Effects Of Recombinant Adenovirus-mediated Gene Transfer Of HIF-1α During Human Mesenchymal Stem Cells Differentiate Into Cardiomyocyte-like In Vitro

Background and objectives:The treatments for ischemic heart diease now mainly include the conventionalmedical treatment, percutaneous coronary intervention and coronary artery bypassgrafting. However, a significant proportion of patients having symptoms refractory tomedical treatment, are unsuitable for conventional revascularization therapies. So it isnecessary to find an alternative strategy. Nowadays, the study on theory andtechnology of stem cells is more deeply and has initiators achievement. Theseimportant findings have become the reasonable base on repairing and rebuildinginjured heart.Cellular cardiomyoplasty has been proposed as an alternative strategyfor cardiac regeneration. A variety of cell types have been proposed as usefulcandidates. Human mesenchymal stem cells(hMSCs) are a group of cells with highlycapability of self-renewal and potentials of multilineage differentiation.hMSCs areeasy to be got and cultured in vitro and suitable for self-transplantation, which makesthem an appropriate source of cells for cell therapy, hMSCs were serial passaged for4 months by Makino, and the immortalized cells were achieved. By the treatment of5-aza, part of those cells were differentiated into the cardiocytes-like cells resemble to fetal cadiocytes. Many researches show that embro and adult stem cells can beinduced into cardiomyocyte-like cells, which supported the theory foundation of useof hMSCs for the cellular transplantion into the heart.Hypoxia inducible factor 1 (HIF-1) is a transcriptional factor that plays a key rolein the cellular adaptive response to hypoxia. It controls various physiological andpathological processes such as angiogenesis and erythropoiesis by modulating thetranscription of multiple target genes including VEGF. It has been demonstrated inthe preclinicial studies that gene therapy with constitutively active form of HIF-1αmay result in physiologically functional neovascularization. In recent years,neovascularization potentials of HIF-1αcoupled with the treatment of ischemic heartdisease has spurred considerable interests in better understanding theneovascularization of HIF-1αand its potential therapeutic applications. Therefore,HIF-1αhas been considered the gene having extensive treatment perspective.Adenoviral vectors have significant advantages over other viral vectors in the wayssuch as the ease of preparation, broad spectrum of infection, high viral titer and notinducing mutation. It has been one of the best vectors mediated gene transfer intherapeutic angiogenesis.However, in vivo studies on physiological role of HIF-1αin 5-acacytine tohMSCs have not been reported yet. Under these premises, this study is designed toconstruct the recombinant adenovirus vectors termed as Ad-LacZ, Ad-HIF-1α_nature,Ad-HIF-1α₅₆₄, Ad-HIF-1α_564/402 and Ad-HIF-1α_564/803 in order to get high levels ofgene expression in mammalian cells, to culture hMSCs, to induce hMSCs tocardiomyocytes by 5-azacytidine, to investigate the effects of recombinant adenovirusencoding HIF-1αinduced hMSCs by 5-azacytidine, and to explore the role of HIF-1αoverexpression in ischemic heart disease. It will provide a basis for studyingtherapeutic angiogenesis in ischemic heart disease and the clinical application in the future.MethodThe first part of this study: Recombinant adenovirus Ad-LacZ, Ad-HIF-1α_nature,Ad-HIF-1α₅₆₄, Ad-HIF-1α_564/402 and Ad-HIF-1α_564/803 were produced by homologousrecombination in HEK293 cells, amplified also in HEK293 cells on a large scale, andthese virus titres was measured respectively by End-point Dilution Assay, andpurified by ultracentrifugation in CsCl step gradient solutions. PCR assay was used toidentify the augmented of these recombinant adenovirus. At OD₂₆₀ the amount ofviral particles was determined, the purity of these purified recombinant adenoviruswas also evaluated by electronmicroscope, and the transfection efficiency wasassessed by X-gal staining.The second part of this study: (1), Isolation and Cultivation of hMSCs: Bonemarrow samples were obtained from healthy adult human donors at the NanfangHospital of Southern Medicial University. Each donor was obtained information con-sent. hMSCs were separated by centrifugation in Ficoll solution (density 1.077g/ml)followed by the method of M.F. Pittenger's. Then they were seeded in DMEM-LGsupplemented with 10% fetal bovine serum at a concentration of 1×10⁶ cells/cm².Cultures were maintained at 37℃in a humidified atmosphere camber containing CO₂cells. The medium was changed in 72 hours, nonadherent was removed with changein medium, and twice weekly thereafter. When the cultures reached nearly 90% ofconfluence, cells were trypsinized with 0.25% trypsin, washed, resuspened at a ratioof 1:2. The serum lots selected for hMSCs outgrowth from marrow aspiration werechosen for their optimum ability to promote the growth of an adherent, well-spreadcolony-form cell. (2), Immunophenotyping of hMSCs: Detections of cell surfaceantigens were cultures of hMSCs using flow cytometry. Cells performed on Passages1, 5 and 10 were detached by 0.25% trypsin, washed, recovered by centrifugation at density of 1×10⁵ cells/ml. Aliquots (100μl) of suspension were incubated withrespective 20μl of fluorescence conjugated mouse anti-human monoclonal antibodiesas FITC-Anti-HLA-DR/CD13-PE;FITC-CD45/CD34-PE;FITC-CD44/CD29-PE andisotype negative controlled mouse anti mouse antibodies IgGI-FITC,IgG1-PE(Beckon Dickinson). All incubations with antibodies performed for 20 min at roomtemperature, washed with PBS. Each sample was analyzed by collecting at least10,000 events on a Beckton Dickinson Vantage instrument using CellQaest Software.(3), Committed Differentiation of hMSCs into adipoblast: For these study, cells ofnearly 90% conflunce on passage 5 cultures were treated in adipogenesis medium(1μmol/L dexamethasone, 0.5mmol/L 1-methyl-3-isobutyl xanthine (IBMX),10μg/ml insuline) Controlled cells were treated with regular medium. The mediumwere changed every third or forth day. On the 14 days, the dishes were stained withOil Red O, observed under a light microscope. (4), Committed Differentiation ofhMSCs into osteoblast: Cells of nearly 90% conflunce on passage 5 cultures weretreated in osteogenesis medium (0.1μmol/L dexamethasone, 10mmol/Lβ-glycerophosphate, 50μmol/L Vit.C). Controlled cells were treated with regularmedium. On 28th day, the dishes were stained by chinalizarin and Von Kossa's.The third part of this study: For the study, cells of nearly 90% conflunce onpass-age 5 cultures were treated in cardiomyocytes inducing medium (10μmol/L5-azacytidine, 2mmol/L L-Glutamine, 10%FCS and DMEM-LG). Controlled cellswere treated with cardiomyocytes medium(10μmol/L 5-azacyditine, 2mmol/LL-Glutamine, 5%FCS and DMEM-LG). The medium were changed every third orforth day. Cells were harvested at 28 days after inducement, andimmunocytochemistry method was used to identify antigen of muscle tissue andcardiac muscle, such asα-Actin, Connexin 43 and cardiac special troponin T (cTnT).The fourth part of this study: hMSCs infected with Ad-LacZ were examined for transfection efficiency through X-gal staining on days 2 after infection. The optimalmultiplicity of infection (MOI). To infect hMSCs with Ad-LacZ, Ad-HIF-1α_nature,Ad-HIF-1α₅₆₄, Ad-HIF-1α_564/402 and Ad-HIF-1α_564/803 at the optimal multiplicity ofinfection (MOI), and then to exam the cTnI of the cardiomyocyte-like cells by ELISAon days 14, 28 and 32 after induced by 5-azacytidine. Cells were harvested at 28 daysafter inducement, and Western blot analysis were used to detecte the expression ofHIF-1αand VEGF.ResultThe first part of this study: Plaque titration on HEK293 cells showed titers of1.99×10¹² pfu/ml, 6.31×10¹² pfu/ml, 3.98×10¹² pfu/ml, 10.0×10¹² pfu/ml and 1.26×10¹²pfu/ml for Ad-LacZ,Ad-HIF-1α_nature, Ad-HIF-1α₅₆₄, Ad-HIF-1α_564/402å’ŒAd-HIF-1α_564/803, respectively, by End-point Dilution Assay. The titers purified by ultra-centrifugation in CsCl step gradient solutions were (7.755±0.477)×10¹¹OPU/ml,(5.077±0.188)×10¹¹OPU/ml, (6.848±0.129)×10¹¹OPU/ml, (6.292±0.260)×10¹¹OPU/ml