Fibrin Patch-based Transplantation Of IGF-I Engineered MSCs In A Swine Model Of Postinfarction Left Ventricular Remodeling

PART I:Isolation and Identification of the Mesenchymal Stem Cells Derived from Swine Bone MarrowObjective:The objective of this study is to establish the method for isolation, culture and proliferation of mesenchymal stem cells (MSCs) derived from swine bone marrow to cardiomyogenic differentiation with the exposure to different concentration of 5-azacytidine (5-Aza).Method:(1) MSCs were obtained from aspirates of swine bone marrow blood, and layed over the Percoll, after gradient centrifugation at 1500 rpm for 30 min, all of the mononuclear cells were plated in culture dish with DMEM+10%FBS and incubated at 37 ℃ with 5% CO2 in a fully humidified atmosphere. After 2 hours, cells began to adhere to the bottom and gradually increasedly afterwards. Many adherent cellular configurations displayed mostly fibroblast-like cell with shuttle shape or flat shape. (2) MTT method drew the MSCs growth curve of P2, P5 and P8. (3) The trypan-blue staining identified cell activity. (4) Morphology, structure and biological characteristics of cells were detected by an inverted phase contrast microscope. The phenotype was identified with immunofluorescence and flow cytometry sorting via MSCs cell membrane of CD44 and CD90. (5) 5-Aza can induce swine bone marrow mesenchymal stem cells of P2 and P5 into cardiomyocyte in vitro, and then statistics and calculate transformation efficiency of cardiomyocyte.Result:(1) The combination of gradient centrifugation and attachment culture method was an effective method to isolate MSCs which had a higher proliferation ability; success of the isolation culture of swine bone marrow mesenchymal stem cells. The growth curves of primary and passaged cells have the same shape "S", The sub-culturing demonstrated that MSCs begin to die after 8 passages. Fresh complete medium was added and replaced every 3 or 4 days, many adherent cellular configurations displayed mostly fibroblast-like cell with shuttle shape or flat shape. (2)The trypan-blue staining test showed that cell activity was P2>P5>P8. (3) The immunohistochemical test revealed that the positive expression of CD44 and CD90 on the cells is higher than 80%. (4) The conversion rate of MSCs to cardiomyocyte decreased with the increasing passage number at the same concentration of 5-Aza, and the number of cardiomyocytes of P2 was significantly higher than P5.Conclusion: The experiment successfully culture bone mesenchymal stem cells in vitro.5-Aza can induce swine bone marrow mesenchymal stem cells into cardiomyocyte in vitro. Effect of derivation is obvious utilizing P2 MSCs with 10 μmol/L 5-Aza. growth curves of primary and passaged cells have the same shape "S", The sub-culturing demonstrated that MSCs begin to die after 8 passages. Fresh complete medium was added and replaced every 3 or 4 days, many adherent cellular configurations displayed mostly fibroblast-like cell with shuttle shape or flat shape. (2)The trypan-blue staining test showed that cell activity was P2>P5>P8. (3) The immunohistochemical test revealed that the positive expression of CD44 and CD90 on the cells is higher than 80%. (4) The conversion rate of MSCs to cardiomyocyte decreased with the increasing passage number at the same concentration of 5-Aza, and the number of cardiomyocytes of P2 was significantly higher than P5.PART II:Construction of Lentiviral Vectors Specific to Swine IGF-I Gene and the Preliminary Research of Its Infection to the Swine Bone Marrow Mesenchymal Stem CellsObjective: The objective of this study is to construct a recombinant lentiviral vector overexpressing and interference IGF-I, and we observe the effect that 5-Aza inducing overexpressing and interference IGF-I MSCs into cardiomyocyte utilizing real-time PCR, Western blotting and Immunohistochemical methods.Method:Firstly, we made a recombinant plasmid called pCDH-GFP/pLKO.1-TRC by inserting swine IGF-I DNA segment into pCDH-GFP designed up-stream/down-stream primer with BamHl/EcoRI estriction sites of this plasmid pCDH-GFP-IGF-I in the polymerase chain reaction (PCR). Making use of the feature that enzyme Xhol are isocaudarner, we combine the fusion gene GFP-IGF-I with plasmid pCDH digested by enzyme BamHI and EcoRI. The correct clones were picked for PCR to be identified, then sended for sequencing. Secondly, co-transfected the 293T with the recombination pCDH-GFP-IGF-1 and psPAX2, pMD2.G, which were mixed wim lipofectamine 2000. Viral supernatant was harvested at 48h,72h and stored at 4℃. At last, the lentivirus encoding GFP-IGF-I gene infected MSCs, and their expression were observed by fluorescence expression, we found the effect that 5-Aza inducing overexpressing and interference IGF-I of MSCs into cardiomyocytes which changed IGF-I, GATA-4, NKX2.5, β-MHC and MEF-2c by utilizing real-time PCR, Western blotting and Immunohistochemical methods.Results:The reconstructed transfer pCDH-GFP-IGF-I plasmids were identified by PCR amplification and sequencing. The length of GFP-IGF-I gene was 393bp, and enzyme digestion demonstrated that it was the right one. This produced lentiviral