Current Perspectives on Imaging Cardiac Stem Cell Therapy Joseph C. Wu 1 , M. Roselle Abraham 2 , and Dara L. Kraitchman 3 1 Department of Medicine (Cardiology) and Radiology, Stanford University School of Medicine, Stanford, California; 2 Department of Medicine (Cardiology), Johns Hopkins University, Baltimore, Maryland; and 3 Department of Radiology, Johns Hopkins University, Baltimore, Maryland Molecular imaging is a new discipline that makes possible the noninvasive visualization of cellular and molecular processes in living subjects. In the field of cardiovascular regenerative ther- apy, imaging cell fate after transplantation is a high priority in both basic research and clinical translation. For cell-based ther- apy to truly succeed, we must be able to track the locations of delivered cells, the duration of cell survival, and any potential ad- verse effects. The insights gathered from basic research imaging studies will yield valuable insights into better designs for clinical trials. This review highlights the different types of stem cells used for cardiovascular repair, the development of various imaging modalities to track their fate in vivo, and the challenges of clinical translation of cardiac stem cell imaging in the future. Key Words: molecular imaging; cardiovascular disease; stem cells J Nucl Med 2010; 51:1–9 DOI: 10.2967/jnumed.109.068239 Coronary artery disease is a progressive disease with high morbidity and mortality rates in the Western world. After myocardial infarction, the limited ability of the surviving cardiac cells to proliferate renders the damaged heart susceptible to unfavorable remodeling processes and morbid sequelae such as heart failure. For now, heart transplantation is the only viable treatment option for patients with end-stage heart failure. Given the persistent shortage of donor heart organs, stem cell therapy has emerged as a promising candidate for treating ischemic heart disease because it provides a virtually unlimited source of cardiomyocytes, endothelial cells, and other differentiated cell types to be used in all stages of cardiac repair (1,2). Despite the potential of stem cells, several fundamental questions remain unanswered in the field of cardiac stem cell therapy. For instance, what is the long- term fate of the transplanted cells—do they integrate, proliferate, and differentiate? What are the optimal cell type, cell dosage, delivery route, and timing of injection? Thus, the successful introduction of potentially therapeutic stem cells into patients requires concurrent techniques that provide noninvasive assessment of the survival, distribu- tion, and pharmacokinetics of these cells. This review will present an overview of the different stem cells currently being investigated, the different imaging modalities avail- able to track stem cells, the hurdles facing the field, and some perspectives on the future of stem cell imaging. DIFFERENT TYPES OF STEM CELLS There are many potential stem cell sources for myocar- dial repair. The 3 main types of cells are adult stem cells, embryonic stem cells (ESCs), and induced pluripotent stem (iPS) cells. At present, the most clinically applicable cell type is adult stem cells, which include skeletal myoblasts (3,4), bone marrow stem cells (5–7), mesenchymal stem cells (8), endothelial progenitor cells (9), and cardiac progenitor cells (10–14). Autologous skeletal myoblasts were the first cell type to be used clinically for cell-based cardiac repair (3). Skeletal myoblasts are attractive candi- dates because they can be cultured and expanded ex vivo from muscle biopsies, and they survive well after trans- plantation because of their strong resistance to ischemia. Skeletal myoblast transplantation has been shown to pro- vide functional benefit in animal models of infarction (15), but a recent large placebo-controlled, randomized trial in humans did not demonstrate sustained efficacy as defined by the primary endpoint of global ejection fraction (16). Transplantation of bone marrow stem cells has been shown to improve heart