SUPPLEMENTARY MATERIALS AND METHODS
Animals
During surgical procedures, the mice were anesthetized with 2-5% isoflurane in oxygen
for induction and 0.25-4% isoflurane in oxygen for maintenance. The left anterior
descending coronary artery was ligated 2 mm from the left auricular appendage. After
inducing ischemia, the cells (3×105 cells in a 30 µL mixture of cryopreservation medium
and saline) were injected in the peri-infarct borders (2 injections, 10 µL each) and in the
central portion of the ischemic myocardium (1 injection, 10 µL). In control mice, 30 µL of
vehicle (an equal parts mixture of cryopreservation medium and saline) was injected into
the same locations as the cell-treated hearts (10 µL per injection site). Post-operatively
the mice were given the analgesic Keterolac, which was injected subcutaneously at the
dose of 5 mg per kg and administered for 3 days post-operatively.
Echocardiography was also conducted in a blinded fashion to assess heart
function, using a Sequoia C256 system (Acuson, Mountain View, CA) equipped with a
13-MHz linear array transducer (15L8).[1, 2] For the echocardiography, mice were
sedated with an induction dose of 3% isoflurane gas in oxygen for 1 minute and a
maintenance dose of 1.25 to 1.50% isoflurane gas in oxygen. Mice were restrained in
the supine position for the scanning procedure. Two dimensional images were acquired
at the midpapillary muscle level. Short-axis images were used for measurements of EDA
and ESA. Regional left ventricular contractility was calculated as the percentage of
fractional area change (FAC %) = [(EDA − ESA) ÷ EDA] × 100.
Cells
RACs and SACs were isolated from human rectus abdominus skeletal muscle biopsies
using the preplate technique.[3] Tissues were procured from 13, 57, and 70 year-old
male donors. After the tissue was rinsed in saline solutions (50 to 250 mg of tissue per
isolation), all visually apparent and accessible non-muscle tissue (i.e., adipose,
connective and fascia) was trimmed from the biopsy. The tissue was mechanically and
enzymatically dissociated to yield mononuclear human skeletal muscle-derived cells that
were further refined by adhesion rates to tissue culture plastic using the preplate
technique. Cells that adhered rapidly (i.e., RAC fraction) were separated from cells that
adhered slowly (i.e., SAC fraction) for each isolation by transferring the supernatant
containing non-adherent cells in the supernatant to a new flask. The RAC fraction
adhered within 2 hours while the SAC fraction adhered within 2 days. After isolation, the
RAC and SAC populations were independently expanded in culture without additional
preplating to prepare enough cells for intramyocardial injections and cell characterization
using a proprietary growth medium from Cook MyoSite, Inc. The RACs and SACs were
readily expanded ex vivo to yield at least 5×106 cells within 2 weeks after tissue
dissociation for injections and testing.
Following expansion of the cell populations, cells were suspended in serum-free
cryopreservation medium containing dimethyl sulfoxide and cryopreserved as individual
treatment vials for each cell population for each mouse (3.0 × 105 cells in 15 µL of
cryopreservation medium per vial). Cell number and viability was measured with the
Guava Viacount Assay (Guava Technologies, Hayward, CA) on a Guava PCA flow
cytometry system (Guava Technologies). Additional vials were prepared for post-thaw
analysis. For control, vials containing only 15 µL of cryopreservation medium were also
prepared. All aliquots were frozen and stored at −70 to −90 °C until the time of injection.
Shortly before injection, the frozen cell aliquot was thawed and diluted in half with saline.
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Investigators performing the infarction and cellular transplantation procedures were
blinded to the contents of the vials.
Gene expression profile
Total RNA was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA) and was
reverse transcribed to cDNA (Applied Biosystems, Foster City, CA). Gene expression
was measured on a 7900HT Real-Time PCR System (Applied Biosystems) using
Taqman Low Density Arrays (Applied Biosystems) for target genes listed below. All
target genes were normalized to the reference housekeeping gene IPO8
(Hs00183533_m1), which was selected as the endogenous control based on stable
expression across multiple human muscle-derived cell populations using a Human
Endogenous Control Array (Applied Biosystems). Relative quantity of gene expression
was calculated as total amount of RNA based on the comparative ΔCT (i.e., relative
expression level to endogenous control) and ΔΔCT methods (i.e., relative expression
level to the RAC population for each donor).
