Transplantation of bone marrow-derived endothelial progenitor cells and hepatocyte stem cells from liver fibrosis rats ameliorates liver fibrosis

AIM To explore the effectiveness for treating liver fibrosisby combined transplantation of bone marrow-derived endothelial progenitor cells(BM-EPCs) and bone marrow-derived hepatocyte stem cells(BDHSCs) from the liver fibrosis environment.METHODS The liver fibrosis rat models were induced with carbon tetrachloride injections for 6 wk. BM-EPCs from rats with liver fibrosis were obtained by different rates of adherence and culture induction. BDHSCs from rats with liver fibrosis were isolated by magnetic bead cell sorting. Tracing analysis was conducted by labeling EPCs with PKH26 in vitro to show EPC location in the liver. Finally, BM-EPCs and/or BDHSCs transplantation into rats with liver fibrosis were performed to evaluate the effectiveness of BM-EPCs and/or BDHSCs on liver fibrosis.RESULTS Normal functional BM-EPCs from liver fibrosis rats were successfully obtained. The co-expression level of CD133 and VEGFR2 was 63.9% ± 2.15%. Transplanted BM-EPCs were located primarily in/near hepatic sinusoids. The combined transplantation of BM-EPCs and BDHSCs promoted hepatic neovascularization, liver regeneration and liver function, and decreased collagen formation and liver fibrosis degree. The VEGF levels were increased in the BM-EPCs(707.10 ± 54.32) and BM-EPCs/BDHSCs group(615.42 ± 42.96), compared with those in the model group and BDHSCs group(P < 0.05). Combination of BM-EPCs/BDHSCs transplantation induced maximal up-regulation of PCNA protein and HGF m RNA levels. The levels of alanine aminotransferase(AST), aspartate aminotransferase, total bilirubin(TBIL), prothrombin time(PT) and activated partial thromboplastin time in the BMEPCs/BDHSCs group were significantly improved, to be equivalent to normal levels(P > 0.05) compared with those in the BDHSC(AST, TBIL and PT, P < 0.05) and BM-EPCs(TBIL and PT, P < 0.05) groups. Transplantation of BM-EPCs/BDHSCs combination significantly reduced the degree of liver fibrosis(staging score of 1.75 ± 0.25 vs BDHSCs 2.88 ± 0.23 or BMEPCs 2.75 ± 0.16, P < 0.05).CONCLUSION The combined transplantation exhibited maximal therapeutic effect compared to that of transplantation of BM-EPCs or BDHSCs alone. Combined transplantation of autogenous BM-EPCs and BDHSCs may represent a promising strategy for the treatment of liver fibrosis, which would eventually prevent cirrhosis and liver cancer.

HNF-4α determines hepatic differentiation of human mesenchymal stem cells from bone marrow

AIM: To investigate the differentiation status and key factors to facilitate hepatic differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). METHODS: Human MSCs derived from bone marrow were induced into hepatocyte-like cells following a previously published protocol. The differentiation status of the hepatocyte-like cells was compared with various human hepatoma cell lines. Overexpression of hepatocyte nuclear factor (HNF)-4α was mediated by adenovirus infection of these hepatocyte-like cells. The expression of interesting genes was then examined by either re-verse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR methods. RESULTS: Our results demonstrated that the differentiation status of hepatocyte-like cells induced from human MSCs was relatively similar to poorly differentiated human hepatoma cell lines. Interestingly, the HNF-4 isoform in induced MSCs and poorly differentiated human hepatoma cell lines was identified as HNF4γ instead of HNF-4α. Overexpression of HNF-4α in induced MSCs significantly enhanced the expression level of hepatic-specific genes, liver-enriched transcription factors, and cytochrome P450 (P450) genes. CONCLUSION: Overexpression of HNF-4α improves the hepatic differentiation of human MSCs from bone marrow and is a simple way of providing better cell sources for clinical applications.

