Myocardial fibrosis was observed with Masson's staining. RESULTS: Compared with other groups, the results of echocardiography and hemodynamic showed that the left ventricular functions in BMSCs+MCA group improved significantly (P<0.05). The BMSCs numbers in the myocardium in BMSCs+MCA group were significantly higher than those in BMSCs group (P<0.05). The level of BNP was significantly lower in BMSCs+MCA group than that in BMSCs group (P<0.05). Compared with other groups, the expression of GATA-4, Cx43 and cTNI was significantly increased in BMSCs+MCA group. CONCLUSION: Combination of MCA with BMSCs transplantation improves the cardiac functions, possibly due to the enhancement of BMSCs survival and the increase in the protein expression of GATA-4.     

Biological characterics of human fetal cord blood mesenchymal stem cells

AIM: To investigate the biological characterics of human second-trimester fetal cord blood mesenchymal stem cells (MSC) and its application prospects in utero gene transfer/therapy (IUGT). METHODS: Nuclear cells separated from cord blood were cultured in DMEM medium. Surface antigens of the MSC were analyzed by the FACScan flow cytometry. Adipogenic and osteogenic mediums were used to assess the differentiation ability of the cells. Adenovirus vector deliver green fluorescent protein gene (Ad-GFP) was used to transfected the MSC and the expressing of GFP was detected by fluorescent microscope. The MSC were injected into the liver of newborn rat. The immunofluorescence analysis was conducted to determine the presence of double-positive CD105⁺/CD166⁺ cells in different organs of rats. MSC were subcutaneous injected into the human-nonobese diabetes/severe combined immunodeficiency disease (NOD/SCID) mice and carcinogenesises of the MSC in vivo were detected by pathological diagnosis. RESULTS: MSC could be separated from fetal cord blood. These cells were uniformly positive for CD29, CD44, CD59, CD105, CD166 and negative for CD34, CD45, CD80, CD86, HLA-DR. The cells had the abilities to differentiate into adipogenic and osteogenic cells in vitro, expressed the GFP at high levels (56.32%±3.28%). The MSC were located at different organs after injected into the newborn rats and didn't have carcinogenicity in vivo. CONCLUSION: Human second-trimester fetal cord blood MSC is an promising target cells in fetal IUGT.     

Effect of portal vein transplantation with allogeneic adipose tissue-derived mesenchymal stem cells on liver fibrosis in SD rats

AIM: To explore the influence of adipose tissue-derived mesenchymal stem cells (ADMSCs) transplantation on hepatic fibrosis in rats. METHODS: ADMSCs from abdominal lipid tissues were extracted, cultured and passaged. The hepatic fibrosis rat model was built up and randomly divided into 3 groups: hepatic cirrhosis group (n=14); portal vein transplantation group (n=11) and caudal vein transplantation group (n=14). Computer tomography(CT) perfusion index, histological scores and microvessel density were detected and compared after transplantation of ADMSCs among the 3 groups. RESULTS: After transplantation of ADMSCs, the total hepatic blood perfusion, especially portal vein perfusion, significantly increased in portal vein transplantation group determined by CT perfusion scan (P<0.05), but slightly increased in caudal vein transplantation group. The histological scores showed significant alleviation of fibrosis evidence in portal vein transplantation group, and slightly change of adipose degeneration in caudal vein transplantation group. Microvessel density decreased significantly in portal vein transplantation group as compared to the other 2 groups (P<0.05). CONCLUSION: Transplantation of ADMSCs greatly helps the alleviation of hepatic fibrosis. Portal vein transplantation benefits more than caudal vein transplantation.     

