Imaging - Circulation

Imaging

Transplantation and Tracking of Human-Induced PluripotentStem Cells in a Pig Model of Myocardial Infarction

Assessment of Cell Survival, Engraftment, and Distribution by HybridSingle Photon Emission Computed Tomography/Computed Tomography of

Sodium Iodide Symporter Transgene Expression

Christian Templin, MD; Robert Zweigerdt, PhD; Kristin Schwanke, PhD; Ruth Olmer, PhD;Jelena-Rima Ghadri, MD; Maximilian Y. Emmert, MD; Ennio Muller; Silke M. Kuest, MD;

Susan Cohrs, PhD; Roger Schibli, PhD; Peter Kronen, DVM; Monika Hilbe, DVM;Andreas Reinisch, MD, PhD; Dirk Strunk, MD; Axel Haverich, MD; Simon Hoerstrup, MD, PhD;Thomas F. Luscher, MD; Philipp A. Kaufmann, MD; Ulf Landmesser, MD*; Ulrich Martin, PhD*

Background—Evaluation of novel cellular therapies in large-animal models and patients is currently hampered by the lackof imaging approaches that allow for long-term monitoring of viable transplanted cells. In this study, sodium iodidesymporter (NIS) transgene imaging was evaluated as an approach to follow in vivo survival, engraftment, anddistribution of human-induced pluripotent stem cell (hiPSC) derivatives in a pig model of myocardial infarction.

Methods and Results—Transgenic hiPSC lines stably expressing a fluorescent reporter and NIS (NISpos-hiPSCs) wereestablished. Iodide uptake, efflux, and viability of NISpos-hiPSCs were assessed in vitro. Ten (�2) days after inductionof myocardial infarction by transient occlusion of the left anterior descending artery, catheter-based intramyocardialinjection of NISpos-hiPSCs guided by 3-dimensional NOGA mapping was performed. Dual-isotope single photonemission computed tomographic/computed tomographic imaging was applied with the use of 123I to follow donor cellsurvival and distribution and with the use of 99mTC-tetrofosmin for perfusion imaging. In vitro, iodide uptake inNISpos-hiPSCs was increased 100-fold above that of nontransgenic controls. In vivo, viable NISpos-hiPSCs could bevisualized for up to 15 weeks. Immunohistochemistry demonstrated that hiPSC-derived endothelial cells contributed tovascularization. Up to 12 to 15 weeks after transplantation, no teratomas were detected.

Conclusions—This study describes for the first time the feasibility of repeated long-term in vivo imaging of viability and tissuedistribution of cellular grafts in large animals. Moreover, this is the first report demonstrating vascular differentiation andlong-term engraftment of hiPSCs in a large-animal model of myocardial infarction. NISpos-hiPSCs represent a valuable toolto monitor and improve current cellular treatment strategies in clinically relevant animal models. (Circulation. 2012;126:430-439.)

Key Words: imaging � induced pluripotent stem cells � iPS cell � myocardial infarction in pig� sodium iodide symporter (NIS)

Stem cell–based therapies are being actively explored as apotentially innovative therapeutic strategy for various ge-

netic and acquired diseases. Recently, the possibility of repro-gramming human somatic cells into human-induced pluripotent

stem cells (hiPSCs) that are able to differentiate into all celllineages present in the heart1–4 has opened novel opportunitiesfor myocardial repair. With respect to the potential therapeuticapplication of pluripotent stem cell derivatives, major progress

Received December 23, 2011; accepted May 21, 2012.From the Cardiovascular Center, Department of Cardiology (C.T., T.F.L., U.L.), Department of Cardiac Imaging (J.G., E.M., S.M.K., P.A.K.), and

Clinic for Cardiovascular Surgery, Department of Surgical Research (M.Y.E., S.H.), University Hospital Zurich, Zurich, Switzerland; Leibniz ResearchLaboratory for Biotechnology and Artificial Organs, Cardiothoracic, Transplantation, and Vascular Surgery, Hannover Medical School, Hannover,Germany (C.T., R.Z., K.S., R.O., A.H., U.M.); Center for Radiopharmaceutical Science ETH-PSI-USZ, Paul Scherrer Institute, Villigen, Switzerland(S.C., R.S.); Veterinary Anesthesia Services–International, Winterthur, Switzerland (P.K.); Center for Applied Biotechnology and Molecular Medicine(P.K.), Musculoskeletal Research Unit, Equine Department (P.K.), Institute of Veterinary Pathology (M.H.), Vetsuisse Faculty ZH, University of Zurich,Zurich, Switzerland; and Stem Cell Research Unit, Medical University of Graz, Graz, Austria (A.R., D.S.).

*Drs Landmesser and Martin contributed equally to this work.The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.