Cardiomyocyte-like differentiation of human bone marrow mesenchymal stem cells after exposure to 5-azacytidine in vitro

Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073g/mL Percoll and propagated in the right cell culturing medium as previously described. The phenotypes of hMSCs were characterized with the use of flow cytometry. The hMSCs were cultured in cell culture medium (as control) and medium mixed with 5-aza for cellular differentiation. We examined by immunohistochemistry at 21 days the inducement of desmin, cardiac-specific cardiac troponin I (cTnI), GATA 4 and connexin-43 respectively. Results The hMSCs are fibroblast-like morphology and express CD44+ CD29+ CD90+ / CD34- CD45- CD31- CD11a. After 5-aza treatment, 20-30% hMSCs connected with adjoining cells and coalesced into myotube structures after 14days. Twenty-one days after 5-aza treatment, immunofluorescence showed that some cells expressed desmin,GATA4, cTnI and connexin-43 in 5,10 μmol/L 5-aza groups, but no cardiac specific protein was found in neither 3μmol/L 5-aza group nor in the control group. The ratio of cTnI positively stained cells in 10 μmol/L group was higher than that in 5 μmol/L group (65.3 ± 4.7% vs 48.2 ± 5.4%, P ＜ 0.05). Electron microscopy revealed that myofilaments were formed. The induced cells expressed cardiac-myosin heavy chain (MyHC) gene by reverse transcription-polymerase chain reaction (RT-PCR). Conclusions Theses findings suggest that hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration. (J Geriatr Cardiol 2004;1(2) :101-107. )

The genomic landscapes of histone H3-Lys9 modifications of gene promoter regions and expression profiles in human bone marrow mesenchymal stem cells

Mesenchymal stem cells （MSCs） of nonembryonic origins possess the proliferation and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms could be critical for determining the fate of stem cells, and MSCs derived from different origins exhibited different expression profiles individually to a certain extent. In this study, ChiP-on-chip was used to generate genome-wide histone H3-Lys9 acetylation and dimethylation profiles at gene promoters in human bone marrow MSCs. We showed that modifications of histone H3-Lys9 at gene promoters correlated well with mRNA expression in human bone marrow MSCs. Functional analysis revealed that many key cellular pathways in human bone marrow MSC self-renewal, such as the canonical signaling pathways, cell cycle pathways and cytokine related pathways may be regulated by H3-Lys9 modifications. These data suggest that gene activation and silencing affected by H3-Lys9 acetylation and dimethylation, respectively, may be essential to the maintenance of human bone marrow MSC self-renewal and multi-potency.

Overexpression of vascular endothelial growth factor enhances the neuroprotective effects of bone marrow mesenchymal stem cell transplantation in ischemic stroke

Although bone marrow mesenchymal stem cells(BMSCs)might have therapeutic potency in ischemic stroke,the benefits are limited.The current study investigated the effects of BMSCs engineered to overexpress vascular endothelial growth factor(VEGF)on behavioral defects in a rat model of transient cerebral ischemia,which was induced by middle cerebral artery occlusion.VEGF-BMSCs or control grafts were injected into the left striatum of the infarcted hemisphere 24 hours after stroke.We found that compared with the stroke-only group and the vehicle-and BMSCs-control groups,the VEGF-BMSCs treated animals displayed the largest benefits,as evidenced by attenuated behavioral defects and smaller infarct volume 7 days after stroke.Additionally,VEGF-BMSCs greatly inhibited destruction of the blood-brain barrier,increased the regeneration of blood vessels in the region of ischemic penumbra,and reducedneuronal degeneration surrounding the infarct core.Further mechanistic studies showed that among all transplant groups,VEGF-BMSCs transplantation induced the highest level of brain-derived neurotrophic factor.These results suggest that BMSCs transplantation with vascular endothelial growth factor has the potential to treat ischemic stroke with better results than are currently available.

