PRE-CLINICAL RESEARCH
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
John Terrovitis, MD*,
Keng Fai Kwok, BS ,
Riikka Lautamäki, MD, PhD ,
James M. Engles, MS, MBA,
Andreas S. Barth, MD, PhD*,
Eddy Kizana, MBBS, PhD*,
Junichiro Miake, MD, PhD*,
Michelle K. Leppo, BS*,
James Fox, BS,
Jurgen Seidel, PhD,
Martin Pomper, MD, PhD ,
Richard L. Wahl, MD,
Benjamin Tsui, PhD,
Frank Bengel, MD,
Eduardo Marbán, MD, PhD* and
M. Roselle Abraham, MD*,*
* Department of Cardiology, Johns Hopkins University, Baltimore, Maryland
Department of Diagnostic Imaging Physics, Johns Hopkins University, Baltimore, Maryland
Department of Nuclear Medicine, Johns Hopkins University, Baltimore, Maryland
Department of Radiology, Johns Hopkins University, Baltimore, Maryland
Manuscript received February 26, 2008;
revised manuscript received May 7, 2008,
accepted June 19, 2008.
* Reprint requests and correspondence: Dr. M. Roselle Abraham, Department of Medicine, 720 Rutland Avenue, Ross 844, Baltimore, Maryland 21205 (Email: mabraha3{at}jhmi.edu).
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Abstract
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Objectives: We examined the sodium-iodide symporter (NIS), which promotes in vivo cellular uptake of technetium 99m (99mTc) or iodine 124 (124I), as a reporter gene for cell tracking by single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging.
Background: Stem cells offer the promise of cardiac repair. Stem cell labeling is a prerequisite to tracking cell fate in vivo.
Methods: The human NIS complementary deoxyribonucleic acid was transduced into rat cardiac-derived stem cells (rCDCs) using lentiviral vectors. Rats were injected intramyocardially with up to 4 million NIS+-rCDCs immediately after left anterior descending coronary artery ligation. Dual isotope SPECT (or PET) imaging was performed, using 99mTc (or 124I) for cell detection and thallium 201 (or ammonia 13) for myocardial delineation. In a subset of animals, high resolution ex vivo SPECT scans of explanted hearts were obtained to confirm that in vivo signals were derived from the cell injection site.
Results: NIS expression in rCDCs did not affect cell viability and proliferation. NIS activity was verified in isolated transduced cells by measuring 99mTc uptake. NIS+ rCDCs were visualized in vivo as regions of 99mTc or 124I uptake within a perfusion deficit in the SPECT and PET images, respectively. Cells could be visualized by SPECT up to 6 days post-injection. Ex vivo SPECT confirmed that in vivo 99mTc signals were localized to the cell injection sites.
Conclusions: Ectopic NIS expression allows noninvasive in vivo stem cell tracking in the myocardium, using either SPECT or PET. The general approach shows significant promise in tracking the fate of transplanted cells participating in cardiac regeneration, given its ability to observe living cells using clinically applicable imaging modalities.
Key Words: stem cells • imaging • SPECT • PET
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Abbreviations and Acronyms
| | CDC = cardiac-derived stem cell | | CMV = cytomegalovirus promoter | | CR = contrast ratio | | CT = computed tomography | | LV = left ventricle/ventricular | | MRI = magnetic resonance imaging | | NIS = sodium-iodide symporter | | PET = positron emission tomography | | rCDC = rat cardiac-derived stem cell | | RT-PCR = reverse transcription-polymerase chain reaction | | SPECT = single-photon emission computed tomography |
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Stem cells offer the promise of organ repair on demand. Our pre-clinical studies, and studies by other groups, in small and large animal models indicate that autologous cardiac-derived stem cells (CDCs) can successfully regenerate myocardium in the setting of ischemic damage (1–4). Despite this initial success, an important problem that remains to be tackled is low levels of transplanted cell survival and engraftment (5–7). In vivo stem cell tracking is a prerequisite to understanding stem cell biology, optimizing engraftment, and maximizing the functional benefits of stem cell therapy in the heart (8). The ideal cell-tracking method should be nontoxic, sensitive, and specific for the labeled cells, and should permit long-term follow-up (9).