111.087684/-/DC1.Correspondence to Christian Templin, MD, Department of Cardiology, University Hospital Zurich, Ramistrasse 100, 8091 Zurich, Switzerland. E-mail

[email protected], or Ulrich Martin, PhD, LEBAO-HTTG, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany.E-mail [email protected]

© 2012 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.111.087684

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has been achieved concerning scalable cell production5,6 and moreefficient and specific differentiation into cell types of interest.7

Editorial see p 388Clinical Perspective on p 439

However, there are still major hurdles and risks to over-come with regard to PSC-based heart repair. These includesafety risks, especially the potential of teratoma and tumorformation,2 low cell retention and engraftment rates,8–11 andthe general question of whether engraftment of hiPSCs aftersimple intramyocardial cell injection leads to formation offunctional tissue, such as de novo vasculature or myocar-dium, and results in significant clinical benefits.12 Althoughsome of these issues can be addressed in vitro or in appro-priate small-animal models, others will require exploration inlarge-animal models, which are more similar to humans.13

Transplanted human cardiomyocytes, for example, are un-likely to fully functionally integrate with rodent myocardiumbecause of highly dissimilar beating rates.11 Therefore, mean-ingful assessment of human cells for heart repair must bedemonstrated in large-animal models such as dogs, pigs, ormonkeys.13

Clearly, advanced imaging technologies allowing for longitu-dinal tracking of cellular grafts and ideally enabling monitoringof donor cell survival, proliferation, distribution, or even differ-entiation are crucial for (pre)clinical evaluation of novel cell-based therapeutics.14 At present, transgene-based imaging ap-proaches that fulfill these requirements are mainly restricted tofar-red fluorescence reporters or photon-emitting technologies,in particular those based on luciferase expression. Unfortunately,limited tissue penetration restricts application of these technol-ogies to small-animal models only.15

Most previous large-animal studies applied magnetic reso-nance imaging technology to detect nanoparticle-labeled cells.16

The main disadvantage of this approach is the inability todiscriminate vital donor cells from nanoparticle-loaded celldebris or the recipient’s cells such as macrophages, which canactively incorporate cell debris and released nanoparticles byphagocytosis.16 In other studies, direct radionuclide labeling ofcells was performed (eg, through [18F]FDG, In111 oxine, 64Cu-PTSM, or 99mTc-hexamethylprophylene amine oxine).16 Limi-tations of the latter techniques include isotope half-time– andlabel-retention–dependent loss of signal strength.16 In addition,direct labeling strategies with nanoparticles or radionuclides,particularly for In111, have been reported to affect (stem) cellvitality.16

In a first pilot study to overcome these critical drawbacks,herpes simplex virus thymidine kinase–mediated phosphoryla-tion of [18F]FHBG was evaluated as a transgene-based imagingapproach in large animals,17,18 but detection of transplanted cellswas reported several hours after cell injection only.

Another promising approach has been developed re-cently,19 which permits detection of viable transplanted cellsby positron emission tomography (PET) or single photonemission computed tomography (SPECT) after iodide (123I)or 99mTc radiotracer administration. This technology relies onthe expression of a transgenic sodium iodide symporter(NIS)20 in transplanted donor cells. Physiological expressionof NIS in the adult organism is restricted to the thyroid,

stomach, choroids plexus, and salivary gland and is notdetectable in limbs, brain, or other internal organs such as theheart.20 NIS-mediated iodide accumulation in the thyroid isan active transport process that occurs at the basolateralplasma membrane of the thyroid follicular cells against theiodide electrochemical gradient stimulated by thyroid-stimulating hormone and can be specifically inhibited byperchlorate. Clinically, NIS expression provides the basis forthe effective diagnostic of thyroid cancer and its metastasesby iodide isotope administration.20

The technology provides important advantages with re-spect to clinical application for longitudinal tracking of stemcell grafts. This includes the lack of immunogenicity of thehuman NIS transgene and the fact that SPECT scanning ofradiotracers such as 99mTc and 123I is widely available atrelatively low cost and is approved by responsible regulatoryauthorities such as the Food and Drug Administration. More-over, recent progress in quantification of tracer signals withthe use of clinical SPECT systems21 emphasizes the potentialof this approach for tracking cellular transplants in largeanimals and patients. To date, however, the strategy wasevaluated only in a small-animal model focused on detectionof acute cell retention after administration of cardiac derivedstem cells.19

In the present study, we demonstrate the applicability ofthis technology for in vivo monitoring of cellular grafts in apig model of myocardial infarction. SPECT/computed tomo-graphic (CT) imaging results revealed successful in vivodonor cell labeling by intracoronary iodide administration 5days or 12/15 weeks after intramyocardial cell transplanta-tion. These data were confirmed by immunohistochemistryon harvested tissue, providing unequivocal evidence forlong-term survival, engraftment, and differentiation of trans-planted hiPSC derivatives.

MethodsCulture of hiPSCsConventional culture of hiPSCs was performed as described previ-ously.1 Mass cell generation of NIS transgenic hiPSCs (NISpos-hiPSCs)in suspension cultures was performed as described previously.5,6

Generation of NISpos-hiPSC LinesSee the online-only Data Supplement for details of NISpos-hiPSClines.

Characterization of NISpos-hiPSCsSee Figure 1 and the online-only Data Supplement for details.

Iodide Uptake and EffluxSee the online-only Data Supplement for details.

