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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.