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Journal of the American College of Cardiology Vol. 48, No. 10, 2006© 2006 by the American College of Cardiology Foundation ISSN 0735-1097/06/$32.00P

OCUS ISSUE: CARDIAC IMAGING State-of-the-Art PaperMolecular and Stem Cell Imaging

maging Stem Cells Implanted in Infarcted Myocardiumong Zhou, PHD,* Paul D. Acton, PHD,‡ Victor A. Ferrari, MD†hiladelphia, Pennsylvania

Stem cell–based cellular cardiomyoplasty represents a promising therapy for myocardialinfarction. Noninvasive imaging techniques would allow the evaluation of survival, migration,and differentiation status of implanted stem cells in the same subject over time. This reviewdescribes methods for cell visualization using several corresponding noninvasive imagingmodalities, including magnetic resonance imaging, positron emission tomography, single-photon emission computed tomography, and bioluminescent imaging. Reporter-based cellvisualization is compared with direct cell labeling for short- and long-term celltracking. (J Am Coll Cardiol 2006;48:2094–106) © 2006 by the American College of

ublished by Elsevier Inc. doi:10.1016/j.jacc.2006.08.026

Cardiology Foundation

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oronary heart disease accounts for 36% of all cardiovascu-ar death and is the leading cause of heart failure in the U.S.1). Although post-infarction survival rates have been im-roved in recent years, cardiac dysfunction due to segmental

oss of ventricular mass remains a major problem (2). In fact,rogressive left ventricular remodeling occurs in betweenne third to one half of survivors of acute myocardialnfarction with ejection fractions �40% (3), despite optimal

edical therapies, none of which is able to effectively reversehe detrimental remodeling process (4). Even when im-lantable cardioverter-defibrillators are used, 5-year mortal-ty from ischemic cardiomyopathies exceeds 35% (5). Al-hough cardiac transplantation is the most effective therapy,he disparity between organ demand and supply limits itspplicability. A novel treatment strategy often referred to ascellular cardiomyoplasty” includes local (intramyocardial,ntracoronary) and systemic (intravenous) delivery of fetal/eonatal cardiomyocytes (6,7), skeletal myoblasts (8–10),mbryonic stem cells (11,12), bone marrow-derived mesen-hymal stem cells (BMSCs) (13–19), or hematopoietic stemells (20–22). This strategy attempts to enhance cardiacunction by repopulating the infarcted region with viableardiomyocytes and, therefore, bears great promise forestoration of ventricular function after infarction beyondhat achievable with current medical therapy (23).

More than 6 phase I clinical trials have been conductedsing skeletal myoblasts (24) (for review, see Menasche25]); these trials identified a proarrhythmic risk associatedith myoblast implantation and required implantation of an

nternal cardioverter-defibrillator in patients participating in

From the *Departments of Radiology and †Medicine (Cardiovascular), Universityf Pennsylvania, and ‡Department of Radiology, Thomas Jefferson University,hiladelphia, Pennsylvania. This research was supported by NIH grants R01-S048315 (to Dr. Acton), R21-EB002473 (to Dr. Zhou), and R01-HL081185 (tor. Zhou) and by the Pennsylvania State Department of Health Tobacco Blockrant (to Dr. Zhou).

cManuscript received June 20, 2006; revised manuscript received August 14, 2006,

ccepted August 14, 2006.

he ongoing MAGIC (Magnesium in Coronaries) trial.linical trials using bone marrow cells have demonstrated the

afety and feasibility of the procedure but have yielded mixedesults in terms of therapeutic benefit (Table 1). Althoughignificant improvement of cardiac function has been doc-mented in a few trials (26–29), results from 4 double-linded randomized and placebo-controlled trials are not asromising, either showing moderate (30), short-term ben-fits (31), or no improvement (32,33). Attempts to mobilizeematopoietic stem cells (CD34�) from the bone marrowy granulocyte-colony stimulating factor also yielded am-iguous results. Whereas a small-scale FIRSTLINE-AMIFront-Integrated Revascularization and Stem Cell Libera-ion in Evolving Acute Myocardial Infarction by Use ofranulocyte-Colony-Stimulating Factor) trial showed im-

rovement of left ventricular function (27), negative resultsere obtained from a large-scale double-blinded random-

zed and placebo-controlled trial, REVIVAL (Regenerateital Myocardium by Vigorous Activation of Bone Marrowtem Cells) (34). Issues such as selection of low-riskatients (35) and microvasculature obstruction have beenroposed to explain the negative results. However, thelimination of infused cells from the heart might have been

primary culprit. Studies showed that less than 3% ofnfractionated bone marrow cells were retained in thenfarcted heart within a few hours after intracoronarynfusion (36). Both cell type and delivery route (i.e., intra-oronary, intramyocardial, or intravenous) have been showno affect the retention of cells in the heart (36,37). Unfrac-ionated bone marrow cells used in many trials containarious cell populations, some of which may have adverseffect on cardiac repair (38). Critical issues raised from theselinical trials that need to be addressed include 1) the fate ofngrafted cells and optimization of cell survival; 2) theptimal cell type; and 3) mechanisms of action (25,38–41).xperts in the field questioned whether sufficient pre-

linical data have been gleaned to understand pharmacoki-

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2095JACC Vol. 48, No. 10, 2006 Zhou et al.November 21, 2006:2094–106 Tracking Stem Cells by Imaging

etics, pharmacodynamics, and mechanism to provide theype of solid support on which clinical trials are built (38).maging-based cell-tracking methods can potentially evalu-te the short-term distribution of infused cells (discussed inhe direct labeling method section) or their long-termurvival (discussed in the reporter gene approach section)nd cardiac differentiation status (see the section “Opticalmaging”). Therefore, these methods would play an indis-ensable role in detailed preclinical studies to optimize theell type, delivery methods, and strategies for enhancing cellurvival. Safety and ethical issues imposed on a human studyould limit the clinical translation of those methods that

equire extensive manipulation of cells; however, most directabeling methods discussed in the sections “Magnetic Res-

able 1. Clinical Trials Using Autologous Bone Marrow Cells

Clinical Trial Name(Reference)

