Chapter Materials and Methods 52 2.1 MATERIALS 2.1.1 Cell culture media Dulbecco’s Modified Eagle’s Medium Sigma, USA F12K ATCC, USA Fetal bovine serum Hyclone, USA Super-medium Cell Transplants Singapore Pte., Ltd. 2.1.2 Cell lines FLY-A4 cells FLY-A4 cells packaged with retroviral vector carrying nLac-z reporter gene were kindly gifted by Dr. Colin Porter (Imperial College of London, UK). HEK 293 cells Kindly gifted by Associate Professor Ruowen Ge, Department of Biological Sciences, National University of Singapore, Singapore. HeLa Cells Kindly gifted by Associate Professor Hanry Yu, Department of Bioengineering, National University of Singapore, Singapore. Human fibroblasts Generously gifted by Cell Transplants Singapore Pte., Ltd. Human skeletal myoblasts Generously sponsored by Cell Transplants Singapore Pte., Ltd. Human umbilical vein endothelial cells 53 Kindly gifted by Associate Professor Ruowen Ge, Department of Biological Sciences, National University of Singapore, Singapore NIH 3T3 fibroblasts Kindly gifted by Associate Professor El Oakley Reida, National University Hospital, Singapore. 2.1.3 Animals Wistar Rats A total of 65 female Wistar rats (± 200g) were used for rat heart model of cryoinjury. Yorkshire pigs A total of 63 Yorkshire pigs (30 ± Kg) were used for pig heart model of chronic infarction. 2.1.4 Chemicals Absolute alcohol Hayman, England Acetic acid Merck, Germany Acetone BDH Lab, USA Acid fuchsin Sigma, USA 30% Acrylamide/ Bis Solution Bio-Rad, USA 40% Acrylamide/ Bis Solution Bio-Rad, USA Ammonium Sigma, USA Ammonium persulfate Bio-Rad, USA Aniline blue Sigma, USA Araldite Ted Pella, Inc. USA 54 Atropine sulphate Troy Laboratories, Australia Bisacrylamide Sigma, USA Bovine serum albumin ICN Biomedicals Inc., USA Bromophenol blue Bio-Rad, USA Cephalosporin Virabac, Australia Celestine blue Sigma, USA Cesium chloride Sigma, USA Chloral hydrate Sigma, USA Citric acid Sigma, USA Cyclosporine A Novartis, German 4, 6-diamidino-2-phenylindole Sigma, USA 3, 3’ diaminobenzidine tetrahydrochloride ICN Biomedical Inc., USA Dithiothreitol Bio-Rad, USA Dodecenyl succinic anhydride Ted Pella, Inc. USA Eosin Sigma, USA Ethanol Sino Chemical, Singapore 2-ethoxyethyl acetate Sigma-Aldrich, USA Fluospheres® red polystyrene microsphere (580/605), 15 µm Molecular Probes, USA Fluospheres® scarlet polystyrene microsphere (645/680), 15 µm Molecular Probes, USA Fluospheres® yellow-green polystyrene microsphere (505/515), 15 µm Molecular Probes, USA 55 Formalin (37%) Sigma, USA Formamide Armesco, USA Formadehyde Sigma, USA Frozen medium Jung, Germany Glacial acetic acid Sigma, USA Glutaradehyde (25%) Sigma, USA Glycerol Sigma Chemical CO., USA Hematoxylin Sigma, USA Heparin Leo pharma, Denmark Histowax Leica, Switzerland Hydrogen peroxide AnalaR, UK Isoflurane Abbott Laboratories, USA Isopentane Acros Organic, USA Isopropanol Sigma, USA Ketamaine APEX Laboratories, Italy Lead citrate Sigma, USA Magnesium chloride Sigma, USA Methanol Fisher Scientific, UK Methyl green Lab Vision, USA Mount medium Shandon, USA Nitrocellulose membrane Sigma, USA N, N-dimethyformamide Sigma, USA Non-fat milk powder Anlene, New Zealand 56 Osmium tetroxide Agar Scientific Ltd, England Paraffin wax Leica, Switzerland Penicillin/Streptomycin Sigma, USA Phosphate buffered saline (PBS) NUMI, Singapore Phosphomolybdic acid Sigma, USA Plylene Sigma, USA Polyoxyethylenesorbitan mono oleate (Tween-80) Sigma, USA Potassium chloride Sigma, USA Potassium ferricyanide Sigma, USA Potassium ferrocyanide Sigma, USA Potassium hydroxide Merck, Germany Potassium phosphate (bibasic) Sigma, USA Potassium phosphate (monobasic) Sigma, USA Protein binding dye Bio-Rad, USA Resin Sigma, USA RNAlater RNA stabilization reagent QIAGEN, Germany Scintillation buffer Beckman, USA Sodium chloride (0.9%) NUMI, Singapore Sodium chloride+sodium citrate (20x SSC) NUMI, Singapore Sodium dodecyl sulfate (SDS) NUMI, Singapore Sodium hydroxide Merck, Germany Sodium iodate Sigma, USA Sodium phosphate (bibasic) Sigma, USA 57 Sodium phosphate (monobasic) Sigma, USA Sodium pentobarbital APEX Laboratories, Italy Tc99m-sestamibi Amerchan, UK Tetramethylene-ethylenediaminutese (TEMED) Bio-Rad, USA Thiopentone Biochemie GmbH, Austria Thymidine ICN Biomedical Inc., USA Thymol Sigma, USA 2, 4, 6-tridimethylaminuteso methyl phenol TED PELLA, Inc. USA Tris (2-hydroxymethyl-2-methyl-1,3-propanediol) Fisher Scientific, USA Triton-X 