SUPPLEMENTAL MATERIAL
Detailed Methods
Ethics statement
All animal procedures were conducted in accordance with humane animal care standards outlined in the NIH Guide for the Care and Use of Experimental Animals and were approved by the Johns Hopkins University Animal Care and Use Committee.
Cardiosphere derived cell isolation
Hearts were explanted under sterile conditions, myocardial tissue was cut into pieces (explants) and cultured in complete explant medium or CEM (Iscove’s modified Dulbecco medium/IMDM containing 20% heat-inactivated fetal calf serum, penicillin G, streptomycin, L-glutamine and 0.1mmol/L 2-mercaptoethanol) on fibronectin-coated dishes at 37°C and 5% CO2. After a period ranging from 4-7 days, a layer of fibroblast-like cells arises from adherent explants over which small, round, phase-bright cells migrate. These cells were collected and seeded at ~0.5- 2 x105 cells/mL, on poly-D-lysine-coated plates in cardiosphere growth medium or CGM (35% IMDM/65% DMEM-Ham's F-12 (GIBCO), 2% B27 (GIBCO), 0.1 mmol/L 2-mercaptoethanol(GIBCO), 10 ng/ml EGF(RD Systems), 20 ng/ml bFGF(PeproTech), 4 ng/ml Cardiotrophin-1 (RD Systems), 1 U/ml thrombin (Sigma), 100 U/ml penicillin G, 100 U/ml streptomycin, 2 mmol/l L-glutamine), to yield spherical cell clusters called cardiospheres, which grow in suspension. This process was repeated twice at 5-7d intervals from the same explant. Cardiospheres were harvested and plated on fibronectin-coated flasks in CEM to generate monolayers that we call cardiosphere-derived cells or CDCs. CDCs obtained from the second harvest were passaged twice and frozen for future use. For in vivo and in vitro experiments, these CDCs were thawed and expanded for 2 more passages prior to use.
In vitro FDG labeling.
Tritiated 3H[FDG], a beta emitter was used to measure FDG uptake in vitro. rCDCs (105) were plated in 6 well plates, incubated in glucose-free medium (No Glucose Dulbecco’s Modified Eagle Medium, Invitrogen) for 1 hr and then exposed to 0.2 or 2µCi/ml of 3H-FDG with or without insulin (0.1 U/ml) for 30 or 60 min. Retention of 3H-FDG by CDCs was examined at 15min (initial activity), 1hr and 4hr after radio-labeling. The rCDCs were then trypsinized (400 µL of 0.25% trypsin), washed twice, lysed with 500 µL of NaOH(0.33 mol/L) containing 1% sodium dodecylsulfate and transferred into scintillation vials; 10ml of scintillationfluid (Formula 989; Perkin-Elmer, Inc.) was added and counts were measured in a beta-counter. Two independent experiments were conducted and each condition was tested in triplicate.
Assessment of radiotoxicity
One thousand rCDCs were plated in a 96 well plate, and incubated with 6 doses of 18FDG (0.2, 2.0, 10.0, 25.0, 250.0 and 500 μCi/ml of glucose-free media) for 30min, following which, media was changed to regular growth media (Iscove’s Modified Dubelcco’s Medium, Invitrogen containing 20% foetal bovine serum, Hyclone). The WST assay was performed daily for 6 days per manufacturer’s protocol: 10μl of WST-8 was added to each well for 2 hours and absorbance was measured at 450nm (Spectramax M2, Molecular Devices, Sunnyvale, CA, USA). A standard curve (absorbance versus cell number) was created by performing the assay on known numbers of cells.
PET/CT imaging
PET images were acquired on a GE Healthcare Vista small animal PET system. The system has two detector rings, each with 18 detector modules. The detector modules have LYSO and GSO crystal layers which form a dual layer phoswich. The phoswich elements (1.45*1.45*7mm for LYSO and 1.45*1.45*8mm for GSO) form a 13x13 array on each module. The energy window was set to 400-700 keV in order to minimize coincident gamma ray background. 18FDG images were obtained as dynamic, list mode acquisitions of 60 min and were reconstructed in 10min frames. Resolution of the system was 1.4 mm (full width at half maximum) at the center of the field of view. Images after each tracer administration were acquired at exactly the same position.
Animals were placed supine, head first in the PET scanner. Anaesthesia was maintained by continuous inhalation of 1.5% isoflurane for the duration of the experiment. The animals’ temperature was monitored and controlled using a heating lamp.
For myocardial delineation and accurate quantification of activity exclusively derived from cells retained in the myocardium, in contrast to activity from cells that escaped into the lungs, a perfusion PET scan (20min static acquisition) was performedfollowing injection of 37MBq of 13NH3 through the tail vein at the end of the FDG acquisition.