Patients(n)

OPCARE-AMI (26,28) 20 SignificantMI pati

STAMI* (33), preliminary results (30) 100 No improvmonths

OOST* (31) 60 No improvpatients

TEMI* (32) 66 No improvfunction

IRSTLINE-AMI (27) 30 G-CSF–inLVEF aG-CSF

EVIVAL* (34) 114 G-CSF–insize, LVG-CSF

EPAIR-AMI* preliminary results (30) 204 A 3% impinfusion

ACT (29) 18 Significantchronicmononu

Abbreviations and AcronymsBMSC � bone marrow-derived mesenchymal stem cellD2R � dopamine type 2 receptorECFP � enhanced cyan fluorescent proteinFluc � firefly luciferaseGFP � green fluorescent proteinHSV1-tk � herpes simplex virus type 1 thymidine kinaseMLC2v � ventricular myosin light chain 2MPIO � micrometer-sized superparamagnetic iron

oxideNIS � sodium-iodide symporterPET � positron emission tomographySPECT � single-photon emission computerized

tomographySPIO � superparamagnetic iron oxideTA � transfecting agentTFR � transferrin receptorTK � thymidine kinaseUSPIO � ultrasmall superparamagnetic iron oxide

Denotes double-blinded, randomized, and placebo-controlled trials.FDG � fluoro-2-deoxy-D-glucose; G-CSF � granulocyte colony-stimulating factor; L

nance Imaging” and “Radionuclide Imaging” have alreadypplied in patients or would be suitable for the clinic.

A number of methods are available to visualize cells; ineneral, they can be divided into 2 categories: 1) the directabeling method and 2) the reporter gene approach. Theormer involves using an imaging-detectable probe that cane loaded into cells and would remain intracellular duringracking. This method does not involve extensive manipu-ation of the cells and, therefore, is preferred for clinicalmplementation. It has 2 inherent limitations: labels may beiluted upon cell division, making these cells invisible; and

abels may efflux from cells or may be degraded over time.herefore, this method is suitable for short-term tracking to

nswer the question: “Where do the cells go?” and mayllow monitoring of second or third delivery of cells whenrevious labels disappear. For clinical application, the pri-ary issue is to identify suitable labels that are safe to cells

ven when the intracellular label accumulates to a highoncentration. Contrast media or agents that already haveeen approved for clinical use, such as 2-[F-18]-fluoro-2-eoxy-D-glucose for positron emission tomography (PET)36), [In-111]oxine for single-photon emission computer-zed tomography (SPECT) (42,43), and superparamagneticron oxide (SPIO) particles for magnetic resonance imagingMRI) (43), are among the first being tested in variouslinical trials of cell tracking.

For preclinical applications, in addition to direct labeling,he reporter gene approach is used. This approach involvesnserting a reporter gene(s) into stem cells for the purpose ofracking. Products of reporter gene expression generally cane divided into 3 categories, enzymes, receptors, or trans-orters. Diagrams in Figs. 1A to 1C illustrate how these 3eporters work for radionuclide imaging modalities.

Major Findings

ovement of LVEF and regional wall motion in the infarct zone in acutet 4 months after infusion of bone marrow or blood-derived progenitors.ts of LV function compared with controls in acute MI patients at 6infusion of cells.t of LV systolic function compared with the controls in acute MImonths after intracoronary infusion of cells.t in LVEF in acute MI patients with relatively well-preserved cardiacmonths after injection of cells.

mobilization of mononuclear CD34� cells led to improvement inevention of LV remodeling in acute MI patients at 12 months afterent.mobilization of mononuclear CD34� cells had no influence on infarct

tion, or coronary restenosis in acute MI patients at 4 to 6 months afterent.

ent in LVEF over controls in acute MI patients at 4 months afterlls.ovement in LVEF and increased FDG uptake in the infarcted region inatients at 3 months after intracoronary injection of bone marrowcells.

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2096 Zhou et al. JACC Vol. 48, No. 10, 2006Tracking Stem Cells by Imaging November 21, 2006:2094–106

To prevent loss of the reporter upon cell division, a stableransfection, that is, integration of the reporter gene into cellenome, is necessary; thus, extensive molecular manipula-ion is required (44). Reporter genes would be extremelyseful in assessing survival status of the implanted cellsecause the reporter will be expressed as long as the cells arelive and will be passed to daughter cells upon cell division;his approach has been used to monitor the uncontrolledrowth of embryonic stem cells into a tumor (teratoma)hen a large number of cells are implanted, as shown inigure 1D. If a cardiac specific promoter is used to control

he expression of the reporter (46,47), then the cardiacifferentiation of stem cells in their physiological environ-ent also can be monitored. The reporter gene approachould allow selection for optimal cell type and engraftment

ites that promote survival or differentiation of implantedells.