Most studies have used direct labeling of stem cells with agents such as radionuclides or particles before transplantation (8,9). In this study, we adopted a novel approach of using a reporter gene (the sodium-iodide symporter [NIS]) that is normally expressed in the thyroid, stomach, choroids plexus, and salivary gland (10), but not in the heart, to track CDCs after injection into the heart; the NIS promotes cellular uptake of iodide or pertechnetate (technetium 99m [99mTc]) and sodium ions, driven by the transmembrane sodium gradient (11). Remarkably, after ectopic NIS expression, only cells overexpressing NIS will uptake 99mTc (pertechnetate) or iodine 124 (124I) after intravenous injection of these tracers, permitting noninvasive, longitudinal monitoring of stem cell engraftment by single-photon emission computed tomography (SPECT) and positron emission tomography (PET), respectively. The main advantage of the reporter gene approach over direct stem cell labeling approaches is that expression of the reporter gene and functional competence of the expressed protein are dependent on cell viability, unlike direct labeling where the label can be taken up by cardiac myocytes or inflammatory cells after labeled cell death, thus confounding quantification of engraftment (12–14).
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Methods
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Cells.
Rat cardiac-derived stem cells (rCDCs) were cultured from Wistar Kyoto rats as previously described (1,2) (Online Methods).
Animal model.
Wistar Kyoto rats (n = 31) underwent myocardial infarction by permanent ligation of the left anterior descending coronary artery, after which 1 to 4 million NIS+ syngeneic rCDCs were injected intramyocardially at 2 to 4 sites (Online Appendix).
Lentivirus preparation.
A third-generation lentiviral vector system was used to label the rCDCs (15). Expression of the human sodium-iodide symporter (hNIS) gene was driven by constitutively active promoters, cytomegalovirus promoter (CMV) or CAG (composite promoter consisting of the CMV enhancer and chicken beta actin promoter) (Online Appendix).
Cell proliferation.
A colorimetric proliferation assay was performed to test the effect of ectopic NIS expression on CDC viability/function (Online Appendix). In vitro angiogenesis assays were performed as recommended by the manufacturer (Becton-Dickinson, Franklin Lakes, New Jersey) (Online Appendix). In vitro 99mTc (pertechnetate) uptake assay was performed to examine the function of the ectopically expressed NIS (Online Appendix).
Polymerase chain reaction (PCR).
Reverse transcription (RT)-PCR was performed to confirm NIS expression in myocardium, 24 h after injection of NIS-labeled rCDCs (Online Appendix). Quantitative PCR for the rat SRY gene was performed on days 1 and 8 to confirm CDC engraftment after gender-mismatched rCDC transplantation (Online Appendix).
In vivo imaging.
SPECT/Computed Tomography (CT)
Animals were imaged using a Gamma Medica X-SPECT-CT scanner (Gamma Medica, Northridge, California). SPECT imaging was performed before PET imaging because 124I has a relatively long half-life (4.2 days) and emits gamma rays that would increase background and decrease contrast during SPECT imaging. The radioisotopes 99mTc (7.7 ± 1.7 mCi/285 ± 63 MBq) and 210Tl (1 mCi/37 MBq) were injected via the tail vein (n = 13; 6 received CMV.NIS cells, 5 CAG.NIS, and 2 nontransduced rCDCs), 24 h after CDC injection. In addition, 3 rats injected with 4 x 106 CMV.NIS cells were imaged on days 1, 3, 6, and 10 post-transplantation for longitudinal cell tracking. Images were acquired 1 h after tracer injection, since this time point has been associated with peak myocardial accumulation of 99mTc in a previous detailed time course study (16). Dual isotope SPECT was performed to shorten scan time. Details regarding the imaging protocol are provided in the Online Methods. Resolution of the SPECT system was 1.7 mm (full width at half maximum), at the center of the field of view (with a radius of rotation of 5 cm and a pinhole aperture of 1 mm). X-ray CT was performed on the same SPECT/CT system (Online Appendix).
PET/CT
124I (3.70 ± 1.45 mCi/137 ± 54 MBq) was injected via the tail vein (n = 5; 3 received CAG.NIS CDCs and 2 CMV.NIS CDCs) 24 h after SPECT imaging and 48 h after CDC transplantation. A static 30-min acquisition was obtained 1 h after tracer injection. Ammonia-13 1 mCi (37 MBq) was injected after completion of the iodine acquisition, and was followed by a 20-min static acquisition. Since the ammonia scan was intended only for myocardial delineation and not for quantification of perfusion, the high background from persisting 124I in the blood pool was not of concern, although it could have slightly diminished the contrast between myocardium and left ventricular (LV) cavity.