100 Bio-Rad, USA Trypan blue Sigma, USA Trypsin-EDTA Sigma, USA Visipaque NYCOMED, Norway X-gal Sigma, USA Xylene Merck, Germany 2.1.5 Proteins, antibodies and kits Proteins α-fibroblast growth factor (α-FGF) Sigma, USA Molecular weight color markers (6.5~45kDa) Sigma, USA Recombinant human Ang-1 R & D System, USA Recombinant hVEGF165 Chemicon, Inc., USA Primary antibodies Mouse anti-human cardiac troponin I antibody Serotec, UK 58 Mouse anti-human CD56- PE BD PharMingen, USA Mouse anti-human connexin-43 antibody Sigma, USA Mouse anti-human MHC I Neomarkers, USA Mouse anti-human MHC II Cybus Biotechnology, USA Mouse anti-human VEGF antibody BD PharMingen, USA Mouse anti-pig CD4+ antibody BD PharMingen, USA Mouse anti-pig CD8+ antibody BD PharMingen, USA Mouse anti-pig monocyte/ granulocyte antibody BD Biosciences, USA Mouse anti-fast myosin heavy chain antibody NeoMarkers, USA Mouse anti-skeletal actin NeoMarkers, USA Mouse anti-slow myosin heavy chain antibody Chemicon Int., USA Mouse anti-α smooth muscle actin antibody Sigma, USA Rabbit anti-Ang-1 antibody Chemicon Inc. USA Rabbit anti-desmin antibody Sigma, USA Rabbit anti-human VEGF antibody Chemicon Inc., USA Rabbit anti-vWF-VIII antibody Dako, Denmark Secondary antibodies Goat anti-mouse IgG-FITC Sigma, USA Goat anti-rabbit IgG-FITC Sigma, USA Goat anti-Rabbit IgG-HRP Chemicon Int., USA Goat anti- Rabbit IgG-TRITC Sigma, USA Rabbit anti-mouse IgG-TRITC Chemicon Int., USA Rabbit anti-mouse IgG-HRP Chemicon Int., USA 59 Rabbit anti-pig IgG-FITC US Biological, USA Kits BrdU labeling and detection kit II Roche Diag GmbH, USA DAPI/Antifade Qbiogene, France DNeasy isolation kit QIAGEN, Germany Human VEGF sandwich ELISA kit Chemicon Int., USA QIAGEN OneStep RT-PCR kit QIAGEN, Germany TaqPCR master mix kit QIAGEN, Germany Total Y-chromosome DNA probe Qbiogene, France Total RNA isolation kit QIAGEN, Germany Ultravision detection system LAB VISION, USA 2.1.6 Apparatus Adhesive coating histology slides Histobond, USA Biocoat matrigel matrix plate BD, USA CD-6 cellagen discs ICN Biomedicals, Inc., USA Collagen coated 225 cm3 flask Costar, USA Glass filtration apparatus China Glass syringe (20 ml) China Glassware (cylinder, beaker etc) Becton Dickinson, USA Glass chamber slide Lab-Tek, NUNC, Denmark Histosette I Simport, Canada Microfilter (0.22µm) Millex, USA Normal culture flasks (25 and 75 cm3) NUNCLONTM, Denmark 60 Polypropylene centrifugal tube (15 and 50 ml) Corning, USA Polysine coated glass slides Esco, USA Poretics polycarbonate filter, 10.0 µm OSMONICS Inc., USA Power supply (capacity 200V, 500mA) Bio-Rad, USA Spectrophotometer cuvet Sigma, USA Sterile pipette Costar, USA Tissue culture plate (6, 24 and 96 wells) NUNCTM Surface, Denmark Ultraclear centrifuge tubes (10 ml) Beckton Dickinson, USA 2.1.7 Surgical accessories Anesthesia instruments Aesculap, USA Anesthesia machine Datex Ohmeda, Finland Catheter Arrow, USA Electrosurgical generator Bovie X-10, UK Forceps Aesculap, USA Gauze China Retractor Aesculap, USA Rodent ventilator Harvard Apparatus, USA Scalpel blade Aesculap, USA Scissors Aesculap, USA Suture (Merck, Prolene) Johnson & Johnson, Belgium 2.1.8 Equipment 12 mHz echocardiographic probe Agilent Tech. USA Aquasonic 100 ultrasound transmission gel Agilent Tech. USA 61 2.2.2.2 Pig heart model of chronic myocardial infarction Female Yorkshire pigs (each weighing 30± Kg) were subjected to coronary artery ligation. The schematic representation of xenomyoblast transplantation study using pig heart model of chronic infarction is shown in Flow Chart (Ye et al., 2004). Development of pig heart model Pigs were tranquilized by intramuscular injection of mixture containing ketamaine (20 mg /kg) and Atropine sulphate (0.05mg/ kg) followed by intravenous injection of thiopentone (20mg/ kg) for intubation. Pigs were maintained on mixture of oxygen and isoflurane (2.5%, Abbott Laboratories) by inhalation (Dräger, Germany). Saline infusion was maintained intravenously throughout the surgical procedure. Pigs were placed in a right lateral decubitus position and the chest area was shaved and prepared in a sterile condition. A limited left side thoracotomy (5-7 cm) was performed between 4th and 5th intercostal space. The pericardium was incised to expose LCx coronary artery. A branch of LCx coronary artery around the origin was ligated using a 4-0 prolene suture. The successful occlusion was further confirmed by angiography and electrocardiography. The pericardium was sutured with 4-0 prolene, chest was closed and animals were returned to their cages to recover. All pigs were maintained on 40mg/ kg cephalosporin for days post operation. Coronary angiography was performed to select coronary artery and confirm successful occlusion of coronary artery. Right or left carotid artery was isolated and catheter was inserted until it reached left coronary artery sinus. Angiographys were taken before and after coronary artery ligation. 