This review describes methods for cell tracking based onheir corresponding imaging modalities: MRI, radionuclideomographic imaging techniques focusing on PET andPECT, and optical imaging. For each imaging modality,irect labeling methods as well as reporter gene approaches areiscussed, in addition to their utility in clinical and preclinical

igure 1. Diagrams of detection of (A) enzyme-based (e.g., herpes simpleype 2 receptor [D2R]), and (C) transporter-base (e.g., sodium-iodide sym-18–labeled tracers or by single-photon emission tomography using I-123edica). In A, 9-[3-fluoro-1-hydroxy-2-(propoxymethyl)]guanine tracers t

hereas unbound ones will diffuse out of cells. In B, tracers binding to D2ill be washed out. In C, tracers will be transported into and out of cells

hrough organification (see the section “Radionucleotide Imaging” for deillion (107) murine embryonic stem cells transfected with a truncated vers

ositron emission tomography was performed at day 4 and weekspropoxymethyl)]guanine was injected intravenously for visualization of Heceiving cells, but control animals had background activities only. Quantifirom week 2 to week 4, corresponding to proliferation of embryonic stem cao et al. [45] with permission).

ell tracking applications. i

AGNETIC RESONANCE IMAGING

uperparamagnetic iron oxide particles. Superparamag-etic iron oxide particles are well known for their ability toenerate MR contrast. For diagnostic MRI, 2 acronyms aresed for iron-based magnetic particles: SPIO particles andltra-small SPIO (USPIO) particles; both consist of arystalline iron oxide core coated with polymers such asextran, polyethylene glycol, and starch; SPIOs, with a hydro-ynamic diameter �30 nm, are taken up by the reticuloen-othelial system in the liver and spleen, leading to remark-ble signal loss in these tissues. The USPIOs (�30 nmiameter) can escape the initial uptake by liver and spleennd reach other targets, such as lymph nodes (48), and canerve as a blood pool agent (49). They are obtained fromPIOs by size exclusion or other fractionation procedures;ompared with SPIOs, USPIOs have lower T2 relaxivitiesnd longer blood half life, both as the result of their smallerarticle size. Synthesis of SPIO and USPIO particles, theiragnetic properties (e.g., relaxivities) and clinical applica-

ions are reviewed recently by Lawaczeck et al. (50).Two compounds within the SPIO family have been ap-

roved for clinical use: one compound under the name oferidex (Berlex Biosciences, Ricmond, Virginia; or ferumox-

type 1 thymidine kinase [HSV1-tk]), (B) receptor-based (e.g., dopaminer [NIS]) reporter genes detected by positron emission tomography usinginted from Acton and Zhou [44] with permission from Edizioni Minervae bound and metabolized by the HSV1-TK will be trapped inside the cells,cell surface will contribute to the imaging signal, whereas unbound ones

S; cells from nonthyroid tissues, however, cannot retain the iodine inside(D) In vivo assessment of cell survival and proliferation over time. Ten

f HSV1-tk were injected into the myocardium of a noninfarcted nude rat;, 3, and 4. Approximately 1 mCi [F-18]9-[3-fluoro-1-hydroxy-2--tk–expressing cells. Positive signal was observed at week 1 in animals

n of imaging signals showed a drastic increase of thymidine kinase activityto intracardiac and extracardiac tumors, that is, teratoma (reprinted from

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he name of Resovist (or SH-U555A; Schering, Berlin, Ger-any) in Europe and Japan. Both compounds are manufac-

ured for intravenous administration and are targeted at (met-static) tumors in the liver and spleen.

Besides their role in clinical diagnostic imaging, SPIOsnd USPIOs have become a useful tool for cell tracking inhe brain (51–53), heart (15,54–56), and other organs57,58). A recent clinical trial in European Union demon-trated for the first time in humans tracking of therapeuticendritic cells labeled with Feridex (43). Although it rep-esents a clinically applicable method to label cells usingeridex or other SPIOs approved for clinical imaging (59),

his approach will require U.S. Food and Drug Adminis-ration approval. Furthermore, when injected intravenously,eridex or other SPIOs is primarily taken up by the

eticuloendothelial system (e.g., Kupffer cells), which me-abolizes the excess iron; nonphagocytic stem cells, however,ay not have this capability; therefore, effects of iron

verloading on stem cell proliferation and differentiationhould be investigated before the clinical application oferidex or other SPIOs.Endocytosis is a major mechanism for intracellular uptake

f SPIO particles into various types of cells such as T cells60) and macrophages (61). Transfection agents (TAs) suchs poly-L-lysine (59), cationic liposome (62), or protamineulfate (63) forms complexes with SPIO particles in theabeling solution and greatly improve the efficiency ofndocytosis in cells that cannot avidly phagocytose SPIOs.n addition, these agents seemed to protect the cells fromPIO particles, which otherwise were found to be toxic toonphagocytic cells at high concentrations (62). Other wayso enhance endocytosis-mediated intracellular accumulationf SPIO particles include covalent conjugation of SPIOs tontibodies or to cell penetrating peptides such as HIVransactivator (tat) peptide (58,64) or proteins (65).ntibody-linked SPIOs enter into cells via receptor-ediated endocytosis (57,66) whereas an adsorptive endo-

ytosis (67) is thought to mediate the uptake of tat peptide-onjugated SPIOs. Metabolic fate of SPIO particles arexpected to be similar after endocytosis-mediated uptake.s demonstrated in the case of SPIO-TA complexes, aftereing endocytosed, they are localized to endosomes (68,69),hich are later fused with lysosomes, where the SPIO isegraded (68). The long-term viability, growth and apopto-ic indexes of Feridex-TA–labeled human BMSCs wereound unaffected as compared with the unlabeled cells (70).

owever, it is controversial over whether the differentiationf BMSCs was affected by SPIO particles or by the type ofA used: it has been shown that chondrogenesis of BMSCsas inhibited by Feridex-poly-L-lysine labeling (71) but noty Feridex-protamine labeling (72). No studies on effects ofPIO labeling on cardiac differentiation of stem cells haveeen reported so far.In addition to endocytosis, the electroporation procedure

73) has been investigated to achieve instant intracellular
elivery of SPIOs, therefore eliminating the need for c
ransfection agents, conjugating antibodies and peptides andell culture procedures all together. Because the SPIOarticles are directly internalized to cytoplasm but notnsulated in membrane vesicles such as endosomes, their

etabolic fate may not be the same as those being endocy-osed.

