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Supplementary materials and mhetods
Cell lines
All cell lines were cultured in an incubator at 37°C with a mixture of air and 5% CO2. Reagents
were purchased from Invitrogen unless otherwise stated.
Neonatal rat cardiomyocytes (rCMC) were obtained with a modification of an original
protocol.32 Cells were cultured in 4:1 Dulbecco's modified Eagle's medium (DMEM)-Medium199
supplemented with 5% fetal bovine serum (FBS), 5% horse serum (HS), L-glutamine (L-glu) and
penicillin/streptomycin (pen/strep). Cells were treated with mitomycin C (MitC, Sigma) to prevent
fibroblast cell growth. Final cell populations contained more than 95% CMCs, as assessed by
immunofluorescence analysis.33
The mouse myoblast C2-C12 cell line (ATCC) is a striated muscle cell model; these cells
were cultured in DMEM containing 20% FBS, L-glu, and pen/strep. Cells were sub-cultured two or
three times before use. Differentiation into myotubes was induced at ~70% confluence by replacing
the high serum medium with DMEM containing a mixture of bovine insulin, human transferrin, and
sodium selenite (ITS, Sigma).
The human embryonic kidney cell lines, HEK-293 and 293T, were kindly provided by Dr
F.L. Graham (Istituto di Ricerche di Biologia Molecolare-IRBM, Rome, Italy). 293T cells
(originally called 293tsA1609ne) were grown in Iscove's modified Dulbecco's medium (IMDM)
supplemented with 10% FBS (Hyclone), L-glu and pen/strep. They derive from HEK-293 cells
transformed with sheared Type 5 Adenovirus DNA, and transfected with the tsA 1609 mutant gene
of SV40 Large T Antigen and the Neor gene of E. coli.
Mouse fibroblast NIH-3T3 cells (ATCC) and mouse embryo fibroblasts (MEF, Specialty
Media) were cultured in DMEM containing 10% FBS (Hyclone), L-glu and pen/strep.
Visceral endoderm-like cells (End2) were kindly provided by Prof. C. Mummery and
cultured as described13 in 1:1 DMEM/F12 supplemented with 7.5 % FBS (Hyclone), sodium
pyruvate (NaPy), non-essential amino acids (NEAA), Glutamax and pen/strep.
The mouse embryonic stem cell (mESC) lines34,35 used in this study are feeder-independent
and were cultured on gelatin-coated plates. Cells were maintained in Glasgow MEM/BHK medium
containing 10% FBS, 0.23 % sodium bicarbonate, MEM essential amino acids, 1mM NaPy, 100
mM 2-mercaptoethanol, and L-glu. This medium is referred to as cultivation medium. Leukemiainhibitory factor (LIF, 1000 units/ml, Chemicon International) was added to maintain the
pluripotent, undifferentiated state.36 To induce differentiation, cultivation medium without LIF was
used. When embryo bodies (EBs) formed, they were transferred to bacteriological plates and
maintained in suspension for 3 days in cultivation medium supplemented with 0.1% DMSO. EBs
were maintained in suspension two days more with cultivation medium, then were allowed to settle
onto gelatin-coated plates in the presence of cultivation medium without LIF or DMSO. Medium
was changed every two days. Contracting areas appeared within 16-18 days.
Human embryonic stem cell (hESC) line 7 (hESC7, purchased at passage 11) and 8 (hESC8,
purchased at passage 18) were obtained from Prof. D. Melton. Maintenance, passaging and
differentiation
were
performed
following
the
supplied
protocols10
(http://www.mcb.harvard.edu/melton/hues). Briefly, hESCs were cultured on MitC-mitotically
inactivated mouse embryonic fibroblasts (MEFi) in KO-DMEM supplemented with 10% serum
replacement (KO-SR), 10ng/ml basic fibroblast growth factor (bFGF), 12ng/ml recombinant human
LIF, Glutamax and pen/strep. Cells were adapted to trypsin passaging before being used in
experiments. Differentiation was induced by culturing the cells in suspension (starting on what is
referred to as day 0) in the absence of hLIF and bFGF; this allowed them to form cystic EBs.10
When specified, 5M 5-aza-2'-deoxycytidine (AZA) was added at each media change (i.e. every
third day) starting from day 1. After 7-10 days, EBs were passaged onto plates coated with gelatin
and cultured in differentiation medium supplemented with 1% KO-SR.
