Angiogenesis and myogenesis using human skeletal myoblasts for cardiac repair 1

The purified skeletal myoblasts were cultured and propagated in cell culture flasks pre-coated with collagen, using Super-medium at 37oC in 5% CO2 incubator until the required number was

Chapter 2 Materials and Methods

2.1 MATERIALS

2.1.1 Cell culture media

Dulbecco’s Modified Eagle’s Medium Sigma, USA

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)

Generously gifted by Cell Transplants Singapore Pte., Ltd

Human skeletal myoblasts

Generously sponsored by Cell Transplants Singapore Pte., Ltd

Human umbilical vein endothelial cells

Kindly gifted by Associate Professor Ruowen Ge, Department of Biological

Sciences, National University of Singapore, Singapore

Atropine sulphate Troy Laboratories, Australia

3, 3’ diaminobenzidine tetrahydrochloride ICN Biomedical Inc., USA

Fluospheres® red polystyrene microsphere

Fluospheres® scarlet polystyrene microsphere

Fluospheres® yellow-green polystyrene microsphere

Formalin (37%) Sigma, USA

Osmium tetroxide Agar Scientific Ltd, England

Polyoxyethylenesorbitan mono oleate (Tween-80) Sigma, USA

Sodium chloride+sodium citrate (20x SSC) NUMI, Singapore

Sodium phosphate (monobasic) Sigma, USA

Tetramethylene-ethylenediaminutese (TEMED) Bio-Rad, USA

2, 4, 6-tridimethylaminuteso methyl phenol TED PELLA, Inc USA

Tris (2-hydroxymethyl-2-methyl-1,3-propanediol) Fisher Scientific, USA

2.1.5 Proteins, antibodies and kits

Proteins

Molecular weight color markers (6.5~45kDa) Sigma, USA

Primary antibodies

Mouse anti-human cardiac troponin I antibody Serotec, UK

Mouse anti-human CD56- PE BD PharMingen, USA Mouse anti-human connexin-43 antibody Sigma, USA

Mouse anti-pig monocyte/ granulocyte antibody BD Biosciences, USA

Mouse anti-fast myosin heavy chain antibody NeoMarkers, USA

Mouse anti-slow myosin heavy chain antibody Chemicon Int., USA

Mouse anti-α smooth muscle actin antibody Sigma, USA

Secondary antibodies

Rabbit anti-pig IgG-FITC US Biological, USA

Kits

2.1.6 Apparatus

Normal culture flasks (25 and 75 cm3) NUNCLONTM, Denmark

Polypropylene centrifugal tube (15 and 50 ml) Corning, USA

Poretics polycarbonate filter, 10.0 µm OSMONICS Inc., USA

Power supply (capacity 200V, 500mA) Bio-Rad, 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

2.1.8 Equipment

Aquasonic 100 ultrasound transmission gel Agilent Tech USA

CO2incubator Forma Scientific., USA

Laminar flow work station (Class II) Elman, Singapore

LS 6500 multi-purpose scintillation counter Beckman, England

Microwave oven Sanyo, Japan

Mini trans-blot electrophoretic transfer cell Bio-Rad, USA

Sonos 5500 echocardiographic machine Agilent Tech USA

Universal hood (Gel document system) Bio-Rad, USA

Vingmed Vivid 5 ultrasound machine General Electric, USA

2.1.9 Computer software

Coulter flowcytometric software Scripps Res Institute, USA

Microsoft office 2000 Microsoft, USA

2.2 METHODS

2.2.1 In Vitro studies

2.2.1.1 Cell culture

FLY-A4 cells, HEK 293 cells, HeLa cells, Human fibroblasts, and NIH 3T3 fibroblasts

Cells were cultured in DMEM supplemented with 10% FBS (10% DMEM) at

37oC in 5% CO2 incubator

Human umbilical vein endothelial cells

HUVEC were cultured in F12K culture medium supplemented with 10% FBS,

1% penicillin/ streptomycin, 20 units/ ml heparin and 20 ng/ ml α-FGF at 37oC in 5%

CO2 incubator Cells were regularly passaged at 80% of confluence

Human skeletal myoblasts

Myoblasts from human male donor skeletal biopsies were prepared and expanded

in vitro according to the in-house Standard Operating Protocol (SOP) (Cell Transplants Singapore Pte Ltd, Singapore) and trade secrets with a license of the United States Patent