The PET images were acquired on a small animal PET system (VISTA, GE Healthcare, Piscataway, New Jersey). The complete PET imaging protocol is described in the Online Appendix. Resolution of the system was 1.4 mm (full width at half maximum) at the center of the field of view (17). Images after each tracer administration were acquired at exactly the same position. Coregistration of PET and CT images was performed using rigid body transformation and is described in the Online Methods (Online Fig. 1).
Ex vivo SPECT imaging.
To validate the results obtained by in vivo imaging and to confirm the origin of the in vivo signal, a high resolution ex vivo SPECT scan was performed (n = 5) after the completion of the in vivo experiment (Online Appendix).
Image analysis.
All images were analyzed using AMIDE software (18). A volume of interest was drawn to include the bright spot at the cell injection site, for each animal. The same volume of interest was then placed inside the LV cavity to obtain signal intensities in the blood pool. Contrast ratio % was defined as: 100 x ([signal in the cells] – [signal in blood pool])/signal in blood pool. A detailed description of the signal quantification protocol we employed is provided in the Online Methods.
Statistical analysis.
Values are reported as mean ± SD. Repeated measures analysis of variance was used for comparison of cell proliferation rates (NIS+ vs. nontransduced CDCs) at different time points. The paired t test was used to compare contrast ratio (CR) between SPECT and PET images and percent uptake of injected dose between SPECT images. Statistical analysis was performed using Graph Pad Prism software, version 4 (Graph Pad Software, La Jolla, California). A value of p < 0.05 was chosen for statistical significance.
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Results
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Immunostaining and RT-PCR.
Ectopic expression of hNIS in transduced rCDCs was confirmed by immunostaining with a human-specific NIS antibody (Fig. 1A). In addition, expression of hNIS messenger ribonucleic acid was detected by amplification of a 353-bp band using gene specific primers for RT-PCR on messenger ribonucleic acid isolated from hearts of animals injected with NIS+ rCDCs.
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Figure 1 Confirmation of hNIS-Expression
(A) Immunostaining confirmed the expression of human sodium-iodide symporter (hNIS) (green) in rat cardiac-derived stem cells after lentiviral transduction (nuclei were counterstained by blue Hoechst dye). (B) hNIS messenger ribonucleic acid was detected by reverse transcription-polymerase chain reaction in rat hearts after transplantation of sodium-iodide symporter (NIS)+ rat cardiac-derived stem cells (band at 353 base pairs [bp] white arrow, I). The white arrow for II indicates the lack of a specific band in a rat heart injected with the same number of NIS– cells. (C) Confirmation of hNIS activity in transduced cells. NIS transduction promoted in vitro technetium 99m (pertechnetate) uptake by NIS+ rat cardiac-derived stem cells; this uptake was abolished by the specific NIS blocker sodium perchlorate (100 µmol/l) (2 independent experiments, each condition tested in triplicate). ctr = control.
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In vitro 99mTc (pertechnetate) uptake.
Functionally, NIS+ cells accumulated 99mTc (pertechnetate), an effect that was inhibited by the specific NIS blocker, sodium perchlorate (uptake: 6.0 ± 0.9% vs. 0.07 ± 0.05%, without and with blocker, respectively) (Fig. 1C); nontransduced cells did not demonstrate any 99mTc uptake. We attribute the low in vitro uptake of 99mTc to rapid tracer efflux from the transduced cells (19,20). We believe that this property would be favorable for the use of NIS as a reporter gene, since the transient intracellular presence of the radiotracer would reduce the risk of radiotoxicity to the stem cells (20).
Cell viability and function.
Overexpression of hNIS did not significantly affect rCDC viability or proliferation, when compared with that of nontransduced control cells (p = 0.718 by repeated measures analysis of variance) (Fig. 2A). Likewise, cells overexpressing hNIS did not demonstrate any decrease in their cardiogenic potential, determined by additional transduction of cells with a lentiviral construct expressing firefly luciferase under transcriptional control of a cardiac-specific promoter (NCX1 or cardiac sodium-calcium exchanger) (Fig. 2B) (21) or in their angiogenic capacity (determined by their ability to form vascular tubes in an angiogenesis assay) (Figs. 2C to 2E).
