Online Appendix for the following
JACC: Cardiovascular Interventions article
TITLE: Reparative Effects of Allogeneic Mesenchymal Precursor Cells Delivered
Transendocardially in Experimental Nonischemic Cardiomyopathy
AUTHORS: Peter J Psaltis, MD, PhD, Angelo Carbone, BSc, Adam J Nelson, BMedSc,
Dennis H Lau, MD, Troy Jantzen, PhD, Jim Manavis, BSc, Kerry Williams, Silviu Itescu,
MD, PhD, Prashanthan Sanders, MD, PhD, FACC, Stan Gronthos, PhD, Andrew CW
Zannettino, PhD, Stephen G Worthley, MD, PhD, FACC
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Expanded Materials and Methods
Preparation of ovine BM MPC
Rambouillet-derived BM MNC were incubated with 20 µg/ml of the mouse IgG
monoclonal antibody, STRO-3 (anti-ovine non-tissue specific alkaline phosphatase)
(Division of Hematology, IMVS, Adelaide, SA), for 1 h at 4°C. After washing, the cells
were incubated with sheep anti-mouse IgG-Dynabeads (four beads per cell) on a rotator
for 1 h at 4°C. Positive cells binding to beads were removed using the MPC-1 magnetic
particle concentrator (Dynal, Oslo, Norway), according to the manufacturer’s
recommended protocol.
Colony efficiency was assayed by incubating the different MNC fractions
(unfractionated, positive, negative) in alpha Modification of Eagle's Medium (αMEM)
(SAFC Biosciences, Inc., Lenexa, Kansas), supplemented with 20% (v/v) fetal calf serum
(FCS; SAFC Biosciences), 2 mM L-glutamine (SAFC Biosciences), 1 mM sodium
pyruvate (SAFC Biosciences), 100 µM L-ascorbate-2-phosphate (WAKO Pure Chemical
Industries Ltd, Osaka, Japan) and penicillin (50 i.u./mL) / streptomycin sulphate (50
µg/mL), at densities of 1x103 to 1x105 per cm2, in triplicate, in six-well plates. On day 14,
plates were washed twice with phosphate buffered saline (PBS), stained with 0.1% (w/v)
toluidine blue (in 1% paraformaldehyde (PFA) w/v in PBS) for at least 1 h and finally
rinsed in tap water. Aggregates of ≥50 cells were scored as CFU-F using an Olympus SZPT dissecting light microscope (Olympus Optical CO Ltd, Tokyo, Japan).
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STRO-3Positive MNC preparations were used to establish primary MPC cultures which
underwent further in vitro culture in αMEM supplemented with 10% FCS (v/v).
Single-color flow cytometric analysis
Passage 4 (P4) cultures of MPC were liberated by trypsin/EDTA digest. Samples of
2x105 cells were washed and resuspended in primary mouse IgG mAb (anti-CD29,
CD44, CD166, CD45, CD14, CD31 or isotype negative control 1B5) for 1 h at 4°C. After
washing, cells were incubated in goat anti-mouse IgG-FITC (20 µg/mL) for 45 min at
4°C, then washed twice and fixed for flow cytometric analysis, using an Epics®-XL-MCL
flow cytometer (Beckman Coulter, Fullerton, CA) and EXPO32 software. A total of
1x104 events were collected per sample as list mode data. Positivity for antibody staining
was defined as the level of fluorescence greater than 99% of what was observed with the
isotype-matched control antibody.
In vitro differentiation of ovine MPC
Passage 4 MPC were also cultured under inductive conditions for mineralization,
adipogenesis and chondrogenesis as detailed previously (1,2). Capacity to form bone
matrix was determined in the presence of 100 µM L-ascorbate-2-phosphate, 10-7 M
dexamethasone and 3 mM inorganic phosphate. After four weeks of induction, cultures
were fixed with 4% PFA and then stained with alizarin red S solution (Sigma Aldrich
Inc., St Louis, MO).