Recently, micrometer-sized SPIO (MPIO) particles (�1o a few micrometers in diameter) have been proposed forore sensitive detection and have been investigated in a

umber of preclinical studies. These particles contain �20%o 60% magnetite by weight and are made by a specialrocess whereby a mixture of magnetite and polystyrene/ivinyl benzene is dispersed in water, and the suspension isolymerized to trap the magnetite in the polymer matrix;he magnetite is dispersed throughout the beads. Initiallyeveloped for magnetic cell separation (74), MPIO particlesre approximately 35-fold larger in diameter and more than0,000 times larger in volume than the USPIOs; by far,hese particles have the highest sensitivity for MRI detec-ion (17,69,75,76).

The detectability of labeled cells depends on a number ofactors, including magnetic field strength, how much ironccumulates inside each cell (labeling efficiency), the num-er of cells implanted, relaxivity of the IO particle, andpatial resolution of the image. Feridex-labeled stem cellsere readily detected by MRI when 107 to 108 stem cells

ach containing approximately 16 pg of Fe (59) werenjected directly into myocardium of a swine as demon-trated in Figures 2A and 2B, reprinted from Kraitchmant al. (15). For stem cells labeled with MPIO particles, anfficiency of 0.2 � 0.18 ng iron oxide per cell (equivalent to43 � 130 pg Fe per cell) was reported by Hill et al. (77);ote that cellular iron oxide content in this work is indirectlyuantified through fluorescence label of the particle; detec-ion of 105cells implanted into a pig heart was reported at a.5-T scanner as shown in Figure 2C, reprinted from Hill etl. (77).

Using SPIO labels, the detection of a small number ofells or single cells by MRI has been achieved in the rat andouse brain (53,78,79). In contrast, detection of single or

mall numbers of cells in the heart by MRI is an evenreater challenge because of cardiac motion and limitedntracellular concentration of imaging probes. For T2/T2*-ased MRI detection, the signal loss at the boundary ofeart and lung as the result of susceptibility difference alsoould pose an interference as demonstrated in Figure 2D:

he T2* values of the lateral myocardium wall were very closeo those of labeled cells at the injection site. Injectionserformed under X-ray, NOGA cardiac mapping system, orR guidance will ensure the mid-myocardium location of

mplanted cells; however, potential cell migration or wallhinning may push these cells in close proximity to thepicardial side of the lateral wall.erritin and transferrin receptor. Although iron is an

ndispensable participant in numerous physiological pro-
esses and biochemical reactions in the body such as

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ransportation of oxygen in the blood, free iron ions areoxic to cells, and the body has an elaborate set of mecha-isms to bind iron in various tissue compartments. Ferritin

s such a metalloprotein and is found mainly in the liver,hich can store approximately 4,500 iron ions in a hollow

hell made of 24 identical subunits. Ferritin genes have beenroposed as a reporter gene for MRI detection: throughransfection by an adenoviral vector, overexpression oferritin genes inside cells leads to intracellular iron accumu-ation and MR contrast as the result of shortening of T2/T2*80,81).

Transferrin is a plasma protein for iron transport. Eachransferrin molecule has the ability to carry two iron ionsFe3�) and is taken up by a transferrin receptor (TFR) onhe cell surface; consequently, iron molecules are trans-orted inside the cell. An engineered TFR (82,83) has beenroposed as a reporter gene; this TFR gene expresses anpproximate 5-fold excess of transferrin receptors on the cellurface and lacks the iron regulatory region; therefore, TFRxpression is not regulated by intracellular iron concentra-ion; for TFR overexpressing cells to accumulate sufficientron for imaging, transferrin proteins are covalently linkedo USPIO particles and the complex is injected intrave-ously; gradient echo MRI then detects the signal loss (ashe result of T2/T2* decrease) induced by uptake ofransferrin-USPIO by cells overexpressing TFR. Comparedith the ferritin reporter system, the requirement for cova-

ent attachment of USPIOs to transferrin is cumbersomeut increases the detection sensitivity because of the largerarticle size of USPIOs compared with ferritin. Iron over-

oading due to the overexpression of a mutated TFR thatacks the iron regulatory region may disrupt cellular metab-lism, leading to toxicity as evidenced by elevated reactivexygen species level in the cell (84). The feasibility of usingerritin and the TFR reporter system for cell tracking in theouse brain at 1.5-T and 7-T magnetic field strength has

ecently been investigated (84).adolinium chelates. In contrast to the negative image

ontrast (hypointensity) generated by USPIO/SPIO/MPIOarticles by T2/T2* mechanism, gadolinium III [Gd(III)]

igure 2. Detection using magnetic resonance imaging (MRI) at 1.5 T,yocardium of a swine; MRI was performed 24 h after injection. (B) Mag

15] with permission). (C) Detection using MRI at 1.5 T of micrometer-mplanted in the myocardium of a swine (reprinted from Hill et al. [77] wcale immediately to the right; T2* values of micrometer-sized superparamt the heart/lung boundary. Obviously, the reduction of T2* at myocardiuentricle; RV � right ventricle.