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Figure 2 Effect of hNIS Overexpression on Cell Viability, Proliferation, and Function
(A) There was no difference in viability and proliferation of NIS+ versus nontransduced rat cardiac-derived stem cells. (B) Additional transduction with a lentiviral construct expressing firefly luciferase under the transcriptional control of a cardiac-specific promoter (NCX1, cardiac sodium-calcium exchanger) indicated no decrease in the cardiogenic potential of hNIS-expressing cells compared with control cells. (C to E) Angiogenesis and vascular tube formation assays. Representative images of human vascular endothelial cells as positive control cells (C), nontransduced rat cardiac-derived stem cells (D), and rat cardiac-derived stem cells transduced with hNIS (E). The ability to form vascular structures was preserved after transduction with hNIS. Abbreviations as in Figure 1.
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In vivo SPECT.
Thalium 201 perfusion scans lead to adequate visualization of the viable myocardium in all experiments. A large perfusion deficit due to infarction was seen in the middle and apical segments of the anterior and anterolateral walls and septum. Cells expressing hNIS were identified as a region of increased tracer uptake within the perfusion deficit area (Figs. 3A to 3C), in all animals on day 1 post-transplantation (11 of 11, 2 injected with 106 cells and 9 with 2 x 106 cells), whereas, in animals that received control, nontransduced CDCs (n = 2, 1 injected with 106 cells and 1 with 2 x 106 cells), the only cardiac 99mTc (pertechnetate) signal was derived from the blood pool (Figs. 4A to 4C). 99mTc (pertechnetate) uptake of the hNIS cells (% of injected dose) was 0.08 ± 0.07% (n = 11); animals injected with CMV.NIS transduced cells had a significantly higher pertechnetate uptake than animals injected with CAG.NIS transduced cells (0.12 ± 0.07% vs. 0.03 ± 0.03%, p = 0.027), in concordance with our in vitro data comparing the 2 promoters, using firefly luciferase as the reporter gene (Online Appendix, Online Fig. 2). The CR was also higher when CDCs transduced with CMV.NIS were used (70 ± 40% with CMV.NIS cells vs. 28 ± 29% for CAG.NIS cells, p = 0.085), indicating higher expression levels of NIS in CMV.NIS cells and consequently higher pertechnetate uptake. These results underscore the importance of the promoter and consequently transgene expression levels on the outcome of in vivo imaging. Importantly, CR between injection site and myocardium and lung was high (115 ± 49% and 202 ± 121%, respectively), indicating lack of specific uptake of pertechnetate by tissues that do not normally express NIS. In fact, CR between the region of the myocardium containing the perfusion deficit (corresponding to the injection site) and the LV cavity was –18 ± 10% in the animals injected with nontransduced cells; this indicates a brighter signal in the blood pool than in the hypoperfused infarcted myocardium.
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Figure 3 Dual Isotope SPECT/CT of an Animal Injected With hNIS-Expressing Cells
(Red) Technetium 99m uptake; (green) thallium 201 (201Tl) uptake. There is a clear intramyocardial region (yellow arrows) corresponding to the rat cardiac-derived stem cell injection site within a perfusion deficit. Noninfarcted myocardium appears green due to 201Tl uptake. The liver was also visualized due to uptake of 201Tl. (A) Transverse, (B) coronal, and (C) sagittal slice orientation. CT = computed tomography; hNIS = human sodium-iodide symporter; SPECT = single-photon emission computed tomography.
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Figure 4 Dual Isotope SPECT/CT of an Animal Injected With Nontransduced Cells
(Red) Technetium 99m uptake; (green) thallium 201 uptake. The only Tc signal in the cardiac region is derived from the blood pool (atrial and ventricular cavities). (A) Transverse, (B) coronal, and (C) sagittal slice orientation. Abbreviations as in Figure 3.
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In animals (n = 3) injected with 4 x 106 CDCs that underwent serial SPECT imaging, CDCs were identified on day 1, 3, and 6 post-injection (CR was 452 ± 29%, 196 ± 71%, and 131 ± 66%, on days 1, 3, and 6, respectively) (Fig. 5), but not on day 10, where the CR was 1.1 ± 14%, between the area of brightest signal in the myocardium and the LV cavity. These results indicate that NIS can be used for longitudinal stem cell tracking after transplantation.