88 Purified human skeletal myoblasts in cell culture Ad-vector carrying hVEGF165 and/ or Ang-1 Fluorescent immunostaining for angiogenic gene expression of skeletal myoblasts transduced with Ad-vector carrying angiogenic genes Processing muscle biopsies for myoblast generation Male human donor Five ml DMEM with or without cells was injected into pig infarct heart at weeks after development of model. + nLac-z retroviral vector Cardiac function analysis by SPECT or Echo at weeks after treatment. Pig heart was harvested at or 12 weeks after treatment Skeletal myoblasts survived in pig heart Increased blood vessel density Flow Chart 3. Schematic representation of myoblast transplantation using pig heart model of chronic infarction. 89 89 ECG was performed to monitor the electrophysiological changes during the development of pig heart model, myoblast transplantation and weeks after myoblast transplantation. After shaving the hair on two upper limbs and left hind limb, ECG leads were attached to designated limb and ECG was recorded during the surgical procedure. Three weeks after development of animal model, pigs were randomized into groups: DMEM group (divided into two subgroups: group-1 and group-2), Ad-null myoblast group, non-transduced myoblast group, hVEGF165 myoblast group, Ang-1 myoblast group and Bic-myoblast group. Because Singapore was declared as severe acute respiratory syndrome (SARS) infected area from March 2003 to July 2003, Echo was used for DMEM group-2, Ad-null myoblast group, Ang-1 myoblast group and Bicmyoblast group, instead of SPECT nuclear imaging for DMEM group-1, non-transduced myoblast group and hVEGF165-myoblast group. Experimental design The experimental design for pig heart model of chronic infarction is outlined in Flow Chart 4. Skeletal myoblast transplantation into pig heart Three weeks after ligation, pigs were anesthetized again using the same protocol as described in Section 2.2.2.2 (page 88). Five ml of DMEM with or without x 108 skeletal myoblasts were intramyocardially injected into the center (5 injections) and periphery (20 injections) areas of the infarction with a 27-gauge needle at 0.2 ml each injection. The total injection time was 15 minutes. Pigs were maintained on 40mg/ Kg cephalosporin for days as prevention of infection and faster wound healing after operation. They were kept on intramuscular administration of Cyc-A at a daily dose of mg/ Kg to avoid immune rejection of the transplanted human skeletal myoblasts, starting from days before and until weeks after cell transplantation. 90 DMEM Group-1 n=6 Non-transduced myoblast group n=5 Ligation Ligation Ad-null myoblast Group n=5 Ang-1myoblast Group n=7 Bic-myoblast Group n=11 harvest harvest Cyc-A Non-transduced myoblst harvest harvest Cyc-A hVEGF165 -myoblasts harvest harvest Ligation Cyc-A DMEM injection harvest harvest Ligation Cyc-A Ad-null myoblast harvest harvest Ligation Cyc-A Ang-1 myoblast harvest harvest Ligation Cyc-A Bic-myoblast harvest harvest harvest harvest hVEGF165-myoblast Ligation group n=8 DMEM Group-2 n=6 Cyc-A DMEM injection Normal group n=6 Summary of the procedures weeks ECG angiography weeks ECG, SPECT or Echo microsphere weeks ECG, SPECT or Echo microsphere; histology Microsphere histological studies Flow Chart 4: Experimental design for pig heart model of chronic infarction. Black arrow refers to the time of development of pig heart model; Green arrow refers to the start of immunosuppressive therapy using Cyc-A; Red arrow refers to the time of treatment. 