All primers and probes were purchased from Applied Biosystems.
(gene symbol–assay reference number):
PAX7–Hs00242962_m1
PAX3–Hs00240950_m1
DES–Hs00157258_m1
CDH15–Hs00170504_m1
MYOD1–Hs00159528_m1
MYOG–Hs00231167_m1
MYF5––Hs00271574_m1
MYF6–Hs00231165_m1
MYH2–Hs00430042_m1
VWF-Hs00169795_m1
CNN1-Hs00154543_m1
GPD1-Hs00193386_m1
DLK1-Hs00171584_m1
PTPRC-Hs00236304_m1
COL4A1-Hs01007469_m1
FN1-Hs00415006_m1
FGF2–Hs00266645_m1
PDGFB–Hs00234042_m1
ANGPT1–Hs00181613_m1
VEGFA–Hs00900055_m1
HGF–Hs00300159_m1
TGFB1–Hs00998133_m1
IGF1–Hs01547656_m1
NGF–Hs00171458_m1
BDNF–Hs00538277_m1
GDNF–Hs00181185_m1
NRG1–Hs00247624_m1
Myogenic assays
Myogenic purity analysis was performed on cellular suspensions using a PE-conjugated
CD56 antibody (1:50, BD Pharmingen, San Diego, CA). A PE-conjugated IgG isotype
2
antibody (BD Pharmingen) was used to stain cells for control. Nonviable cells were
excluded from CD56 flow cytometry analysis by 7-Amino Actinomycin D (7-AAD)
staining (Guava Technologies). The Guava PCA flow cytometry system measured the
percentage of cells expressing CD56.
Cell proliferation and survival assays
For cell survival assays, cells were cultured in the live cell imaging system for 72 hours
in culture medium supplemented with propidium iodide (PI, 1:500, Sigma, St. Louis, MO)
to stain dead cells and either hydrogen peroxide (300 µM, Sigma) or tumor necrosis
factor alpha (TNF-α, 10 ng/mL, Sigma) to induce cell death by oxidative and
inflammatory stresses, respectively. Brightfield and fluorescent images were captured
repeatedly at 10-minute intervals in the live cell imaging system, which contains an
incubation chamber for controlling temperature, humidity and CO2 gas levels. The
culture plates remained undisturbed on the live cell imaging system for the entire 72hour period. The percentage of viable cells (i.e., PI exclusion) was measured from
images acquired at 12-hour intervals using imaging software.
Histology and immunohistochemistry
Mice were sacrificed at either 3 days or 2 weeks after cell transplantation. We harvested
the hearts, froze the tissue in 2-methylbutane (Sigma) pre-cooled in liquid nitrogen, and
serially sectioned the hearts at a thickness of 8 µm from the apex to the base.[2] Thawed
tissue sections were fixed for 5 minutes in cold acetone prior to immunostaining, rinsed
and blocked with an appropriate serum.
Sections were stained for fast skeletal myosin heavy chain (MYH) expression.[4]
For MYH staining, the MOM kit (Vector, Burlingame, CA) was used according to
manufacturer’s instructions to reduce any background caused from the use of a
secondary anti-mouse IgG antibody on murine tissue. Mouse monoclonal MYH antibody
(MY-32 clone, Sigma) was used at a concentration of 1:400 and then incubated with an
Alexa Fluor 488 conjugated donkey anti-mouse IgG (1:400, Molecular Probes, Eugene,
OR). To detect host cardiomyocytes, slides were subsequently incubated with goat anticardiac troponin I (cTnI, 1:20,000; Scripps, San Diego, CA) at room temperature for 2
hours followed by the addition of either Alexa Fluor 555 conjugated donkey anti-goat IgG
(1:200, Molecular Probes) or anti-goat IgG biotinylated, then followed by StreptavidinCy5 (Zymed Laboratories, South San Francisco, CA).
