Supplementary Material And Methods
Generation of a cardiac-specific lentiviral vector. The three-plasmid expression system
used to generate lentiviral vectors by transient transfection comprises a packaging
plasmid, pCMVDR8.74, designed to provide the HIV proteins needed to produce the virus
particle; an envelope-coding plasmid, pMD.G, for pseudotyping the virion with VSV-G; and
a self-inactivating (SIN) transfer vector plasmid. The transfer vector plasmid contains the
enhanced GFP marker gene. The cardiac troponin (TNNI3_340b) proximal promoter
sequence was amplified by PCR from pGL3.TNNI3.luc and cloned to obtain the plasmid
pRRLcPPT.TNNI3_340b.EGFP.WPRE. An additional DNA sequence of 118 bp (cPPT) is
situated before the EGFP cassette. The cardio-specific enhancer in the cardiac muscle actin protein promoter was cloned upstream from the TNNI3 promoter to generate
pRRLcPPT.hEnAct_846b-TNNI3_340b.EGFP.WPRE.1
Stocks of the TNNI3-GFP lentivirus were obtained from the supernatants of 293T
cells co-transfected with the three plasmids necessary for viral production.1
Generation of reprogramming-OSKM lentiviral vectors. The cDNA for the open reading
frames of OSKM genes were amplified from commercial clones (by Open Biosystems –
Euroclone, Huntsville, AL, USA) by direct PCR using specific primer sets carrying either
NheI or BclI restriction site. Primer sequences are listed in Table S1. After sequence
verification, PCR fragments were then each cloned by standard protocols into lentiviral
vectors containing GFP and neomycin resistance driven by the PGK promoter
(PGF_GFP_Neo). The original vector was kindly provided by Prof. A. Brivanlou (The
Rockefeller University, New York, USA). The resulting lentiviral constructs showed
expression of the four genes of interest (OSKM) in frame with GFP and neomycin by T2A
peptidase site. The primers used for gene expression assays by RT-PCR are given in
Table S3.
Generation of reprogramming-OSK lentiviral vectors. Lentiviral vectors employed for
the induction of reprogramming via OSK factors harbored bicistronic plasmids expressing
Oct4 and either Sox2 or Klf4. The original vector was kindly provided by Prof. L. Naldini
(Fondazione San Raffaele, Milan, Italy). Low passage 293T cells (Cell Biolabs) were used
to produce lentiviruses expressing each pair of transgenes, using the pPAX2 and pVSV-G
packaging constructs and a calcium phosphate transfection protocol. Supernatants
containing OSK lentiviruses were collected 48 hr later, filtered, and used freshly right after
preparation.
Myocardial cell cultures. CMs and CFs were isolated from hearts of 1-day-old CD-1
neonatal mice (Charles River Laboratories). After excision, whole hearts were transferred
into ice-cold HBBS and the blood gently squeezed out. The hearts were then minced into
small pieces in 0.1 mg/ml trypsin (Sigma) supplemented with 20 g/ml deoxyribonuclease
I (DNAse I), and left to pre-digested overnight at 4C. The following day, trypsin was
removed and the myocardial pieces incubated in 0.5 mg/ml collagenase (Worthington
Biochemical) solution containing DNAse I (Sigma) with intermittent pipetting along with
shaking at 37C in a water bath for 3 min. The supernatant, containing free cells, was then
collected and kept on ice. The digestion step was repeated three times. Cell suspensions
from each digestion were pooled, filtered through a 70m strainer, and centrifuged at 800
rpm for 5 min at 4C. The cell pellet was then resuspended in a maintenance medium (CM
medium) containing high glucose DMEM (Sigma) supplemented with 10% horse serum
(Invitrogen), 5% fetal bovine serum (FBS, Invitrogen), 10 mM HEPES (Invitrogen), 2 mM
L-glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml). The cells were plated
and incubated at 37C for 3 hr to allow the differential attachment of non-myocytes,
constituted mostly of CFs. The CM enriched suspension was then plated on 0.1% gelatincoated dishes at a density of 1.5x104 cells per cm2. To inhibit non-muscle cell proliferation,
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100 M 5-bromo-2’-deoxyuridine (BrdU) was added to the medium. After 24 hr, the
medium was replenished with fresh medium without BrdU.