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Figure 5 Longitudinal Tracking of hNIS-Expressing Cell by SPECT
(Upper panel) Day 1 after cell injection; (middle panel) day 3 after cell injection; (lower panel) day 6 after cell injection. Arrows point to signal from engrafted cells. Display settings are identical in all images. Abbreviations as in Figure 3.
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In vivo PET.
Ammonia 13 perfusion scans generated clear images of the viable myocardium in 5 of 5 animals that underwent small animal PET. All animals received 2 x 106 cells (2 received CAG.NIS and 3 CMV.NIS cells). Injection sites were identified in 3 of 5 animals (2 had received CMV.NIS and 1 CAG.NIS cells) (Figs. 6D to 6F and 7A to 7C). CR obtained by PET was low (18 ± 8%) in this particular experimental setting (dedicated small animal scanner and 124I as tracer), suggesting limitations of 124I as a positron emitting tracer. Coregistration of the PET images with CT confirmed signal localization in myocardium (Fig. 7).
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Figure 6 SPECT and PET Imaging of an Animal Injected With hNIS-Expressing Cells
(A to C) SPECT/computed tomography 24 h after cell injection. Red: technetium 99m uptake; green: thalium 201 uptake. White arrows point to signal from hNIS expressing rat cardiac-derived stem cells. (A) Transverse, (B) coronal, and (C) sagittal slice orientation. (D to F) PET of the same animal at 48 h after cell injection. Red: iodine 124 uptake; green: ammonia 13 uptake. White arrows point to signal from hNIS-expressing cells. (D) Transverse, (E) coronal, and (F) sagittal slice orientation. PET = positron emission tomography; other abbreviations as in Figure 3.
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Figure 7 Successful PET/CT Coregistration Based on the 18Fluoride PET Images
Red: iodine 124 (124I) uptake. White arrows point to signal from hNIS-expressing rat cardiac-derived stem cells. The stomach takes up 124I and is visualized in the image (yellow arrows). Noninfarcted myocardium and liver (black arrows) take up ammonia 13 and appear green in the perfusion scan. (A) Transverse, (B) coronal, and (C) sagittal slice orientation. CT = computed tomography; other abbreviations as in Figures 3 and 6.
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Ex vivo SPECT.
An ex vivo cardiac SPECT scan was performed in 5 animals after the completion of in vivo imaging in order to verify that signal was indeed from cells and not from the blood pool. A region of increased 99mTc (pertechnetate) uptake that was similar in appearance to the in vivo signal was identified within the myocardium, but the CR was considerably higher (323 ± 293% vs. 70 ± 40%) due to lack of signal from the blood pool (Online Fig. 3).
RT-PCR.
Quantitative PCR confirmed that rapid cell loss occurred within the first week after intramyocardial CDC implantation. We found that 13.2 ± 3.6% of the injected cells were retained on day 1, and 2.8 ± 1.8% on day 8 after transplantation (Fig. 8). Based on these PCR results, we estimate that the threshold for CDC detection by SPECT is approximately 150,000 cells.
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Figure 8 CDC Engraftment by Quantitative PCR
Column histogram reveals percentage cell engraftment on days 1 and 8 relative to baseline. Rapid cell loss occurs within 8 days after intramyocardial cell delivery. CDC = cardiac-derived stem cell; PCR = polymerase chain reaction.
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Discussion
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Tracking stem cell fate after transplantation is essential for elucidation of the mechanisms underlying stem cell engraftment and resultant functional benefit. This is the first report using hNIS as the reporter gene to track stem cells in the heart by SPECT and PET imaging. The advantages of this approach are that it is translatable into the clinical setting because it employs a transgene that is nonimmunogenic and the radiotracer 99mTc with SPECT scanning, both of which are widely available and Food and Drug Administration–approved.
To date, most cell-tracking studies have used in vitro cell labeling with iron nanoparticles followed by in vivo magnetic resonance imaging (MRI) (22–24). Despite the significant advantages of MRI (high sensitivity, excellent anatomical and functional data), we and others recently showed that iron-derived signals can persist in the myocardium and can be detected by MRI long after cells have been destroyed, generating false-positive signals (12–14). Genetic labeling, however, offers the important advantage that only cells expressing the transgene will generate a signal (25,26).