Human bone marrow mesenchymal stem cell transplantation attenuates axonal injury in stroke rats

Previous studies have shown that transplantation of human bone marrow mesenchymal stem cells promotes neural functional recovery after stroke, but the neurorestorative mechanisms remain largely unknown. We hypothesized that functional recovery of myelinated axons may be one of underlying mechanisms. In this study, an ischemia/reperfusion rat model was established using the middle cerebral artery occlusion method. Rats were used to test the hypothesis that intravenous transplantation of human bone marrow mesenchyrnal stem cells through the femoral vein could exert neuroprotective effects against cerebral ischemia via a mechanism associated with the ability to attenuate axonal injury. The results of behavioral tests, infarction volume analysis and immunohistochemistry showed that cerebral ischemia caused severe damage to the myelin sheath and axons. After rats were intravenously transplanted with human bone marrow mesenchymal stem cells, the levels of axon and myelin sheath-related proteins, including microtubule-associated protein 2, myelin basic protein, and growth-associated protein 43, were elevated, infarct volume was decreased and neural function was improved in cerebral ischemic rats. These findings suggest that intravenously transplanted human bone marrow mesenchymal stem cells promote neural function. Possible mechanisms underlying these beneficial effects include resistance to demyelination after cerebral ischemia, prevention of axonal degeneration, and promotion of axonal regeneration.

Effect of Intracoronary Infusion of Bone Marrow Mononuclear Cells or Peripheral Endothelial Progenitor Cells on Myocardial Ischemia-reperfusion Injury in Mini-swine

Objective To simulate and assess the clinical effect of intracoronary infusion of bone marrow mononuclear cells or peripheral endothelial progenitor cells on myocardial reperfusion injury in mini-swine model. Methods Twenty-three mini-swine with myocardial reperfusion injury were used as designed in the study protocol. About （3.54±0.90）×10^7 bone marrow mononuclear cells （MNC group, n=9） or （1.16± 1.07）× 10^7 endothelial progenitor cells （EPC group, n=7） was infused into the affected coronary segment of the swine. The other mini-swine were infused with phosphate buffered saline as control （n=7）. Echocardio- graphy and hemodynamic studies were performed before and 4 weeks after cell infusion. Myocardium infarc- tion size was calculated. Stem cell differentiation was analyzed under a transmission electromicroscope. Results Left ventricular ejection fraction dropped by 0% in EPC group, 2% in MNC group, and 10% in the control group 4 weeks after cell infusion, respectively （P〈0.05）. The systolic parameters increased in MNC and EPC groups but decreased in the control group. However, the diastolic parameters demonstrated no significant change in the three groups （P〉0.05）. EPC decreased total infarction size more than MNC did （1.60±0.26 cm2 vs. 3.71±1.38 cm2, P〈0.05）. Undermature endothelial cells and myocytes were found under transmission electromlcroscope. Conclusions Transplantation of either MNC or EPC may be beneficial to cardiac systolic function, but might not has obvious effect on diastolic function. Intracoronary infusion of EPC might be better than MNC in controlling infarction size. Both MNC and EPC may stimulate angiogenesis, inhibit flbrogenesis, and differentiate into myocardial cells.

Neuronal differentiation effects of vascular endothelial factor on bone marrow stromal cells

BACKGROUND：Studies have demonstrated that bone marrow stromal cells （BMSCs） undergo neuronal differentiation under certain in vitro conditions.However,very few inducers of BMSC differentiation have been used in clinical application.The effects of vascular endothelial growth factor （VEGF） on in vitro neuronal differentiation of BMSCs remain poorly understood.OBJECTIVE：To investigate the effect of VEGF on neuronal differentiation of BMSCs in vitro,and to determine the best VEGF concentration for experimental induction.DESIGN,TIME AND SETTING：In vitro comparative study was performed at the Central Laboratory and Laboratory of Male Reproductive Medicine,Shenzhen Hospital of Peking University from October 2008 to August 2009.MATERIALS：Recombinant human VEGF165 was purchased from Peprotech Asia,Rehovot,Israel.Neuron-specific enolase （NSE） was purchased from Beijing Biosynthesis Biotechnology,China.METHODS：BMSCs were harvested from adult Sprague Dawley rats.The passaged cells were pre-induced with 10 ng/mL basic fibroblast growth factor for 24 hours,followed by differentiation induction with 0,5,10,and 20 ng/mL VEGF,respectively.MAIN OUTCOME MEASURES：Morphological changes in BMSCs prior to and following VEGF induction.Expression of NSE following induction was determined by immunocytochemistry.RESULTS：Shrunken,round cells,with a strong refraction and thin bipolar or multipolar primary and secondary branches were observed 3 days after induction with 5,10,and 20 ng/mL VEGF.However,these changes were not observed in the control group.At 10 days after induction,the number of NSE-positive cells was greatest in the 10 ng/mL VEGF-treated group （P〈 0.05）.The number of NSE-positive cells was least in the control group at 3 and 10 days post-induction （P〈 0.05）.Moreover,the number of NSE-positive cells was greater at 10 days compared with at 3 days after induction （P〈 0.05）.CONCLUSION：Of the VEGF concentrations tested,10 ng/mL induced the greatest number of neuronal-like cells in vitro from BMSCs.

