Methods
Reagents
Dulbecco’s modified Eagle medium (DMEM), fetal-bovine serum, penicillin,
streptomycin, trypsin, sodium pyruvate, and phosphate-buffered saline (PBS) were
obtained from Gibco BRL (Grand Island, NY). DAPI (4,6-diamino-2-phenylindole) was
obtained from Invitrogen. Superparamagnetic iron oxide (SPIO) particles (average size:
0.9 μm) were obtained from Bangs Laboratories (Fishers, IN). The particles contained
magnetite cores encapsulated with styrene/divinyl benzene and coated with Dragon-green
fluorophore.
Induction of myocardial ischemia/reflow injury in vivo
Male Fisher-344 rats (200-250 g) were used. Rats were randomly divided into four
groups of 8 animals each: (1) MI group (serum-free medium-treated); (2) Ox group (MI
with hyperbaric oxygen treatment); (3) MSC (MI with MSC transplantation); (4)
MSC+Ox (MI with MSC and HBO treatment). Rats were anaesthetized with ketamine
(50 mg/kg, i.p) and xylazine (5 mg/kg, i.p) and maintained under anesthesia using
isofluorane (1.5-2.0%) mixed with air. During the surgical procedure, the absence of the
pedal reflex was used as an indication that a surgical plane of anesthesia has been
maintained. Myocardial infarction (MI) was created by ligating the left-anteriordescending (LAD) coronary artery for 1 h. After 1 h of ischemia, the ligation was
released, flow was restored (reperfusion), and the chest cavity was closed. After ligation
of the LAD coronary artery, successful infarction was confirmed, for all groups, by an ST
elevation on the electrocardiograms. After reinstallation of spontaneous respiration,
animals were extubated and allowed to recover from anesthesia. No mortality was
observed in any of the groups at the end of the study. All the procedures were performed
with the approval of the Institutional Animal Care and Use Committee of The Ohio State
University and conformed to the Guide for the Care and Use of Laboratory Animals (NIH
Publication No. 86-23).
Isolation, culture and characterization of MSCs
Fisher-344 rats (200-250 g) were euthanized by CO2 inhalation. After shaving the hind
limbs and wiping with 70% ethanol, an incision was made around the perimeter of the
hind legs using sterile scissors, and the skin was detached from the muscles. The muscles
and connective tissue from both tibia and the femur were removed, and the ends of the
bones were cut, using a sterile bone cutter, to expose the bone marrow. Bones were
flushed with DMEM media using a sterile 22 G needle connected to a 10-ml syringe in a
100-mm culture dish. After collecting the bone marrow in the dish, the suspension was
aspirated, at least three times, using a 22 G needle. The uniformly-suspend cells were
transferred to a 50-ml centrifuge tube. The cells were centrifuged at 200 g for 5 minutes
at room temperature. After aspirating and discarding the supernatant, the cell pellet was
re-suspended in growth media and transferred to a T75 flask. The cells were cultured
using DMEM with GlutaMax (4500 mg/l glucose) supplemented with 10% heatinactivated fetal bovine serum, 100 U/ml penicillin, and 100-µg/ml streptomycin. The
total growth media in the flask was 20 ml. The T-75 flasks were incubated at 37ºC, with a
mixture of air and 5% CO2 in a humidified chamber. After 3 days of culture, 10 ml of
pre-warmed fresh growth media was added to each flask. The culture media was changed
every 2-3 days until scattered colonies of spindle-shaped cells were visualized by
microscopy. Once the cells reached to passage 3, they were detached using accutase and
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the purity of the cells was determined by flow cytometry (Supplemental Figure 1) using
CD29, CD44 as positive markers (≥ 85%), and CD14 and/or CD45 as negative markers
(≤ 10%).
Characterization of MSC phenotype
MSCs of the second and third passages were analyzed using flow cytometry to
characterize the phenotype of the cells. The cells (1×106) were incubated with antibody or
isotype control for 45 min on ice. Cell aliquots were incubated with fluorescein
isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal antibody against
CD44 (Chemicon), CD14 (Chemicon), CD29 (integrin β1; Biolegend), and CD45 (BD
Pharmingen). Aliquots of cells were also stained with respective isotype controls, such as
IgG conjugated to FITC or PE. Flow data were acquired using a FACS Calibur (BD) and
analyzed using CellQuest software (BD).
