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Online Appendix for the following JACC article
TITLE: Noninvasive Quantification and Optimization of Acute Cell Retention by In
Vivo Positron Emission Tomography After Intramyocardial Cardiac-Derived Stem
Cell Delivery
AUTHORS: John Terrovitis, MD, Riikka Lautamäki, MD, PHD, Michael Bonios, MD,
James Fox, BS, James M. Engles, MS, MBA, Jianhua Yu, BS, Michelle K. Leppo, BS,
Martin G. Pomper, MD, PHD, Richard L. Wahl, MD, Jurgen Seidel, PHD, Benjamin M.
Tsui, PHD, Frank M. Bengel, MD, M. Roselle Abraham, MD, Eduardo Marbán, MD,
PHD
APPENDIX
Animal model. Female WKY rats (n=85 total) underwent left thoracotomy in the 4th
or 5th intercostal space under general anesthesia (isoflurane inhalation, 4% for
induction and 2.5% for maintenance). The heart was exposed and myocardial
infarction was produced by permanent ligation of the left anterior descending
coronary artery, using a silk 5.0mm suture, immediately before cell injection. CDCs
(2 million, suspended in 150μl of PBS) were injected directly into the myocardium, at
two sites into the infarct, using a 28G needle. Subsequently, the chest was closed and
the animals were transported to the PET scanner. For PET imaging, animals were
placed supine, head first in the scanner. Anesthesia was induced by 4% inhalation of
isoflurane for 2 minutes and was maintained by continuous inhalation of 1.5%
isoflurane for the whole duration of the experiment. The animals’ temperature was
monitored and controlled using a heating lamp. Animal care was in accordance to
Johns Hopkins University guidelines.
In vivo imaging. PET images were acquired on a GE VISTA (GE Healthcare,
Piscataway, New Jersey, USA) small animal PET system. The energy window was set
to 400-700 keV window in order to minimize coincident gamma ray background.
Coincidence events were rebinned in the Fourier space and reconstructed using a 2D
OS-EM algorithm, with 4 updates for 18FDG and 16 updates for 13NH3 images. The
reconstructed PET volume is a 175x175x61 (axial direction) matrix, with a voxel size
of 0.39x0.39x0.78mm (axial direction). Images of the rats were obtained either as
dynamic, list mode acquisitions of 60 min reconstructed in 10min frames, or as
consecutive, repeated 10-min static acquisitions.
The radioactivity contained by the labeled cells was always in the range of 1μCi or
less, (reflecting the very low dose used for labeling in order to prevent radiotoxicity).
In order to reliably and accurately determine the activity of the cells before and after
injection (residual) , a static PET acquisition of the syringe containing the labeled
cells (5min) was obtained immediately before cell injection. After cell injection, the
same syringe was imaged again (same imaging parameters), to calculate the net
injected radioactivity (that corresponds to the exact cell number delivered in every
animal). Static 5-minute acquisitions yielded adequate number of counts to allow
statistically reliable quantification. This was considered to be superior to dose
calibrator measurements because the detection system was identical to in vivo
measurements.
After the completion of the 60min 18FDG acquisition, a perfusion PET study using
13
NH3 (ammonia) was performed, with the animal kept at exactly the same position.
37 MBq of 13NH3 were injected intravenously in the tail vein and a 20min static
acquisition was performed. The purpose of this scan was myocardial delineation and
accurate quantification of activity exclusively derived from cells retained in the
myocardium, in contrast to activity from cells that migrated to other organs.
After perfusion scan, 37MBq of [18F]-fluoride was injected in order to facilitate the
co-registration of PET and CT images obtained with the different scanners. After the
completion of the PET acquisitions, the animal was moved into the CT scanner
(restrained on the same bed) and CT images were obtained.