Mesenchymal Stromal Cells Derived from Human Embryonic Stem Cells, Fetal Limb and Bone Marrow Share a Common Phenotype but Are Transcriptionally and Biologically Different

Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to determine the optimal source of MSCs. We investigated if this biological heterogeneity in MSCs from different sources results in different mechanisms for their differentiation. In this study, we compared the gene expression patterns of phenotypically defined MSCs derived from three ontogenically different sources: Embryonic stem cells (hES-MSCs), Fetal limb (Flb-MSCs) and Bone Marrow (BM-MSCs). Differentially expressed genes between differentiated cells and undifferentiated controls were compared across the three MSC sources. We found minimal overlap (5% - 16%) in differentially expressed gene sets among the three sources. Flb-MSCs were similar to BM-MSCs based on differential gene expression patterns. Pathway analysis of the differentially expressed genes using Ingenuity Pathway Analysis (IPA) revealed a large variation in the canonical pathways leading to MSC differentiation. The similar canonical pathways among the three sources were lineage specific. The Flb-MSCs showed maximum overlap of canonical pathways with the BM-MSCs, indicating that the Flb-MSCs are an intermediate source between the less specialised hES-MSC source and the more specialised BM-MSC source. The source specific pathways prove that MSCs from the three ontogenically different sources use different biological pathways to obtain similar differentiation outcomes. Thus our study advocates the understanding of biological pathways to obtain optimal sources of MSCs for various clinical applications.

Vascular endothelial growth factor/platelet-derived growth factor receptor pathway is involved in bone marrow mesenchymal stem cell differentiation and directional migration toward gliomas

BACKGROUND： Vascular endothelial growth factor （VEGF） induces bone marrow-derived mesenchymal stem cell （BMSC） differentiation into vascular endothelial-like cells and promotes BMSC migration toward gliomas. However, the molecular mechanisms by which VEGF induces BMSC differentiation and migration remain poorly understood. OBJECTIVE; To investigate the role of platelet-derived growth factor （PDGF） receptor （PDGFR） in BMSC differentiation and migration induced by VEGE DESIGN, TIME AND SETTING： A parallel, controlled, in vitro experiment was performed at the Molecular Neurobiology ＆ Neural Regeneration and Repairing Laboratory, Anhui Provincial Hospital of Anhui Medical University, China from June 2008 to March 2009. MATERIALS： U87 glioma cells were purchased from Shanghai Institutes for Biological Sciences; mouse anti-human PDGFR and VEGF receptor （VEGFR） monoclonal antibodies were purchased from Peprotech, USA. METHODS： Isolated BMSCs were precultured with neutralizing antibody for VEGFR-1, VEGFR-2, PDGFR-α, and PDGFR-β to block biological activity of related receptors, followed by induced differentiation with 50μg/L VEGF. BMSCs induced with 50μg/L VEGF alone served as the VEGF-induced group. The control group remained untreated. MAIN OUTCOME MEASURES： Cell surface markers were identified by flow cytometry; BMSC surface cytokine receptor expression was detected by reverse transcription-polymerase chain reaction; the Transwell model was used to observe cell migration. RESULTS： After blocking the PDGFR, VEGF did not induce BMSC cell surface marker CD-31 or von Willebrand factor （vWF） expression. However, inhibition with VEGF receptor blocking agents, VEGF induced BMSCs to express CD-31 and vWE Following inhibition of the PDGFR, the number of cells migrating through the polycarbonate membrane Transwell chamber was decreased, as well as the number of BMSCs migrating to glioma cells. However, through the use of VEGF receptor blocking agents, the number of migrating cells remained unchanged. VEGF preculture increased the number of BMSCs migrating to gliomas. CONCLUSION： VEGF interacts with PDGFRs on the BMSC surface to attract BMSC directional migration and induce BMSC differentiation. The VEGF/PDGFR pathway participates in BMSC directional migration to glioma. VEGF pretreatment increased efficiency of BMSC migration to glioma.