91 Regional blood flow study using fluorescent microspheres After anesthesia and intubation, pigs were placed in dorsal decubitus position. Right or left carotid artery was isolated and catheter was inserted until it reached left ventricle. At the same time, right or left superficial femoral artery was isolated and prepared for taking blood sample. Microsphere solution (1.4 x104 microspheres/ kg body weight) was injected through the catheter into LV during the second ten seconds of one minute while 10 ml blood sample was drawn in the same minute. The red, yellow-green and scarlet fluorescent microspheres were sequentially injected at weeks (3 weeks after development of model), and 12 weeks after treatment. Pig heart samples weighing gram each were collected from center and peri-infarct regions at 12 weeks after treatment. The samples were collected in triplicate from each area. The pig number allocated for microsphere study is illustrated in Table 2.2. Table 2.2 Number of pigs allocated at 2, and 12 weeks Groups weeks 12 weeks* Total DMEM group-1 (3) Non-transduced myoblast group (3) (4) DMEM group-2 Ad-null myoblast group (3) hVEGF165-myoblast group weeks Ang-1-myoblast group (3) Bic-myoblast group (4) 11 * The number in bracket is the number of pigs allocated for microsphere study 92 The microspheres in tissue and blood samples were recovered by digestion and negative pressure filtration. The fluorescent dye was extracted by ethoxyethyl acetate from the microspheres and measured on a Luminescence Spectrometer at wavelengths of 580/ 605 nm for red fluorescence, 505/ 515 nm for yellow-green fluorescence and 645/ 680 nm for scarlet fluorescence. Regional blood flow was calculated by using following relation: Q (ml/minute/g) = (Fltissue / Flblood) x R (ml/minute)/g In this relationship, Fltissue was the fluorescent density of organ piece; Flblood was the fluorescent density of reference blood flow sample; R was the withdrawal rate of the reference blood flow sample. Euthanasia of pigs After treatment, pigs were sacrificed at pre-determined time intervals of and 12 weeks after treatment. The animals were sedated and heart was stopped by intravenous injection of sodium pentobarbitone (50mg/ Kg). The marked area of heart muscle was removed. The heart muscle was chopped into 10x 10 mm pieces and labeled in order. The heart tissue was preserved alternative in 10% formalin or frozen in liquid nitrogen cooled isopentane. The animal number assigned for harvesting is listed in Table 2.2. Preparation of pig heart tissue Tissue sections at 6~10 µm thickness (cryosection) or µm thickness (paraffin section) were cut (see Appendix for details) and used for histochemical and immunohistochemical studies. Hematoxylin/ Eosin staining and Masson Trichrome staining were carried out to visualize the infarction using the protocol as described in Appendix 4. The pig heart 93 sections were used for nLac-z and BrdU expression to identify the transplanted skeletal myoblasts. Survival of skeletal myoblasts in pig heart After explantation of the hearts, the site of myoblast transplantation was processed for histochemical staining, PCR, and FISH analysis to identify the survival of the donor skeletal myoblasts. nLac-z expression of pig heart: Cryosections of pig heart at 8-10 µm thickness were stained for nLac-z expression as described in section 2.2.1.6 (page 80). nLac-z positive donor myoblasts per 100x microscopic field were counted from 6-8 tissue sections of pig heart at and 12 weeks after myoblast transplantation. BrdU expression of pig heart: Pig heart cryosections at 8-10 µm thickness were histochemically stained for BrdU expression as described in section 2.2.1.6 (page 80). PCR for human Y-chromosome expression in pig heart: Pig heart tissue samples from remote, peri and centre of the infarct were collected at 2, and 12 weeks after cell transplantation and used for PCR analysis for human Y-chromosome. Skeletal myoblasts were used as a positive control. Total DNA was isolated according to manufacturer’s instruction using DNeasy tissue kit (Appendix 5). The PCR analysis for human Ychromosome was carried out using TaqPCR master mix kit. DNA template of each sample (1 µg) was added into the master mix and 30 cycles were performed. Primer sequences for PCR amplification of human Y-chromosome was the same as described in section 2.2.2.1 (page 85), pig ϒ-actin (Sun et al., 1996) forward 5’- GGAAGCTCTGCATTGTGGAGT-3’; reverse 5’-TGTCGTTTTTCTGAGAACAGGG3’. The same protocol of thermal cycle was performed as described in section 2.2.2.1 94 (page 85) with annealing temperature at 63oC for Y-chromosome and 56oC for pig ϒactin. FISH analysis of pig heart: Cryosections with µm thickness was cut from pig heart tissue transplanted with human skeletal