Ex Vivo Cardiac SPECT/CT ImagingFor assessing the SPECT/CT detection limit in terms of prelabeledNISpos-hiPSCs, cells were incubated for 90 minutes with 1 MBq 123Iper 1�106 cells. After vigorous washing, serial cell dilutions comprisinga total of 1�104, 1�105, 5�105, 1�106, 3�106, 6�106, or 1.2�107

labeled cells were injected into the lateral and septal walls of the leftventricle. The 123I signal was visualized through a dual-detectorhybrid SPECT/CT scanner (Infinia Hawkeye, GE Healthcare). Tomimic in vivo imaging, heart-derived 123I signals were recordedthrough a dissected pig chest that was placed above the heart tomimic signal attenuation.

Templin et al Long-Term In Vivo Imaging of iPSCs in Pig Hearts 431

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Large-Animal ModelFemale Landrace pigs (Large White Duroc, Sidler, Zurich, Switzer-land; n�13; weight, 25–30 kg; aged 8–10 weeks) were investigatedin the present study. Animals were immunosuppressed with cyclo-sporine A (Novartis Pharmaceuticals, East Hanover, NJ; 10 mg/kgbody wt daily) and prednisolone (Pfizer, Zurich, Switzerland; 2.5mg/kg body wt daily) starting 5 days before hiPSC transplantationuntil animals were euthanized. Furthermore, all animals received2�400 mg/d dronedarone at the first preoperative and operative dayand 2�200 mg/d from the first postoperative day until the end of thestudy. All procedures were approved by the responsible local authoritiesand performed according to the Guide to the Care and Use ofExperimental Animals published by the US National Institutes of Health(National Institutes of Health publication 85–23, reviewed 1996). Adetailed description of the study design is shown in Figure 2.

Induction of Myocardial InfarctionMyocardial infarction was created by inflating the balloon for 180minutes in the mid segment of the left anterior descending coronary

artery, followed by reperfusion. See the online-only Data Supple-ment for details.

Electromechanical MappingThree-dimensional NOGA mapping (Biologics Delivery Systems,Biosense Webster, Johnson & Johnson, Irwindale, CA) was per-formed 10�2 days after induction of myocardial infarction to assesselectrophysiological tissue viability patterns and wall motion abnor-malities. The catheter was placed through the right carotid sheathacross the aortic valve into the left ventricle. By navigating thecatheter along the endocardium, the local intracardiac electrogramtogether with the wall motion was recorded from its tip at multipleendocardial sites, and detailed 3-dimensional electromechanicalmaps of the left ventricle were generated. The unipolar voltagevalues were color coded (visualizing infarct zones as red and viabletissue as green to purple) and superimposed on the 3-dimensionalgeometry of the map. NOGA mapping and injections were per-formed as described previously.22

Figure 1. Generation and functional characterization of sodium iodide symporter transgenic human induced pluripotent stem cells(NISpos-hiPSCs). A, Map of the bicistronic vector pCAG_rNIS_IRES2_Venus_nucmem used for the generation of transgenic hiPSCclones. B, Fluorescence-activated cell sorting analysis of Venus expression in several independent rNISpos-hiPSC clones. Individualclones showed a stable level of transgene expression for up to 23 passages. C, Overlay of fluorescence and phase contrast imagerevealing the expected nuclear Venus localization of a typical NISpos-hiPSC colony cultured on Matrigel (clone 6, 6 passages afterclonal selection). D, Detection of Venus by immunohistochemistry on a section of paraformaldehyde-fixed NISpos-hiPSC aggregatesderived from suspension culture and the respective isotope control. For immunostaining, an anti-green fluorescent protein antibodywas used, which cross-reacts with the yellow green fluorescent protein variant Venus. Notably, fixation and immunohistochemistrytreatments lead to an apparently cytoplasmic Venus-specific staining, which is in contrast to the direct Venus fluorescence in viablecells that is strictly restricted to the nuclear membrane, as shown in C. E, Kinetics of 125I uptake by NISpos-hiPSCs (clone 6), inhibitionby 100 �mol/L perchlorate, and NISneg-hiPSC controls (mean�SEM, triplicate measurements). Iodide uptake reached a steady statewithin 90 minutes with a maximum concentration of �100-fold above background in negative controls. 125I accumulation in NISpos-hiPSCs was completely abolished by perchlorate, suggesting specificity of the signal. F, Kinetics of iodide retention in NISpos-hiPSCs(mean�SEM, triplicate measurements). hCBiPS indicates human cord blood iPS.

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Intramyocardial Cell InjectionshiPSCs (1�108) were labeled with 100 MBq of 123I (1 MBq�1�106

cells) for 90 minutes before cell injection. Subsequently, cells werewashed twice in phosphate-buffered saline and were resuspended inphosphate-buffered saline as the delivery medium, which wassupplemented with fluorospheres (Molecular Probes) to enableunequivocal identification of the cell injection sites under ultravioletlight during tissue sampling in euthanized animals (Figure I in theonline-only Data Supplement). Cell injection was performed with an8F MyoStar injection catheter (Biosense-Webster) with a 27-gaugeneedle, as follows: Three sites each with 8 single 250-�L injectionswere selected (left ventricle anterior wall: 50 million control cells[human mesenchymal stem cells {MSCs}]; lateral and septal wall:50 million NISpos-hiPSCs or 50 million NISpos-hiPSCs mixed with50 million human MSCs). Control animals received phosphate-buffered saline injections at corresponding injection sites. Exactpositions of individual injection sites were documented by NOGAmapping.