Bone marrow-derived mesenchymal stem cells regulate the function of Th17/ Treg in peripheral blood of severe asthmatic children

AIM：To investigate the regulatory function of bone marrow-derived mesenchymal stem cells (MSCs) on T helper 17 cells (Th17) and regulatory T cells (Treg) in peripheral blood of severe asthmatic children. METHODS：MSCs were isolated, cultured and identified in vitro. MSCs digested with mitomycin were cocultured with T lymphocytes (TLC) at different ratios (1∶1, 1∶2, 1∶10 and 1∶20) from severe asthmatic children for 72 h. The proliferation of TLC was measured by CCK-8 method. In the coculture system of the 1∶2 ratio and the single TLC system, the supernatant levels of interleukin-17 (IL-17) and transforming growth factor-β (TGF-β) were measured by ELISA. The mRNA expression of retinoic acid-related orphan nuclear receptor C (RORC) and forkhead box protein 3 (Foxp3) in TLC was detected by qRT-PCR. RESULTS：After cocultured with MSCs, the proliferation of TLC decreased significantly in a dose-dependent manner (P<0.05). It also showed decreases in IL-17 (3 799±441 vs 4 890 ±373, P<0.05) and RORC mRNA level (1.21±0.14 vs 3.85±0.48, P<0.05), while an increase in TGF-β level (209±32 vs 117±26, P<0.05) was observed. No influence on the mRNA expression of Foxp3 was found (P>0.05). CONCLUSION：MSCs suppresses Th17 polarization of naive peripheral blood CD4+ T cells and matures Th17 cells secreting IL-17, which may effectively revise Th17/Treg imbalance of asthma.     

Biological characters of mesenchymal stem cells of human umbilical cord blood

AIM：To study the isolation,expansion and purification of mesenchymal stem cells (MSCs) from human umbilical cord blood (UCB),and investigate some biological identities of MSCs.METHODS：(1) MSCs of UCB,adult bone marrow (BM) and fetus BM were isolated by centrifugation with Ficoll,and the different kinds of MSCs were observed everyday.(2) Surface markers of MSCs were identified by flow cytometry.(3) The level of HGFs (TPO,SCF,FLT-3L,IL-6) secreted by different sources of MSCs was checked by ELISA method.RESULTS：(1) No difference in morphology of the colonies between UCB MSCs and BM MSCs was observed.However,the mononuclear cells needed in culture of UCB MSCs was about 3 times more than that in culture of BM MSCs.The times of UCB MSCs colony formation and confluencing were longer than that of BM in primary culture.(2) After passaged,there was no significant difference in the proliferation rates of 3 kinds of MSCs.Only 4 of 15 UCB samples contained a homogeneous population of MSCs.(3) UCB MSCs shared the same markers with BM MSCs.Neither hematopoietic marker nor immunologic recognition antigens were expressed.(4) The level of hematopoietic growth factors (HGFs) secreted by 3 kinds of MSCs was similar.CONCLUSIONS：(1) MSCs were isolated from UCB,but the amount of MSCs in UCB was smaller than that in BM,and just seldom samples of UCB contained homogeneous MSCs.(2) MSCs from UCB and BM shared the same biological characteristics,such as proliferation ability,surface markers,immunophenotypes and HGFs secretion.     

Effects of cytokines on renovation of acute renal failure in mouse with bone marrow derived mesenchymal stem cell transplantation

AIM: To observe the effects of cytokines on renovation of acute renal failure (ARF) in mouse with bone marrow derived mesenchymal stem cell (MSC) transplantation. METHODS: ARF animal model was induced in mouse by subcutaneous injection of cisplatin. Mice were randomly assigned into 3 groups: normal control group, ARF group and MSC group. After 24 h cisplatin injection, animals were injected intravenously with MSC in MSC group. Animals were sacrificed at 1 d, 4 d, 7 d, 14 d and 28 d after cisplatin injection. The blood urea nitrogen (BUN) and serum creatinine (Scr) were measured. The renal morphologic changes were scored with Paller’s criterion on hematoxylin and eosin (HE) stained sections. The mRNA and protein expressions of HGF, BMP-7, TNF-α and IL-10 were detected by RT-PCR and immunohistochemistry method. RESULTS: After 4 d of cisplatin injection, the BUN and Scr values in MSC group were significantly lower than those in ARF group (P<0.01). After 7 d and 14 d, the values of BUN and Scr in MSC group were still lower than those in ARF group (P<0.01, P<0.05). The renal morphologic scores of MSC group were also lower than those of ARF group. After 7 d, the expressions of HGF, BMP-7 and IL-10 were higher in MSC group than those in ARF group, the expression of TNF-α in MSC group was lower than that in ARF group. CONCLUSION: MSC promotes the recovery of acute renal failure induced by cisplatin. The mechanism may partly depend on paracrine of growth factor and amelioration of inflammatory.     