To detect proliferating cells within the engraftment regions, tissue was co-stained
for MYH and proliferating cell nuclear antigen (PCNA). The human anti-human PCNA
antibody (PCNA, 1:400, US Biological, Swampscott, MA) was applied at room
temperature for 2 hours followed by the addition of goat anti-human IgG 555 (1:800,
Molecular Probes). Donor cell proliferation index was determined as the number of
PCNA-positive cells that was present in the MYH-positive engraftment region. Nuclei
were stained with 4'-6-diamidino-2-phenylindole (DAPI, 100 ng/mL, Sigma).
Tissue was stained with Masson’s trichrome staining kit according to
manufacturer’s instructions (IMEB, San Marcos, CA). Scar area fraction was defined as
the ratio of scar area to the cardiac LV muscle area and was averaged from 5 sections
per heart. The area of infarct scar and the area of cardiac LV myocardium were
measured using a digital image analyzer (Image J, National Institute of Health,
Bethesda, Maryland).
To measure capillary density, we stained heart muscle sections with the antimouse CD31 (PECAM-1) antibody (BD Pharmingen, San Jose, CA).[1] Capillary density
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within the infarct area was determined from the number of CD31-positive structures per
mm2 using Image J software.
The terminal dUPT nick end-labeling (TUNEL) assay was performed using the
ApopTag® Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Temecula, CA)
according to manufacturer’s instructions.[2] The TUNEL stain was visualized with the
VIP substrate kit (Vector Laboratories, Burlingame, CA). TUNEL-stained tissues were
then counterstained with anti-cTnI (1:20,000, Scripps) and processed with the
Vectastain® Elite ABC Kit (Vector Laboratories) and the DAB (3,3’- diaminobenzidine)
substrate kit (brown stain, Vector) to visualize cTnI-positive myocytes. The apoptotic
index was determined from the total number of apoptotic cardiomyocytes in four high
power fields (HPF, 400x magnification).
We also performed dual label immunostaining for Ki-67 and cTnI expression.[2]
For Ki-67 immunohistochemical staining, deparaffinized sections were immersed in
preheated sodium citrate buffer and were incubated overnight at 4°C with a rat
monoclonal anti-mouse Ki-67 antibody (1:50, Dako, Glostrup, Denmark) that does not
cross-react with the human Ki-67 protein. Positive reaction was visualized with the
Vectastain® Elite ABC Kit (Vector) and the SG substrate kit (blue stain, Vector)
according to manufacturer’s instructions. These sections were then counterstained with
goat anti-cTnI (1:20,000, Scripps) and processed with Vectastain® Elite ABC Kit and the
3-amino-9 ethycarbazole (AEC) substrate kit (red stain, Vector) according to
manufacturer’s instructions. The number of mitotically active endogenous
cardiomyocytes within the peri-infarct region was measured in 8 HPF (400x
magnification).
Supplementary References
1.
2.
3.
4.
Oshima H, Payne TR, Urish KL, Sakai T, Ling Y, Gharaibeh B, et al. (2005).
Differential myocardial infarct repair with muscle stem cells compared to
myoblasts. Mol Ther 12: 1130-1141.
Okada M, Payne TR, Zheng B, Oshima H, Momoi N, Tobita K, et al. (2008).
Myogenic Endothelial Cells Purified from Human Skeletal Muscle improve
Cardiac Function after Transplantation into Infarcted Myocardium. J Am Coll
Cardiol 52: 1869-1880.
Gharaibeh B, Lu A, Tebbets J, Zheng B, Feduska J, Crisan M, et al. (2008).
Isolation of a slowly adhering cell fraction containing stem cells from murine
skeletal muscle by the preplate technique. Nat Protoc 3: 1501-1509.
Payne TR, Oshima H, Sakai T, Ling Y, Gharaibeh B, Cummins J, et al. (2005).
Regeneration of dystrophin-expressing myocytes in the mdx heart by skeletal
muscle stem cells. Gene Ther 12: 1264-1274.
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