NO 5,130,141 and a license of the Singapore Patent NO 34490 (WO 96/18303) Cell production was in compliance with current Good Manufacturing Practice and International Organization for Standardization 9001 Briefly, the left rectus femoris muscle was preconditioned under local anesthesia prior to biopsy Two days later, approximately 5 g of muscle tissue was harvested through a small skin incision The muscle biopsy was immediately dissociated with 0.1% collagenase and 0.2% crude trypsin in PBS Cells were cultured in the Super-medium (Cell Transplants Singapore Pte Ltd, Singapore) The culture yielded up to 500 millionskeletal myoblast Throughout the culture, the cells were tested negative for sterility (14-day test) and gram stain

(absence of gram positive or negative bacteria) according to certified laboratory analysis The purified skeletal myoblasts were cultured and propagated in cell culture flasks pre-coated with collagen, using Super-medium at 37oC in 5% CO2 incubator until the required number was obtained

Skeletal myoblasts were regularly passaged every 48-72 hours to prevent the myoblasts from fusion in vitro After washing with PBS for 3 times, skeletal myoblasts were detached by 0.05% trypsin containing 0.02% EDTA The detached cells were washed 3 times with centrifuging at 1500 RPM for 5 minutes each Then the cells were re-suspended into new flask in Super-medium and incubated at 37oC 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 After washing and air drying, cells were fixed in 100% 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 anti-rabbit IgG-FITC for 1 hour for detection of desmin The excess antibody was washed with PBS The slides were air dried in the dark, and counter-stained with DAPI and observed under

fluorescent microscope The proportion of PE and FITC positive skeletal myoblasts was calculated by counting 5 microscopic fields for stained and unstained cells on 5 slides

Cytofluorimetry of non-transduced myoblasts for desmin and CD56 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 100 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 were added into designated tubes After one and half hours incubation, cells in designated tube were washed and incubated with 1: 200 goat anti-rabbit IgG-FITC for 1 hour at room temperature for detection of desmin After thorough washing with PBS, cells were re-suspended in 0.5 ml PBS and analyzed using Coulter flowcytometric software by a staff that was blinded to this experiment This experiment was performed in triplicate and the data were analyzed using WinMDI version 2.8

Fluorescent immunostaining and cytofluorimetry of genetically modified myoblasts for desmin and CD56 expression

To assess the effect of viral vector transduction on skeletal myoblasts, viral vector (adenoviral and retroviral vectors) transduced myoblasts were processed by dual fluorescent immunostaining and cytofluorimetry for desmin and CD56 expression using the same protocol as described above

2.2.1.3 Skeletal myoblast transduction with Ad-vector carrying angiogenic gene

Skeletal myoblasts were transduced with monocistronic Ad-vector carrying hVEGF165 or Ang-1, or Bic Ad-vector simultaneously carrying hVEGF165 and Ang-1

Monocistronic Ad-vector carrying hVEGF 165 or Ang-1

The monocistronic Ad-vectors carrying hVEGF165 or Ang-1 were kindly provided

by Associate Professor Ruowen Ge, Department of Biological Sciences, National University of Singapore The replication deficient Ad-vector carrying either hVEGF165 or Ang-1 gene was driven by immediate early human cytomegalovirus promoter For propagation of the virus, HEK293 cells were cultured in DMEM supplemented with 2% FBS (2% DMEM) At 80-90% confluence, HEK293 cells were infected with virus and following full development of CPE, cells were harvested and lysed by 5 repeated freezing/ thaw cycles The cell lysate and the supernatant were purified using cesium chloride gradient ultra-centrifugation as described in Appendix 1

Adenoviral bicistronic vector carrying both hVEGF 165 and Ang-1

Ad-Bic vector carrying hVEGF165 and Ang-1 was kindly provided by Associate Professor Ruowen Ge, Department of Biological Sciences, National University of Singapore, Singapore The construction of this Ad-Bic was similar to that reported by

Bett et al (1994) The Ad-bic containing Ang-1 and hVEGF165 genes were separated by IRES sequence and driven by a common murine cytomegalovirus (MCMV) promoter The end product was an E1-deficient recombinant virus carrying the hVEGF165 and Ang-

1 genes For propagation of the virus, HEK293 cells were cultured in 2% DMEM At 90% of confluence, HEK293 cells were infected with virus Following full CPE development, cells were harvested and lysed by 5 repeated freezing/ thaw cycles The viral vector from cell lysate and the supernatant were purified using cesium chloride gradient ultra-centrifugation as described in Appendix 1