Since a dual modality PET/CT scanner was not available to us, we used a separate
microCT scanner for this purpose. X-ray computed tomography was performed on a
Gamma Medica X-SPECT (Gamma Medica, Northridge, CA, USA), a bi-module
SPECT/CT live small animal imaging system. An X-ray tube of tube voltage 75kVp
was used; 512 projections were acquired over a 360 degree range. The projections
with 1184x1120 isotropic pixels (100μm) were reconstructed into a CT volume of
5123 isotropic voxels (170μm3).
Co-registration of PET and CT images was performed using rigid body
transformation with manually identified bone as landmarks. Co-registered CT volume
was then converted to an attenuation map using a bi-linear transformation scheme. 2D
OS-EM reconstruction with attenuation correction based on the attenuation map was
then performed, for attenuation-corrected quantification results.
Image analysis. A volume of interest (VOI) was drawn to include the bright spot at
the cell injection site or the radioactivity within the syringe, before and after the
injection. VOI activities (of syringes containing cell suspensions and in vivo cardiac
images) were decay corrected; in addition in vivo images were rescaled to correct for
underestimation due to attenuation by using the calculated error obtained from the co-
registered CT studies, thus enabling accurate measurement of the percentage of the
net injected dose (%ID) retained intramyocardially in vivo (%ID=100 * [Activity
within VOI / (Activity in syringe before injection-Activity in syringe after injection)].
Quantification of engraftment by real time PCR. Quantitative PCR was performed
1 hr after cell injection in 6 animals (cells in PBS group) and in 16 at 21 days after
cell injection (8 FG and 8 cells in PBS group) in order to validate the results obtained
by PET but also compare medium term engraftment in these groups. We injected cells
isolated from male donor WK rats into the myocardium of female recipients and
quantified engrafted donor cell numbers, as a function of time, by real-time PCR,
using the SRY gene located on the Y chromosome as target.
The whole heart was weighed, homogenized and genomic DNA was isolated from
aliquots of the homogenate corresponding to 12.5mg of myocardial tissue, using the
DNA Easy minikit (Qiagen), according to the manufacturer’s protocol. The TaqMan®
assay (Applied Biosystems) was used to quantify the number of transplanted cells
with the rat SRY gene as template (forward primer: 5'-GGA GAG AGG CAC AAG
TTG GC-3', reverse primer: 5'-TCC CAG CTG CTT GCT GAT C-3', TaqMan probe:
6FAM CAA CAG AAT CCC AGC ATG CAG AAT TCA G TAMRA, Applied
Biosystems) (4). For absolute quantification of gene copy number, a standard
curve was constructed with samples derived from multiple log dilutions of genomic
DNA isolated from male rat CDCs. All samples were spiked with 50ng of female
genomic DNA to control for any effects this may have on reaction efficiency in the
actual samples. The copy number of the SRY gene at each point of the standard
curve is calculated based on the amount of DNA in each sample and the total mass of
the rat genome per diploid cell (http:www.cbs.dtu.dk/databases/DOGS/index.html).
All samples were tested in triplicates. For each reaction, 50ng of template DNA was
used. Real time PCR was performed in an ABI PRISM 7700 instrument. The result
from each reaction, copies of the SRY gene in 50ng of genomic DNA, was expressed
as the number of engrafted cells/heart, by first calculating the copy number of SRY
gene in the total amount of DNA corresponding to 30mg of myocardium and then
extrapolating to the total weight of each heart (since there is one copy of the SRY
gene per cell).
Histology. In 6 animals (3 of the cells in PBS group and 3 of the FG group) 2 million
cells transduced by a lentiviral vector over-expressing eGFP were injected after the
induction of myocardial infarction. At 3 weeks, the animals were euthanized and the
hearts were harvested and frozen in OCT compound. Sections every 50μ of the infarct
and infarct border zone area (10μ thickness) were prepared and immunocytochemistry
for eGFP and Troponin I were performed, using a chicken anti-eGFP (Abcam,
Cambridge, MA, USA) and a rabbit anti-human Trop I (Santa-Cruz, CA, USA)
primary antibody respectively.