roduces positive contrast (enhancement) on T1-weighted c

R images. Although the relaxivity is very similar amonghe various small molecular Gd(III) chelates currently inlinical use, they differ in 2 structural features: ionic versusonionic and linear versus macrocyclic (85), for example,dDTPA (Magnevist; Berlex Biosciences) is a linear and

onic chelate, whereas gadoteridol (ProHance; Bracco-Bykulden, Konstanz, Germany) is a nonionic and macrocyclic

helate. Because of their relatively low longitudinal relaxiv-ty (4 mmol l�1s�1), high intracellular accumulation is theey for the utility of these contrast media as labeling agents.t has been estimated that the number of internalizedd(III) chelates on the order of 107 to 108 per cell is

ecessary for MRI visualization of cells (86). The structuraleatures mentioned previously may determine their ability toenetrate the cell membrane and potential toxicities to theells exposed to high concentrations during incubation. Ashe result of the nonparticulate nature of the Gd chelates,inocytosis (used by cells for absorption of extracellularuids) instead of phagocytosis would be a major mechanismor their cellular uptake; recent studies show that pinocytosisppears to be a very effective way for labeling stem cells byncubating cells with high concentrations of Gd(III) che-ates for a period of a few hours (87). Compared withinocytosis, which is a form of endocytosis (88) and inhich Gd(III) chelates are sequestered in membrane vesi-

les, electroporation procedure results in intracellular distri-ution of Gd molecules and a greater longitudinal relaxationate constant (R1) and higher MR signal intensity of labeledells compared with pinocytosis (89). A class of macrocyclicanthanide chelates has been synthesized that label cells bynserting 2 hydrophobic alkyl chains into cell membranesith the hydrophilic metal binding site facing the extracel-

ular medium (90). By association only with the cell mem-ranes, these labels are unlikely to be metabolized by cellularnzymes that lead to release of toxic free Gd ions; further-ore, Gd chelate is more accessible by extracellular water

esulting in a higher relaxivity.-19 – based labeling agents. In addition to the nuclei of-1, some other nuclei, including F-19, P-31, and C-13

lso have MR phenomena and can be detected; the multinu-

ridex-labeled mesenchymal stem cells (107 to 108 cells) injected into thetion of view inside the yellow box in A (reprinted from Kraitchman et al.superparamagnetic iron oxide–labeled mesenchymal stem cells (105 cells)rmission). (D) The color map corresponds to T2* values indicated on thec iron oxide–labeled cells are very close to those of lateral myocardial wall

air interface is a source of interference for detection of cells. LV � left

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or spectrometers); for clinical scanners, capability of nu-leus other than H-1 is also available as optional packages.he gyromagnetic ratio of F-19 nuclei is approximately 90%f the proton; therefore, it is a sensitive nucleus for MRetection; furthermore, there is no background F-19 signalecause F-19 is not a natural metabolite in mammals. Thenatomical localization of the F-19 signal is achieved byverlaying F-19 images on H-1 images acquired sequen-ially. F-19-based compounds such as perfluoropolyetherave been used for tracking dendritic cells in animals (91).reliminary results on F-19 MR imaging of stem cells

abeled with perfluoro-nanoparticles are promising (92,93).

ADIONUCLIDE IMAGING

ecause of their exquisite picomolar (10�11 to 10�12 mol/l)ensitivity (94), PET and SPECT imaging modalities areble to detect tracer quantity of radioisotopes for studyingiological processes in living subjects. Technological devel-pments of both PET and SPECT have led to the imple-entation of specialized systems for small animal imagingith much greater spatial resolution (1–2 mm) (95–103),hich has dramatically advanced the field of cell tracking in

nimal models in vivo.adioisotope-based cell labels. Radioactive isotopes such

s [In-111]oxyquinoline (oxine) (42) and [T-99m]examethylprophylene amine oxime (104) have been used inhe nuclear medicine clinic for labeling autologous whitelood cells, which are subsequently infused back to theatients for localization of inflammatory sites. In general,adioisotopes with a relatively long decay half-life are usedo track cells during a period of several hours or even days,

igure 3. (A) In vivo single-photon emission computed tomography (SPEdual-energy window detects simultaneously Tc-99m (pseudo-colored inagnetic resonance image (grey) (reprinted from Shen et al. [113] with k

f heart slices obtained after In-111 and Feridex double-labeled stem cellsf iron for localization of stem cells (reprinted by permission of the Son-111–labeled bone marrow–derived mesenchymal stem cells to infarctedPECT imaging is shown. For each panel, sagittal (left) and coronal (right)
f cells in the lung is indicated by strong In-111 signal in the lung (reprinted fright ventricle.
or instance In-111 (T1/2 � 2.8 days) for SPECT andu-64 (T1/2 � 12.7 h) for PET (105). The isotope is

arried into the cells via a lipophilic chelator, which governshe initial extraction of the tracer into the cells. Once insidehe cells, a trapping mechanism reduces the lipophilicityf the molecule, and the isotope is retained. After a shortncubation period, the cells are washed to remove anynbound activity and are injected into the host. Labelingfficiencies close to 100% are not uncommon, althoughellular concentrations of the isotope depend on factors,ncluding cell type, incubation time, and concentrations. Forell tracking in the heart, [In-111]oxine (106–110), [In-11]tropolone (111), [Tc-99m]exametazime (112), andF-18]fluoro-2-deoxy-D-glucose (36) have been used. Theiodistribution of In-111-labeled stem cells after intrave-ous (106), intramyocardial, intracoronary, and interstitialetrograde coronary venous delivery has been reported (37):t was found that most delivered cells were not retained inhe heart for each delivery modality, which raised theoncern that proangiogenic or other physiological effectsonveyed by these cells may have a negative impact onissues or organs that are not the target of these cells.