80-Ad-vector propagation

All Ad-vectors used in this study were propagated in HEK293 cells At ~80% of confluence, HEK293 cells were infected with Ad-vector Cells were maintained for 72-96 hours in 10% DMEM At stipulated time, the supernatant from HEK293 cells was removed and purified using cesium chloride gradient ultra-centrifugation as described in Appendix 1

Ad-vector titer

Ad-vector titration was carried out using an end-point assay described by Quantum Biotechnology, USA Approximately 1x104 HEK293 cells/ well were plated in 96-well tissue culture plate in 100 µl 2% DMEM on the day of experiment This was followed by adding 100 µl purified viral stock in 2% DMEM with dilution ranging from

10-5 to 10-13 per ml Each dilution was applied to 10 wells and another 16 wells were added with 100 µl of 2% DMEM as negative controls Cells were incubated for 10 days

at 37°C and observed daily for CPE development At the end of incubation, the tissue culture infectious dose-50 (TCID50) was calculated using the formula: TCID50/ ml =

the ratios (always starting from the first 10-1 dilution) Furthermore, PFU per milliliter was calculated by subtracting the TCID50/ml from 0.7 Log The assay was repeated at least three times to determine the viral titer

Screening for replication-competent Ad-vector

Purified Ad-vectors were screened for the presence of replication-competent viruses using method described by Hehir et al (1996) with slight modifications On the

day of experiment, HeLa cells were plated into 24-well tissue culture plate at a density of 2x104 cells/ well The viral stocks were serially diluted and applied from 1010 or 109 to

101 PFU/ ml concentration into each well For each purified viral clone, duplicate sets were prepared; one set was daily monitored for 10 days for CPE development and the other set was terminated by day 4 post infection, and infected cells were collected for subsequent experiment The infected cells were lysed by 5 cycles of freezing and thaw in

200 µl PBS The cell lysate was cleared of cell debris by centrifugation at 4383 g for 4 minutes Aliquots of 100 µl of the supernatant were applied to A549 cells plated in 24-well plate (5x104 cells/ well) and subsequently monitored for an additional 10 days for CPE development

Optimization of skeletal myoblast transduction with Ad-vectors

Skeletal myoblasts were cultured at 1x105 cell density in 6-well tissue culture plate Cells were exposed to either Ad-VEGF165 or Ad-Ang-1 or Ad-Bic or Ad-null, at titers ranging from 200 PFU/ myoblast to 4000 PFU/ myoblast The supernatant containing Ad-vector was filtered through 0.22 µm microfilter into each flask and incubated at 37oC in 5% CO2 incubator Transduction was allowed to continue for 2, 4, 8 and 24 hours At pre-determined time after transduction, the viral infection medium was replaced with fresh Super-medium for 24 hours The transduction procedure was repeated three times to achieve optimum transduction efficiency This experiment was performed

in triplicate

2.2.1.4 In vitro characterization of Ad-vector transduced myoblasts

After transduction with respective Ad-vector, skeletal myoblasts were qualitatively and quantitatively assessed for angiogenic gene expression in vitro The expressed angiogenic proteins were assessed for biological activity

Immunostaining for hVEGF 165 or Ang-1 expression

The Ad-VEGF165 or Ad-Ang-1 transduced myoblasts were seeded and grown on glass chamber slides (1 x 105 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 reduce non-specific background staining due to endogenous HRP The non-specific binding sites were blocked by incubation with Ultra V block (Ultravision detection system) for 5 minutes at room temperature followed by incubation with 1: 200 dilution of mouse anti-human VEGF, or 1: 100 rabbit anti-Ang-1 antibodies at room temperature for overnight

On the following day, the excess antibody solution was discarded After three times washing with PBS, cells were incubated with 1: 400 dilution of rabbit anti-mouse IgG-HRP or 1: 200 goat anti-rabbit IgG conjugated HRP for 1 hour The antigen-antibody

reaction was visualized using DAB visualization system (Ultravision detection system)

For fluorescent immunostaining, cells were incubated with 1: 400 goat anti-mouse IgG-FITC secondary antibody (for hVEGF165) or 1: 200 goat anti-rabbit IgG-TRITC secondary antibody (for Ang-1) for 1 hour after applying respective primary antibodies as described above After thorough washing, the slides were air dried in the dark and counter stained with DAPI and observed under fluorescent microscope The FITC or

