Supplementary Methods
Detailed Methods
Flow cytometry analysis of CDCs
CDCs were characterized by flow cytometry as described3, 4. Briefly, cells were incubated
with FITC, PE or APC-conjugated antibodies against CD45, CD90, CD105, CD117 (ckit), ABCG2, DDR2, alpha-SMA, MDR1, CD140b, and Sca1 (from BD Biosciences,
R&D Systems, eBioscience, or Cedarlane) for 60 minutes. Isotype-identical antibodies
served as negative control. Quantitative analysis was performed using a CyAN flow
cytometer with Summit software (Beckman Coulter).
Mouse myocardial infarction model and cell injection
Acute myocardial infarction was created in male SCID-beige mice (10-12 weeks old), as
described3, 4. Briefly, immediately after ligation of the left anterior descending artery with
9-0 Prolene, hearts were injected at four points in the infarct border zone with a total of
40 µl of one of the following interventions: phosphate-buffered saline (Control, n=11
mice), 1x105 NF donor CDCs (n=19 mice), 1x105 MI CDCs (n=21 mice), or 1x105 HF
CDCs (n=24 mice).
TTC staining
The mouse hearts were excised and sectioned perpendicularly to the long axis into 4
sections (approximately 2 mm thick). The 2-mm sections were then placed in individual
wells of a 24-well cell culture plate, and incubated with 1% 2,3,5-triphenyltetrazolium
chloride (TTC, GFS Organic Chemicals) solution for 15 min at 37°C. Each section was
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visualized with a dissection microscope and photographed with a digital camera (Canon).
The viable area (red) was quantified using NIH ImageJ software.
Heart morphometry
For morphometric analysis, animals in each group were euthanized 3 weeks post-MI
(after cardiac function assessment), and the hearts were harvested and frozen in OCT
compound. Cryo-sections every 100 μm (5 μm thick) were prepared. Masson’s trichrome
staining was performed as per manufacturer’s instructions (HT15 Trichrome Staining
(Masson) Kit; Sigma-Aldrich). Images were acquired with a PathScan Enabler IV slide
scanner (Advanced Imaging Concepts, Princeton, NJ). From the Masson’s trichromestained images, infarct wall thickness was measured with NIH ImageJ software3, 4 and
volumetric analyses of viable mass, scar mass and scar percentage were performed1.
Cardiac function assessment
Mice underwent echocardiography 4 hours (baseline) and 3 weeks post-MI using Vevo
770TM Imaging System (VISUALSONICSTM, Toronto, Canada)3, 4. Hearts were imaged
two-dimensionally in long-axis views at the level of the greatest left ventricular (LV)
diameter. LV ejection fraction (LVEF) was measured with VisualSonics V1.3.8 software
from views taken through the infarcted area. Studies were read and analyzed by an
experienced echocardiographer who was blinded as to study group identity.
Paracrine assay
To compare the production of various growth factors and cytokines, cells were seeded in
6-well culture plates at densities of 1x106 cells/ml in FBS-free Iscove's Modified
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Dulbecco's Medium (IMDM) for 3 days. The supernatants (conditioned media) were
collected and the concentrations of VEGF, bFGF, HGF, IGF-1, SDF-1, MCP-3, IL-6, and
leptin were measured with human-specific ELISA kits (R&D Systems Inc.), according to
the manufacturer's instructions. Matrix metalloproteinase (MMP) activities of CDCconditioned media were measured by InnoZyme™ MMP-2/MMP-9 Activity Fluorogenic
Assay kit (EMD Chemical Group). Additional conditioned media was used for other in
vitro studies (cardiomyocyte survival and tube formation assays).
Western blot analysis of myocardial levels of SDF-1
Animals (n=4 per group) were sacrificed 24 hr after MI and cell injection. Myocardial
tissue samples were lysed in lysis buffer supplemented with proteinase inhibitors cocktail
(Roche) and homogenized with a rotor stator homogenizer. Homogenates were
centrifuged at 12,000 RCF for 15 minutes at 4°C, supernatants were collected and
protein content was quantified by Lowry assay (BioRad). The equivalent of 15 μg of
total protein per lane was loaded onto 12% Precise Protein gels, and then transferred to
PVDF membranes. Membranes were blocked with 5% non-fat milk and incubated
overnight with primary antibodies against SDF-1 (Abcam) and GAPDH (Cell Signaling
Technology). Subsequently, the appropriate horseradish peroxidase-conjugated secondary
antibodies were used, and then the blots were visualized by using SuperSignal West
Femto maximum sensitivity substrate (Thermo Scientific) and exposed to Gel Doc XR
System (BioRad Lab. Inc.).Quantitative analysis was performed by ImageJ software, and
expressions were normalized to GAPDH.