Double potentialities of tumoricide and hematopoiesis of human bone marrow cells activated by IL-2 and IL-3

The biomodulative and hematopoietic potentialities of IL-2 and IL-3 activatedbone marrow(ABM)cells from patients with lung adenocarcinoma were studied in vitro.Human bone marrow(BM)cells could be activated by IL-2 in culture for 7d.TheseIL-2 ABM cells had higher cytolytic activities against cells of H 7402 cell line and freshautologous adenocarcinoma cells and maintained the cytotoxicities longer than IL-2 acti-vated peripheral blood lymphocytes(APBLs),a point of possible importance in adoptiveimmunotherapy for cancer patients.The IL-2 ABM cells also had similar number ofBFU-E and CFU-GM to that had fresh BM cells if 1L-3 was added 48h alter IL-2 inculture.The IL-2 and IL-3 ABM cells might be used to eliminate tumor cells and tosupply reconstitutive elements of BM for autologous bone marrow transplantation.

An experiment study of osteogenesis of Ad-VEGF165 transfected human bone marrow mesenchymal stem cells in vitro

Objective ：To evaluate the effect of osteogenic potential on human marrow mesenchymal stem cells （hMSCs） transferred with human vascular endothelial growth factor（VEGF） gene by adenovirus, methods：hMSCs were isolated from human marrow, cultured in vitro and randomly divided into 3 groups ：Ad-VEGF165 group： adding 1×10^10 OPU/ml Ad-VEGF in hMSCs culture fluid after incubating 24 hours, changing into ordinary complete culture and continuing culturing; Positive control group： Cultured hMSCs with 1 nmol/L dexamethasone, 10 mmol/L glycerophosphate and 50 mg/L vitamin C ,exchanging this conditioned medium twice a week; blank control group：no special treatment but culturing hMSCs in DMEM.To evaluate osteogenesis competence, Von Kossa＇s staining and a quantitative alkaline phosphates （ALP） activity analysis were performed after 2 weeks treatment. Results：The calcified nodes formed after 2 weeks treatment in Ad-VEGF165 group and Positive control group but not in blank control group. ALP activities in Ad-VEGF165 group ,Positive control group and blank control group were （7.91 ± 0.90）u/L, （8.18 ± 0.76 u/L） and （3.46 ± 0.49）u/L respectively. The differences were no statistical significance between Ad-VEGF165 group and positive control group （P 〉 0.05）, but Ad-VEGF165 group and Positive control group were significantly different with blank control group （P 〈 0.05）. Conclusion：Adenovirus mediated VEGF165 gene can transfect hMSCs and promote osteogenesis of hMSCs.

Effects of transplantation of human bone marrow mesenchymal stem cells in rats with liver failure

Objective: To investigate the effect of hepatic differentiation of human bone marrow mesenchymal stem cells (HBMSCs) induced in vitro and transplanted into rats with liver failure via portal vein, and observe the changes of liver function and pathological tissue. Method:After passage to the 6th generation in vitro, the hepatic differentiation was induced by HGFand EGF inducible factors. CCL4 acute liver failure model in rats were established, and randomly divided into 5 groups transplanted with differentiated stem cells via portal vein. These five groups included HGF-differentiated HBMSCs transplantation, EGF-differentiated HBMSCs transplantation, EGF+HGF-differentiated HBMSCs transplantation, non-differentiated HBMSCs transplantation, and non-HBMSCs transplantation. Liver function and pathological changes were detected. Results: Rats models survival, serum albumin, aminotransferase and coagulation indexes were observed at 12 h, 72 h, 7 d, 1 month and 2 months after treatment. The results showed that the survival and albumin, aminotransferase and coagulation function of rats were improved significantly after treatment in HGF-differentiated, EGF-differentiated, EGF+HGF-differentiated and non-differentiated transplantation groups, compared tothe non-HBMSCstransplantation group(P<0.05), while no significance was observed in above four groups(P>0.05).Pathological changes was ameliorated in the liver of rat models in HGF-, EGF-, EGF+HGF- and non-differentiated transplantation groups, compared to the non-HBMSCs transplantation group. Conclusion: Liver-differentiated BMSCs transplanted into rats with liver failure could effectively improve liver function and survival rate.

