Online Appendix for the following September 11 JACC article
TITLE: Recipient Age Determines the Cardiac Functional Improvement Achieved by Skeletal
Myoblast Transplantation
AUTHORS: Chung-Dann Kan, MD, Shu-Hong Li, MSC, Richard D. Weisel, MD, Shun Zhang,
BSC, Ren-Ke Li, MD, PHD
Appendix E1: Supplementary Methods
In Vitro Study
SKMC cultures: SKMCs were isolated from young and aged rats by digestion of skeletal muscle
using 0.2% collagenase type II with 0.25% trypsin. The isolated cells were cultured on laminincoated dishes with Iscove’s modified Dulbecco’s medium (IMDM) containing 20% FBS, 0.1
mmol/l β-mercaptoethanol, 100 IU/ml penicillin, and 100 μg/ml streptomycin, then incubated at
37oC in a 5% CO2 humidified incubator. The medium was changed every 3 days, and sub-culture
was carried out at 60% confluency to prevent myogenic differentiation. Myotube formation was
induced by culturing 80% confluent myoblasts in IMDM medium containing 5% FBS for seven
days.
Cell growth study: SKMCs (5 × 105) from both age groups were seeded into 100-mm laminincoated dishes. The total cell numbers were counted with a cell counter at each passage from 1 to
1
5. Counts obtained at each passage were averaged, and an index of cell proliferation was
calculated for each group as the increase in cell numbers between each passage.
Immunohistochemical staining: Cultured SKMCs were identified using antibodies against
myosin heavy chain and desmin. Fixed cells from both age groups were first treated for 10
minutes with 10% normal horse serum, then incubated with the appropriate antibody (goat
polyclonal anti-myosin heavy chain antibody (Santa Cruz, 1:50 dilution) for myosin heavy chain;
monoclonal anti-desmin antibody (Sigma, 1:2000 dilution) for desmin) in a moist chamber, for 2
hours at room temperature. The cells were next incubated for 30 minutes with biotinylated
secondary anti-mouse or -goat IgG antibodies (Vector Labs Inc.), and HRP-conjugated Ultra
Streptavidin (ID Labs Inc.). Colour development was accomplished with freshly prepared
NovaRed solution (Vector Labs Inc.), and cells were counterstained with Mayer’s hematoxylin.
After dehydration with graded alcohols and clearing with xylene, the samples were mounted in
Permount (Fisher).
In Vivo Study
Myocardial infarction: Young and aged rats were anesthetized with isoflurane, intubated, and
ventilated with positive pressure using a Harvard ventilator. In each rat, the left anterior
descending artery was ligated through a thoractomy, and the incision was closed with 3-0 Ethilon
2
sutures. Rats received antibiotics (150,000 IU/kg) and Buprenorphine hydrocholoride
(0.01mg/kg) intramuscularly after surgery.
Cell preparation: Since the focus of the current study was on the effect of recipient age, rather
than cell donor age, on the efficacy of cell therapy, recipients from both age groups received
highly-myogeneic SKMCs isolated from young donor rats. To track the implanted cells in the
recipient myocardium, they were incubated for 48 h with 10 M bromodeoxyuridine (BrdU,
Sigma) before transplantation. Labeling efficiency was determined by randomly selecting five
dishes for immunohistochemical staining with BrdU according to manufacturer’s protocols. On
the day of cell transplantation, cultured SKMCs were detached from the culture dish using 0.05%
trypsin in PBS solution, centrifuged, and resuspended in 0.05 ml serum free culture media for
implantation. A small proportion of the cells were stained using 0.4% trypan blue solution, in
order to calculate the percentage (of total cells counted) that were damaged. The viability of
SKMCs cultured from young donors was 98 ± 1.6% prior to transplantation.
Cardiac function: Before and one week after coronary ligation (prior to cell transplantation) and
four weeks after cell transplantation, cardiac function was measured using echocardiography.
Each rat was sedated with 2% inhaled isoflurane, and the procedure was performed on the left
hemithorax. Short axis views and an M-mode tracing of the left ventricle (LV) were recorded
from the position which provided the best view of the entire infarct area, with a sweep speed of
200 mm/s. The infarct size was recorded as the maximal length of the akinetic wall seen in enddiastole in the short axis view. Percent fractional area change (%FAC) was calculated as [(LV
3
end-diastolic area) − (LV end-systolic area)]/(LV end-diastolic area). LV diastolic dimension
(LVDd) and LV systolic dimension (LVDs) were measured from the M-mode tracing. LV enddiastolic volume (LVEDV) was calculated as 7.0 × LVDd3/(2.4 + LVDd), LV end-systolic
volume (LVESV) as 7.0 × LVDs3 / (2.4 + LVDs), and percent LV ejection fraction (%EF) as
(LVEDV − LVEDS)/LVEDV ×100. All data were averaged over three measurements.
Cardiac function was also evaluated at 4 weeks after cell transplantation using a
pressure–volume catheter. Each rat was anesthetized, and the heart exposed. A pressure-volume
catheter-tipped pressure transducer (3F, Millar Instruments) was inserted from the apex.
Volumes were measured by conductance, and end-systolic and end-diastolic values were
recorded. Pressures were measured, and the stroke work was calculated as the area within the
pressure–volume loops.
Histology and Immunohistochemistry: After the functional measurements were taken at 4 weeks
after cell transplantation, hearts were arrested by intracoronary perfusion with 5 ml of 20% KCl
solution, perfused with 10% formaldehyde formalin, quickly excised, and immersed in 10%
formaldehyde. The fixed hearts were cut into 2 mm thick slices, embedded in paraffin, and
sectioned for histological studies.
Heart sections were stained with Masson trichrome to assess cell density in the central
infarcted region, and with von Willebrand factor to identify vascular structures in the peripheral
infarcted region (infarct border zone). Five fields from each of five sections per animal were
randomly selected for analysis, and photographed. Cells and blood vessel structures were
quantified from the digital images using Image-J share software (National Institutes of Health).
Cell densities were expressed as the number of nuclei per 0.2 mm2; vascular densities were
4
expressed as the number of blood vessels per 0.2 mm2. To identify the transplanted SKMCs, the
sections were immunostained with monoclonal antibodies against BrdU, and analyzed similarly.
Implanted cell survival in the central infarcted region was expressed as the number of BrdU
positive nuclei per 0.2 mm2.
To evaluate collagen preservation in the central infarcted region, heart sections were
stained with Verheoff’s Van Geison stain. Four fields from each of five sections per animal were
randomly selected for analysis, and photographed. The photographs were subjected to
computerized image analysis to quantify the percentage of each section [(stained area / total scar
area) × 100] labeled with Verheoff’s Van Geison-stained collagen.
Stem Cell Factor (SCF) Protein Levels: SCF levels were measured using a commercially
available ELISA kit (R&D system). Briefly, infarcted myocardium (central and peripheral
infarcted regions combined) from animals in all four groups (N = 4/group) were collected at
seven days after cell or media implantation. The samples were homogenized, and total protein
was extracted from the powdered tissue in lysis buffer (20 mM Tris [pH7.4], 150 mM NaCl, 1
mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM Na pyrophsphate, 1 mM -glycerolphosphate, 1
mM Na3VO4, 1 g/ml leupeptin, 1 g/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride)
for 1 hour on ice. After centrifugation at 10,000 g for 10 min, the supernatant was collected and
protein concentration was determined using a Bio-Rad DC protein assay kit, according to the
manufacturer’s instructions. SCF levels were quantified using an ELISA kit according to the
manufacturer’s instructions, and normalized by pg/mg of total protein.
5