Effects of PD98059 on the differentiation from mesenchymal stem cells to osteoblasts

AIM: To investigate the effects of PD98059 on the differentiation from mesenchymal stem cells to osteoblasts.METHODS: hMSC were separated from human marrow and expanded in cuture medium. hMSC were induced with dexamethasone, β-glycerophosphate, vitamin C which acted as osteoblast differentiation inducer. PD98059 was added into the osteoblasts induction medium. The cells were assayed with cell morphology, alkaline phosphatase (AP) activity and calcium deposition. RESULTS: The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. After induced with osteoblasts induction medium, the cells showed changes in cell morphology from spindle-shape to cuboidal and polygonal. The AP activity increased gradually and reached the peak in 12 days, then decreased. Many scattered tangerice calcium nodes were observed. PD 98059 significantly inhibited AP activity and calcium deposition in a dose-dependent manner. A striking observation of the present study was that a few adipocytes appeared in cultures that were treated with PD 98059 and osteogenic differentiation medium. CONCLUSION: These results indicated that osteogenic diferentiation from the hMSCs was related to the activation of the ERK.     

Transfection of mesenchymal stem cells with human heme oxygenase-1 gene improves survival under conditions of serum-free and hypoxia

AIM: To investigate the effects of human heme oxygenase-1 (HO-1) gene transfection on mesenchymal stem cells (MSCs) survival under the conditions of serum-free and hypoxia. METHODS: MSCs were acquired from the bone marrow of adult rats. The cells were isolated, purified, cultured, and transfected with Adv-HO-1. The expression of GFP was detected by immunofluorescence. Cell apoptosis was detected by nuclear DAPI staining and FACS. The concentrations of VEGF, HGF and b-FGF in the culture supernatant were measured by ELISA. The caspase-3 protein level and activity of cardiomyocytes cultured with the supernatants from different MSCs under the condition of serum-free and hypoxia were assayed by Western blotting and fluorimetry, respectively. RESULTS: HO-1-MSCs exhibited strong expression of GFP. The fragmented or condensed chromatin diminished in HO-1-MSCs compared with the MSCs lacking exogenous transfection of HO-1 gene. A lower proportion of apoptosis was observed in HO-1-MSCs compared with MSCs under the conditions of serum-free and hypoxia (P<0.01). The expressions of VEGF, HGF and b-FGF in the supernatants of HO-1-MSCs were higher than those in MSCs (P<0.01). A significant reduction of caspase-3 level and activity in the cardiomyocytes treated with the supernatants from HO-1-MSCs was observed, compared to that treated with supernatants from MSCs (P<0.01). CONCLUSION: HO-1 improves the MSCs survival under the conditions of serum-free and hypoxia. Several cytokines released by HO-1-MSCs may protect the cardiomyocytes against apoptosis.     

Effects of exosomes derived from hypoxia-preconditioned umbilical cord mesenchymal stem cells on function of endothelial cells

AIM To investigate the effect of exosomes derived from hypoxia-preconditioned human umbilical cord mesenchymal stem cells (hUCMSCs) on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs). METHODS hUCMSCs and HUVECs were isolated, cultured and identified. Exosomes derived from hUCMSCs were extracted by ultracentrifugation. The morphological change of exosomes was observed under transmission electron microscope. The particle size and concentration of exosomes were detected by nanoparticle tracking analysis, and the surface specific marker proteins of exosomes were determined by Western blot. hUCMSCs were divided into normoxia group and hypoxia group. The viability of hUCMSCs was measured by CCK-8 assay. HUVECs were divided into control group, normoxic exosome group and hypoxic exosome group. The proliferation of HUVECs was detected by EdU assay. The migration ability was detected by cell scratch assay and Transwell experiment. Tube formation ability was evaluated by tube formation experiment. RESULTS Compared with normoxia group, hypoxia pretreatment enhanced the viability and exosome release of hUCMSCs. Compared with normoxic exosome group, hypoxic exosomes enhanced the proliferation, migration and tube formation of HUVECs. CONCLUSION Exosomes derived from hUCMSCs under hypoxia enhances the proliferation, migration and tube formation of HUVECs.     