TRITC positive cells were calculated by counting 5 microscopic fields for stained and unstained cells on 5 slides

Dual immunostaining for hVEGF 165 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 105 per glass chamber), using Ad-null transduced myoblasts as a negative control After fixing with 50% acetone/ methanol for 10 seconds, the non-specific binding sites were blocked by Ultra V block for 5 minutes at room temperature followed by incubation with 1: 200 dilution of mouse anti-human VEGF and 1: 100 rabbit anti-Ang-1 antibodies at room temperature for overnight After thorough washing, cells were incubated with 1: 400 goat anti-mouse IgG-FITC (for hVEGF165) and 1: 200 goat anti-rabbit IgG-TRITC (For Ang-1) for 1 hour After thorough washing, the slides were air dried in the dark and counter-stained 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 carrying angiogenic genes were 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 hVEGF 165 or Ang-1 expression: QIAGEN One-step RT-PCR kit

was used for RT-PCR analysis of skeletal myoblasts for hVEGF or Ang-1 expression

using human GAPDH as house-keeping gene Briefly, total RNA (0.5 µg) of each sample was added into each master mix The respective 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 30 cycles were performed followed by final extension at 72 oC for 10 minutes

Table 2.1 RT-PCR primers and annealing temperature

forward 5'-CGGTGAATATTGGCTGGGGAATGAG-3' Human Ang-1

(576bp) (64oC)

reverse 5'-TCACCGCCTCGGCTTGTCACA-3' forward 5’-TTCCACGGCACAGTCAA-3’

Pig GAPDH

(576bp) (54oC)

reverse 5’-GCAGGTCAGGTCCACAA-3’

forward 5’-TTCTTGTGCAGTGCCAGCCTCGTC-3' Rat GAPDHb

(743bp) (63oC)

reverse 5'-TAGGAACACGGAAGGCCATGCCAG-3'

RT-PCR analysis for hVEGF 165 and Ang-1 expression: RT-PCR protocol as described

above was used for analysis of concurrent hVEGF165 and Ang-1 expression from Ad-Bic

transduced myoblasts Primers of hVEGF165 and Ang-1 were mixed and added into master mix simultaneously The reverse transcription was 50oC for 30 minutes followed

by initial PCR activation step for 15 minutes at 95 oC The denature temperature was

94oC for 0.5 minute The annealing temperature was 62oC for 0.5 minute The extension was 72oC for 1 minute Totally 30 cycles were performed followed by final extension at

72 oC for 10 minutes

Western blot analysis

Preparation of cell lysates: The Ad-null, Ad-VEGF165, Ad-Ang-1 and Ad-Bic transduced myoblasts were grown in 6-well tissue culture plates at a cell density of 2x105 cells/ well After 48 hours, supernatant and transduced myoblasts were collected for Western blot analysis Cells were suspended in 0.5 ml PBS and lysed by repeated freezing and thaw at least 5 times The supernatants and cell lysates were kept at –20oC until used Protein concentration was determined using spectrophotometer as described in Appendix 3

SDS-polyacrylamide gel electrophoresis: The 10% SDS-polyacrylamide gel

electrophoresis was cast using Minigel apparatus The gel was prepared as follows Briefly, the gel was cast by mixing 2.5 ml 30% acrylamide (30% acrylamide/ 0.8% bisacrylamide) with 1.8 ml 1.5 M Tris-Cl/ 0.4% SDS (pH 8.8) and 3.1 ml water and 100

µl 10% ammonium persulfate with 10 µl TEMED A 3.9% stacking gel was prepared by adding 0.65 ml 30% acrylamide with 1.25 ml 0.5 M Tris-Cl/0.4% SDS (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, 100 mM dithiothreitol, 4% bromophenol blue) for 1 minute

before loading into the stacking gel The gel was run at 100 voltage with amperage at 100mA for 2 hours in 1 x SDS electrophoresis buffer (25 mM Tris, 192 mM glycine and 0.1% SDS) A pre-stained protein molecular weight marker (6.5-45kDa) was used as a reference