Exploring the Mechanism of CircRNA-vgll3 in Osteogenically Differentiated Human Bone Marrow Mesenchymal Stem Cells

Objective:To explore the mechanism of circRNA-vgll3 in osteogenic differentiation of human bone marrow mesenchymal stem cells.Methods:BMSCs cells were transfected with circRNA-vgll3,and divided into circRNA-vgll3 high-level group,circRNA-vgll3 low-level group,and negative control group(circRNA-vgll3 not transfected)according to the amount of transfection.The proliferation and apoptosis of BMSCs osteoblasts in each group were analyzed,and the alkaline phosphatase(ALP)activity,type I collagen gray value,bone morphogenetic protein 2(BMP-2),Runx2 protein,and mRNA expression levels were detected.Results:The circRNA-vgll3 low-level group had a significant inhibitory effect on the proliferation of BMSCs osteoblasts,and the apoptosis rate of the circRNA-vgll3 low-level group was significantly higher than that of the circRNA-vgll3 high-level group(P<0.05);ALP activity,type I collagen gray value,BMP-2,Runx2 protein,and mRNA expression levels in the high-level circRNA-vgll3 group were significantly higher than those in the low-level circRNA-vgll3 group,and the difference was statistically significant(P<0.05).Conclusion:Overexpression of circRNA-vgll3 can promote the osteogenic differentiation ability of BMSCs,while low expression of circRNA-vgll3 can inhibit the osteogenic differentiation ability of BMSCs.The main mechanism of action is that circRNA-vgll3 can affect osteogenic differentiation by regulating the Runx2 protein.

Transfection of bone marrow mesenchymal stem cells using green fluorescence protein labeled hVEGF165 recombinant plasmid mediated by liposome

Objective:To study the role of bone marrow mesenchymal stem cells(BMSCs)in construction of vascularized engineered tissue.Methods:hVEGF165 was amplified via RT-PCR before recombinant with pShuttle-green fluorescence protein;green fluorescent protein(GFP)-CMV.Then the recombinant shuttle plasmid was transfected into BMSCs with Lipofectamine^(TM)2000 for packaging and amplifying.hVTGF165 mRNA expression in BMSCs cells was tested.Results:The sequence of hVEGFI65 in pShutlle-GFP-hVFGF165 plasmid was confirimed by double-enzyme cleavage method and sequencing.hVECF165 was highly expressed in BMSCs.Conclusions:The GFP/hVECF165 recombinant plasmid vector was constructed successfully and expressed effectively in host cells,which may be helpful for discussing the possibility of the application of VEGF165-BMSCs in tissue engineering and ischemic disease cure.

Bone marrow cells produce nerve growth factor and promote angiogenesis around transplanted islets