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RT-PCR
We examined the expression of cardiac and extracellular matrix genes (GATA4, MEF2C,
and LAMB1) by qRT-PCR. Briefly, cells were collected by scraping after 3 days of
culture, and total RNA was extracted using RNeasy Micro Kit (Qiagen). The specific
primers and probes for GATA4, MEF2C, and LAMB1 were obtained from Applied
Biosystems, and RNA expression levels were quantified with the ABI Prism 7900HT
Sequence Detection System (Applied Biosystems) according to the manufacturer’s
protocol. Experiments were performed in triplicate, including no template and reverse
transcriptase minus controls. The assays used 50 ng of RNA per sample, and the
housekeeping gene GAPDH was used to normalize all samples.
TUNEL assay
To quantify CDCs’ resistance to oxidative stress in vitro, NF donor, MI, or HF CDCs
were seeded on fibronectin-coated 4-chamber culture slides. After 24 hours of culture,
cells were incubated with 100 µM H2O2 in the medium for another 24 hours, after which
cells were fixed and apoptotic cells detected by terminal deoxynucleotidyl transferase
dUTP nick end labeling (TUNEL) assay using the In Situ Cell Death Detection Kit
(Roche Diagnostics, Mannheim, Germany), according to the manufacturer's instructions.
To explore the anti-apoptotic effects of SDF-1, recombinant human SDF-1 (R&D
Systems; 20 nM) or SDF-1 inhibitor AMD3100 (Sigma-Aldrich; 40 μM) were added into
the culture. Similarly, TUNEL staining was performed on heart sections of animals
sacrificed 24 hrs after MI and cell injections.
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Tube formation assay
The pro-angiogenic effects of CDC-conditioned media were studied by endothelial cell
tube formation assay. Human umbilical vein endothelial cells (HUVECs; from ATCC)
were seeded onto growth factor-reduced MatrigelTM (BD Biosciences) in 96-well plates at
a density of 2x104 cells per well. 100 μL of conditioned media from NF donor, MI, and
HF CDCs were added into the wells. Complete vascular growth media (ATCC; with proangiogenesis growth factors) and basal vascular media (ATCC) were used as positive and
negative controls, respectively. After 6 hours, the wells were imaged with a Nikon white
light microscope. The accumulative tube length was then measured with Image-Pro Plus
software (version 5.1.2, Media Cybernetics Inc., Carlsbad, CA).
NRCM culture and immunocytochemisty
Neonatal rat cardiomyocytes (NRCMs) were derived as described and plated onto
fibronectin-coated chamber slides. 12 hr after seeding, conditioned media from NF donor,
MI, and HF CDCs were added into the culture. Plain IMDM was used as a negative
control. Recombinant rat SDF-1 (Sigma-Aldrich; 20 nM) or SDF-1 inhibitor AMD3100
(Sigma-Aldrich; 40 μM) was added to the cultures to reveal the effects of SDF-1 on
cardiomyocyte survival. After 3 days, NRCMs were fixed and stained for cardiac marker
(alpha sarcomeric actin [αSA]) and DAPI for nuclei. Images were taken with a confocal
microscope (Leica TCS SP5 X). αSA-positive cells per field were counted from the
images.
Histology
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All animals were sacrificed at 3 weeks (after echocardiography study), and excised hearts
were cryo-sectioned (5 µm thickness). Heart cryosections were then fixed with 4% PFA,
blocked/permeabilized with Protein Block Solution (DAKO, Carpinteria, CA) containing
1% saponin (Sigma-Aldrich), and then stained with the following antibodies: mouse antihuman nuclei antigen (HNA; Millipore), rabbit anti-von Willebrand factor (Abcam),
rabbit anti-c-kit (Sigma-Aldrich), rabbit anti-Ki67 (Abcam), rat anti-CD34 (Abcam),
chicken anti-CXCR4 (Sigma-Aldrich), and mouse or rabbit anti-alpha sarcomeric actin
(Sigma-Aldrich or Abcam). FITC, Texas-Red, or Cy5 secondary antibodies were obtained
from Abcam. Images were taken by a Leica TCS SP5 X confocal microscopy system.
Statistical analysis
Results are presented as mean ± SD unless specified otherwise. Comparisons between
any 2 groups were performed using 2-tailed unpaired Student’s t test. Comparisons
among more than 2 groups were performed using one way ANOVA followed by post-hoc
Bonferroni test. Differences were considered statistically significant when p<0.05.
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