myoblasts (confirmed by nLac-z expression) and denatured in 70% formamide/ 2x SSC (pH7.2) at 72oC for minutes. This was followed by dehydration in -20oC ethanol series 70%, 90% and 100% for minute each. The hybridization mixture containing µl pig chromosome-10 probe (Red fluorescence, gifted by Dr. Jiri Rubes, Veterinary Research Institute, Czech Republic) and 10 µl human total Y-chromosome DNA probe (green fluorescence) was denatured at 72oC for minutes. Immediately after chilling on ice, the probe mixture was applied onto the tissue and covered with glass cover-slip, sealed and hybridized at 37oC for overnight. On the second day, slide was washed with 50% formamide/ 2x SSC (pH 7.2) for 10 minutes twice at 42oC followed by 2x SSC washing for minutes in duplicate. After air drying in the dark, samples were mounted with 10 µl anti-fade solution containing 0.24 µg/ml DAPI and observed under fluorescent microscope. Characterization of differentiated skeletal myoblasts in pig heart Pig heart samples containing human skeletal myoblasts (confirmed by cryosection for nLac-z expression) were used for immunohistochemical studies to characterize the differentiation of skeletal myoblasts after transplantation into heart. Immunostaining for skeletal myosin heavy chain, specific human cardiac markers (troponin I and connexin43), and human MHC I & II expression was carried out. General protocol for immunohistochemical staining: In order to characterize skeletal myoblasts in pig heart, immunohistochemical studies were carried out using various antibodies. The general procedure for immunostaining using cryosections was performed 95 as follows. The cryosections were fixed in -20oC ethanol for 10 minutes. After washing, sections were incubated with 3% hydrogen peroxide for 15 minutes to reduce nonspecific background staining due to endogenous peroxidase. The non-specific binding sites were blocked using Ultra V Block for minutes at room temperature followed by incubation with individual antibody at respective concentration (Table 2.3, page 103). On the second day, biotinylated goat anti-polyvalent (from Ultravision Detection System) was applied for 10 minutes at room temperature followed by incubation with streptavidin peroxidase for 10 minutes at room temperature. After thorough washing, DAB substrate buffer (from Ultravision Detection System) was applied on slide and observed under light microscope. Expression of skeletal muscle specific proteins in pig heart: The cryosections of pig heart tissue positive for nLac-z expression were used for dual immunostaining for fast or slow isoforms of skeletal myosin heavy chain. Paraffin sections of pig heart tissue where myoblast survived (confirmed by nLac-z expression) were assessed for skeletal muscle actin expression. The procedure was performed as described in Section 2.2.2.2 (page 95) using 1: 100 specific mouse anti-skeletal myosin heavy chain fast or slow isoform antibodies or 1: 50 mouse anti-skeletal actin antibody. Human cardiac troponin I or connexin-43 expression in pig heart: Paraffin sections of pig heart tissue where myoblast survived (confirmed by nLac-z expression) were assessed for human cardiac troponin I or connexin-43 expression using human heart tissue section (Kindly provided by Associate Professor Ming Teh) as a positive control. After dewaxing procedure, immunostaining was performed as described in Section 2.2.2.2 (page 95) using 1: 50 specific mouse anti-human troponin I and connexin-43 antibodies. Immunostaining of nLac-z positive myofibers with anti-pig IgG antibody: Cryosections of pig heart tissue positive for nLac-z expression were dually immunostained with 96 specific anti-pig IgG-FITC antibody using DMEM injected pig heart tissue as positive control. Rat heart tissue incubated with anti-pig IgG-FITC antibody and DMEM injected pig heart tissue without incubation of anti-pig IgG-FITC antibody were used as negative controls. Samples were fixed with 100% methanol for 15 minutes at -20oC. After blocking with Ultra V Block for minutes at room temperature, 1: 1000 dilution of rabbit anti-pig IgG-FITC antibody was applied on cryosections for 30 minutes at room temperature. After thorough washing, samples were air dried and counter stained with DAPI and observed under fluorescent microscope. Immunostaining for human MHC I & II expression in pig heart: Cryosections of pig heart tissue positive for nLac-z expression