In all experiments using mouse or human ESCs, the day on which ESCs were dispersed into
suspension was considered day 0. This follows the nomenclature given by Kehat I.,3 which
considers the day of suspension as the day when differentiation starts.
Processing, isolation and immunostaining of cardiosphere-forming cells from murine heart
biopsies was performed as extensively described previously.26
Transient transfection and dual luciferase assay
Plasmids (pGL3.SV40.luc and pGL3.CMV.luc) expressing luciferase under the control of strong
non-tissue-specific promoters (i.e. simian virus 40 or cytomegalovirus, respectively) were obtained
commercially (Promega).
pGL3.hTNNI3_340b.luc was obtained by PCR amplification of residues 837 to 1174 (270/+70) of GenBank TM accession number X907807 with primers that introduced an EcoRV site at
the 5’ end and a BamH1 site at the 3’ end [primers: forward (5 -GGGATATCT
CCTTGTGTGAGGGAGTGG -3 ), reverse (5 -GGGGGATCCGGGTGACCTTCAGGGTCC-3 );
restriction sites underlined]. The resulting amplification product was cloned into pGL3 basic cut
with Sma1 and Bgl2.
rCMCs, C2.C12 (myoblasts and myotubes), and NIH-3T3 cells were grown in their
respective milieus and transfected in parallel. Sub-confluent cell cultures were co-transfected using
Lipofectamine, following the supplied instructions: DNA/lipid complexes were prepared by mixing
1 µg of a luciferase reporter plasmid (pGL3.hTNNI3_340b.luc, pGL3.SV40.luc or pGL3.CMV.luc)
and 0.1g of Renilla luciformis luciferase-reporter plasmid (pRL.TK, Promega). All transfections
included a background sample. Fifty hours later, cells were harvested and luciferase activity
determined with the Dual Luciferase Reporter Assay (Promega) in a TD20 luminometer (Turner
Designs), following the manufacturer’s instructions. Luciferase activity was calculated by
subtracting the level measured in the background sample and then normalizing to transfection
efficiency as measured by the activity deriving from the co-transfected pRL.TK. The data so
obtained for each sample was used in the specified ratios.
Intra-myocardial/quadriceps injection of plasmid-DNA and luciferase assay
Mice were anesthetized with a ketamine (100mg/Kg)-xylazine (2.5 mg/Kg) mixture, administered
i.p., and connected to a rodent ventilator, after tracheal intubation. hearts were exposed and injected
twice with 10l 3g/l of plasmid DNA solution in PBS (pGL3.hTNNI3_340b.luc or
pGL3.SV40.luc). A total of 60g was injected. Injection into the free wall of the left ventricle (LV)
was performed with a 32-gauge needle while the heart was beating, under visual guidance.
Intra-quadriceps injection was performed without surgery. A total amount of 60g/leg of
plasmid DNA was injected (in two injections of 30g/50l pGL3.hTNNI3_340b.luc or
pGL3.SV40.luc). All experiments included a PBS injection as negative control.
Mice were sacrificed 3 days after surgery. Heart and quadriceps from each mouse were
excised and used for luciferase assay. Mice were treated in accordance with European guidelines.
Tissue extracts were preparated and normalized for protein concentration by Bradford assay
(BIORAD). The luciferase activity of tissues extracts was measured using a Luciferase Assay Kit
(Promega) and a TD-20/20 luminometer (Turner Designs) according to the manufacturer’s
instruction and was calculated by subtracting the level measured in the negative control samples.
Lentiviral vector production and titration
The three-plasmid expression system used to generate lentiviral vectors by transient transfection
was performed as previously described.9,37
The transfer vector plasmid backbone containing the enhanced green fluorescence protein
(EGFP) reporter gene driven by the human phosphoglycerate kinase (hPGK) or cytomegalovirus
promoters (pRRLcPPT.hPGK.EGFP.WPRE and pRRLcPPT.CMV.EGFP.WPRE, respectively)
have been described before for assembling 'advanced' third-generation lentiviruses.9
The mouse cardiac troponin (mTNNI3_ 427b) proximal promoter sequence was amplified
by PCR from an original genomic clone. A 427bp PCR fragment, corresponding to nucleotides 301/+126 of the previously published TnIc promoter (GeneBank
TM
accession number Z22784)8
was generated [primers: forward (5’-CCATCGATCTGCAGTTCAGTGAG-3’) and reverse (5’-
CGGGATCCTGATCTCCAGAGGC-3’) restriction sites underlined]. The BamHI site, but not the
ClaI site, was preserved in the new construct. The pRRLcPPT.CMV.EGFP.WPRE construct 9 was
cut with BamHI and ClaI to remove the CMV promoter sequence, and the mouse TnIc promoter
fragment inserted.