A dual isotope SPECT study of In-111-labeled cardio-yoblasts and [Tc-99m]sestamibi enabled the imaging of

oth cells and perfusion deficit in the infarcted regionimultaneously (109); the SPECT images were registeredith the high-resolution MR images, which provides de-

ailed myocardial anatomy for localization of cells, as shownn Figure 3A (113); the autoradiographic confirmation ofn-111 signal with the histological staining of stem cells areresented in Figures 3B to 3D. As a step further from

imaging of In-111–labeled stem cells implanted in the infarcted rat heart;w) and In-111 (blue) signals; the SPECT images were coregistered on a

ermission of Springer Science and Business Media). (B) Autoradiographsmplanted in the infarcted heart of a rat; (C and D) Prussian blue stainingof Nuclear Medicine from Zhou et al. [109]). (E and F) Homing of(dog) after intravenous injection at day 1 (E), day 2 (F) and day 7 (G) byof fused SPECT (color) and CT (grey) images are shown. Initial retention

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etection of a large number of cells implanted in the heart,he monitoring of stem cell homing using SPECT has beenchieved in small animals using a Gamma camera (108) andn large animals using a clinical SPECT scanner (110). Ashown in Figures 3E to 3G, after being doubly labeled byn-111 and Feridex, BMSCs were intravenously injectednto a dog model, and their homing to the heart wasetected by the use of SPECT; however, the use of MRIas not able to detect these small numbers of cells.lthough the minimal number of In-111-labeled cells that

an be detected in the heart in vivo is not known, a recenttudy using phantom suggested that 10,000 cells could beetected in a 16-min scan with appropriate backgroundorrection (111).

One concern of radioactive labeling agents is the potentialadiation damage to the cell as demonstrated in a study inhich hematopoietic progenitor cells were labeled with

In-111]oxine and injected into the cavity of the leftentricle heart in a rat model of myocardial infarct (108);lthough gamma imaging revealed homing of the progeni-or cells to infarcted myocardium, significant impairment ofroliferation and function of labeled cells was observed. Aecent study by Jin et al. (111) comparing In-111 incubationctivity and cell viability at day 14 suggests that 100%iability can be achieved provided the incubation activity is0.9 MBq.Rapid efflux of labels out of cells over the course of time

105,109) leads to label loss from viable cells. In a SPECTtudy using [In-111]oxine-labeled cardiomyoblasts (109),ne third of the original In-111 signal was obtained 72 hfter injection when corrected for radio decay of In-111.abel loss introduces errors to the detection of survivingells because one cannot distinguish whether reduction ofn-111 signal is due to loss of labels from viable cells or isue to death or remove of cells from the injection site.herefore, the direct labeling approach is more suitable for

he short-term tracking of injected cells whereas a reporterene approach, discussed next, would allow monitoring ofurvival of implanted cells over a longer period of time.eporter genes for PET/SPECT detection. A number of

eporter genes have been developed for PET and SPECTmaging (44,114). Herpes simplex virus type 1 thymidineinase (HSV1-TK) or its mutant form HSV1-sr39tk (115)xpresses a viral thymidine kinase (note that tk representshe gene whereas TK represents the protein). Thymidineinase phosphorylates a range of substrates, including thy-idine (the natural substrate), analogs of pyrimidine, and

cycloguanosines. The monophosphates resulted from TKeaction are converted by cellular enzymes to di- andriphosphates, which are trapped inside the cells, ashown in Figure 1A; in contrast, cells not expressing TKill not metabolize or retain the tracer. A variety ofyrimidine analogs and acycloguanosine derivatives, e.g.,-[3-Fluoro-1-hydroxy-2-(propoxymethyl)]guanineFHBG), have been designed for better tracer retention and
etection by PET and SPECT imaging of HSV1-tk ex- b
ression (see review by Tjuvajev et al. [116]). The metab-lites of TK, if present at a sufficiently high concentration,esult in cell death; therefore, HSV1-tk has been used as auicide gene in many gene therapy protocols (117–119) or asnegative selection marker to eliminate TK-expressing cells

n standard molecular biology protocols. By taking advan-age of the high sensitivity of PET or SPECT imagingodalities, it is possible to administer tracer quantities of

he substrate labeled with a positron or single-photonmitting radioisotope for detection of HSV1-tk expressionnd, as such, toxicity to TK-expressing cells should beegligible: the feasibility of utilizing HSV1-tk as a reporterinstead of a suicide) gene has been demonstrated (120–27). HSV1-tk reporter gene has been examined in preclin-cal studies for evaluations of gene therapy directed at the

yocardium (128,129).The concept of reporter gene is applicable to cell tracking.

urviving cells can be tracked when a promoter of viralrigin (e.g., the promoter of cytomegalovirus [CMV]) issed to control the expression of the reporter gene; such aeporter gene is constitutively active as long as the cell islive and is minimally regulated by physiological processesn the cell. Positron emission tomograpic imaging detectionf cardiomyoblasts (H9c2 cells) that are transiently trans-ected with CMV-HSV1-tk and were subsequently injectednto rat myocardium has been demonstrated (130). How-ver, to monitor the survival or proliferation of implantedells over the course of time, it is necessary to stablyntegrate the reporter gene into genomes of implanted cells.table transfection will ensure that the reporter will not beiluted upon cell division (45). However, the reporterrotein, when it is of exogenous origin and is exposedutside cells, may induce immune reactions from the host,eading to elimination of reporter signals.

Dopamine type 2 receptor (D2R) is a receptor-basedeporter gene and is one of the few systems that could besed for cell tracking in the brain because the reporterrobe, for instance, [F-10]fluoroethylspiperone (FESP),eadily crosses the intact blood-brain barrier. It binds to the2R with high affinity and has been shown to provide a

uantitative measure of D2R expression in living animals131). To overcome the potential problems that the occu-ancy of the ectopic D2R by the endogenous agonist mayncrease levels of cellular cyclic adenosine monophosphatecAMP), leading to physiologic consequences on the targetissue/cells (132), mutant strains of the D2R that do notctivate the signal pathway have been developed for PETmaging (133). In the brain, the presence of endogenous

2R, particularly in the striatum, would provide a largeackground signal and make it difficult to resolve theresence of the D2R reporter system. A similar problemay apply to the myocardium, in which expression of a

etectable level of D2R is reported (134).The sodium-iodide symporter (NIS), as shown in Figure

C, has been proposed as a reporter gene (135–137) and has
een applied to cardiac imaging (138,139). The NIS occurs