In order to facilitate co-registration of PET images with CT and to allow subsequent attenuation correction, 37mBq of free 18F (fluoride) were injected in all animals after completion of the 13NH3 acquisition, as previously described.(1)
After a period of 5 mins, required for adequate uptake of fluoride by the bones, a 5 min static acquisition was obtained. The purpose of this scan is to obtain images with distinguished radioactivity uptake in bones that serve as co-registration landmarks. The animal was held in the same bed position for all the above scans. After completion of the PET acquisitions, the animal was moved into the CT scanner (restrained on the same bed) and CT images were obtained.
Coincidence events were re-binned in the Fourier space and reconstructed using a 2D OSEM (ordered-subset expectation maximization) algorithm, with 8 updates for 18FDG, and 16 updates for 13NH3 images. The reconstructed PET volume is a 175*175*61 (axial direction) matrix, with a voxel size of 0.39*0.39*0.78mm3 (axial direction).
X-ray computed tomography was performed on a Gamma Medica X-SPECT(Gamma Medica, Northridge, CA, USA), a bi-module SPECT/CT 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 (170μm).
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.
PET Image analysis. All images were analyzed using AMIDE software.(2) For quantification of signal in the heart, a volume of interest (VOI) was drawn to include the bright spot at the site of cell injection. For quantification of activity in the lungs, 2 VOIs were drawn, one for each lung. The two large VOIs of the lungs also included activity from the heart, which was then subtracted from the calculated lung activity. Background activity was measured in an area outside the body of the animal, scaled to the volume of the heart and lung VOIs and subtracted from the respective activities. VOI activities (of the syringe containing cell suspension and in vivo cardiac and lung images) were decay corrected; in addition in vivo images were rescaled to correct for attenuation, thus permitting accurate measurement of percentage of the injected dose (%ID=100 * [Activity within VOI / (Activity in syringe before injection-Activity in syringe after injection)], corresponding to in vivo CDC retention intra-myocardially and in the lungs.
Fluorescence microscopy
Two-photon microscopy was used for imaging on a micrometer scale. The following are the advantages of multi-photon excitation 1) fluorescence excitation occurs only in the narrow focal plane of the objective lens, thus eliminating out of focus fluorescence in the collected image, 2) the long wavelength of the incident light permits deeper penetration of the tissue with less damaging effects outside the focal plane, 3) multiple fluorophores are stimulated by a single excitation wavelength with no cross- talk between excitation and emission bands. Here, 2-photon excitation was provided by a Tsunami femtosecond mode-locked TiSa pulsed laser (Spectra Physics) pumped by a 10W Millenia X solid state laser.
In order to localize CDCs after intra-myocardial injection, two millionrCDCs were labelled with the membrane dye, CellTracker CM-Di(3) (1,1’-Dioctadecyl 3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate; Invitrogen)and then injected into the infarcted region (n=4), immediately after the establishment of reperfusion. Immediately following cell injection, 300 μl of a 4% Thioflavine S(4, 5) solution (Sigma) was injected into the right ventricular cavity (in order to visualize the vasculature). The heart was excised 1.5 min later, flash frozen in liquid nitrogen and then fixed in 4% paraformaldehyde and stored at 40C (to prevent re-distribution of the thioflavin-S).(5)The myocardium was imaged through a Nikon 20X lens with a 1.0 numerical aperture. For measurement of simultaneous Di-I and Thioflavin-S fluorescence, an excitation wavelength of 730 nm was used. Emitted lightwas collected by 2 photomultiplier tubes fitted with narrowbandpass filters of 560-650 nm for DiI and 500-550 nm for Thioflavin S. 512x512 pixel images were collected,digitized at 8-bit resolution,and stored directly on the hard disk.
For capillary imaging, adult rats (n=2) were injected intraperitoneally with heparin (125 IU/kg body wt) fifteen minutes prior to excising the heart. The ascending aorta was cannulated and hearts were perfused in the Langendorff mode using Tyrode solution containing (in mmol/l) 134 NaCl, 4 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 10 HEPES, 11 D-glucose, and 2 CaCl2 (Adjusted to pH=7.35 with 1mol/l NaOH). The heart was immobilized by perfusing with the excitation-contraction uncoupler blebbistatin (6-8)(10 µmol/L; Sigma) dissolved in Tyrode solution. Subsequently, endothelial cells were labeled by perfusion with tetramethylrhodamine methyl ester ( TMRM)(9-11)(50nM) and Thioflavin S was used to visualize the capillaries: the heart was perfused with 1% Thoflavin Ssolution (pH 7.3) for 10 seconds and images were acquired during the washout phase using a Nikon 20X lens, and an excitation wavelength of 740 nm. 512x512 pixel images were collected,digitized at 8-bit resolution,and stored directly on the hard disk.