function in animal studies (5,17), and no serious complications have been reported in clinical trials to date, but long-term benefit has not been demonstrated consistently (18). Further, the mechanisms by which these stem cells exert their effects remain poorly characterized (1). In particular, the reported capacity for bone marrow stem cells to transdifferentiate into cardiomyocytes and thereby regenerate functional myocardium remains contro- versial (5,17,19–21). Mesenchymal stem cells are another attractive therapeutic candidate because they are capable of multilineage differentiation (22) as well as possessing reported immunoprivilege status (23). In large-animal models, allogeneic porcine mesenchymal stem cells have Received Sep. 22, 2009; revision accepted Dec. 21, 2009. For correspondence or reprints contact: Joseph C. Wu, 300 Pasteur Dr., Grant S140, Stanford, CA 94305. E-mail: [email protected]COPYRIGHT ª 2010 by the Society of Nuclear Medicine, Inc. jnm068239-sn n 2/25/10 IMAGING OF CARDIAC STEM CELL THERAPY • Wu et al. 1S Journal of Nuclear Medicine, published on April 15, 2010 as doi:10.2967/jnumed.109.068239 by on June 19, 2020. For personal use only. jnm.snmjournals.org Downloaded from
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Current Perspectives on Imaging CardiacStem Cell Therapy
Joseph C. Wu1, M. Roselle Abraham2, and Dara L. Kraitchman3
1Department of Medicine (Cardiology) and Radiology, Stanford University School of Medicine, Stanford, California;2Department of Medicine (Cardiology), Johns Hopkins University, Baltimore, Maryland; and 3Department of Radiology,Johns Hopkins University, Baltimore, Maryland
Molecular imaging is a new discipline that makes possible thenoninvasive visualization of cellular and molecular processes inliving subjects. In the field of cardiovascular regenerative ther-apy, imaging cell fate after transplantation is a high priority inboth basic research and clinical translation. For cell-based ther-apy to truly succeed, we must be able to track the locations ofdelivered cells, the duration of cell survival, and any potential ad-verse effects. The insights gathered from basic research imagingstudies will yield valuable insights into better designs for clinicaltrials. This review highlights the different types of stem cells usedfor cardiovascular repair, the development of various imagingmodalities to track their fate in vivo, and the challenges of clinicaltranslation of cardiac stem cell imaging in the future.
J Nucl Med 2010; 51:1–9DOI: 10.2967/jnumed.109.068239
Coronary artery disease is a progressive disease withhigh morbidity and mortality rates in the Western world.After myocardial infarction, the limited ability of thesurviving cardiac cells to proliferate renders the damagedheart susceptible to unfavorable remodeling processes andmorbid sequelae such as heart failure. For now, hearttransplantation is the only viable treatment option forpatients with end-stage heart failure. Given the persistentshortage of donor heart organs, stem cell therapy hasemerged as a promising candidate for treating ischemicheart disease because it provides a virtually unlimitedsource of cardiomyocytes, endothelial cells, and otherdifferentiated cell types to be used in all stages of cardiacrepair (1,2). Despite the potential of stem cells, severalfundamental questions remain unanswered in the field ofcardiac stem cell therapy. For instance, what is the long-term fate of the transplanted cells—do they integrate,proliferate, and differentiate? What are the optimal celltype, cell dosage, delivery route, and timing of injection?
Thus, the successful introduction of potentially therapeuticstem cells into patients requires concurrent techniques thatprovide noninvasive assessment of the survival, distribu-tion, and pharmacokinetics of these cells. This review willpresent an overview of the different stem cells currentlybeing investigated, the different imaging modalities avail-able to track stem cells, the hurdles facing the field, andsome perspectives on the future of stem cell imaging.
