Hepatocyte Growth Factor Genetically Modified Bone Marrow-derived Mesenchymal Stem Cells Transplantation Promote Angiogenesis

Part I Construction of recombinant lentiviral vector carrying human hepatocyte growth factor geneObjective:To construct a third-generation lentiviral vector carrying human hepatocyte growth factor (hHGF) for transfection.Methods:Lentiviral vector plasmid carrying human HGF (pRNAT-U6.2-hHGF) was generated by removal of green fluorescence protein (GFP) gene from pRNAT-U6.2 plasmid by NheI/XbaI digestion and ligation of human HGF cDNA, which was identified by restriction endonuclease analysis, PCR and DNA sequence analysis. Recombinant plasmid pRNAT-U6.2-hHGF was co-transfected into 293T cells with the helper plasmids pMDLg/pRRE, pMD2G-VSVG and pRSV-Rev using the TELEVECTOR Lentiviral Vector Easy-high Production kit. The recombinant lentiviral stock (Lenti-HGF) was generated, concentrated, and then the titer was determined.Results:The hHGF cDNA was identical to that of GeneBank. Recombinant lentiviral plasmid pRNAT-U6.2-hHGF was successfully constructed, and the titer of Lenti-HGF was 4×108Tu/ml.Conclusion:Recombinant third-generation lentiviral vector plasmid carrying hHGF (pRNAT-U6.2-hHGF) was constructed successfully. PartⅡExpression of human hepatocyte growth factor in primary rat bone marrow-derived mesenchymal stem cells and its biological activityObjective:To explore the usability of lentiviral vector mediated human hepatocyte growth factor (hHGF) gene delivery into bone marrow-derived mesenchymal stem cells (BM-MSCs), and to observe its expression and biological activity.Methods:(1) Primary rat BM-MSCs was isolated, expanded and purified by taking advantage of their differential adherence to dishes combined with Percoll density gradient centrifugation technique. MSC phenotype was analyzed by flow cytometry. Meanwhile, potency of MSCs differentiation into osteocytes and adipocytes were determined by alizarin red S staining and oil red O staining. (2) The third passage MSCs were transfected by high titer lentiviral stocks expressing human HGF (Lenti-HGF) or GFP (Lenti-GFP), and the transfection efficiency was assessed by fluorescent microscope observation and flow cytometry analysis. The concentration of hHGF in cell culture medium supernatants was determined by enzyme linked immunosorbent assay (ELISA). (3) Biological activity of hHGF secreted from Lenti-HGF infected BM-MSCs was determined by MTT cell proliferation assay, Transwell chamber migration assay and Hoechst staining.Results:(1) Plenty of highly purified BM-MSCs were achieved by the combined methods. (2) Bright green fluorescence in the MSC was observed under the fluorescent microscope after 72h transfected with Lenti-GFP. The best multiplicity of infection (MOI) was 50, and the transfection efficiency reached about 75%. Furthermore, ELISA showed that hHGF secretion could be stably maintained more than 14 days since the level of hHGF in the culture supernatant from the Lenti-HGF transduced BM-MSCs was significantly higher than that of Lenti-GFP transduced BM-MSCs at day 5 (P<0.01). (3)The hHGF secreted from transduced MSCs showed the proproliferative, promigratory, antiapoptotic effects for human umbilical vein endothelial cells (HUVECs) and promigratory effect on vascular smooth muscle cells (VSMCs) in vitro. Conclusion:The recombinant lentivirus (Lenti-HGF) can mediated transduction into BM-MSCs with a highly efficient and long lasting expression of the transgene. Key words:Hepatocyte growth factor; Mesenchymal stem cell; Lentivirus; ApoptosisPartⅢHepatocyte growth factor genetically modified bone marrow-derived mesenchymal stem cells transplantation promote angiogenesisObjective:To investigate whether a new strategy that combines MSCs transplantation and ex vivo human hepatocyte growth factor (hHGF) gene transferring with lentiviral vectors was more therapeutically efficient than MSCs cell therapy alone in a rat hindlimb ischemia model.Methods:One day after resection of left femoral artery, Sprague-Dawley (SD) rats were randomly divided into three groups:(a) HGF genetically modified MSCs (HGF-MSCs) transplantation (HGF-MSC group, n=8), (b) BM-MSCs transplantation (MSC group, n=8), (c) PBS injection (PBS group, n=8). After creating hindlimb ischemia, an interval of 7 days was permitted for postoperative recovery. Then a total of 5×106 HGF-MSCs, MSCs obtained from same SD rats or PBS of same volume were injected into the ischemic thigh muscles of rats at five different points in each group. The ischemic thigh muscle were analyzed for capillary density, transplanted cell differentiation, T lymphocyte infiltration and the expression of HGF and VEGF protein at one or three weeks after treatment. Serum levels of HGF in HGF-MSC group before and 3 weeks after transplantation were determined by ELISA.Results:Three weeks after transplantation, angiogenesis was significantly enhanced by both MSC group and HGF-MSC group, while capillary density was highest in the HGF-MSC group (P<0.05). The number of transplanted cell-derived endothelial cells was higher in the HGF-MSC group than in the MSC group since one week after treatment (P<0.05). In treated ischemic hind limb muscles, allogeneic HGF-MSC and MSC injection were associated with comparatively few T lymphocyte infiltration in PBS group (P>0.05). The expression of angiogenic growth factors like HGF and VEGF in local ischemic muscles undergoing intramuscular cell transplantation was more abundant in HGF-MSC group than the other two groups (P<0.05). Serum levels of HGF in HGF-MSC group before and 3 weeks after transplantation have no significant difference (P>0.05).Conclusions:The present study shows that HGF gene-modified MSC transplantation therapy induced more potent angiogenesis and collateral vessel formation than MSC cell therapy alone. Stem cell-based angiogenic gene therapy may be a new therapeutic strategy for the treatment of severe ischemic cardiovascular disease.