Supporting Information
Quantitative Proteomics Reveals Differential Regulation of Protein Expression in Recipient
Myocardium after Trilineage Cardiovascular Cell Transplantation
Ying-Hua Chang1,2, Lei Ye3,4,, Wenxuan Cai1,5, Yoonkyu Lee2, Huseyin Guner2, Youngsook Lee1,5,6,
Timothy J. Kamp1,5,6,7, Jianyi Zhang3,4,8,9,*, Ying Ge 1,2,5,6,10,*
1
Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, USA;
2
Human Proteomics Program, University of Wisconsin-Madison, Madison, WI, USA;
3
Division of Cardiology, Department of Medicine, University of Minnesota, Minneapolis, MN, USA;
4
Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA;
5
Molecular and Cellular Pharmacology Training Program; University of Wisconsin-Madison, Madison,
WI, USA;
6
Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison, Madison, WI, USA;
7
Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA;
8
Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA;
9
Department of Electrical and Computer Engineering, University of Minnesota, Minneapolis, MN, USA;
10
Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA

Current address: National Heart Research Institute Singapore, National Heart Centre Singapore.
Running title: Proteomics Reveals Mechanisms after Cell Therapy
*Address for Correspondence: Ying Ge, PhD. University of Wisconsin-Madison, 1300 University
Ave., SMI 130, Madison, WI 53706,Tel: 608-263-9212, Fax: 608-265-5512.E-mail: ge2@wisc.edu.
Jianyi (Jay) Zhang, MD., PhD. ,University of Minnesota, 420 Delaware Street SE,Minneapolis, MN
55455, Tel: 612-625-7403,Fax: 612-626-4411.E-mail: zhang047@umn.edu
1
Supplemental Methods
Generation of cells
The hiPSC-CMs, -SMCs, and -ECs used in this study were developed from GRiPS, an iPSC line created in
house.1 The iPSC line was reprogrammed from human male, neonatal, dermal fibroblasts by transfecting
cells with the Sendai virus (GRiPS) coding for OCT4, SOX2, KLF4, and C-MYC before altering it to
constitutively express green fluorescence protein. A hiPSC growth medium was used to culture cells, along
with irradiated mouse embryonic fibroblasts, which were passaged every 6-7 days.2 hiPSCs were
differentiated into CMs, SMCs and ECs according to the previously described method. 3, 4, 5 hiPSC-SMCs
were characterized according to the expression of α-smooth muscle actin (SMA), SM22, and calponin
whereas hiPSC-ECs were characterized based on the expression of CD31, CD144, and vWF-8.6
Swine heart model of ischemia reperfusion (I/R) injury and treatment
This study was performed in female Yorkshire swine (~13 kg, Manthei hog farm, Elk River, MN). Those
from the Sham group underwent the necessary surgical procedures for I/R injury excluding the ligation step
and were allowed to recover without the experimental treatments. The swine model of myocardial I/R injury
was previously described.1 The swine were anesthetized with inhalation of 2% isoflurane, intubated, and
then ventilated with a respirator along with supplemental oxygen. The body temperature, ECG, blood
pressure, and arterial oxygen saturation of the specimens were monitored throughout surgery. A left
thoracotomy was performed, and the roots of the 1st and 2nd diagonal coronary arteries from the left
anterior descending coronary artery (LAD) were occluded for 60 min before being reperfused. If ventricular
fibrillation occurred, electrical defibrillation was the immediate response. Drugs used during the open-chest
surgery included Lidocaine (2 mg/kg iv bolus before ligation followed by 0.05 mg/(kg•min) iv for 70 min)
and Amoidorane (5 mg/kg iv bolus before ligation followed by 0.04 mg/(kg•min) iv for 70 min). The chest
was then closed in layers and animals were allowed to recover. Standard post-operative care, including
analgesia, was administrated until animals ate normally and became active. Animals in the CM+EC+SMC
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group were treated by injecting 2 million hiPSC-CMs, 2 million hiPSC-ECs, and 2 million hiPSC-SMCs
(6 million cells total) directly into the injured myocardium. The immunosuppression drug, cyclosporine,
was administered to animals to reduce the likelihood of immune rejection in animals treated with the hiPSCderived cells at the dosage of 15 mg/kg. Assessment of CD11b+ cell density suggested that the immune
response was elevated and delayed in the animals received trilineage cardiac cell transplantation
(CM+EC+SMC). However, the increase was not significant.1 Tissue samples from the periscar border
zone in the MI and CM+EC+SMC groups, as well as the corresponding tissue in the Sham group, were
harvested for further analysis.