DIFFERENT TYPES OF STEM CELLS
There are many potential stem cell sources for myocar-dial repair. The 3 main types of cells are adult stem cells,embryonic stem cells (ESCs), and induced pluripotent stem(iPS) cells. At present, the most clinically applicable celltype is adult stem cells, which include skeletal myoblasts(3,4), bone marrow stem cells (5–7), mesenchymal stemcells (8), endothelial progenitor cells (9), and cardiacprogenitor cells (10–14). Autologous skeletal myoblastswere the first cell type to be used clinically for cell-basedcardiac repair (3). Skeletal myoblasts are attractive candi-dates because they can be cultured and expanded ex vivofrom muscle biopsies, and they survive well after trans-plantation because of their strong resistance to ischemia.Skeletal myoblast transplantation has been shown to pro-vide functional benefit in animal models of infarction (15),but a recent large placebo-controlled, randomized trial inhumans did not demonstrate sustained efficacy as definedby the primary endpoint of global ejection fraction (16).Transplantation of bone marrow stem cells has been shownto improve heart function in animal studies (5,17), and noserious complications have been reported in clinical trialsto date, but long-term benefit has not been demonstratedconsistently (18). Further, the mechanisms by which thesestem cells exert their effects remain poorly characterized(1). In particular, the reported capacity for bone marrowstem cells to transdifferentiate into cardiomyocytes andthereby regenerate functional myocardium remains contro-versial (5,17,19–21). Mesenchymal stem cells are anotherattractive therapeutic candidate because they are capable ofmultilineage differentiation (22) as well as possessingreported immunoprivilege status (23). In large-animalmodels, allogeneic porcine mesenchymal stem cells have
Received Sep. 22, 2009; revision accepted Dec. 21, 2009.For correspondence or reprints contact: Joseph C. Wu, 300 Pasteur
Dr., Grant S140, Stanford, CA 94305.E-mail: [email protected] ª 2010 by the Society of Nuclear Medicine, Inc.
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been shown to reduce infarct size, increase ejectionfraction, and improve myocardial blood flow (24). Thereare ongoing clinical trials using both autologous andallogeneic mesenchymal stem cell transplantation for myo-cardial regeneration (25). Endothelial progenitor cells aretypically defined as cells that show endothelial character-istics, including uptake of Dil-acetylated low-density lipo-protein, and the expression of typical endothelial markerproteins including vascular endothelial growth factorreceptor-2 (VEGFR-2/KDR), endoglin (CD105), von Wil-lebrand factor, and platelet endothelial cell adhesionmolecule-1 (CD31). Endothelial progenitor cells have alsobeen used in clinical trials involving patients with chronicleft ventricular dysfunction, although the beneficial effectwas less than that for bone marrow stem cells (26). Morerecently, several studies have confirmed the presence ofresident cardiac progenitor cells in the myocardium (10–14). These cardiac progenitor cells can be isolated andexpanded ex vivo and can also differentiate into cardio-myocytes, smooth muscle cells, and endothelial cells underthe appropriate culturing conditions. Clinical trials involv-ing cardiac progenitor cells began in 2009.
Besides adult stem cells, another potential source oftherapeutic cells is ESCs. ESCs are capable of pluripotentdifferentiation into all 3 germ layers (ectoderm, meso-derm, and ectoderm), whereas most adult stem cells arecapable only of multipotent or unipotent differentiation(27). ESCs are also capable of unlimited self-renewal,whereas most adult stem cells have a limited capacity todivide and eventually become senescent—a phenomenoncommonly known at the Hayflick limit (28). ESC-derivedcardiomyocytes (29,30) and ESC-derived endothelialcells (31,32) have been shown to improve cardiac func-tion after transplantation in rodent models of myocardialinfarction. However, significant hurdles must be over-come before future clinical trials can take place, becauseof the issues of potential immunogenicity (33,34) andtumorigenicity (35,36), not to mention the ethical andpolitical controversies associated with ESC research inthe United States.
Unlike ESCs, iPS cells avoid the ethical and politicalproblems because they are derived from the patient’s ownautologous cell source (37). iPS cells can be reprogrammedfrom human fibroblasts into an ESC-like phenotype usingdifferent transcription factors such as Oct 4, Sox 2, Nanog,Klf4, Lin 28, and c-Myc as originally described indepen-dently by Yamanaka (38) and Thomson (39). Besides thepatient’s skin cells, other starting cell sources can bekeratinocytes (40), blood (41), or fat stromal cells (42).Similar to ESC-derived cardiomyocytes, iPS cells havebeen differentiated into cardiomyocytes (43), and injectionsof human iPS cells into immunocompetent mice (44) withmyocardial infarction have been shown to improve cardiacfunction. In summary, several cell types exist with potentialfor cardiovascular repair. Molecular imaging will likelyplay an important role in improving our understanding of
their safety and efficacy under a preclinical model andeventually in clinical settings in the future.