Western-blot analysis
Molecular signaling cascades involved after ischemia/reperfusion injury were determined
by Western-blot analysis in cardiac tissues homogenates. Western blots for NOS3
signaling were performed with the tissue homogenates prepared from the anterior wall of
the left ventricles from rats of all four groups. At four weeks after treatment rats in MI,
Ox, MSC and MSC+Ox were anesthetized and sacrificed. Control rats (non I/R/Sham,
n=4) were also used. The hearts were rapidly excised, rinsed in ice-cold PBS (pH 7.4)
containing 500 U/ml heparin, to remove red-blood cells and clots, frozen in liquid
nitrogen and stored at -80oC until analysis. Heart tissues were homogenized and the
tissue homogenate was incubated for 60 min on ice, followed by microcentrifuging at
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10,000 g for 15 min at 4oC. Aliquots of 75 µg of protein from each sample were boiled in
Laemmli buffer (Bio-Rad Laboratories, Hercules, CA) containing 1% 2-mercaptoethanol
for 5 min. The protein was separated by SDS-PAGE, transferred to polyvinylidene
difluoride (PVDF) membrane, and probed with primary antibodies for HIF-1α (Novus
Littleton, CO), NOS3 and phospho-NOS3 (ser-495/1177) (Cell Signaling; Beverly, MA).
Data were expressed as percent of the expression in the control group.
Reverse transcriptase PCR
Snap-frozen cardiac tissue was homogenized using mortar and pestle (cooled to
temperature in a liquid nitrogen bath). TRIZOL (1 ml/50 mg) was mixed with the sample
and the tissue was transferred to the pestle for grinding, until only a layer of very fine
dust remained. The powder was transferred to 15-ml falcon tubes filled with TRIZOL
solution using an RNase-free spatula. The mixture was then mixed thoroughly using a
vortex. After homogenization, the solutions were transferred to Eppendorf tubes and left
in TRIZOL at room temp for 5 min. Afterwards, 200 μl of chloroform per 1 ml of
TRIZOL was added and mixed thoroughly. The tissues were again left at room
temperature for 10 min, and then centrifuged at 16,000g for 15 minutes at 4°C. The upper
aqueous and colorless layer was transferred to a fresh Eppendorf tube filled with 75 μl of
LiCl (lithium chloride). One ml of chilled ethanol was then added and the solution was
kept at -20°C for 2-3 hours. The Eppendorf tube was centrifuged at maximum speed
(>16,000g) for 15 minutes at 4°C. The supernatant was discarded and 250 μl of 70%
ethanol was added.
The Eppendorf tube was then kept at room temperature for 2
minutes. Afterwards, the tube was again centrifuged at 7,500 RPM for 5 minutes at 4°C,
after which the supernatant was discarded and the pellet was resuspended in RNA grade
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water until completely dissolved. Single strand c-DNA was synthesized for incubation
using sense and anti-sense primers and a revert aid TM h minus first strand cDNA
synthesis kit (Fermentas, MD). The resulting cDNA was diluted 1:10 before proceeding
with the PCR reaction. PCR was conducted in a Mastercycler gradient (Brinkmann
Instruments). Each PCR reaction used 50-µl cDNA, 2.5 U Taq polymerase (Eppendorf
Scientific), 0.2 mM dNTPs and 0.5 µM primer. PCR products were resolved on 2%
agarose gel containing 0.01% (v/v) ethidium bromide and visualized using an UV
illuminator.
RT-PCR primers:Name Sequence
β-actin forward 5′-CACCCGCGAGTACAACCTTC-3′
β-actin reverse 5′-CCCATACCCACCATCACACC-3′
NOS3 forward 5′-ACGTGGAGATCACCGAGCTC-3′
NOS3 reverse 5′-GTGCTCATGTACCAGCCACTG-3′
Immunoprecipitation
Proteins were isolated from cardiac tissue samples using a Total Protein Extraction kit
from Millipore according to the manufacturer’s protocol. After cell lysis, the extracted
DNA was fragmented by passing the lysed suspension through a needle attached to a 1ml syringe a total of 5-10 times. The lysates were pre-cleared to help reduce non-specific
binding of proteins to agarose or sepharose beads. Pre-clearing with an irrelevant
antibody or serum will remove proteins that bind immunoglobulins non-specifically. In
our experiment, 50 μl of GAPDH antibody of the same species (rabbit) was added for 3 h
to pre-clear the lysate. NOS3 antibody was then added to the pre-cleared lysate. The
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lysate was incubated on ice with the antibodies overnight and 100 μl of bead slurry was
added to the solution. The bead and lysate mix was incubated for 4 h at 4oC with gentle
agitation. The mixture was spun at 14,000xg for 10 minutes at 4°C. The bead pellet was
discarded and the supernatant was saved for immunoprecipitation. In order to increase the
yield, The beads were then washed 1 or 2 more times in lysis buffer and the supernatants
were collected.
Figure Legends
Supplemental Figure 1. Characterization of MSCs. FACS analysis was performed in
isolated MSCs at passage 3 to assess the purity. Most of the MSCs were positive for
CD29 & CD44 (Positive Markers) and negative for CD14 & CD45 (negative markers).
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