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Adipogenesis was induced in the presence of 0.5 mM methylisobutylmethylxanthine, 0.5
µM hydrocortisone, and 60 µM indomethacin. After three weeks of induction, cultures
were fixed with 4% PFA and stained with oil red O solution (MP Biomedicals, NSW,
Australia).
Chondrogenesis was assayed by culturing cell aggregates in the presence of serumdepleted media supplemented with 10 ng/ml transforming growth factor-β1 (TGF-β1)
over 4-6 weeks. Resulting pellets were fixed in 4% PFA (w/v in PBS), paraffinembedded and histologically stained with 1% alcian blue (w/v) dissolved in 3% glacial
acetic acid (v/v).
General anesthesia and post-procedural care of sheep
All procedures were performed under general anesthesia and mechanical ventilation, with
continuous monitoring of end-tidal CO2, pulse oximetry, electrocardiography and intraarterial blood pressure. Anesthetic induction was achieved by mask inhalation of
isoflurane (4% in 100% oxygen), followed by endotracheal intubation and maintenance
of anesthesia by inhalation of a mixture of isoflurane (2-3%) in 100% oxygen. Animals
were mechanically ventilated with tidal volume of 10 ml/kg, to maintain end tidal CO2 at
approximately 40 mmHg. Post-operative care after invasive surgeries included
subcutaneous administration of 1-2 mg/kg Ketoprofen for analgesia and 0.1 mL/kg
Terramycin (oxytetracycline 200 mg/mL) for antibiotic prophylaxis.
Cardiac magnetic resonance
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Prior to cardiac magnetic resonance (CMR), anesthetized sheep had the wool over their left
parasternal region shaved and the skin thoroughly cleansed with warm water, detergent and
ethanol, to improve electrocardiogram lead adherence. They were then positioned in dorsal
recumbence inside the CMR scanner and mechanical ventilation was commenced to ensure
adequate breath-holds during image acquisition.
Cine images were electrocardiogram-gated and consisted of steady state free precessionbased sequences with the following parameters: repetition time (TR)/echo time (TE)
52.05 ms/1.74 ms; flip angle 70°; matrix 256x150; 25 phases per cardiac cycle; FOV 380
mm with slice thickness 6 mm and inter-slice gap 4 mm through the ventricles. Heart rate
was typically 90-100 bpm (R-R’ interval 667-600 msec) during imaging and therefore the
breath-hold times were between 8 and 12 sec for all animals.
Ventricular ejection fractions and chamber volumes were determined in blinded fashion,
with Argus software (Leonardo workstation, Siemens). End-diastolic and end-systolic
images were chosen as the maximal and minimal, mid-ventricular, cross-sectional areas
in a cinematic display. Short axis endocardial and epicardial borders were traced
manually for each slice in end-diastole and end-systole. These areas were multiplied by
the slice thickness (10 mm) and added together to obtain the EDV and ESV, respectively.
Papillary muscles were included for the volume measurements. Care was taken not to
include atrial slices at end-systole secondary to apical movement of the base of the heart
during LV contraction. Ejection fraction was calculated by the formula: EF (%)=(EDVESV)/EDV x100.
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Transthoracic echocardiography
Transthoracic echocardiography was performed serially during doxorubicin dosing to
monitor changes in LV dimensions and contractile function and to titrate dose number.
Images were obtained once animals had been fully anesthetized and mechanically
ventilated for 30 min and placed in the right lateral decubitus position. Two dimensionalM-mode measurements of LV end-diastolic dimension (EDD) and end-systolic
dimension (ESD) were taken from right parasternal short axis views, just basal to the
insertion of the papillary muscles. For each parameter, measurements from three separate
consecutive cardiac cycles were averaged. Left ventricular fractional shortening (FS) was
derived from the equation: FS (%)=(EDD-ESD)/EDD x100.