IMAGING TECHNOLOGIES FOR TRACKINGSTEM CELLS
Two primary methods have emerged for stem celllabeling using noninvasive imaging. Direct labeling strate-gies using radioactive tracers and iron particles have beenthe most widely adapted for radionuclide imaging andMRI, respectively (45–52). Fewer reports have been pub-lished using nanoparticles such as quantum dots (53,54).The second major method of stem cell labeling uses thetransfection of stem cells to express a protein, receptor, orenzyme that can be detected by noninvasive reporter geneimaging. That technique has been performed primarilyusing SPECT (55) and PET (56–61). However, a fewexamples using MRI (62,63), ultrasound (64), and otherimaging modalities (65–67) have been used in preclinicalstudies. The primary advantage of direct labeling tech-niques is the simplicity and therefore the minimal manip-ulation of the cells that is required. The primarydisadvantage of direct labeling techniques is that the labelcan become physically decoupled from the stem cell suchthat the detection of the label may no longer representengrafted stem cells. In contrast, the primary advantage ofreporter gene techniques is that the reporter gene is usuallydetected only in living cells. In addition, if the cells arerapidly dividing, the reporter gene should be imparted tothe daughter cells such that the stem cells can still bedetected over time in later generations. However, trans-fection of the cells with reporter genes is a more arduousprocess than direct labeling methods.
Each imaging modality has specific advantages anddisadvantages with respect to delivery and tracking of stemcells for cardiovascular applications ( ½Fig: 1�Fig. 1). Radionuclideimaging techniques excel at detecting small numbers ofcells because they lack background signals. However, bothanatomic imaging and interactivity are poor with radionu-clide. Optical imaging techniques are well suited forreporter gene techniques in small animals but suffer fromthe inability to detect cells deep within the body, limitingtheir clinical applicability. MRI provides superb anatomicdetail of soft tissue but lacks the sensitivity to detect smallnumbers of cells. Interventional techniques with MRI arestill in the developmental stages because of the need tocreate MRI-compatible devices. Nevertheless, the lack ofionizing radiation with MRI is another advantage overconventional radiographic and radionuclide techniques.
Although echocardiography provides a safe, noninvasive,and inexpensive method to rapidly evaluate cardiac func-tion, methods to label stem cells for tracking are only nowbeing explored. Contrast-enhanced ultrasound techniquesusing site-specific microbubbles have been applied forimaging angiogenesis and more recently for imaging cellengraftment as well (68). In a recent study, Kuliszewskiet al. transfected bone marrow-derived endothelial progenitor
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cells to express a unique marker protein (H-2Kk) on the cellsurface (64). Through attachment of the monoclonalantibody against H-2Kk onto the outer surface of micro-bubbles, endothelial progenitor cell–targeted microbubbleswere created. In vivo contrast-enhanced ultrasound imagingof endothelial progenitor cells engrafted into the vascula-ture within Matrigel (BD Biosciences) plugs was demon-strated. The real-time interactivity of echocardiography andlack of ionizing radiation favor the development of thisimaging modality for stem cell delivery and tracking.
Similarly, the real-time interactivity of x-ray angiogra-phy has made it the method of choice for minimallyinvasive cardiovascular stem cell therapeutic trials. Butthe high toxicity of most radiopaque contrast agents haslimited the feasibility of stem cell tracking. New emergingstrategies may be able to overcome these problems.However, at present, other imaging modalities as describedabove have been more extensively developed both forpotential translation to the clinical realm and for optimizingstem cell therapeutic regimes.