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aturally in high concentrations in the thyroid, with loweroncentrations in salivary glands, stomach, thymus, breast,nd other tissues. The symporter is a membrane glyco-rotein and provides an active transport mechanism forodium and iodine ions into cells. The expression of theymporter can be imaged with radioactive iodine, such as-123 for SPECT or I-124 for PET, or other tracers, whichind to the same site, such as [Tc-99m]pertechnetate. Aroad range of cell types and delivery mechanisms have beensed with the NIS reporter. The NIS is not immunogenic,nd its endogenous expression is limited to a few tissues,llowing it to be used in a variety of imaging applications.owever, the use of NIS as a reporter system has been

ampered somewhat by the efflux of the radiotracers fromonthyroid tissues. In the thyroid, organification of the

odine occurs, catalyzed by the enzyme thyroperoxidase,hich effectively traps the iodine after it is transported into

he tissue. Coexpression of this enzyme with the NISeporter gene may be required to improve the retention ofadioactive iodine in transfected cells (140).

An additional benefit of the reporter gene approach is theossibility of linking a therapeutic gene to the reporter,llowing the system to monitor both cell trafficking andene therapy simultaneously (141). For example, skeletalyoblasts have been used to deliver a therapeutic gene, such

s vascular endothelial growth factor, which may aid in theormation of new vessels in the region being regenerated byhe grafted myoblasts (142). Coupling a reporter gene totem or progenitor cells would allow tracking of not only cellelivery and survival, but also the expression of the thera-eutic gene.A major disadvantage of reporter gene approaches for cell

racking is the requirement for molecular manipulations ofhe cells under study. When a virus or a viral vector is usedor transfection, immune reactions may be induced as aesult of the expression of viral proteins or even the reporterrotein itself when nonmammalian proteins, such as HSV1-tk,re used, leading to loss of long-term expression of the reporter.entiviral vector is preferred for stable integration of re-orter genes into stem cells because it can transfect bothividing and nondividing cells and lead to high levels ofransgene expression (143,144). Other potential concernsegarding long-term efficacy of the method include reporterene silencing, in which reporter gene expression decreasesver time with progressive cell differentiation. Lentiviralectors (145) and certain pharmacologic interventions (146)ave been proven to reduce the gene silencing problem.

PTICAL IMAGING

uminescence and fluorescence. Two optical imagingechniques, luminescence and fluorescence, have been usedor stem cell tracking in vivo (147–151). Luminescence inhe form of light (wave length in the range of 400–700 nm)s generated from an enzymatic reaction catalyzed by, e.g.,
refly luciferase (Fluc); when cells expressing Fluc are p
upplied with D-luciferin, a substrate of the enzyme, cellsill emit luminescence. Although weak, this light energy

an be captured by a sensitive charge-coupled device cameran a light-sealed box. In addition to Fluc, other luciferases,uch as Renilla luciferase with its substrate, coelenterazine,ave also been utilized as reporter system (152,153). Be-ause of limited tissue penetration of luminescence and theact that luciferase is a nonmammalian protein, this imagingodality is limited to small animal models. In addition,

uminescence detection relies on a continuous wave tech-ique (154), leading to a diffusive image on the animalurface, unlike PET/SPECT/MR imaging modalities, inhich data are acquired in a tomographic fashion. Still,

uminescence imaging is extremely useful for high-hroughput screening because of its high sensitivity and itstraightforward imaging procedures. Increasing the amountf substrate (e.g., D-luciferin) for an enzymatic reactione.g., catalyzed by Fluc) will increase the amount of theroduct (e.g., luminescence) even when the concentration ofhe enzyme (Fluc) remains the same; therefore, the detec-ion threshold of the luciferase reporter system can beignificantly reduced by administering an excess amount of-luciferin, which does not appear to be toxic to animals

ven in large doses.Fluorescence is generated by excitation of a fluorophore;

reen fluorescent protein (GFP) and its variants are theost commonly used optical reporters. Tomographic detec-

ion of fluorescence is feasible; however, because of highhoton absorbance and scattering in the visible range, onlyear infrared (�700 nm) fluorophores can be detected when

ocated a few centimeters underneath the surface. Shallowenetration is a severe limitation for in vivo detection evenn small animals, not to mention in humans. Attachment offluorophore to a stem cell would allow detection of stem

ells by histology using conventional microscopes. Furtherevelopments in optical imaging techniques, such as quan-um dots (155), diffuse optical tomography (156,157), andptical coherence tomography (158), offer tremendous en-ancements to currently available technology; their applica-ions, however, are primarily limited to preclinical re-earches.tem cell differentiation monitored by cardiac-specific

eporter genes. A reporter gene permits longitudinal mon-toring of fundamental biological processes (e.g. differenti-tion) within the context of physiologically authentic envi-onments. Monitoring of cardiac differentiation of stemells by reporter genes is made possible when the reporter isxpressed as a consequence of a cardiac specific genexpression. In practice, this monitoring is achieved by usingromoter sequence of the cardiac gene to drive the expres-ion of the reporter, rendering the latter under the tran-criptional control of the former. Promoters of ventricularyosin light chain 2 (MLC2v) and cardiac alpha myosin

eavy chain gene have been used to convey cardiac speci-city to reporter genes (159,160). Meyer et al. (46) used the
romoter sequence of MLC2v gene to drive the expression

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f enhanced cyan fluorescent protein (ECFP, a variant ofFP); when stem cells are transduced with MLC2v-ECFP

eporter, their cardiac differentiation as determined byxpression of MLC2v gene was found to be parallel to theetection of ECFP fluorescence (46). Cardiac specific GFPeporter (or its variants) would be extremely useful forcreening or selection of stem cells undergoing cardiacifferentiation by fluorescent microscopy or flowcytometry161). To monitor stem cells undergoing cardiac differen-iation in vivo, luminescent (e.g., Fluc) or NIS reporternder cardiac specific promoters would be suitable; in vivouminescent imaging of MLC2v-Fluc expression in the

ouse heart has been achieved (47), suggesting that thispproach is feasible for monitoring cardiac differentiation oftem cells in small animals. Gamma camera and micro-PETmaging of cardiac alpha myosin heavy chain–NIS expres-ion in the heart in a transgenic mouse model has beenemonstrated recently (139); this reporter gene is moreelevant to clinical application than Fluc because of theonimmunogenic nature of NIS and the availability oflinical counterparts of the imaging modalities.