Cardiac catheterization and hemodynamic measurements
An 8Fr sheath was used to access the left external jugular vein and a 7.5Fr thermodilution
catheter was passed for obtaining right atrial, right ventricular, pulmonary arterial and
pulmonary capillary wedge pressures which were recorded in end-expiration. Cardiac
outputs were obtained at end-tidal CO2 40 mmHg, from triplicate thermodilution
measurements, using iced 5% dextrose boluses (10 mL).
Left ventricular systolic and end-diastolic pressure measurements were obtained using a
5Fr pigtail catheter passed retrogradely into the LV from the femoral arterial access site.
Electromechanical mapping
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Sheep were secured in dorsal recumbence, femoral arterial access obtained and
intravenous heparin (100 IU/kg) administered. Briefly, a 7Fr NogaStar® mapping catheter
(Biologics Delivery Systems Group, Cordis Corporation) was used to sample endocardial
points from each region of the LV chamber, using well-established criteria for
endocardial-catheter tip contact and stability, as well as point density (3). Points were
acquired with the map displaying local activation times, with fill threshold 15 mm. Local
activation time was determined for each endocardial point, as the time difference between
a selected reference point on the surface electrocardiogram and the steepest, negative
intrinsic deflection in the unipolar intracardiac electrogram. The total time to activate the
LV was therefore measured as the time interval between the endocardial points with
earliest and latest activation.
Local unipolar voltage (UV) amplitudes were recorded as the maximum peak-to-peak
voltage values from the unipolar intracardiac electrograms.
Regional mechanical function was represented by the linear local shortening ratio (LLS),
which was determined by a validated formula that averages the change in distance
between an index point and all its recorded surrounding points from late diastole to
maximum systole. To account for non-uniformity of sampled points, this formula
incorporates a weighting function to emphasize points within a distance of 8-15 mm.
The in-built calculations used for LLS were:
(a) For LLS between two points “i” and “j”:
LLSij=[Ledij -Lesij]/Ledij
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(b) For the average LLS of a given point “p” relative to all neighboring points “j=1…n”:
LLSp=∑j=1…n{LLSpjxWpj[Ledpj]}/∑j=1…n{Wpj[Ledpj]}.
The weight value (Wpj[Ledpj]) is a function of the point density in a defined region, the
volume of the heart and the distances between points at end-diastole.
Immunohistochemistry
Histological sections were dewaxed using xylene and rehydrated with decremental
concentrations of ethanol. Antigen retrieval was performed using citrate buffer (pH 6.0).
Slides were allowed to cool and were washed twice in phosphate buffered saline (PBS,
pH 7.4), before quenching the endogenous peroxidase activity. Non-specific proteins
were blocked using normal horse serum for 20 min. Primary antibodies were applied
overnight at 20°C, consisting of mouse anti-human desmin (0.625 µg/mL) (Dako,
Denmark), mouse anti-human Ki67 (2 µg/mL) (BioGenex Laboratories, San Ramon,
CA), mouse anti-human αSMA (0.03 µg/mL) (Sigma Aldrich Inc., St Louis, MO), and
rabbit anti-GFP (0.5 µg/mL) (InvitrogenTM, Carlsbad, CA). After repeated washing in
PBS, secondary incubations were performed with either biotinylated anti-rabbit or antimouse immunoglobulin (2 µg/mL) (Vector Laboratories Inc., Burlingame, CA) for 1 h at
20°C, followed by further washes and incubation with a streptavidin-conjugated
peroxidase tertiary (Pierce, Rockford, IL) for 1 h at 20°C. Sections were then visualized
using diaminobenzidine tetrahydrochloride, washed, counterstained with hematoxylin,
dehydrated, cleared and mounted.
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All slides were photographed with the The NanoZoomer Digital Pathology System
(Hamamatsu Photonics, Japan) and analyzed blinded to study group.