FIGURE 1. Schematic for noninvasiveimaging of stem cell fate in myocar-dium. Four different techniques includemagnetic particle labeling, radionuclidelabeling, quantum dot labeling, andreporter gene labeling. First 3 tech-niques are considered physical labeling,whereas last technique is consideredgenetic labeling. SPIO 5 superpara-magnetic iron oxide; IFP 5 iron fluo-rescent particles. (Reprinted withpermission of (45).)
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FIGURE 2. (A–C) Detection of 18F-FDG–labeled cardiac-derived stem cells(CDCs) in rat heart by small-animal PET/CT. CDCs were labeled with 74 kBq of18F-FDG per milliliter and injected intra-myocardially after ligation of mid leftanterior descending coronary artery.PET was performed immediately aftercell transplantation. Myocardium(green) was delineated by intravenousinjection of 37 MBq of 13N-NH3. Cells(red) were visualized within perfusiondeficit by PET. Transverse (A), coronal(B), and sagittal (C) image orientationsare shown. (D–F) SPECT/CT of sodium-iodide symporter-transduced CDCs inrat heart. CDCs were transduced withlentivirus expressing sodium-iodidesymporter driven by constitutively ac-tive promoter, cytomegalovirus, andinjected intramyocardially after ligationof mid left anterior descending coronaryartery. SPECT/CT dual-isotope imagingwas performed 24 h after cell trans-
plantation. Myocardium (green) was delineated by intravenous injection of 201Tl. Transplanted cells (red) were identified withinperfusion deficit by SPECT after intravenous injection of 99mTc. Transverse (D), coronal (E), and sagittal (F) image orientationsare shown.
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In all studies of adult stem cell transplantation, the gainin cardiac function, when identified, has been modest, withreported increases of left ventricular ejection fractionversus placebo usually being about 5% (26,69–71). Possi-ble reasons for the marginal benefit are low transplantedcell engraftment and low levels of differentiation intofunctioning cardiac myocytes. Augmenting transplantedcell engraftment could improve long-term functional ben-efit by increased differentiation of stem cells into cardiacmyocytes, increased recruitment of endogenous stem cells,and beneficial effects on surviving cardiac myocytes viaparacrine mechanisms. A combination of fundamentalwork on the determinants of cell engraftment in the acuteand chronic infarct settings, and molecular imaging tech-niques that provide information about cell fate, cardiacfunction, and infarct size, is needed to maximize cardiacregeneration and minimize the risk of complications suchas ventricular arrhythmias (16,72–75). In vitro studiesindicate that cell type, cell number, and the underlyingarchitecture are important determinants of arrhythmogene-sis (76,77). Hence, quantification and localization of cellengraftment using molecular imaging techniques would beuseful in minimizing adverse events in clinical studies ofcell transplantation.
Although MRI, bioluminescence, and nuclear imaginghave been used to track adult stem cells in vivo aftertransplantation, only nuclear and bioluminescence imagingallow quantification of engraftment. Recently, preclinicalbioluminescence imaging studies have been performed forassessment of important clinical questions such as theoptimal timing of stem cell delivery (78), direct comparisonof various stem cell types (79–81), and determination of thetemporal kinetics of bone marrow stem cell homing aftersystemic delivery (67). Bioluminescence imaging, however,is limited to small animals. PET of 18F-FDG–labeled stemcells is an attractive option because PET permits quantifi-cation of engraftment in vivo as well as translation intolarge-animal models and humans. However, because thehalf-life of 18F is about 110 min, this technique can be usedonly to interrogate acute biodistribution and cell retentionafter transplantation. Studies using a variety of techniques,including direct cell radiolabeling, genetic labeling withreporter genes, and real-time quantitative polymerase chainreaction, have revealed that acute myocardial cell retentionwas less than 10% with 48 h irrespective of the cell typeand delivery route (45,46,49,72,82–84). An improved un-derstanding of the determinants and functional conse-quences of varying acute cell retention is needed todesign new, effective cell delivery strategies.