CT AngiographyFor cardiac CT angiography, 1 mL/kg body wt iodixanol (Visipaque320 mg/mL; GE Healthcare, Bucks, UK) at a flow rate of 4 to 5 mL/sfollowed by 50 mL saline solution was injected into the ear vein viaa 20-gauge catheter. All scans were performed with the 64-detectorCT component (LightSpeed VCT, GE Healthcare) of a SPECT/CTscanner with prospective ECG triggering, as established and de-scribed in detail previously.23 Images were analyzed on an externalworkstation (AW 4; GE Healthcare).

Full-body CT scanning was performed on the same CT scannerwith the use of 100 mL iodixanol.

SPECT ImagingFor assessment of myocardial perfusion, 99mTC (10 MBq/kg) wasinjected into the ear vein, and images were acquired over 5 minuteson the SPECT part of a hybrid SPECT/CT scanner (Discovery NMCT 570c, GE Healthcare) integrating a 64-slice CT device and acadmium-zinc-telluride gamma camera. The cadmium-zinc-telluridecamera has been extensively described recently.24,25 Briefly, it is agamma detector with a pinhole collimator and 19 stationary detectormodules positioned around the chest. For perfusion imaging, asymmetrical energy window at 140 keV (�5%) was used, and fordual peak acquisition with 123I, a window at 159 keV (�5%) wasadded.

For mid-term (5 days after transplantation) and long-term (12–15weeks after transplantation) detection of transplanted hiPSCs, weperformed intracoronary injection of 123I into each main coronaryartery (left anterior descending, left circumflex, right coronaryarteries) with the use of an over-the-wire balloon catheter during 3coronary occlusions, each lasting 2 to 3 minutes. Between occlu-

sions, the coronary artery was reperfused for 2 minutes. Afterward,animals underwent SPECT/CT imaging as described above.

See Figure II in the online-only Data Supplement for details.

(Immuno)Histological AnalysisSee details in the online-only Data Supplement.

ResultsFunctional NIS Expression in TransgenichiPSC CloneshiPSCs (clone hCBiPSC2)1 were cotransfected with thebicistronic vector pCAG_rNIS_IRES2_Venus_nucmem (Fig-ure 1A) and the plasmid �MHCneoPGKhygro,26 enablingselection of hygromycin-resistant transgenic cell clones. Sixindependent Venuspos clones (designated rNIS clones 1, 2, 4,5, 6, 7) were analyzed for stability of transgene expression.Flow cytometry and fluorescence microscopy revealed robustand stable Venus expression for up to 23 passages withline-specific variations (Figure 1B and 1C). Venus-specificimmunohistological assessment was also performed on par-affin sections of suspension culture–derived cell aggregatesand differentiated cells in embryoid bodies. In contrast to theexpected Venus fluorescence signal in the nucleus of viablecells (Figure 1C), cytoplasmic staining was noted after fixationand immunohistochemistry (Figure 1D, left); however, stainingspecificity was proven by isotype (Figure 1D, right) and non-transgenic cell controls (data not shown). Quantitative reversetranscription polymerase chain reaction revealed coexpression ofthe NIS transgene in Venus-expressing clones (data not shown).Venus protein expression was demonstrated in undifferentiatedas well as differentiated cells of the NISpos-hiPSC clone 6,including hiPSC-derived CD31pos endothelial cells (Figure III inthe online-only Data Supplement). Further analyses and all celltransplantation experiments were conducted with the use of theNISpos-hiPSC clone 6.

Transgenic cells and control hiPSCs were analyzed for NIS-specific iodide uptake by in vitro incubation with 125I, an isotopewith a half-life of 13 hours. Intracellular labeling efficiencydepends on the effective half-life of an applied isotope and thekinetics of the respective isotope accumulation of the cells. 125Iuptake in NISpos-hiPSCs reached a plateau after �90 minutes.After vigorous washing, 125I accumulation was �100 timeshigher than for nontransgenic hiPSC control cells (Figure 1E).Importantly, iodide uptake was completely blocked by thespecific NIS inhibitor perchlorate. Typically, the majority ofaccumulated 125I was released to the medium within 2 hours(Figure 1F). Notably, neither NIS expression nor radiolabelingof NISpos-hiPSCs affected cell viability (Figure IV in theonline-only Data Supplement).

Successful SPECT/CT-Based Detection of Down to1�10

5 123

I-Labeled NISpos-hiPSCs in ExplantedPig HeartsEx vivo cardiac SPECT/CT was performed to determine thedetection limit for 123I-prelabeled NISpos-hiPSCs after in-tramyocardial cell injection. Equal volumes of a serial dilu-tion of labeled cells were applied. As shown in Figure 3,SPECT/CT enabled detection of a minimum of 1�105 cellsinjected into explanted pig hearts. Notably, 123I tracer uptake

Figure 2. Study design. MI indicates myocardial infarction; CsA,cyclosporine A; PBS, phosphate-buffered saline; SPECT-CT,single photon emission computed tomography/computedtomography; and iPSCs, induced pluripotent stem cells.