Effects of cotransplantation of umbilical cord blood and mesenchymal stem cells by intra-bone marrow injection on hematopoietic reconstitution in a rat model

AIM: To investigate the effects of cotransplantation of mesenchymal stem cells (MSCs) and umbilical cord blood (UCB) by intra-bone marrow (IBM) injection on the hematopoietic reconstitution and recovery of bone marrow MSCs in the recipients. METHODS: Wistar female rats were transplanted with fetal and neonatal peripheral blood (FNPB) and BrdU-labeled MSCs separated from BMNCs of F344 rats. The MSCs were infused by IBM injection in bilateral tibiae or intravenous injection (IV), while the FNPB was all via IBM route. The survival rate, reconstitution of hematopoietic and immunological function, engraftment level of HSCs and recovery of bone marrow (BM)-MSCs in recipients were monitored. The origins of BM-MSCs of recipients were examined by immunofluorescence assay. RESULTS: (1)The survival rate in the two cotransplantation groups was 100% at day 60, while that in FNPB group was only 66.7%. (2)The counts of peripheral blood cells and BM hematopoietic stem/progenitor cell colonies of the recipients were better in cotransplantation groups than those in FNPB group, especially in the FNPB (IBM)+MSC (IBM) group. (3)No significant difference between of engraftment level of HSCs in the two cotransplantation groups was observed. The percentage of RT1A1 cells subset in FNPB (IBM)+MSC (IBM) group was much higher than that in FNPB group (P<0.05). (4)At day 30, the growth characteristic of recipient BM-MSCs was still below normal, but that in FNPB (IBM)+MSC (IBM) group was the best of all the experiment groups (P<0.05). (5)The donor MSCs coexisted with host MSCs in only a few recipient rats. CONCLUSION: The cotransplantation of MSCs and FNPB can accelerate the recovery of recipient BM-MSCs and hematopoietic reconstitution, promote the engraftment level of HSCs. Cotransplantation by IBM route is safe and has better effects on hematopoietic reconstitution than by IV route.     

Effects of myocardial extracts on transdifferentiation of bone marrow-derived mesenchymal stem cells of rats

AIM: To investigate the effects of myocardial extracts of rats on transdifferentiation of bone marrow-derived mesenchymal stem cells (MSCs). METHODS: After establishing stable culture methods for rat MSCs, they were induced by high level of myocardial extracts on day 3 of primary culture for 1 week. Immunocytochemistry technique was used to detect MHC and troponin-T, and the expression of Nkx2.5, α-MHC and ANP genes were identified by RT-PCR technique 3 weeks later. RESULTS: MSCs of rats expressed cardiac MHC and troponin-T proteins, Nkx2.5 and α-MHC genes after induction. However, they neither expressed ANP gene nor formed myotubes and intercalated disk. CONCLUSION: Myocardial extracts can induce MSCs to transdifferentiate into cardiomyocytes.     

Effects of nicotine on proliferation and migration of human umbilical cord mesenchymal stem cells