were used to assess the expression of human MHC I and II in pig heart. Human skeletal myoblasts and human peripheral blood samples were used as positive controls. The blood samples were smeared on polysine coated microscopic glass slides and air dried. The immunohistochemical staining was performed as described in Section 2.2.2.2 (page 95) using specific mouse anti-human MHC I (1: 100) or human MHC II (1: 50) antibodies. Pig immune response to human skeletal myoblasts Immunostaining of pig heart for identification of pig monocyte, granulocyte and T lymphocytes in areas where skeletal myoblasts survived were carried out. Furthermore, cytoflorimetric analysis of pig serum was performed for detection of pig anti-human skeletal myoblast antibodies. Isolation of pig mononuclear cells from blood: Five ml pig blood samples were collected from the ear vein. One ml of 10% acetic acid was added to the blood sample for minutes to cause red blood cells lysis. The samples were then centrifuged at 1500 RPM 97 for minutes and the pellet washed x3 with PBS for minutes each. The pellet was resuspended in 0.5 ml PBS and smeared on coated glass slides and air dried. The remaining pig mononuclear cells were used as positive controls for identification of immune cells in pig heart tissue where skeletal myoblasts survived. Immunostaining for identification of pig monocyte or granulocyte: The nLac-z positive pig heart sections were dually immunostained for identification of pig monocyte/ granulocyte. The procedure was performed as described in Section 2.2.2.2 (page 95) using 1: 100 mouse anti-pig monocyte/ granulocyte antibody. Immunostaining for identification of pig CD4+ or CD8+ T lymphocytes: The nLac-z positive pig heart sections were dually immunostained for identification of pig CD4+ or CD8+ T lymphocytes. The procedure was performed as described in Section 2.2.2.2 (page 95) using 1: 100 mouse anti-pig CD4+ or CD8+ antibodies. Cytofluorimetry of pig serum for detection of pig anti-human skeletal myoblast antibody: Pig serum was obtained at the following time points to detect the presence of pig anti-human skeletal myoblast antibody: before cell transplantation as baseline and days, 1, 2, and 12 weeks after myoblast transplantation. Five ml of blood samples were collected from ear vein at each time point and centrifuged at 2000 RPM twice to remove blood cells. The serum was kept at –80oC until analysis. Glass tubes containing 1x106 human skeletal myoblasts each were used. Skeletal myoblasts were fixed in -20oC methanol for 15 minutes followed by blocking using 1% BSA for hour at room temperature. One hundred µl pig sera (original or diluted 4x) was added into each designated glass tube and incubated for one and half hours at room temperature. After washing, resuspended cells were incubated with 1: 1000 dilution of 98 rabbit anti-pig IgG-FITC for another one and half hours at room temperature. After thorough washing, cells were resuspended in 0.5 ml of PBS and analyzed using Coulter flowcytometric software by a researcher who was blind to this experiment and the data were analyzed using WinMDI version 2.8. This experiment was performed in triplicate. Assessment of neovascularization in pig heart Pig heart tissues from hVEGF165-myoblast group, Ang-1 myoblast group and Bicmyoblast group were immunostained for hVEGF165 and Ang-1 expression and confirmed by RT-PCR analysis. For blood vessel density analysis, pig heart tissue sections were immunostained for vWF-VIII and SMA expression. Immunostaining for hVEGF165 or Ang-1 expression in pig heart: Pig heart tissues of hVEGF165-myoblast and Ang-1 myoblast groups at weeks after treatment were used for immunostaining for hVEGF165 and Ang-1 expression. DMEM injected and Ad-null myoblast transplanted pig hearts were used as negative controls. Tissue sections positive for nLac-z expression were fixed with 50% acetone/ methanol for minute followed by 10 minutes incubation with 3% hydrogen peroxide. The non-specific binding was blocked with Ultra V Block for minutes followed by incubation with 1: 50 mouse antiVEGF or rabbit anti-Ang-1 for overnight. On the second day, biotinylated goat antipolyvalent buffer was applied on sections for 10 minutes followed by incubation with streptavidin peroxidase buffer for another 10 minutes at room temperature. After thorough washing, DAB substrate buffer was applied on slide to visualize the expression of hVEGF165 or Ang-1 expression and observed under bright field microscope. 