In a recent study, PET of 18F-FDG–labeled cardiac stemcells ( ½Fig: 2�Fig. 2) in a rat model of myocardial infarctionrevealed that large numbers of intramyocardially injectedcells were trapped in the lungs acutely, an effect that was
FIGURE 3. Ablation of teratoma formation with HSVttk asboth PET reporter gene and suicide gene. (A) Immunodefi-cient animals were injected with undifferentiated mouseESCs stably expressing triple-fusion reporter gene construct(Fluc-mRFP-HSVttk). Treatment of control animals withsaline resulted in formation of multiple teratomas by week5. (B) In contrast, study animals treated with ganciclovir (50mg/kg of body weight) for 2 wk showed abrogation of bothbioluminescence and PET signals. (Reprinted with permis-sion of (56).)
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more pronounced during ischemia–reperfusion (85). An-other study using the same animal model indicated thatacute myocardial cell retention could be doubled by de-creasing the ventricular rate with adenosine administrationor by epicardial application of fibrin glue (86). Together,these results suggest that the coronary microvasculature andcontractility play an important role in acute cell retentioneven after intramyocardial cell injections. Future studiesincorporating advances in tissue engineering and PET havethe potential of greatly improving transplanted cell re-tention and possibly the functional consequences of celltherapy.
Important insights into in vivo stem cell biology can begleaned from longitudinal interrogation of cell fate aftertransplantation. A recent study in a rat model of celltransplantation used the human sodium-iodide symportergene as a reporter gene for longitudinal stem cell trackingby SPECT and PET (Fig. 2B) (84). Sodium-iodide sym-porter transports iodine in conjunction with sodium ionsinto cells and is highly expressed in the thyroid, salivarygland, choroid plexus, stomach, and lactating mammary
gland (87) but is not expressed in the heart, thus permittingdetection of transplanted cells expressing this gene by PETor SPECT, after intravenous administration of iodine orpertechnetate (99mTc). The main significance of this studylies in the potential for clinical translation because per-technetate SPECT is a widely available, clinically approvedimaging modality. The principal downside of using thisreporter gene for cell tracking is low signal in the acutesetting after cell transplantation, which could be related toedema at the injection site or impaired energetics in theinjected cells.
As stated previously, pluripotent stem cells (e.g., ESCsand iPS cells) have generated significant interest because oftheir self-renewing capacity and pluripotent potential. Ingeneral, the 3 stages of cardiac development can be broadlycategorized as undifferentiated ESCs, differentiated beatingembryoid bodies, and differentiated ESC-derived cardio-myocytes. In 2006, the initial proof-of-principle study used
FIGURE 4. Imaging fate of transplanted ESC-derived cardiomyocytes and ESC-derived endothelial cells. (A) Human ESC-derived cardiomyocytes stably expressing Fluc-eGFP double-fusion reporter gene were injected into ischemic myocardium ofimmunodeficient SCID mice. Longitudinal bioluminescence imaging showed that signal activity fell drastically within first 3 wk oftransplantation and remained stable thereafter, with no evidence of tumorigenesis. (Reprinted with permission of (29).) (B)Mouse ESC-derived endothelial cells stably expressing Fluc-eGFP double fusion reporter gene were injected into ischemicmyocardium of syngeneic SV129 mice. Longitudinal bioluminescence imaging showed similar pattern of acute donor cell loss,with about 1% signal intensity (relative to day 2) at 8 wk. Control animals injected with phosphate-buffered saline showed noimaging signals, as expected. (Reprinted with permission of (31).) CM 5 cardiomyocyte; EC 5 endothelial cell.
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undifferentiated murine ESCs stably expressing a triple-fusion reporter gene construct with firefly luciferase (Fluc;bioluminescence), monomeric red fluorescent protein(mRFP; fluorescence), and herpes simplex virus truncatedthymidine kinase (HSVttk; PET reporter gene) to track cellfate in vivo (56). Both bioluminescence and PET imagingshowed that undifferentiated ESCs are capable of causingboth intracardiac and extracardiac teratomas (i.e., tumorsconsisting of all 3 germ layers), which can be ablated bytreatment with ganciclovir that targets HSVttk-expressingcells (½Fig: 3� Fig. 3). A follow-up study has shown that injection ofmouse ESC-derived beating embryoid bodies can also leadto teratoma formation with delayed onset (88). Indeed,intramyocardial injections of as few as 100,000 humanESCs have been shown to cause teratoma formation inimmunodeficient mice (36).