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was detected through a dissected pig chest that had beenplaced above the heart to mimic signal attenuation in vivo. Onthe basis of these results, 5�107 prelabeled NISpos-hiPSCswere subsequently applied in vivo per injection area with ahypothetical detection threshold of �2% persistent cells.

Prelabeled NISpos-hiPSCs Were Detected for up to5 Hours After Catheter-Based IntramyocardialDelivery Into Distinct Regions of Infarcted HeartsAs indicated in the study design (Figure 2), myocardialinfarction was induced in 13 animals, 10 of which wereenrolled in the study. Ten days after transient occlusion of theleft anterior descending coronary artery, SPECT/CT imagingrevealed perfusion defects in the apex and mid segment of theanterior and septal walls in all treated animals (Figure 4, line1). This was also confirmed by NOGA mapping demonstrat-ing loss of electric activity at the apicoseptal, apicobasal, andapicolateral myocardial regions (red color). Brown pointsrepresent the locations of the NOGA-guided intramyocardialinjections at the border zone of infarction (Figure 4, line 2,and Figure V in the online-only Data Supplement). One to 3hours after injection of 123I-prelabeled NISpos-hiPSCs (5�107

hiPSCs or 5�107 hiPSCs coadministered with 5�107 humanMSCs, respectively, per injection area), intensive 123I signalswere detected by SPECT/CT imaging in the septal and lateralwalls of the left ventricle, corresponding exactly to theinjection site as recorded by NOGA mapping. As expected,123I-treated control cells injected into a separate region of thesame heart did not result in a detectable SPECT signal(Figure 4, line 3). Notably, MSC coadministration consider-ably increased signal intensity, suggesting improved hiPSCretention in the myocardium after transplantation (Figure 4and Video I in the online-only Data Supplement). The signalintensity was highest in the first hour after cell transplantationand diminished successively. At 3 to 5 hours after injection,signal intensity was substantially reduced (Figure VI in theonline-only Data Supplement) and became undetectable after24 hours (data not shown). Imaging of control animals treatedwith phosphate-buffered saline did not show any SPECTsignal (data not shown). As expected, immunohistologicalstaining of tissue sections of the region where NISpos-hiPSCsand human MSCs had been coinjected showed an injectionchannel filled with rounded cells containing a fraction of

Venuspos and OCT4pos cells (Figure 4). A semiquantitativeanalysis of the obtained 123I signals in the 2 pigs that had beeneuthanized after day 1 follow-up demonstrated that theNISpos-hiPSCs cotransplanted with MSCs gave a 6.8-fold/1.7-fold stronger 123I signal than the separately transplantedNISpos-hiPSC cells (Figure VII in the online-only DataSupplement). No detectable 123I activity was associated withthe transplanted NISneg control cells (Table I in the online-only Data Supplement).

Long-Term Detection of Engrafted NISpos-hiPSCDerivatives for up to 15 weeks by SPECT/CTImaging After Intracoronary Iodide AdministrationFor detection of transplanted NISpos-hiPSC derivatives at 5days and 12 to 15 weeks after intramyocardial cell injection,123I was infused by intracoronary injection into the leftanterior descending, left circumflex, and right coronary ar-teries. One hour after tracer injection, the resulting 123Isignals were assessed. As expected, on SPECT/CT scanning,a very strong uptake was observed in the thyroid, whereas aweaker uptake was detected in the stomach (data not shown).Interestingly, in the heart, 123I signals were detected exclu-sively in regions where NISpos-hiPSCs had been cotrans-planted with human MSCs (Figure 5). In independent hearts,123I accumulation was detected either in the lateral heartregion or in the septal left ventricular wall, always correlatingwith the area of NISpos-hiPSC/MSC coinjection. In contrast,no 123I signal could be detected in regions where only NISpos-hiPSCs or control cells had been injected.

In vitro coculture experiments under hypoxic conditionswere performed to investigate whether antiapoptotic effectsof MSCs might support the survival of NIS-hiPSCs. Interest-ingly, MSCs significantly decreased the rate of NIS-hiPSCapoptosis. This effect was dependent of both secreted factorsand cell-cell contact (Figure VIII in the online-only DataSupplement).

After SPECT/CT, hearts were euthanized for tissue sampleanalysis. In correlation with SPECT/CT imaging and NOGAmapping of myocardial infarction, histological analyses ofrespective heart regions (collected 5 days and 15 weeks aftercell transplantation) revealed typical signs of infarction andsubsequent fibrosis, including widespread replacement of myo-cardium by loose to densely arranged cells with fibroblast

Figure 3. Ex vivo single photon emission computed tomography/computed tomography of pig hearts transplanted with sodiumiodide symporter transgenic human induced pluripotent stem cells (NISpos-hiPSCs). 123I-prelabeled NISpos-hiPSCs were trans-planted into pig hearts ex vivo to determine the minimal number of detectable NISpos-hiPSCs. Depicted are single photon emis-sion computed tomographic/computed tomographic images of pig hearts after injections of equal volumes with different amountsof cells as indicated: 1�1.2�107; 2�6�106; 3�3�106; 4�1�106; 5�5�105; 6�1�105; 7�1�104. Injected NISpos-hiPSCs weredetectable down to 1�105 cells.