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Supplementary Figure Legends
Supplementary Figure 1. Flow chart of study protocol
All animals underwent creation of a 3 cm pericardial window (PW) to avoid
inflammatory pericardial effusion, 1-2 weeks prior to commencement of doxorubicin
dosing (for further details see reference (4)). CMR=cardiac magnetic resonance.
IC=intracoronary. IM=intramyocardial. MPC=mesenchymal precursor cells.
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Supplementary Figure 2. NOGA® XP-guided intramyocardial injections
A-C: In a preliminary study, electromechanical navigation was used to guide
multisegmental injections of gadolinium-DTPA into the left ventricular (LV)
myocardium of healthy sheep. Sheep were imaged by CMR 4-6 h later to verify uptake of
contrast into the myocardium. A right lateral view of a representative electrical activation
map demonstrates the distribution of injection sites (brown circular markers) around the
LV (A). Delayed-enhancement CMR (horizontal long axis) confirms intramyocardial
gadolinium-DTPA along the anterior and posterior walls of the LV (B). Corresponding
injection sites are highlighted on the electroanatomical map by white rings (A) and on
CMR by yellow asterisks (C). Figure A-C reproduced from reference (3) with permission
from Springer Science & Business Media. D-E: A cell-injection track is shown in the
mid-myocardium 6 h after transendocardial transplantation of mesenchymal precursor
cells (Hematoxylin & eosin). Note the dense clustering of cells and the presence of minor
petechial hemorrhage (white arrows).
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Supplementary Figure 3. Isolation of ovine BM MPC by STRO-3
Bone marrow was aspirated from Rambouillet sheep to prepare single cell suspensions of
unfractionated (Unf), STRO-3Positive (STRO-3+) and STRO-3Negative (STRO-3-)
mononuclear cells (MNC). A: The recovery of colony forming units-fibroblast (CFU-F)
was enhanced three-fold by STRO-3 enrichment, while STRO-3Negative MNC exhibited
minimal clonogenic potential (n=3 donors; ANOVA P<0.01; *P<0.05 by Tukey posttest). B: Representative photomicrographs are shown of STRO-3-MPC cultured under
inductive osteogenic (alizarin red S stain) (left) and adipogenic (oil red O stain) (right)
conditions, confirming multilineage developmental potential (arrows denote examples of
differentiation). C: Representative flow cytometric histograms demonstrate that ex vivo
cultured ovine STRO-3-MPC express mesenchymal stromal cell-associated markers
(CD29, CD44 and CD166) and lack expression for hematopoietic (CD45, CD14) and
endothelial markers (CD31). No fill=1B5. Black fill=mAb of interest.
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Supplementary Figure 4. Effect of MPC on left ventricular size and function
Relative changes from pre-injection to 8-week follow-up are plotted for left ventricular
ejection fraction (EF) (A,B) and fractional shortening (FS) (C,D) against chamber size in
end-diastole and end-systole. Data are shown as mean±SEM % relative change. *P<0.05
for intergroup comparison. EDD=end-diastolic dimension. EDV=end-diastolic volume.
ESD=end-systolic dimension. ESV=end-systolic volume.
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References for Supplementary File
1.
Psaltis PJ, Paton S, See F, et al. Enrichment for STRO-1 expression enhances the
cardiovascular paracrine activity of human bone marrow-derived mesenchymal
cell populations. J Cell Physiol May;223:530-40.
2.
Gronthos S, Zannettino AC, Hay SJ, et al. Molecular and cellular characterisation
of highly purified stromal stem cells derived from human bone marrow. J Cell Sci
2003;116:1827-35.
3.
Psaltis PJ, Worthley SG. Endoventricular Electromechanical Mapping - The
diagnostic and therapeutic utility of the NOGA® XP Cardiac Navigation System.
J Cardiovasc Trans Research 2009;2:48-62.
4.
Psaltis PJ, Carbone A, Nelson A, et al. An ovine model of toxic, nonischemic
cardiomyopathy--assessment by cardiac magnetic resonance imaging. J Card Fail
2008;14:785-95.
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