99 RT-PCR for hVEGF165 and Ang-1 expression in pig heart: Pig heart tissues of hVEGF165-myoblast, Ang-1 myoblast and Bic-myoblast groups at 2, and 12 weeks after treatment were collected for RT-PCR for hVEGF165 and Ang-1 expression. Ad-VEGF, Ad-Ang-1 and Ad-Bic transduced myoblasts were used as positive controls, while Adnull myoblast transplanted pig heart was used as a negative control. Total RNA of pig heart tissue was isolated according to manufacturer’s instruction using Total RNA Isolation Kit as described in Appendix 2. RT-PCR analysis of hVEGF165 and Ang-1 expression in pig heart was carried out using One-step RT-PCR kit. The same protocol as described in section 2.2.1.4 (page 73) was performed. One µg of RNA template of each sample was added into master mix and 30 cycles were performed. Primers for RT-PCR amplification of hVEGF165, Ang-1, and pig GAPDH are listed in Table 2.1 (page 73). Quantitative histological assessment for vascular density in pig heart: Tissue sections were fixed with –20oC ethanol followed by blocking with Ultra-V Block for minutes, 1: 500 rabbit anti-vWF-VIII and 1: 400 mouse anti SMA were applied on tissues for overnight. After thorough washing, 1: 500 goat anti rabbit IgG conjugated with TRITC (for vWF-VIII) and 1: 400 goat anti mouse IgG-FITC (for SMA) were applied for one and half hours. After thorough washing and air drying, the slides were observed under fluorescent microscope. A total of 15-18 slides from each group were used to assess the blood vessel density based on vWF-VIII and SMA fluorescent immunostaining. Three pictures from peri-infarction region were taken from each slide. The blood vessel density was calculated based on positive for TRITC (for vWF-VIII) or FITC (for SMA) 100 respectively. The mature blood vessel index is calculated as described in Section 2.2.2.1 (page 87). Pig heart function assessment Rest SPECT nuclear imaging or Echo were performed to assess pig heart function at weeks after treatment (3 weeks after the development of the animal model) and weeks after treatment. Pig heart function assessment using rest SPECT nuclear imaging: One hour after intravenous bolus injection of 15 mCi Tc99m-sestamibi, pigs were placed in prone position in a dual detector camera system (ADAC Laboratories, USA). The rest SPECT nuclear imaging was processed in a blinded fashion using integrated ADAC Pegasys Computer. The rest circumferential count profiles were constructed at the mid ventricular level by dividing the mid ventricular short axis in to 60 angular segments centered on the ventricular cavity. The radiation count was measured for each of the segment and normalized with the maximal number of counts assigned value as 100. The normalized counts per segment were plotted versus the angular position of the segment and used to assess the extent of ischemia and perfusion. The data of ejection fraction was obtained to assess the left ventricular contractility improvement. The data of wall thickness, wall motion and perfusion of infarcted segment, were obtained to assess the changes after treatment. Pig heart function assessment using echocardiography: After anesthesia and intubation, pigs were placed in a right lateral decubitus position. Echo of pig heart was performed using Vingmed Vivid ultrasound machine by one researcher who was blind to the animal intervention procedure. From M-mode Echo of the left ventricle, measurements of 101 LVEDD and LVESD were obtained. The LVEF and LVFS were calculated as described in section 2.2.2.1 (page 87). Investigators who were blind to the animal groups made all the assessments. The measurements were averaged between to consecutive cardiac cycles. 2.3 Statistical analysis Statistical analysis was performed using SPSS (version 11.0). All data were presented as mean ± standard error means (SEM) and analyzed by analysis of variance (ANOVA) between groups. Intra-group comparison at 0, and 12 weeks after treatment was carried out using paired student t test. P[...]