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phenotype, and moderate presence of granulocytes and lympho-cytes. Immunohistochemistry confirmed persistent engraftmentof NISpos-hiPSCs. Anti-Venus–specific staining demonstratedthe presence of Venuspos hiPSC derivatives 5 days and 12/15weeks after cell injection. Notably, after 12/15 weeks, thehistological images suggest that hiPSC derivatives had adoptedan endothelial phenotype lining numerous vessels in the injectedheart regions (Figure 5, right, and Figure IX in the online-onlyData Supplement). Complementing semiquantitative analyseson the capillary densities of the respective heart regions sug-gested an increased capillary density in the regions of NISpos-

hiPSC-MSC versus NISpos-hiPSC injection (Figure X in theonline-only Data Supplement). Interestingly, in contrast to anal-ysis 5 hours after cell administration, no OCT4-specific cellstaining could be detected at 5 days or 12/15 weeks (data notshown). In addition, no teratoma or other obvious tumor forma-tion was detected in the euthanized recipient animals duringautopsy.

DiscussionThe availability of transgene-based imaging technologies inlarge animals that enable nontoxic, specific, sensitive, non-

Figure 4. In vivo cardiac hybrid single photon emission computed tomography/computed tomography (SPECT/CT) demonstrates suc-cessful induction of myocardial infarction and suggests long-term survival of sodium iodide symporter transgenic human induced pluri-potent stem cell (NISpos-hiPSC) grafts. Representative images of a heart of 1 of 3 recipient animals that were euthanized 6 hours aftercell injection are shown. Line 1: SPECT/CT in vivo imaging of the left ventricle demonstrates a loss of myocardial perfusion (blue) in theanteroapical and septal walls after occlusion of the left anterior descending coronary artery. Noninfarcted myocardium appears orangecolored, indicating normal 99mTC-tetrofosmin uptake. Line 2: Three-dimensional NOGA mapping of the left ventricle recorded duringcell injection. NOGA colors represent unipolar voltage values: red�scar, green to blue�viable tissue. Cell injection sites in the lateral(NISpos-hiPSCs�mesenchymal stem cells), septal (NISpos-hiPSCs), and anterior (nontransgenic mesenchymal stem cells) walls areshown as brown dots. Line 3: SPECT/CT imaging of left ventricle 1 hour after catheter-based intramyocardial cell injection demonstrat-ing intense 123I signals (yellow) in the lateral and septal walls that correspond exactly to the injection sites (each injection area 5�107

hiPSCs), as recorded by NOGA mapping; control cells did not result in a detectable radiotracer signal (anterior wall). Coadministrationof mesenchymal stem cells markedly increased signal intensity (lateral wall). Immunohistochemistry: Depicted are immunohistologicalsections of the lateral ventricle wall showing a cell injection channel 6 hours after cell application filled with cotransplanted human mes-enchymal stem cells and Venuspos NISpos-hiPSCs stained for Venus and OCT4 (each with brown color).


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invasive, and serial long-term imaging of viable grafts is ofutmost importance for preclinical evaluation of stem cell–based therapies.

In the present study, we have established a reporter geneapproach that enables in vivo monitoring of long-term sur-vival of hiPSC derivatives in a pig model of myocardialinfarction. Notably, this is the first report demonstrating thefeasibility of repeated longitudinal in vivo imaging of cellviability, proliferation, and tissue distribution of cellulargrafts in internal organs of large animals.

Similar to luciferase imaging in small animals, viable cellsexpressing NIS are able to accumulate 123I and to evoke adetectable SPECT signal, thereby preventing false-positivedetection of dead cells, cellular debris– bound label, orphagocytosing macrophages. Although the ratio of the inten-sity of the specific 123I signal in our NISpos-hiPSCs comparedwith NISneg-hiPSCs as determined in vitro is lower than thetypical signal-to-background ratio in the case of luciferasereporter systems, long-term surviving NISpos-hiPSC deriva-tives evoked a 123I signal of considerable intensity in the areasof intramyocardial cell injection. This may be explained bythe relatively low absorption of the 123I signal through softand hard tissue and is in contrast to the very limited tissuepenetration of luciferin-emitted light, which, even in smallanimals with low tissue thickness, leads to significant loss ofsignal.

Ex vivo injection of 123I-labeled NISpos-hiPSCs revealeda detection limit of �1�105 cells in isolated pig hearts.Because 123I signals were recorded through an isolated pigchest, one can expect signal intensities in vivo in the samerange as those detected in hearts assessed ex vivo. Althoughnot directly comparable, because (1) lentiviral vectors andanother tracing approach were used; (2) another stem celltype, namely, cardiac stem cells, was applied; and (3) signalretention through pig chest is slightly different from retentionthrough rat chest, the determined detection limit is in thesame range as for cell transplantation into rat hearts. Here,Terrovitis et al19 estimated 1.5�105 as the minimal detectablecell number. Further advantages of human NIS-SPECT ver-sus herpes simplex virus thymidine kinase–PET imaging arewide availability of the necessary tracers, lack of need forspecialized radiosynthesis facilities, and lack of immunoge-nicity of the human NIS, which may be of critical importancefor long-term monitoring in particular.