ENSITIVITY AND SPATIALESOLUTION: MULTIMODALITY APPROACH

ell tracking demands high sensitivity and spatial resolutionrom imaging modalities because of the microscopic targets,hat is, stem cells. It is generally thought that opticalmaging has the greatest sensitivity: the detectable concen-ration of the tracer ranges from 10�15 to 10�17 mol/l; thiss followed by PET and SPECT imaging (10�11 to 10�12

ol/l) whereas MRI is thought to have the lowest sensitivity10�5 mol/l) (94). The concentration-based sensitivity as-umes the target size is comparable with the image resolu-ion: a 1-cm resolution (equivalent to 1000 �l voxel size) isommon for a clinical PET scanner whereas submilimeteresolution (voxel size ranging from 1 �l to 10�3 �l)outinely is obtained on a clinical MRI scanner. When amall number of cells or single cells are the target, themaging probe is concentrated in a small volume fraction ofvoxel; as such, the aforementioned assumption is no longeralid. An estimation of detection limit that includes theontribution of spatial resolution recently was proposed byeyn et al. (79,162) and leads to the conclusion that the

etectability of SPIO-labeled single cells would be similar toicro-PET detection of a few hundred cells labeled with

Cu-64]pyruvaldehyde-bis(N4-methylthiosemicarbazone).owever, to the best of our knowledge, detection of a single

r a small number of stem cells in the heart has not beeneported likely because of various technical challenges dis-ussed in the section “Super Paramagnetic Iron Oxidearticles.” Recent results on detection of MPIO-labeledacrophages that were homed to the heterotypically trans-

lanted heart (76) shed promising light on this issue.A multimodality reporter system expressing a trifusion

rotein consisting of red fluorescent protein, Renilla lucif- v

rase, and the HSV1-TK enzyme (153) offers the possibilityf using the particular imaging technique that best suits thepplication: fluorescence for microscopy of cells or for cellorting in vitro, bioluminescence for high sensitivity imag-ng in vivo in small animals, and PET or SPECT whenuantitative accuracy is important, or tomographic localiza-ion is necessary. The use of multiple imaging modalitiesould take advantage of high-detection sensitivity providedy optical and radionuclide imaging and, at same time,uperior spatial resolution afforded by MRI or other high-esolution modalities (e.g., CT). The feasibility of localiza-ion of stem cells detected by highly sensitive PET orPECT imaging to the anatomical context described byRI has been demonstrated in Figure 3A and also in

igures 3E to 3G, in which monitoring of stem cellsoming to the heart has been demonstrated using a multi-odality approach to achieve high detection sensitivity and

igh spatial resolution. Because in vivo imaging also plays aignificant role in evaluation of cardiac functions, for exam-le, global and regional contractile functions, perfusion, andiability, multimodality imaging would combine stem cellracking with evaluation of functional recovery resulted fromellular cardiomyoplasty.onclusions. The clinical promise for stem cell therapy in

schemic heart disease will not be fully attained without anmproved understanding of stem cell biology and mecha-isms of repair and regeneration. Early work in cardiacellular therapy has focused on more fundamental topics,ncluding assessing stem cell survival and optimizing im-lantation techniques; however, rigorous hypothesis testingnd clinical applications will rely heavily on noninvasivemaging techniques to guide these investigations. Theevelopment of imaging tools that allow serial assessment ofhe subject with high spatial resolution and the ability toontemporaneously determine cellular viability representne of the current goals for these applications. Opticalmaging techniques provide high spatial resolution andermit tracking of stem cells but are limited to preclinicalse. Nuclear techniques, including reporter genes and directellular radiolabeling, afford very good detectability butore limited spatial resolution. Magnetic resonance imag-

ng methods permit good spatial resolution but limitedetectability. A multimodality approach using combinedET or SPECT and MRI agents may ultimately proveost useful in clinical settings.In the September 21, 2006 issue of the New England

ournal of Medicine, the results of 3 clinical trials involvingtem cell therapy for myocardial infarction were released.he studies reported variable results with respect to im-rovement in left ventricular function following cellularherapy. In the Schachinger et al. (163) study, a statisticallyignificant—but clinically minimal—benefit in ventriculo-raphic ejection fraction measurement was seen in the groupreated with bone marrow cells at 4 months post-therapy.he ASATAMI trial (164) showed no improvement in left

entricular ejection fraction following injection of bone

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arrow cells, however, infarct size was reduced, and re-ional wall motion was improved in the treatment group. Inhe TOPCARE-CHD trial (165), there was a small (2.9%)mprovement in left ventricular ejection fraction, but theatients in this study had a history of remote, rather thancute, infarctions which makes the findings even moreignificant. These reports underscore the need for a greaternderstanding of the mechanisms underlying stem celliology and cellular reparative therapy, and their potentialses in the post-infarction state.

eprint requests and correspondence: Dr. Rong Zhou, Molec-lar Imaging Laboratories, Department of Radiology, Universityf Pennsylvania, B6 Blockley Hall, 422 Curie Boulevard, Phila-elphia, Pennsylvania 19104. E-mail: [email protected].

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imaging stem cells implanted in infarcted myocardium · stem cell–based cellular cardiomyoplasty...

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