... primer sequences for detection of hVEGF165, Ang -1 and human GAPDH are listed in Table 2 .1 The reverse transcription was 50oC for 30 minutes followed by initial PCR activation step for 15 minutes at 95oC The denature temperature was 94oC for 0.5 minute The annealing temperature was 64oC (for hVEGF165), 60oC (for Ang -1) and 65oC (for human GAPDH) for 0.5 minute The extension was 72oC for 1 minute In total... with DAPI and observed under fluorescent microscope The FITC or 71 TRITC positive cells were calculated by counting 5 microscopic fields for stained and unstained cells on 5 slides Dual immunostaining for hVEGF165 and Ang -1 expression For co-expression of hVEGF165 and Ang -1 from Ad-Bic transduced myoblasts, Bic -myoblasts were seeded and grown on glass chamber slides (1 x 10 5 per glass chamber), using Ad-null... expression Non-transduced myoblasts or human fibroblasts (as a negative control) were trypsinized and resuspended in glass tubes (1x106 cell/ tube) The cells were fixed in 20oC methanol for 10 minutes followed by incubation with 0 .1% Triton-X 10 0 for another 10 minutes After blocking the non-specific sites with 1% BSA for 1 hour at 37oC, 1: 50 rabbit anti-desmin antibody and/ or mouse anti -human CD56-PE antibody... 5'-GTAGTGCCACTTTATCCCATTCAG-3' reverse Human VEGF165 (576bp) (64oC) 5'-CGGTGAATATTGGCTGGGGAATGAG-3' reverse Human GAPDH (302bp) (65oC) forward 5'-TAGGAACACGGAAGGCCATGCCAG-3' RT-PCR analysis for hVEGF165 and Ang -1 expression: RT-PCR protocol as described above was used for analysis of concurrent hVEGF165 and Ang -1 expression from Ad-Bic 73 transduced myoblasts Primers of hVEGF165 and Ang -1 were mixed and added into master... washing and air drying, cells were fixed in 10 0% methanol for 10 minutes at –20oC followed by incubation with 0 .1% Triton-X100 for 10 minutes The non-specific binding sites were blocked by incubation for 1 hour with 1% BSA at 37oC followed by incubation with 1: 50 rabbit anti -human desmin antibody or mouse anti -human CD56-PE for over-night After thorough washing, cells were incubated with 1: 200 goat... IgG-FITC (for hVEGF165) and 1: 200 goat anti-rabbit IgG-TRITC (For Ang1) for 1 hour After thorough washing, the slides were air dried in the dark and counterstained with DAPI and observed under fluorescent microscope The proportion of FITC and TRITC positive myoblasts was calculated from the ratio, counting 5 microscopic fields for stained and unstained cells on 5 slides RT-PCR analysis Skeletal myoblasts. .. analyzed by RT-PCR for angiogenic gene expression at 1, 8, 18 and 30 days after transduction Total RNA was isolated by using the Total RNA Isolation Kit according to manufacture’s instructions as outlined in Appendix 2 RT-PCR analysis for hVEGF165 or Ang -1 expression: QIAGEN One-step RT-PCR kit was used for RT-PCR analysis of skeletal myoblasts for hVEGF165 or Ang -1 expression 72 using human GAPDH as house-keeping... with 5% CO2 2.2 .1. 2 In vitro characterization of skeletal myoblasts The purity of skeletal myoblast culture was assessed for desmin and CD56 expression Fluorescent immunostaining of non-transduced myoblasts for desmin and CD56 expression Non-transduced human skeletal myoblasts or human fibroblasts (as a negative control) were harvested and seeded in microscopic glass chambers and cultured for 24-48 hours... were assessed for biological activity Immunostaining for hVEGF165 or Ang -1 expression The Ad-VEGF165 or Ad-Ang -1 transduced myoblasts were seeded and grown on glass chamber slides (1 x 10 5 per glass chamber), using Ad-null transduced myoblasts as a negative control Cells were fixed with 50% acetone/ methanol for 10 seconds After washing, cells were incubated with 3% hydrogen peroxide for 15 minutes to... (pH6.8) and 3.0 ml water and 30 µl 10 % ammonium persulfate with 6 µl TEMED The protein samples were boiled in 1 x loading buffer (62.5 mM Tris-Cl pH6.8, 10 % glycerol, 2% SDS, 10 0 mM dithiothreitol, 4% bromophenol blue) for 1 minute 74 before loading into the stacking gel The gel was run at 10 0 voltage with amperage at 10 0mA for 2 hours in 1 x SDS electrophoresis buffer (25 mM Tris, 19 2 mM glycine and 0 .1% . incubation with 0 .1% Triton-X 10 0 for another 10 minutes. After blocking the non-specific sites with 1% BSA for 1 hour at 37 o C, 1: 50 rabbit anti-desmin antibody and/ or mouse anti -human CD56-PE. assessed for biological activity. Immunostaining for hVEGF 16 5 or Ang -1 expression The Ad-VEGF 16 5 or Ad-Ang -1 transduced myoblasts were seeded and grown on glass chamber slides (1 x 10 5 . chambers and cultured for 24-48 hours. After washing and air drying, cells were fixed in 10 0% methanol for 10 minutes at –20 o C followed by incubation with 0 .1% Triton-X100 for 10 minutes. The non-specific