Once the proteins were separated, they were electrophoretically blotted onto nitrocellulose membrane using Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell according to the manufacture’s instruction and performed at 500 mA for 2 hours After washing with 10mM Tris/ HCl wash buffer (pH 7.6) containing 0.05% Tween-20, the membrane was incubated in blocking buffer (5% non-fat dry milk, 10mM Tris pH 7.5, 100mM NaCl, 0.1% Tween-20) for 3 hours at room temperature The membrane was then incubated with 1: 1000 dilution of rabbit anti-hVEGF165 and/ or anti-Ang-1 polyclonal antibody for overnight On the second day, the membrane was developed by applying 1: 2000 goat anti-rabbit IgG-HRP The signal was visualized using the DAB substrate system

Human VEGF ELISA

The quantitative detection of hVEGF165 secreted from Ad- hVEGF165 and Ad-Bic transduced myoblasts was carried out using human VEGF Sandwich ELISA kit Skeletal myoblasts were grown in 6-well tissue culture plates at a cell density of 2x105 cells/ well, using non-transduced and Ad-null vector transduced myoblasts as negative controls Cell cultured supernatants from each well were collected at regular time intervals from day 1 for up to 30 days at two days interval and kept frozen at –20oC until used for assay The assay was performed as per instruction of the supplier Briefly, 100 µl samples or standard hVEGF165 were coated into each designated well in triplicate and 25 µl diluted

biotinylated rabbit anti-hVEGF165 polyclonal antibody was dispensed into each well The plate was incubated at room temperature for 3 hours followed by incubation with streptavidin conjugated alkaline phosphatase for 45 minutes after washing The presence

of primary antibody was detected by color reagent system The absorbance was determined at 490 nm using ELISA plate reader

2.2.1.5 Biological activity of hVEGF 165 or Ang-1 from transduced myoblasts

The biological activity of hVEGF165 or Ang-1 was assessed using HUVEC proliferation and Thymidine [H3] incorporation assays

HUVEC proliferation assay

HUVEC were cultured in 6-well tissue culture plate (1x105 cells/ well) in triplicate for each experimental sample After 24 hours culturing with 2% DMEM, fresh Super-medium, conditioned supernatants from non-transduced or Ad-null or Ad-hVEGF165, and Ad-VEGF165 transduced myoblasts supernatant supplemented with anti-hVEGF165 antibody were added into designated wells After 4 days (96 hours), HUVEC were harvested and counted

For assessment of biological activity of concurrent hVEGF165 and Ang-1, HUVEC was cultured in 6-well tissue culture plate (2.5x104 cells/ well) in triplicate for each sample After 24 hours culturing with 2% DMEM, fresh Super-medium and conditioned supernatants from non-transduced or Ad-null or Ad-Bic, and Ad-Bic transduced myoblast cultured supernatant supplemented with anti-hVEGF and/ or anti-Ang-1 antibodies were added into designated well After 4 days (96 hours), HUVEC were harvested and counted Both experiments were performed in triplicate

Thymidine [H 3 ] incorporation assay

Thymidine [H3] incorporation assay using HUVEC was performed using the same protocol as described above except that 2 µCi thymidine [H3] was added into each well After 4 days’ cell culture, the cells were thoroughly washed with ice cold PBS to remove un-incorporated thymidine [H3] The cells were lysed using 0.5 ml 10.25N sodium hydroxide and centrifuged at 5000 RPM for 10 minutes to remove the cell debris The clear supernatant (400 µl) was mixed with 4 ml scintillation buffer (Ready SafeTMBeckham, USA) and radioactivity counts were measured every 10 minutes using Beckman LT-6500 Multipurpose Scintillation Counter

For assessment of biological activity of hVEGF165 and Ang-1 secreted by myoblasts, HUVEC was cultured in 6-well tissue culture plates (4 x 104 cells/ well) in triplicate for each sample After 24 hours culturing with 2% DMEM, the cells were washed twice with PBS, fresh Super-medium, conditioned supernatant from non-transduced, Ad-null transduced, Bic-myoblast transduced myoblast, and Bic-myoblast cultured supernatant supplemented with anti-VEGF and/ or Anti-Ang-1 antibodies were added into designated wells Each well was supplemented with 2 µCi Thymidine [H3] After culturing for 4 days, HUVEC were harvested as described above and radioactivity was counted every 10 minutes using Beckman LT-6500 Multi-purpose Scintillation Counter Both experiments were performed in triplicate

Capillary-like tubular formation by HUVEC

HUVEC were seeded into 6 well Biocoat Matrigel Matrix Plates at a density of 1x105/ well After culturing in 2% DMEM for 24 hours, HUVEC were co-cultured with various cells that were previously cultured in cellagen discs Non-transduced or Ad-null