and (6.193±0.221)×10¹¹OPU/ml for Ad-LacZ, Ad-HIF-1α_nature, Ad-HIF-1α₅₆₄,Ad-HIF-1α_564/402å’ŒAd-HIF-1α_564/803, respectively. The DNAs of the purifiedrecombinant adenoviruses and the HIF-1αgene were extracted to confirm thepresence of recombinant adenoviruses by PCR. The sizes of PCR products were287bp, 380 bp, 460bp and 214bp in excellent accord with expectation, respectively.The Mycoplasma and the Chlamydia were not observed by electronmicroscope. Theoptimal multiplicity of infection (MOI) was 50 pfu/cell by X-gal staining.The second part of this study: (1), Isolation and Cultivation of hMSCs: A smallpercentage of isolated cells were adherent to the flasks and grow as typicallyfibroblastic or spindle shape, which proliferated rapidly. Primary cultures reached90% of confluence in 14 to 20 days. Nonadherent or loosely adherent small roundcells were present in primary cultures, and disappeared soon after passage, and passaged until 7-10 passges. (2), Cell surface antigens of hMSCs: At passage 1, 5 and10, the isolated cultured adherent cells typically expressed CD13,CD29 andCD44, while CD34,CD45 and HLA-DR of them were negative. They comprised asingle phenotypic population. (3), Adipogenesis Differentiation: When hMSCs weregrown in adipogenesis inducing medium, lipid vacuoles accumulation were detectableafter 3 days. Oil Red O staining showed the lipid vacuoles orange red while thenuclear blue, which demonstrated the committed differentiation of hMSCs intoadipoblast. The controlled group showed no lipid vacuoles accamulation. (4),Osteogenesis Differentiation: When hMSCs were grown in osteogenesis inducingmedium, the cells showed typical cuboidal shape. Von Kossa's staining andchinalizarin staining both showed mineral deposition significantly.The third part of this study: Cellular morphology became big, arrayedconsistently and proliferated slowly after induced by 10μmol/L 5-azacytidine. Cellswere identified the antigen of muscle tissue and cardiac muscle by immuno-cytochenistry method at 28 days after inducement. Results showed that part of theinducing cells were positive stained forα-Actin, Connexin 43 and cTnT. The positiverate was (32.27±13.17)%, (29.51±5.22)% and (19.96±4.01)%, respectively. Therewas negative expression in hMSCs with the same number of days which hadn't beeninduced by 5-azacytidine.The fourth part of this study: Over 90% of MSCs were infected at MOI 75OPU/cell, the cTnI of the cardiomyocyte-like by ELISA on days 14,28 and 32 afterinduced by 5-azacytidine for Ad-LacZ,Ad-HIF-1α_nature, Ad-HIF-1α₅₆₄, Ad-HIF-1α_564/402 andAd-HIF-1α_564/803, comparised with the Ad-LacZ, were more significant (p=.000). Thelevel of protein of HIF-1αin the 4 treated groups(Ad-HIF-1α_nature, Ad-HIF-1α₅₆₄,Ad-HIF-1α_564/402 and Ad-HIF-1α_564/803) is higher than that of the Ad-LacZ group. Thelevel of VEGF was the same as the HIF-1α. ConclusionThe first part of this study: Recombinant adenovirus Ad-LacZ, Ad-HIF-1α_nature,Ad-HIF-1α₅₆₄, Ad-HIF-1α_564/402 and Ad-HIF-1α_564/803 were constructed with high titer,high transfection efficiency and low toxicity.The second part of this study: We succeeded in isolation, cultivation adulthuman bone marrow mesenchymal stem cells. (1), They were adherent to the flasksgrow as typically fibroblastic or spindle shape. (2), They expressed CD13,CD29 andCD44, while CD34, CD45 and HLA-DR of them were negative. (3), They can beinduced into adipoblast and osteoblast.The third part of this study: 5-azacytidine can induce human bone marrowmesenchymal stem cells which are purified in vitro culture into cardiomyocytes-likeand induced cells express muscle tissue proteinsα-Actin and Connexin 43 as well ascardiac special troponin T. The DMEM low glucose medium including 5% fetalbovine serum is fit for the culture research of human bone marrow mesenchymal stemcells induced to cardiomyocyte-like in vitro.The fourth part of this study: HIF-1αand its mutation genes can promote totransformating hMSCs induced by 5-azacytidine to cardiomyocytes, which hassupported the theory foundation of use of hMSCs for the cellular transplantion intothe heart.