particles had already infected by observed fluorescence express in MSCs and which efficiency were about over 80%. The results of real-real time PCR, western blotting and immunohistochemical methods showed that overexpression IGF-I MSCs cell group of IGF-I, GATA-4, NKX2.5, β-MHC and MEF-2c expression was significantly higher than the other groups, and the indicators of siRNA-IGF-I-MSCs cell groups was significantly lower than that of other groups.Conclusion:Lentivirus expressing IGF-I was successfully constructed. Overexpression IGF-I MSCs can be conducive to cardiomyocyte differentiation. At last, the lentivirus encoding GFP-IGF-I gene infected MSCs, and their expression were observed by fluorescence expression, we found the effect that 5-Aza inducing overexpressing and interference IGF-I of MSCs into cardiomyocytes which changed IGF-I, GATA-4, NKX2.5, β-MHC and MEF-2c by utilizing real-time PCR, Western blotting and Immunohistochemical methods.Part III:Optimization of the Swine Model of Postinfarction Left Ventricular RemodelingObjective:To evaluate the efficacy, safty and stability of optimized swine model of postinfarction left ventricular remodeling.Method:Forty Female Shanghai white pigs were divided into control group (n=10), optimized group (OPTI, n=15) and LAD ligation group (LAD, n=15). A left thoracotomy was performed via the 4th rib, and myocardial ischemia (MI) was established by selected occlusion of obtuse marginal branches (OM) and/or diagonal branches (D) in OPTI group; and by ligation at one-third distal section of LAD in LAD group. The coronary artery was ligated for 60 min followed by reperfusion. A "open-close" procedure was performed in control group. Magnetic reasonance imaging (MRI) was performed pre-operation and 4 weeks post-operation. After the MRI experiments were completed, the animals were anesthetized, the thoractomy incision was reopened. Hemodynamics was measured. The heart was then explanted and specimens were collected for further study.Results:The mortality was 13% (2/15) in OPTI group and 33% (5/15) in LAD group. There were significant differences in the variations of the scar size (SS) and left ventricular ejection fraction (LVEF) (P<0.01) between two groups. However, no significant difference was found in the regional contractile parameters (Systolic thickening fraction) STF and (Systolic wall stress) SWS between two groups (P>0.05).Conclusion:A more reliable and stable swine model of postinfarction left ventricular remodeling can be established by selected occlusion of obtuse and/or diagonal branches of left coronary system, allowing for further pre-clinical study of cardiac regeneration. incision was reopened. Hemodynamics was measured. The heart was then explanted and specimens were collected for further study.Part IV:Fibrin Patch-based Transplantation of IGF-I Engineered MSCs in a Swine Model of Postinfarction Left Ventricular RemodelingObjective:This study aim to explore the therapeutic potential of IGF-I engineered mesenchymal stem cells (MSCs) for cardiac repair in a swine model of postinfarction left ventricular remodeling.Method:Mesenchymal stem cells (MSCs) obtained from swine bone marrow were engineered to GFP only (cMSCs), over express IGF-I and GFP (oeMSCs), and interference IGF-I and GFP (siMSCs). The MSCs were tested in a swine model of post-infartcion LV remodeling to examine the therapeutic potential. Female Shanghai white pigs underwent coronary artery occlusion of 60 min followed by reperfusion. Animals were randomized to receive fibrin patch vehicle (MI, n=7), fibrin patch seeded with 10 million cMSCs (cMSCs, n=7), fibrin patch seeded with 10 million oeMSCs (oeMSCs, n=7), fibrin patch seeded with 10 million siMSCs (siMSCs, n=7). Animals were followed up for one month and cardiac function was measured with clinic 1.5 Tesla MRI at one week and 4 weeks post operation. Immunofluorescence staining was employed to evaluate myocyte and transplanted cells. GFP expression (GFP), endothelial marker CD31 (CD31) and cardiac marker Troponin T (cTnT) were detected. Immunohistochemistry and Western blot were also employed to evaluate expression of IGF, CD31, Bcl-2 and BAX.Results:At one week postoperation, a significant improvement of global cardiac function in terms of left ventricular ejection fraction (LVEF) was observed in all of cell transplanted groups (P＜0.05 vs MI). A significant improvement of regional thickening fraction in the infarct and border zone was observed (P<0.05 vs MI). At one month post operation, beneficial effects from either type of MSCs transplantation persist. Left ventricular structural and functional abnormalities were less pronounced in hearts in cell-treated group than in MI hearts (P＜0.05), and these improvements were accompanied by declines in scar size. The oeMSCs group displayed a significant increase in vascular density, IGFexpression and Bcl-2 expression comparing to the other groups. The opposite chage was detected in BAX expression (P＜0.05).Conclusion:IGF-I engineering of MSCs augments efficacy in a large animal model of infarction, indicating that this cardio-protective growth factor can be utilized and manipulated for therapeutic strategies.