Online Appendix for the following JACC: Cardiovascular Imaging article
TITLE: Molecular Imaging of Bone Marrow Mononuclear Cell Survival and Homing in Murine
Peripheral Artery Disease
AUTHORS: Koen E.A. van der Bogt, Alwine A. Hellingman, Maarten A. Lijkwan, Ernst-Jan
Bos, Margreet R. de Vries, Juliaan R.M. van Rappard, Michael P. Fischbein, Paul H. Quax,
Robert C. Robbins, Jaap F. Hamming, Joseph C. Wu
APPENDIX
SUPPLEMENTAL METHODS
Preparation and characterization of bone marrow mononuclear cells (MNCs). The long
bones were explanted, washed, and flushed with PBS using a 25-gauge needle to collect bone
marrow. After passing through a 70 μm strainer, the isolate was centrifuged at 1200 rpm for 5
minutes, washed, and resuspended into PBS. To acquire the MNC fraction, the bone marrow
isolate was centrifuged for 40 minutes at 1600 rpm using a 14 mL tube with 3 mL Ficoll-Paque
Premium (GE Healthcare, Piscataway, NJ, USA) gradient and 4 mL cell/saline suspension, as
described (5). MNCs were prepared freshly before application.
Characterization of MNCs by flow cytometry. Cells were incubated in 2% FBS/PBS at 4°C
for 30 min with 1 μL of APC-conjugated anti-CD31 (eBioscience), anti-CD45 (BD Biosciences),
and anti-Gr-1 (BD Biosciences), or PE-conjugated anti-CD34 (eBioscience), anti-CD11b (BD
Biosciences), anti-Flk-1, anti-Sca-1 (both eBioscience), and anti-NK1.1 (BD Biosciences), and
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processed through a FACSCalibur system (BD, San Jose, CA, USA) according to the
manufacturer’s protocol.
Ex vivo ELISA for apoptosis on digested muscle. To further explore short-term effect of cell
therapy on paw perfusion, we performed an apoptosis-specific ELISA on the affected
gastrocnemius muscles. The selected muscle was explanted, digested using a stator-rotator
homogenizer, and lysed. ELISA was performed directly on the supernatant to quantify histoneassociated DNA fragments (mono- and oligonucleosomes), marking early apoptotic cells (Cell
Death Detection ELISA, Roche Applied Science, Indianapolis, IN).
Post-mortem immunohistochemistry. Immunohistochemistry was performed to visualize
smooth muscle cell layers of collateral arteries with an antibody against smooth muscle actin.
Furthermore, with an antibody against GFP, GFP+-MNCs were traced in the ischemic skeletal
muscle. Five µm-thick paraffin-embedded sections of skeletal muscle fixed with 4%
formaldehyde were used. These were re-hydrated and endogenous peroxidase activity was
blocked for 20 minutes in methanol containing 0.3% hydrogen peroxide. Skeletal muscle slides
were stained with monoclonal anti-α smooth muscle actin (mouse anti-human, DAKO, dilution
1:800). Antigen retrieval was not necessary and sections were incubated overnight with primary
antibody. Rabbit anti-mouse HRP (DAKO, dilution 1:300) was used as a secondary antibody.
For the negative control, an isotype control instead of the primary antibody was used. The signal
was detected using NovaRED substrate kit (Vector laboratories) and sections were
counterstained with hematoxylin. Stainings were quantified from randomly photographed
sections using image analysis (ImageJ). For tracing of GFP+-MNCs, adductor muscle slides
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were incubated with anti-GFP (rabbit anti-mouse, Invitrogen, dilution 1:4000) without antigen
retrieval. After overnight incubation, labeling was followed by a biotin-conjugated secondary
antibody (donkey anti-rabbit, dilution 1:300). As a positive control, a slide of GFP+ cardiac
muscle tissue was used.
Statistical analysis. Statistics were calculated using SPSS 16.0 (SPSS Inc., Chicago, IL, USA).
Descriptive statistics included mean and standard error. Comparison between groups was
performed using a 1-way between groups ANOVA, or 1-way repeated measures ANOVA when
compared over time. Significance was assumed according to the Bonferroni-Holm’s procedure
when more than 2 groups were compared. P-values were considered statistically significant if
P<0.05. Correlation was tested using a simple regression model according to standard Excel
software.
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SUPPLEMENTAL FIGURES
Supplemental Figure 1: Quantification of short-term apoptotic rates in gastrocnemius
muscles of MNC treated animals. ELISA for histone-associated DNA fragments in mono- and
oligonucleosomes of digested gastrocnemius muscles revealed an almost 3-fold increase in
apoptosis following left femoral artery ligation and PBS treatment as compared to the healthy
contralateral muscle as well as compared to MNC treated animals (* indicates P<0.05).
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Supplemental Figure 2: Ex vivo confirmation of in vivo MNC distribution patterns. (A)
Graphic in vivo representation of MNC retention in liver, spleen, and bone marrow. Removal of
the skin surprisingly leads to a remarkable reduction of signal intensity from the scarred area. (B)
After explantation of various organs and ex vivo BLI, the signal that was previously observed
from the injured area during in vivo experiments appeared to be a cumulative signal from MNC
retention from skin, subcutaneous tissue, and muscle. (C) To confirm the BLI signals from the
bone marrow, the marrow was flushed from the bone and processed through flow cytometry for
GFP+ donor cells. The flow cytometry results correlated to the BLI results as the recipient bone
marrow indeed contained Fluc+-GFP+ donor MNCs. Scale bars represent BLI signal in
photons/s/cm2/sr.
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Supplemental Figure 3: Influence on vascular occlusion method on cell survival and paw
perfusion. (A) A faster endogenous restoration in paw perfusion is observed in the suture
ligation group as compared to the electro-coagulation group (p=NS, n=4/group). (B) Suture
ligation seems to provide a slightly more favorable environment for transplanted MNC survival,
as non-significant increased BLI signals are observed on day 3 as compared to the electrocoagulation group (p=NS, n=4/group).
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