The human cardiac troponin (hTNNI3_340b) proximal promoter sequence was amplified by
PCR from pGL3.hTNNI3.luc (primers described above) with Taq polymerase (LA Taq, TaKaRa).
The amplification product was digested with EcoRV and BamHI restriction enzymes and cloned
into
the
corresponding
sites
of
pRRLcPPT.__.EGFP.WPRE.
The
plasmid
pRRLcPPT.hTNNI3_340b.EGFP.WPRE thus generated contained the 340bp hTNNI3-proximal
promoter.
PCR primers were chosen to amplify residues 100322-101238 of GenBankTM accession
number AC087457.5 harboring the cardio-specific enhancer in the cardiac muscle alpha-actin
proprotein promoter on human chromosome 15 flanked by Xho I and Sal I restriction sites at the 5'
and 3' ends, respectively.14 The PCR product was cloned into the pCR2 vector with the TA cloning
kit (Invitrogen). The pCR2.hEnAct construct so obtained was digested with XhoI and EcoRV
restriction enzymes (corresponding to residues 100322-101168) and cloned into the corresponding
sites
upstream
of
the
hTNNI3
promoter
to
generate
pRRLcPPT.hEnAct_846b-
TNNI3_340b.EGFP.WPRE.
The fragment containing residues 611-899 of Genbank accession number X03922, annotated
as a potential enhancer in the human cytomegalovirus (hCMV) IE1 gene promoter region was
obtained by cutting pRRLcPPT.CMV_TNNI39 with SnaB1 and EcoR5; subsequent intermolecular
ligation
was
performed
to
replace
hEnAct
to
obtain
pRRLcPPT.hEnCMV_288b_TNNI3.EGFP.WPRE.
pSINF.EF1a.GFP.SAR/HS was kindly provided by Prof. G.R. Hawley and has been already
described.38 Briefly, this is a self-inactivating lentiviral vector that contains cPPT and a central
termination sequence followed by the human elongation factor 1 (EF1a) promoter driving
expression of the EGFP gene. A scaffold attachment region (SAR, from the human interferon-
gene) together with a chromatin insulator (HS, from the 5’ end of the chicken -globin locus) was
incorporated into a lentiviral-vector backbone.
Lentiviral vector stocks were obtained from the supernatants of 293T cells co-transfected
with the 3 plasmids necessary for viral production. These were: the packaging plasmid, pCMV
R8.74, designed to provide the HIV proteins needed to produce the viral particles; the envelopecoding plasmid, pMD.G, for pseudotyping the virion with VSV-G, and; one of the self-inactivating
(SIN)
transfer
vector
plasmids
described
above
pSINF.EF1a.GFP.SAR/HS,
(pRRLcPPT.hPGK.EGFP.WPRE,
pRRLcPPT.mTNNI3_427b.EGFP.WPRE,
pRRLcPPT.hTNNI3_340b.EGFP.WPRE, pRRLcPPT.hEnAct_846b-TNNI3_340b.EGFP.WPRE).
The lentiviral vectors obtained were denominated PGK-LVV, EF1-LVV, mTNNI3-LVV, hTNNI3LVV and hEnAct_TNNI3-LVV, respectively.
In order to determine the transducing unit (TU) concentration of the supernatants from 293T
cell cultures, experiments were performed with rCMCs by adding serial dilutions of supernatant to
1  105 rCMCswell in 24-well plates in the presence of polybrene (4gml). Transduced cells were
analyzed by FACS to evaluate %EGFP-positive events. In a typical titration experiment, only
dilutions yielding 0.2-20% EGFP-positive cells were considered for titer calculations: In this
concentration range, we found a linear correlation between MOI and EGFP expression. To calculate
rCMC-TU/ml, the following math was applied: TU/ml = [(target cell number) X (% GFP-positive
cells)] / (ml of viral supernatant).