AIM:To clarify the mechanism by which bone marrow cells promote angiogenesis around transplanted islets.METHODS: Streptozotocin induced diabetic BALB/ c mice were transplanted syngeneically under the kidney capsule with the following: (1) 200 islets (islet group: n=12), (2) 1-5×106 bone marrow cells (bone marrow group: n=11), (3) 200 islets and 1-5×106 bone marrow cells (islet + bone marrow group: n= 13), or (4) no cells (sham group:n=5). All mice were evaluated for blood glucose, serum insulin, serum nervegrowth factor (NGF) and glucose tolerance (GTT) up to postoperative day (POD) 14. Histological assessment for insulin, von Willebrand factor (vWF) and NGF was performed at POD 3, 7 and 14.RESULTS: Blood glucose level was lowest and serum insulin was highest in the islet + bone marrow group. Serum NGF increased in islet, bone marrow, and islet + bone marrow groups after transplantation, and there was a significant difference (P=0.0496, ANOVA) between the bone marrow and sham groups. The number of vessels within the graft area was signif icantly increased in both the bone marrow and islet + bone marrow groups at POD 14 as compared to the islet alone group (21.2 ± 3.6 in bone marrow, P=0.01, vs islet group, 22.6 ± 1.9 in islet + bone marrow, P = 0.0003, vs islet group, 5.3 ± 1.6 in islet-alone transplants). NGF was more strongly expressed in bone marrow cells compared with islets. CONCLUSION: Bone marrow cells produce NGF and promote angiogenesis. Islet co-transplantation with bone marrow is associated with improvement of islet graft function.

Angiogenic Potency of Bone Marrow Stromal Cells Improved by ex Vivo Hypoxia Prestimulation

Summary: To study the angiogenic potency of hypoxia-prestimulated bone marrow stromal cells (BMSCs) when transplanted into acute myocardial infarction models of rats. BMSCs were cultured under hypoxia condition for 24 h. Their expression of VEGF was investigated. The rat acute myocardial infarction models were made by coronary artery ligation and divided into 3 groups at random. In normoxia group, twice-passaged BMSCs were labeled with Bromodeoxyuridine (BrdU) and then implanted into the infarction regions and ischemic border of the recipients in 4 weeks. The rats in hypoxia group were implanted with hypoxia-prestimulated BMSCs. In control group, the model rats received only DMEM medium injection. Six-weeks after AMI, the infarction regions were examined to identify the angiogenesis and the expression of the VEGF. Our results showed that viable cells labeled with BrdU could be identified in the host hearts. The infarction regions in normoxia and hypoxia groups had a greater capillary density and increased VEGF expression than the regions in control group. The capillary density and VEGF expression in hypoxia group were higher than in normoxia group. It is concluded that the enhanced expression of VEGF in BMSCs could be induced by ex vivo hypoxia stimulation. BMSCs implantation promoted the angiogenesis in myocardial infarction tissue via supplying exogenic VEGF. Angiogenic potency of bone marrow stromal cells was improved by ex vivo hypoxia prestimulation though the enhanced VEGF expression.

VEGF-expressing Bone Marrow Mesenchymal Stem Cells Transplantation Improved Heart Function of Myocardial Infarct Rabbits

Objectives To treat myocardial infarction with MSCs transplantation combined with VEGF gene therapy in rabbits and to study its mechanisms. Methods Forty-eight rabbits were randomly divided into MI group （n=12）, MSCs group （n=12）, VEGF group （n=12）, MSCs＋VEGF group （M＋V group, n=12）. Rabbit myocardial infarction models were founded by the ligation of left anterior descending artery. 107 MSCs were injected into the infarct-zone in four sites 2 weeks later in MSCs and M＋ V group, phVEGF gene were injected in infarct-zone in VEGF group and MSCs transfected with phVEGF gene were injected in M＋V group. Heart function including LVEDP, LVSP, LVDP, -dp/dtmax, ＋dp/dtmax, were measured in vivo. The hearts were harvested at 4 weeks after transplantation and sectioned for HE stain, immunohistochemical stain of BrdU and VIII factor antigen. Results The left ventricular hemodynamics parameters showed that heart function were improved more in M＋V group than MSCs group, MI group and VEGF group. The numbers of BrdU positive cells in M＋ V group（61±8）were more than in MSCs group （44±8, P 〈 0.01）. The numbers of vessels in infarcted zone were more in M＋V group （49±8） than in MSCs group （33±6, P 〈 0.01）,VEGF group（30±8, P 〈 0.01）and Mlgroup （18±4, P〈0.01）. Conclusions VEGF-expressing MSCs transplantation could improve heart function after myocardial infarction, and they were more effective than sole MSCs transplantation. Keeping more MSCs survival and ameliorating the blood supply of infarct-zone might be involved in the mechanisms.