Obviously, SPECT imaging of NIS-expressing cell graftshas limitations. As is the case with every in vivo imagingapproach, there is a detection limit in terms of a minimaldetectable number of cells. Certainly, we cannot excludedistribution of single undifferentiated or differentiated hiPSCderivatives to other organs. Indeed, only if a critical numberof cells accumulates or is reached as a result of local prolifera-

Figure 5. In vivo single photon emission computed tomography/computed tomography (SPECT/CT) imaging of long-term survivingderivatives of sodium iodide symporter transgenic human induced pluripotent stem cells (NISpos-hiPSCs) in pig hearts after 5 days and15 weeks, respectively. Non-prelabeled NISpos-hiPSCs (each site 5�107 cells) could be detected in mid-term (5 days) and long-termfollow-up (15 weeks) �1 hour after intracoronary (i.c.) 123I injection. Immunohistochemical staining of tissue sections of correspondingareas of the left ventricular wall confirmed the presence of Venuspos (stained with cross-reacting anti-green fluorescent protein anti-body) NISpos-hiPSC derivatives. Notably, the vast majority of Venuspos iPSC derivatives were found to represent endothelial cells inte-grated into the cardiac vasculature 15 weeks after cell transplantation. No OCT4pos cells could be detected (data not shown). The 123Isignal intensities as determined by volume of interest analysis of the respective animals shown are as follows: 5 days after injectionanimal, volume of interest�4.3�105 counts (septal region�NISpos-hiPSC�mesenchymal stem cell [MSC] injection); 15 weeks afterinjection animal, volume of interest�1.8�105 counts (lateral region�NISpos-hiPSC�MSC injection).


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tion, or the minimal detection limit at a specific site is achieved,can NIS-expressing engrafted cells be detected. Nevertheless, inour experiments, we were not able to detect any 123I signalsoutside the heart despite the naturally NIS-expressing tissuesites. Autopsies of the euthanized animals 12 to 15 weeks aftercell infusion confirmed these results and did not reveal anysign of tumor or teratoma formation. Together with theabsence of OCT4pos cells 12 to 15 weeks after cell transplan-tation, as analyzed by immunohistochemistry, this raises thequestion of whether the high incidence of teratomas observedin immunoincompetent mice is actually indicative of clinicalapplications or is rather an artifact of species incompatibili-ties that prevent natural control of cell proliferation in thespecific tissue niches. Another potential explanation for theobvious absence of undifferentiated cells and the observedparallel persistence of differentiated vascular NISpos-hiPSCderivatives might be the applied immunosuppressive protocolconsisting of relatively high doses of cyclosporine A andprednisolone. In general, the long-term persistence of hiPSC-derived endothelial cells indicates the efficacy of our immu-nosuppressive protocol, at least in terms of T-cell reactivitytoward differentiated iPSC derivatives. However, there isevidence that cyclosporine A is not effective toward naturalkiller cells,27 which might lead to selective natural killercell–based erasure of undifferentiated PSCs.28 In the case ofprednisolone, recent data suggest that inhibition of naturalkiller cells depends on the mode of activation, therebyindicating that prednisolone treatment might also not fullyprevent natural killer–mediated killing of undifferentiatedhiPSCs.29

Another limitation of this imaging technology is the physio-logical expression of NIS in the thyroid, stomach, choroidplexus, and salivary gland. Because the signals from theseorgans are strong, they may cover up weak signals from adjacentorgans. For instance, although it does not pose a problem inlarger animals such as the pig, the strong thyroid signal mayinterfere with close-by cardiac tracer signals in mice. In addition,the stomach background signal may interfere with tracer signalsin the upper abdomen.

Of course, there is room for further improvement. Higherlevels of NISpos expression in optimized vector systems andselected stable cell clones may lead to further increase of 123Iaccumulation compared with NISneg control cells. In addition,we currently do not know whether potential silencing effectsduring in vivo differentiation of the transplanted NISpos-hiPSCs may have diminished NIS expression in our experi-ments and whether expression systems with reduced silencingcan lead to further increase of the 123I signal.

As mentioned above, immunohistochemical staining ofmyocardial tissue sections harvested 5 days and 12 to 15weeks after cell application showed no OCT4pos cells in theareas of previous cell infusion. Instead, Venuspos NISpos-hiPSC derivatives with endothelial phenotype were detected,which contributed to the intramyocardial vasculature. Nota-bly, the utilized CAG promoter–driven NIS does not allowfollowing differentiation events through SPECT imaging.However, this could be realized by placing NIS under thecontrol of cell type–specific promoters, for instance, the OCT4

promoter5 or the cardiomyocyte-specific �-myosin heavy chainpromoter.30

Of importance for future clinical application of this tech-nology will be the use of well-characterized transgenic cellclones with defined transgene integration through zinc fingeror transcription activator-like effector (TALE) nuclease,31 forinstance, in safe harbor sites such as adeno-associated virusintegration site 1,31 thereby diminishing risks of insertionalmutagenesis and carcinogenesis. Importantly, it must be as-sessed whether the required level of radiation can be reduced:Although the applied 123I dose is on a level similar to thatclinically applied for standard tumor therapies, it is far higherthan for routine thyroid diagnostics, thereby prohibitingclinical application of the developed approach at this stage.Furthermore, and although the conducted minimal invasivecell application by intramyocardial hiPSC injection is of highclinical relevance, it must be evaluated whether similar oreven higher sensitivities and signal-to-noise ratios can beobtained in case of global tracer delivery (eg, through the earveins of the pig).