Together, these studies indicate that highly purified ESC-derived cardiomyocytes are required to minimize the risk oftumor formation for cell-based treatment of myocardialdysfunction. Several studies have shown that transplanta-tion of ESC-derived cardiomyocytes can lead to improvedcardiac function (29,30,89). However, analysis with bio-luminescence imaging indicates that about 90% of cells diewithin the first 3 wk of delivery, which may be one reasonwhy only short-term improvement of cardiac function wasobserved ( ½Fig: 4�Fig. 4) (29). Similarly, limited long-term survivalof ESC-derived endothelial cells was seen after injectionsinto both the heart (31) and skeletal muscles (90). Thus, theproblem of donor cell death is particularly troublesome andmay limit the overall efficacy of stem cell–based therapy,making continuing investigations into cell fate monitoringwith new imaging technologies essential.
Another hurdle facing clinical transplantation of humanESCs is the potential immunologic barrier (91). Theimmune response generated after transplantation is directedtoward alloantigens, which are antigens presenting on thecell surface that are considered nonself by the recipientimmune system (27). Solutions that reduce or eliminate thepotential immunologic response to transplanted allogeneichuman ESCs are needed and are reviewed elsewhere (92).Possible strategies to minimize rejection of human ESCtransplants include forming human leukocyte antigen iso-type human ESC-line banks and creating a universal donorcell by genetic modification. In the meantime, immunosup-pressive drugs will be needed. Indeed, the first proposedclinical trial of human ESC therapy involving injections ofdifferentiated neuronal cells into patients with acute spinalcord injury will also involve immunosuppression. However,using longitudinal bioluminescence imaging analysis,Swijnenburg et al. have reported that the single-drug reg-imen with mycophenolate mofetil, sirolimus, or tacrolimuswas not effective in preventing rejection of human ESCs inimmunocompetent mice (34). The combination of tacroli-mus and sirolimus was found to prolong human ESCsurvival modestly to about 4 wk. Thus, further investiga-tions are clearly needed in this area, along with thedevelopment of iPS cells, which in theory should avoidthe immunogenicity problem because the cells are derivedfrom the patients themselves (37).
CHALLENGES IN CLINICAL TRANSLATION
To date, most academic centers have focused on de-veloping new methodologies for stem cell labeling andtracking and may lack the resources to make good-manufacturing-practice products or perform extensivesafety and efficacy testing. The U.S. Food and DrugAdministration (FDA) has developed a framework for theregulation of stem cells (93). Many techniques are beingdeveloped using clinically approved radiotracers or contrastagents for labeling stem cells. However, because thecellular product that will be labeled is typically different
FIGURE 5. Bioluminescence imaging after intramuscularinjection in medial thigh of rabbit model of peripheral arterialdisease provides ability to assess cell viability in vivo inx-ray–visible encapsulated mesenchymal stem cells, similarto nonencapsulated mesenchymal stem cells. (Reprintedwith permission of (101).)
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FIGURE 6. X-ray angiogram of peripheral hind limb ofrabbit before intervention (left) and after creation of femoralartery occlusion via platinum coil (black arrow). X-ray–visiblemicroencapsulated stem cells injected intramuscularly inmedial thigh appear as radiopacities (white arrows). Quarter(Q) is used for reference measurements. (Reprinted withpermission of (102).)