Cell culture conditions. iPS cell generation was optimized through serial experiments
with different culture conditions: three different basal media (DMEM, KnockOut DMEM,
and M199) were supplemented with either ES-screened fetal bovine serum (Thermo
Scientific HyClone, Logan, UT, USA) or KnockOut Serum Replacement. In addition,
different concentrations and combinations of basic fibroblast growth factor (bFGF),
leukemia inhibitory factor (LIF) 2, and valproic acid (VPA), a histone deacetylase inhibitor,3
were tested (Table S2). Results demonstrated that the most efficient medium for
reprogramming was that composed of KnockOut DMEM plus KnockOut Serum
Replacement, 10 ng/ml bFGF, 1000 U/ml LIF, and 0.5 mM VPA (data not shown).
Gene expression profiling for specific genes. Expression of endogenous and
exogenous genes was analyzed by qRT-PCR. Total RNA was extracted from cells at
different time points of reprogramming and differentiation using Trizol (Invitrogen). 1 g of
total RNA was reverse transcribed to cDNA using SuperScript III Kit (Invitrogen) to be
amplified by qRT-PCR. Syber Green PCR master mix (Applied Biosystems) and primers
specific for cardiac markers of differentiation and markers of pluripotency were used
(Tables S4). Each sample was analyzed in triplicate using the Tetrad 2 (Biorad) and
7900HT qRT-PCR (Applied Biosystems).
DNA microarray for whole-genome gene expression. Total RNA was isolated from
mES cells, CM- and CF-derived iPS cells, and CMs with an isolation kit (Invitrogen). RNA
integrity was assessed by electrophoresis. A total of 400 ng RNA was amplified and
labeled using the Illumina TotalPrep RNA Amplification kit (Ambion) following the
manufacturer’s instructions. A total of 1.5 g of labeled cRNA was then prepared for
hybridization to the BeadChip Array MouseWG-6 v2 (Illumina) for whole-genome
expression. Chips were hybridized on a rocking platform in an oven at 58°C for 16 hr. After
hybridization, the chips were washed using protocols outlined in the Illumina manual. Upon
completion of washing, chips were coupled with Cy3 and scanned in the Illumina
BeadArray Reader. Resulting data were log transformed and uploaded into the Genesifter
program (www.genesifter.com). Differences in gene expression were identified using a
minimal threshold value of a five-fold change with no maximal threshold value.
Western blotting analyses. Lysates of undifferentiated and differentiated CM- and CFderived iPS cells were analyzed by Western blotting with antibodies against a-SARC
(Abcam), MLC 2A (Synaptic Systems), MLC 2V (Synaptic Systems), TNNI (Covance), and
NKX2.5 (R&D System).
Immunohistochemistry and immunocytochemistry. BrdU staining for proliferation
assay was performed with a mouse monoclonal antibody (Roche Applied Science); GFP
was detected with a rabbit or mouse anti-GFP (Molecular Probes) antibody; other
antibodies were an anti-a-SARC polyclonal rabbit antibody (Abcam), an anti-c-kit
polyclonal rabbit antibody (Santa Cruz), an anti-Oct4 polyclonal goat antibody (abcam), a
mouse monoclonal anti-Flk-1 (Santa Cruz), an anti-SSEA-1 monoclonal mouse antigen
(Millipore). Nuclei were stained with DAPI.
Flow cytometry and cell sorting. For FACS analysis, cells were fixed in 4% formalin,
labeled with anti-a-SARC (Abcam), anti-c-kit (Becton and Dickinson), anti-Flk-1 (Becton
and Dickinson), anti-Sca1 (Miltenyi Biotec), anti-Isl1 (Developmental Studies Hybridoma
BAnk from University of Iowa, clone 40.2D6 made by Thomas M Jessel – Columbia
Univeristy, New York), anti-1B10 (Sigma), anti- a-SARC (Abcam), anti-MHC (Abcam),
anti-von Willebrand Factor (vWF, Abcam), or anti-smooth muscle actin (SMA, R&D)
antibodies, and detected with a FACS Canto Cell Analyzer (Becton Dickinson). Cells
incubated with TNNI-GFP lentivirus were sorted for GFP fluorescence with a FACSAria II
Cell Sorter (Becton Dickinson).
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