Tissue sample preparation for mass spectrometry analyses
The sample size of Sham, MI, and CM+EC+SMC groups were 3, 4, and 3, respectively. Approximately,
30-50 mg of tissue was used for protein extraction. All of the protein extraction procedures were
carried out in a cold room (4 °C). The frozen pig heart tissue samples were cut into small pieces
(approximate 2 mm3) and immediately washed twice in cold PBS buffer (10-folds volume of tissue mass)
containing a protease inhibitor cocktail (Roche, Switzerland). The washed tissue pieces were transferred
into HEPES buffer (5-folds volume of tissue mass, 0.25 mM sucrose, 25 mM HEPES at pH 7.4, 50 mM
NaF, 0.25 mM Na3VO4, 0.25 mM PMSF, 2.5 mM EDTA, and 1 mg/mL protease inhibitor cocktail). The
tissue samples were homogenized 5-6 times using a Polytron electric homogenizer (Model PRO200, PRO
Scientific Inc., Oxford, CT, USA) for 5-7 s on ice. The homogenate was centrifuged at 120,000 g for 30
min at 4 °C, and the supernatant was removed to deplete most of blood contamination. The remaining pellet
was extracted with the same HEPES buffer with 0.2% mass spectrometry compatible surfactant, MaSDeS,7
(3 folds volume of tissue mass) and then centrifuged at 16,000 g at 4 °C for 30 min. The protein
concentration of each sample was determined using the Bradford assay (Bio-Rad, Hercules, CA) in
accordance with the manufacturer’s protocol. Each protein sample (10 μg) was reduced with 20 mM
dithiothreitol at 65 °C for 1 hour and alkylated with 25 mM iodoacetamide at room temperature for 1 hour.
The samples were all adjusted to a pH of 8. Modified trypsin (1:50 (w/w) (trypsin/protein) was added to
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each sample, which were then incubated at 37 °C for 20 hours to allow for complete digestion. 5% of
acetonitrile with 1% formic acid was added into each sample to stop the reaction. The samples were
centrifuged at 16,000 g at 4 °C for 1 hour before the supernatants were obtained for further analysis by MS.
MS analyses
MS analyses were performed using a nanoflow UPLC (Waters LC nanoACQUITY, MA, USA) coupled
with a Q Exactive mass spectrometer (Thermo Scientific, Bremen, Germany) with a nanoelectrospray
ion source (Thermo Electron, San Jose, CA). A 180 µm × 20 mm trap column (Symmetry C18) and a 75
µm × 150 mm Waters C18 analytical column (1.7 μm particle BEH) with mobile phases A (0.1 % formic
acid in water) and B (0.1 % formic acid in acetonitrile) were used in the analyses. The pump flow rate was
set to 0.35 μL/min as 1 μg of peptides were contained in the trap column for 10 min. A linear gradient of
5-35%B was set for the first 130 min before a rapid increase to 95% B was applied over the following 20
min. A column temperature of 28 °C was maintained for the duration of the experiment. Electrospray
voltage was set at 1.9 kV. The Orbitrap mass analyzer completed full MS scans for the m/z 300–2000 range
with a 70,000 (at m/z 200) mass resolution. The target value of the automatic gain control (AGC) was
1.00E+06 for a 100 ms maximum injection time. The 15 most intense ions with charge states ≥ 2 were
fragmented in the HCD collision cell using a normalized collision energy of 30%. Tandem MS (MS/MS)
spectra were acquired in the Orbitrap mass analyzer at a mass resolution of 17,500 (at m/z 200) (AGC target
1.00E+05, 30 ms maximum injection time). The ion selection threshold was 3,300 for MS/MS. The repeat
sequencing of peptides was minimized by the dynamic exclusion of sequenced peptides for 20 s.
Protein identification and quantification
All MS/MS samples were analyzed using the SEQUEST-based Proteome Discoverer (Thermo Scientific;
version 1.4). Due to the incompletion of the swine proteome database, we used a combination of reviewed
and un-reviewed Sus scrofa sequences from the Uniprot-Swissprot database (released July, 2014). By
removing all proteins with redundant sequences, 31,775 entries remained in the final database to consider.
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Search settings allowed for two missed cleavages with 10 ppm and 0.02 Da mass tolerances for the
precursor and fragment ions, respectively. The carbamidomethyl of cysteine was set as the fixed
modification, and the variable modifications were deamidated asparagine and glutamine as well as oxidated
methionine. A decoy database was used to perform another search, which filtered using a 1% false discovery
rate (FDR). Peptides with high confidence, rank 1, and delta Cn < 0.1 were selected. The search setting that
allowed distinct proteins was enabled. The Proteome Discoverer plugin, InforSense, was used for collecting
Gene Ontology (GO) data while the AmiGo database was used to obtain additional information regarding
other relevant biological processes for the identified proteins. During quantification, Proteome Discoverer
was used to calculate the area under the curve of each peptide and the average of the three most abundant
distinct peptides, which represented the protein intensity. All protein intensities were presented in Log10
scale. After conversion, normalization was calculated by dividing the intensity of each protein in the sample
by the median protein intensity for the entire sample. The minimum protein intensity of each run minus 3
times the S.D. value was the undetectable protein intensity.