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from the FDA-approved application (e.g., 111In-oxine oflymphocytes), or the route of administration (e.g., intra-coronary) may be different, the regulatory hurdles can bequite complex. To obtain an investigational new drugapplication, one would have to seek approval for the stemcell product and also meet the guidelines for radiopharma-ceuticals or contrast agents. Reporter gene transfection ofcells is similarly covered by relevant FDA guidelines thatmust be met.
Beyond adherence to FDA guidelines, there are othermajor hurdles to clinical translation. Frequently, stem cellsfor cardiovascular applications are administered directly tothe myocardium either using a minimally invasive trans-endocardial approach or during coronary artery bypasssurgery. Both techniques could potentially use new devicesto inject the stem cells. Although devices for gene therapyapplications including stem cell delivery have been de-veloped by several small companies, the lack of preclinicalstudies showing high efficacy has tempered the enthusiasmof major vendors to foster development of such devices. Inparticular, the complexity and cost of device developmentcan be prohibitive for new methods such as MRI guidanceor electromechanical mapping.
In addition to the device and stem cell approval processby the FDA, there is the question of which stem cellproducts are to be used in preclinical animal studies todemonstrate efficacy. Because the final product that will beused in patients will presumably be of human origin, shouldthe animal studies be performed using autologous orallogeneic stem cells? Should human stem cells be usedin an immunosuppressed animal, and if so, which specificregimen of drugs should be used? It is possible thatimmunosuppressed animals could yield different resultsfrom those in immunocompetent animals. Furthermore, sofar most cardiac stem cell trials have used interventionaltechniques for stem cell delivery, that is, intracoronary ortransmyocardial routes (7,71,94–97). Thus, whether pre-clinical work using labeled stem cells should focus onanimals large enough to replicate these preferred deliverymethods must also be determined.
To this end, a hybrid technique has exploited microen-capsulation techniques that provide immunoprotection oftransplanted donor cells (98) with x-ray–based deliverymethods for cell tracking. Radiopaque agents can be addedto the microcapsule to enable visualization by x-rayfluoroscopic and CT imaging (99,100). The incorporationof high concentrations of radiopaque agent in the micro-capsule without inducing toxicity or detrimental effects tothe porous microcapsule is an advantage of the system.X-ray–visible microcapsules can then be used to deliverstem cells during cardiovascular interventions (Figs. ½Fig: 5�5 and
½Fig: 6�6) (101,102). Like direct labeling techniques, microcapsuletracking does not indicate whether the stem cells remainviable. Such a technique could be used in combination withreporter gene transfection of stem cells to deliver stem cellsusing conventional x-ray imaging platforms with follow-upexamination by PET/CT or SPECT/CT ( ½Fig: 7�Fig. 7) (103).
CONCLUSION
Although current imaging tools have illuminated differ-ent facets of stem cell biology in vivo, further efforts areneeded by stem cell biologists and imaging experts todevelop, validate, and accelerate progress in this field. Stemcell tracking requires high sensitivity and high spatialresolution; at present, no single imaging modality is perfectin all aspects. Future efforts should continue focusing onthe development of multimodality imaging approachescapable of answering biologically relevant questions andclinical translation.
ACKNOWLEDGMENT
This work was supported in part by R21HL091453-01A1S1 and RC1HL099117.
REFERENCES
1. Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol.
2005;23:845–856.
2. Wollert KC, Drexler H. Clinical applications of stem cells for the heart. Circ
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FIGURE 7. PET/CT reporter gene im-aging of mesenchymal stem cells inporcine heart. Mesenchymal stem cellswere transduced with adenoviruscontaining cytomegalovirus promoterdriving HSVtk reporter gene in vitro,followed by transplantation into porcinemyocardium through left thoracotomy.Cells could then be visualized after9-(4-18F-fluoro-3-[hydroxymethyl]butyl)-guanine injection, seen in this recon-structed image of left ventricle taken 4 hafter intravenous administration of PETreporter probe. Arrows show localiza-tion of cells at injection site in heart.
%ID 5 percentage injected dose; LV 5 left ventricle; T 5 thoracotomy site. (Reprinted with permission of (103).)
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