Image analysis was performed using Image J (NIH,
Real time quantitative polymerase chain reaction (qPCR)
We injected cells harvested from male rats into female recipients, an experimental design that permitted us to use the male specific SRY gene, located on the Y chromosome, as a target for the real-time PCR.(12) Since there is only one copy of the SRY gene per cell, the number of copies of the SRY gene (determined by real-time PCR) in the recipient heart corresponds to the number of transplanted cells (see supplement data).
Genomic DNAwas isolated from aliquots of the homogenate corresponding to 12.5mg of myocardial tissue, according to the manufacturer's instructions (Qiagen). Real time PCR was performed using the TaqMan® chemistry (Applied Biosystems), with the rat SRY geneas target (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). For absolute quantification of gene copy number, a standard curvewasconstructed 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 curveis calculated based on the amount of DNA in each sample and total mass of the rat genome per diploid cell. (http: All samplesweretested in triplicate. For each reaction, 50ng of template DNA was used.Real-time PCRwas performed in an ABI PRISM 7700 instrument. The result from each reaction, i.e. copies of the SRY gene in50ng of genomic DNA,wasexpressed as the number of engrafted cells/heart, by first calculating the copy number of the SRY gene in the total amount of DNA corresponding to 12.5mg ofmyocardium and then extrapolating to the total weight of each heart.
SUPPLEMENTAL FIGURES
Supplemental Figure 1.Images corresponding to Figure 2b/AI-RM group (different plane, where the perfusion deficit is more obvious): A and C, without the cells, arrow points to the perfusion deficit; B and D, with the signal from cells in this plane.
Supplemental Figure 2. Images corresponding to Figure 2d/CI-R group, (different plane, where the perfusion deficit is more obvious): A,B and C, without the cells, arrows point to the perfusion deficit; D,E and F, with the signal from cells in this plane
Supplemental Figure 3. Coronal and sagittal images of an animal that received CDCsvia the intracoronary route following ischemia-reperfusion. 18FDG signal (arrows) is identified in the heart (cells) and urinary bladder (excreted free 18FDG released from cells). The remaining scattered FDG signal represents noise.
Supplemental Figure 4. Histogram of forward scatter, which reflects cell size reveals 2 mainpopulation of cells, based on cell size.
References
(1)Terrovitis J, Kwok KF, Lautamaki R, Engles JM, Barth AS, Kizana E et al. Ectopic expression of the sodium-iodide symporter enables imaging of transplanted cardiac stem cells in vivo by single-photon emission computed tomography or positron emission tomography. J Am Coll Cardiol 2008;52:1652-60.
(2)Loening AM, Gambhir SS. AMIDE: a free software tool for multimodality medical image analysis. Mol Imaging 2003;2:131-7.
(3)Honig MG, Hume RI. Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures. J Cell Biol 1986;103:171-87.
(4)Dohrmann GJ, Wick KM. Demonstration of the microvasculature of the spinal cord by intravenous injection of the fluorescent dye, thioflavine S. Stain technology 1971;46:321-2.
(5)Schlegel JU. Demonstration of blood vessels and lymphatics with a fluorescent dye in ultraviolet light. Anat Rec 1949;105:433-43, incl 2 pl.
(6)Fedorov VV, Lozinsky IT, Sosunov EA, Anyukhovsky EP, Rosen MR, Balke CW et al. Application of blebbistatin as an excitation-contraction uncoupler for electrophysiologic study of rat and rabbit hearts. Heart Rhythm 2007;4:619-26.
(7)Straight AF, Cheung A, Limouze J, Chen I, Westwood NJ, Sellers JR et al. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science (New York, NY 2003;299:1743-7.
(8)Allingham JS, Smith R, Rayment I. The structural basis of blebbistatin inhibition and specificity for myosin II. Nature structural & molecular biology 2005;12:378-9.
(9)Loew LM, Tuft RA, Carrington W, Fay FS. Imaging in five dimensions: time-dependent membrane potentials in individual mitochondria. Biophysical journal 1993;65:2396-407.
(10)Ehrenberg B, Montana V, Wei MD, Wuskell JP, Loew LM. Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes. Biophysical journal 1988;53:785-94.
(11)Aon MA, Cortassa S, O'Rourke B. The fundamental organization of cardiac mitochondria as a network of coupled oscillators. Biophysical journal 2006;91:4317-27.
(12)Fukushima S, Varela-Carver A, Coppen SR, Yamahara K, Felkin LE, Lee J et al. Direct intramyocardial but not intracoronary injection of bone marrow cells induces ventricular arrhythmias in a rat chronic ischemic heart failure model. Circulation 2007;115:2254-61.