As a first application of the established technology for SPECTimaging of transgenic NIS expression, we have addressed thecommon problem of low cell survival and retention rates aftercell transplantation.8–10 Typically, after intramyocardial cellinfusion, the majority of donor cells are directly flushed backthrough the injection channel10,32 or do not survive the initialphase after transplantation.9 To address this problem, Laflammeet al8 recently described a novel prosurvival cocktail that candramatically increase engraftment of transplanted human embry-onic stem cell–derived cardiomyocytes in an athymic rat model.We have now investigated whether cotransplantation of humanMSCs that release antiapoptotic factors33 and express immu-nomodulatory properties34 may improve the survival andengraftment of transplanted NISpos-hiPSCs after xenotrans-plantation into pigs. Interestingly, SPECT imaging suggesteda positive effect of the cotransplanted human MSCs even at 3hours after cell infusion, showing a markedly stronger traceruptake at the sites where NISpos-hiPSCs had been cotrans-planted with human MSCs than at the injection sites of pureNISpos-hiPSCs. It is questionable whether this very earlyphenomenon can be explained through antiapoptotic effects.It is more likely that other factors, including MSC-mediatedincreased cell aggregation, which may favor engraftment anddiminish the flushing out of transplanted cells, are responsiblefor this effect. However, in the mid and long terms (5 daysand 12–15 weeks), NISpos-hiPSCs could be detected exclu-sively at the site of coinjection, indicating an additionalprofound effect on long-term engraftment of NISpos-hiPSCs.Certainly, deciphering the underlying mechanisms will re-quire extensive additional investigations but will be of utmostimportance to optimize MSC-supported survival of hiPSCderivatives.

ConclusionsThis is the first study demonstrating the usefulness of the NISfor serial long-term tracking of survival, engraftment, anddistribution of cellular grafts in a large-animal model inwhich SPECT/CT imaging was used. Moreover, for the firsttime we demonstrate long-term survival and differentiation of


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hiPSCs in a preclinical pig model of myocardial infarction.The applied 3-dimensional hybrid imaging protocol enablescombined assessment of cardiac anatomy and myocardialperfusion and monitoring of donor cell survival, proliferation,and distribution within 1 imaging modality.

AcknowledgmentsWe are grateful to Birgit Feilhauer, Kristina Mayer, and Julia Osipovafor technical assistance with cell production and labeling; ChristineDePasquale and Eliane Fischer for help with in vitro studies; Paula Grestfor assistance in sectioning tissues; Kati Zlinszky for assistance inimmunohistochemistry; Michele Sidler for excellent animal anesthesia;Maja Muller for assistance in animal care; and Hanspeter Fischer forassistance in NOGA mapping.

Sources of FundingThe study was supported in part by a grant of the Swiss NationalResearch Foundation (Sonderprogramm Universitare Medizin;33CM30–124112/1) and by research grants of the Swiss Life Founda-tion and the Gottfried and Julia Bangerter-Rhvner Foundation as well asby the Cluster of Excellence REBIRTH, German Ministry for Educationand Science (01GN0958), CORTISS Foundation, Austrian ResearchFoundation (grant N211-NAN), and Adult Stem Cell ResearchFoundation.

DisclosuresNone.

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SJ, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R. Genetic engineering ofhuman pluripotent cells using TALE nucleases. Nat Biotechnol. 2011;29:731–734.

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CLINICAL PERSPECTIVECardiac cell replacement therapies may significantly extend current therapeutic options for various cardiac diseases. Therecently developed induced pluripotent stem cells are considered a major breakthrough with respect to the development ofnovel regenerative therapies and combine the advantages of adult and embryonic stem cells, namely, the availability of anautologous, ethically nonproblematic cell source with high potential for proliferation and differentiation into all celllineages of interest. However, evaluation of novel cellular therapies in preclinical large-animal models and patients iscurrently hampered by the lack of suitable imaging approaches that allow long-term monitoring of viable transplanted cells.The present study was therefore designed to evaluate sodium iodide symporter transgene imaging as a novel approach tofollow human induced pluripotent stem cell derivatives in a pig model of myocardial infarction. For the first time, our studydemonstrates the usefulness of a sodium iodide symporter transgene for longitudinal in vivo tracking of survival,engraftment, and distribution of cellular grafts in a large-animal model with the use of single photon emissiontomographic/computed tomographic imaging. Moreover, for the first time we demonstrate long-term survival anddifferentiation of human induced pluripotent stem cells in a preclinical pig model of myocardial infarction. The applied3-dimensional hybrid imaging protocol enables combined assessment of cardiac anatomy and myocardial perfusion andmonitoring of donor cell survival, proliferation, and distribution within 1 imaging modality. The developed approach willcontribute to further optimization of novel cardiovascular cell–based treatment strategies and is of utmost importance forcareful in vivo monitoring of associated risks such as potential tumor or teratoma formation.


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Imaging - Circulation

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