Typical supernatants contained approximately 105-106 rCMC-TU/ml corresponding to 10100 ng of p24 protein/ml as measured by HIV-1 p24 core profile ELISA (Abbott Diagnostics or
NENTM Life Science Products) as described previously.9
Lentiviral transduction
105 cells (CMCs, NIH-3T3 or HEK-293) were plated in 24-well plates. On the day of infection, the
medium was removed and replaced with viral supernatant (at the specified MOI) to which
polybrene had been added. All experiments included a background sample. After overnight
incubation, cells were washed and fresh medium added. 48 h from the start of experiments, cells
were washed with PBS, harvested with trypsin-EDTA and analyzed by FACS. Alternatively,
images of these cells were photographed under a fluorescence microscope.
Undifferentiated mESCs were plated on gelatin-coated plates the day before lentiviral
transduction performed overnight at an MOI of 2. After a media change, cells were cultured as
described above.
hESC7 or 8 (at passage 19 and 21, respectively) were amplified on MEFi feeder layers in 6well plates. Transduction was performed (at the specified MOI) when hESC colonies reached 30%
confluence. After an overnight transduction, cells were washed, fresh medium added, and cells
cultured as described above.
Flow cytometric analysis
Sorting experiments and cytometric analyses were performed using a FACSVantange SE and a
FACSCalibur with CellQuest software (Becton Dickinson Immunocytometry Systems),
respectively. For all FACS analyses, at least 10,000 events were recorded.
Transduced cells (rCMCs, NIH-3T3, or HEK-293) were analyzed by FACS with standard
procedures to evaluate mean fluorescence intensity (MFI). MFI was calculated after subtracting
autofluorescence measured in background samples.
Transduced mESCs were collected using trypsin and %EGFP-expressing cells was
evaluated with standard procedures.
Transduced hESC samples were collected using trypsin and stained by a standard indirect
intracellular staining procedure (BecktonDickinson) using a mouse monoclonal anti-human nuclei
antibody (Chemicon) followed by RPE-conjugated goat anti-mouse IgG (DakoCytomation),
according to the manufacturer’s instructions. All samples were analyzed within 24 h of staining.
Autofluorescence was subtracted from each sample, and the percentage of hESCs expressing EGFP
calculated.
The protocol used for dispersing hEBs into suspension for FACS was developed following
the indications of ES Cell International (ESI_http://www.escellinternational.com). Plates containing
EBs were treated with collagenase IV in PBS (200U/ml) for 10 minutes in the incubator. Vigorous
pipetting was used to dislodge cell clumps from the dish. Cells were then transferred to a 15 ml tube
and spinned down at 600g for 2 minutes. The supernatant was removed and the pellet resuspended
in 0.05% trypsin (Gibco). A single-cell suspension was obtained by vigorous pipetting. Just before
sorting, the cell suspension was filtered through a 70M cell strainer (Becton Dickinson).
RNA extraction and Real-Time PCR
Total RNA was extracted from cells with the RNeasy Mini Kit (Qiagen) according to the
manufacturer’s instructions. RT-PCR reactions were performed using Superscript III reverse
transcriptase, random primers and TaqMan oligonucleotides (Assay on Demand, Applied
Biosystems) for cardiac troponin I (TnIc) (code Hs00165957_m1) and alkaline phosphatase (ALP)
(code Hs00758162_m1), as recommended by the manufacturer, in an ABI PRIS 7000 instrument
(Applied Biosystems). All reactions were performed in duplicate. The amount of target, normalized
to the values obtained for amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
and relative to a calibrator, was determinated by 2-ΔΔCT , where ΔΔCT = ΔCTtarget - ΔCTcalibrator.
ΔCTtarget was the difference in threshold cycles between the target and GAPDH, and CT was a
parameter given by ABI PRISM 7700 Sequence Detector software by negative correlation with an
internal reference dye (ROX). ΔCTcalibrator was the value obtained from a human heart sample.
Immunofluorescence staining
Standard indirect immunofluorescence techniques were used to stain paraformaldehyde-fixed cells.
Anti-cardiac troponin I (Babco) and phycoerythrin-conjugated anti-mouse (DakoCytomation)
antibodies were used. Cells that had not been previously transduced were used to set the exposure
parameters to exclude any background autofluorescence.