AIM: To explore the effects of nicotine on nitric oxide (NO), nitric oxide synthase (NOS), inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), mitochondrial membrane potential and cytokine secretion in human umbilical cord mesenchymal stem cells (MSCs). METHODS: MSCs were treated with different concentrations of nicotine. The content of NO was detected by nitrate reductase method. The activity of NOS and iNOS was mea-sured by ultraviolet spectrophotometry. ROS and mitochondrial membrane potential were detected by flow cytometry. The levels of intercellular adhesion molecule 1（ICAM-1）, stromal cell-derived factor 1 (SDF-1), matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1(TIMP-1), transforming growth factor β1 (TGF-β1), insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF) were determined by ELISA. RESULTS: At 24 h and 36 h after exposure to nicotine, the levels of NO were significantly increased in a dose-dependent manner. However, at 48 h, the levels of NO in 0.8 g/L group and 1.0 g/L group were lower than that in control group. The activity of NOS and iNOS were significantly increased in a dose-dependent manner. The level of ROS increased, while mitochondrial membrane potential decreased. After nicotine treatment, the secretions of SDF-1, TGF-β1, IGF-I and bFGF declined, while the levels of ICAM-1, MMP-9 and TIMP-1 increased. CONCLUSION: Nicotine may affect the proliferation, adhesion and migration of MSCs by increasing the levels of NO, NOS, iNOS and ROS and the production of ICAM-1, MMP-9 and TIMP-1, and decreasing the mitochondrial membrane potential and the secretion of SDF-1, TGF-β1, IGF-I and bFGF.     

Rat mesenchymal stem cells differentiate into neuron-like cells with adrenaline hormones

AIM: To investigate the differentiation from rat mesenchymal stem cells (rMSC) into neuron-like cells. METHODS:rMSC were separated from femur marrow and expanded in L-DMEM culture medium supplemented with 10% FSC. rMSC were induced to differentiate into neurons with L-DMEM/adrenaline,L-DMEM/noradrenaline and L-DMEM/isoprenaline, respectively. Meanwhile, rMSC were cultured in L-DMEM in control group. Nestin, neuron-specific enclose (NSE), glial fibrillary acidic protein (GFAP) were detected by immunocytochemistry. RESULTS: rMSC were expanded as undifferentiated cells in culture from 5 to 22 passages, indicating their differentiated capacity. Simple method induced rMSC to exhibit a neuronal phenotype, expressing positive NSE,nestin, and GFAP, at 5 hours in all group. The undifferentiating cells (control group 53.1%±4.3%), and differentiating cells (treated group: adrenaline 74.7%±2.6%; noradrenaline 75.9%±2.4%; isoprenaline 72.1%±4.4%), expressed characteristics of various neuronal cells, from 5 hours to 6 days. There were neuron-like cells in rMSC cultured in L-DMEM/10%FBS from 7 to 13 passage(66.5%±6.4%). CONCLUSION: It suggests that rat neural stem cells (rNSC) exist in bone marrow, rMSC can be differentiated into various neural cells with adrenaline hormones in vitro.     

Inhibitory effect of hypoxia preconditioning on hypoxia/reoxygenation-induced apoptosis in cardiomyocytes

AIM: To study the role of hypoxia preconditioning (HP) in hypoxia-reoxygenation (HR)-induced apoptosis in neonatal rat cardiomyocytes and the possible mechanisms. METHODS: Cultured neonatal rat cardiomyocytes were divided into three groups: normal group, HP+H/R group and H/R group. Acridine orange (AO) staining was performed to detect morphological changes of apoptotic cells. Apoptosis rates of cardiomyocytes were detected by flow cytometry. Colorimetric assay was used to detect caspase-3 activity. Expression of Bcl-2 protein was detected by immunohistochemistry combined with computer image analysis. RESULTS: Apoptotic cells were detected by AO staining after hypoxia of 6 h followed by 3 h-reoxygenation. The hypodiploid apoptotic peak was detected by flow cytometry with the apoptotic rates of (29.7±5.4)%. A significantly reduced apoptotic rates of (7.8±1.3)% was detected in HP group（P＜0.01）. The caspase-3 relative activity of cardiomyocytes induced by H/R was 5.9±0.8, significantly higher than that of control group. HP markedly reduced caspase-3 relative activity to 2.6±0.5 in contrast with H/R group （P＜0.01）. Bcl-2 protein was positive in normal cardiomyocytes with an A value of 119.4±7.1. The A value of H/R group was 99.6±5.0, significantly lower than that in normal group （P＜0.01）. The A value of HP+H/R group was 126.5±6.2, significantly higher than that in H/R group（P＜0.01）. CONCLUSION: HP inhibits H/R-induced apoptosis of cardiomyocytes by improving the expression of Bcl-2 and reducing caspase-3 activity.