Bioinformatic analysis
The MultiExperiment Viewer (MeV v4.9) was used to perform the hierarchical cluster and heat map
analyses (HCA), and the proteins were then clustered according to their Pearson correlation coefficients.
All values of normalized spectral counts (SCs) from the MS data were transformed to Z scores.8 The Z
scores were calculated as Z = (X - μx)/σx, where X is the individual normalized SCs, μx is the mean of the
normalized SCs for the identified protein across the groups (Sham, MI, CM+EC+SMC), and σx is the
standard deviation associated with μx.8 The ToPPFun9 software was used to enrich certain subcellular
locations and biological processes. The STRING10 and NetworkAnalyst11 databases were used to describe
interactomes. Any GO biological processes and networks with a FDR < 0.01 were considered to be
significant.
Western blot
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Equal amounts of protein (50 µg) from the different samples were loaded and resolved on 12.5% SDSPAGE gels. A Fast Semi-Dry Blotter (Fisher Scientific, Waltham, MA, USA) was used in accordance with
the manufacturer's protocol to transfer the proteins to PVDF membranes. The membranes were then placed
in protein-free blocking buffer (Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature and
incubated with primary antibodies overnight at 4 °C. The membranes were washed with Tris-buffered saline
containing 0.1% tween (TBST) five times before incubation with horseradish peroxidase-conjugated
secondary antibodies for 50 min at room temperature. Before the membranes were developed using
enhanced chemiluminescence detection, they were washed with TBST an additional five times. ImageJ was
used to quantify the intensities of the bands.
Statistical analysis
Statistical analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, IL), and a p value under 0.05
was required for the two-tailed tests to be considered statistically significant. All experiments were
performed in biological triplicates, and the results are shown as the mean ± standard error. All of the
different groups were analyzed using nonparametric tests, including the Kruskal-Wallis test for the
proteomic experiments and the Mann-Whitney U test for the Western blot experiments.
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Supplemental Figures
Supplemental Figure 1. Schematic depicting the global protein profiling of myocardial tissue from the
Sham, MI, and CM+EC+SMC groups. (A) Image showing the treatment of Sham, MI, and
CM+EC+SMC swine. (B) Workflow for the label-free quantitative proteomics approach used for the
identification and quantification of differentially regulated proteins.
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Supplemental Figure 2. The number of identified proteins in each group presented with mean ± standard.
After combining the proteins identified in all the replicates, the total non-redundant protein IDs were 2997,
3154, and 2946 in the Sham, MI, and CM+EC+SMC groups, respectively. The quantitative protein
identification method is highly reproducible across biological replicates and unbiased for any experimental
group.
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Supplemental Figure 3. Quality assessment of the quantitative proteomic analyses. Equal amounts of
proteins in each sample were analyzed by MS. All of the given protein intensities are presented in Log10
scale. After conversion, the intensity of each protein in the sample was divided by the median protein
intensity for the entire sample for normalization. (A) Protein intensity distribution for each sample. (B) The
total protein intensity in each group and (C) the distribution of the coefficients of variation (CVs) for the
quantified protein intensities across the biological replicates. A smaller CV indicates the protein intensities
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across biological replicates were relatively consistent, thus, increasing confidence in the protein
quantification.
Supplemental Figure 4. Representative proteins with no significant differences among different groups.
The proteins were identified in all of groups, including many mitochondrial proteins, cytoskeleton proteins,
housekeeping proteins, and several growth factors. These included transforming growth factor (TGF),
EIF4A, insulin-like growth factor (IGF), elongation factor 1 (EEF1B), STAT3, histone2A, actin, tubulin,
and GAPDH.
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Supplemental Figure 5. Subcellular locations of significantly altered proteins in three different
categories.
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Supplemental Figure 6. Biological processes of significantly altered proteins in three different categories.
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Supplemental Figure 7. Heat map of Category 1 (the proteins that were altered in response to trilineage
cell treatment but showed no difference in expression between the Sham and MI groups) with enriched
function analysis.
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Supplemental Figure 8. Heat map of Category 2 (the proteins with altered expression following MI and
reversed expression after trilineage cell transplantation) with enriched function analysis.
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Supplemental Figure 9. The functions and mechanisms of the proteins that exhibited altered expression
after MI that could not be reversed by cell transplantation (Category 3). (A) Heat map of Category 3 with
enriched functional analysis. (B) Interactome analyses. Proteins with similar functions are present in the
same interactome. For instance, the proteins that regulate catabolic process are connected together and
linked to proteins that regulated extracellular matrix organization. The proteins up-regulated in MI are
indicated in red. The proteins in blue were down-regulated after MI.
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