Further significance of the present study lies in its potential for clinical translation. Pertechnetate SPECT is a widely available, clinically approved, relatively low cost imaging modality. Results obtained by SPECT imaging in small animals can be easily tested in larger animal models or humans, using clinical SPECT systems. Importantly, there has been substantial progress in absolute signal quantification in clinical SPECT systems in recent years, a fact that strengthens even further the role of SPECT for in vivo stem cell tracking (27).
The wild-type or mutated herpes virus thymidine kinase gene is another transgene that has been successfully used for stem cell tracking by SPECT or PET imaging. hNIS has 2 important advantages over herpes virus thymidine kinase: it utilizes widely available tracers, so no specialized radio-synthesis facilities are needed, and it is a human gene, precluding an immune response if applied clinically.
To date, only direct labeling of stem cells by 111Indium-oxine or 99mTc hexamethylpropylenamine oxime (Tc-HMPAO) has been used for stem cell tracking by SPECT (28,29). These techniques only permit stem cell tracking over a short period of time, determined by the tracer half-lives (2.8 days for indium and 5.8 h for 99mTc), and also pose the risk of radiotoxicity to the cells. In the present study, we showed that hNIS expression in CDCs is nontoxic and can promote adequate tracer uptake to allow in-vivo cell imaging. Even in short-term studies, visualizing cells using a reporter gene approach is superior to any direct labeling method, since signal in the former is always derived from viable transplanted cells (high specificity). With direct labeling, a fraction of the signal might be derived from tracer released by dead cells and persisting at the injection site (in the extracellular space or uptaken by other cell types), generating false positives and undermining the biological interpretation of the in-vivo imaging studies.
Despite the widespread availability of SPECT, PET is attractive for stem cell studies because it is more sensitive and the signal is readily quantifiable. Here, we showed the feasibility of tracking CDCs in the myocardium by PET, using hNIS as reporter gene. However, the contrast achieved was suboptimal when compared with our SPECT results. Possible explanations for this unexpected finding include: 1) 124I is not a pure positron emitter; only 25.6% of the activity is emitted as positrons, resulting in significantly lower efficiency for this agent as a PET tracer; 2) the emission of coincident, high-energy gamma rays significantly increases scatter and background activity, leading to considerable image deterioration; and 3) positrons emitted by 124I annihilate at a relatively long distance (approximately 1.4 mm in comparison to 0.2 mm for positrons emitted by 18F) in relation to their site of production, leading to degradation of spatial resolution (30). This adverse characteristic is of greater importance in small animal studies, where small errors in localization are large in relation to the size of the myocardium. Despite the disadvantages, there is potential for improvement: the positron emitter 94mTc-pertechnetate can be used instead of 124I for clinical translation; although 94mTc is not a pure positron emitter, it has a more attractive half-life (52.5 min instead of 4.2 days for 124I) and does not affect thyroid gland function.
Several limitations have to be acknowledged. The present proof-of-concept study was confined to assessment of CDC engraftment, not functional effects of transplantation, in a small animal model; feasibility in a large animal model needs to be demonstrated before definitive conclusions about clinical translatability can be drawn. However, application in larger animals is expected to be less challenging, since a considerably higher number of cells will be injected, resulting in higher tracer uptake and stronger in vivo signals.
Another fact that limits immediate translation of our results into patients is the use of lentivirus vectors that have not yet been approved for human use due to safety concerns. This is not an inherent feature of the method; routine plasmid transfection followed by cytotoxic selection could equally well be used to establish NIS-expressing cell lines.
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Conclusions
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This is the first report of in vivo CDC tracking by SPECT and PET, using the sodium-iodide symporter as the reporter gene. This technique is readily translatable into the clinical realm and could be expected to improve our understanding of in vivo stem cell biology and eventually the functional effects of stem cell transplantation in the heart.
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Appendix
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For an additional Methods section and supplementary Figures 1 to 3, please see the online version of this article.
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Footnotes
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This work was supported by the Donald W. Reynolds Foundation, the National Heart, Lung, and Blood Institute, the National Institutes of Health (NIH U24 CA92871 to Dr. Pomper), and the WW Smith Foundation (to Dr. Abraham). Dr. Barth was supported by a grant from the German Research Foundation (DFG, grant BA 3341/1-1).
Drs. Terrovitis and Marban are currently affiliated with The Heart Institute, Cedars Sinai Medical Center, Los Angeles, California.
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