seeing stem cells at work in vivo

Seeing Stem Cells at Work In Vivo

Amit K. Srivastava & Jeff W. M. Bulte

# Springer Science+Business Media New York 2013

Abstract Stem cell based-therapies are novel therapeuticstrategies that hold key for developing new treatments fordiseases conditions with very few or no cures. Although therehas been an increase in the number of clinical trials involvingstem cell-based therapies in the last few years, the long-termrisks and benefits of these therapies are still unknown.Detailed in vivo studies are needed to monitor the fate oftransplanted cells, including their distribution, differentiation,and longevity over time. Advancements in non-invasive cel-lular imaging techniques to track engrafted cells in real-timepresent a powerful tool for determining the efficacy of stemcell-based therapies. In this review, we describe the latestapproaches to stem cell labeling and tracking using differentimaging modalities.

Keywords Stem cells . Cell labeling . Cellular imaging .

Reporter genes . Nanoparticles

Introduction

Unlike the hydrozoans and planarians, characterized by theirability to remodel tissues and organs after a wound, or am-phibians who can repair a damaged limb in just 70 days, thehuman body is extremely poor at repair and regeneration ofdamaged cells. A possible solution to this problem, stem celltransplantation, was introduced in 1945, the year of the first

atomic bomb explosion inWorldWar II. Leon Jacobson at theUniversity of Chicago demonstrated that protecting micespleens with a lead shield could protect them from the lethaleffects of ionizing radiation on bone marrow. His team furtherreported a similar effect with the mice femur. Jacobson andcolleagues eventually found that protection against ionizingradiation could also be achieved by intravenous bone marrowinfusion [1–3]. Initially, it was believed that this protectionwas due to some unknown humoral factor. However, in the1950s, Barnes and Loutit [4] and Main and Prehn [5] showedevidence that the protection from radiation was the result ofbone marrow stem cells.

Stem cell-based therapies hold great promises in regenera-tivemedicine because of their inherent biological propertiesof plasticity, self-renewal, and migration. Edmund B. Wilsonfirst mentioned the term “stem cell” in his book, The Cell inDevelopment and Inheritance, in 1896. Stem cells can beclassified as (i) pluripotent embryonic stem cells derived fromthe inner mass of the blastocyst, (ii) multipotent adult stem cellsfound throughout the body after development, (iii) oligopotentprogenitor cells, the “mid-point” between stem cells and fullydifferentiated cells, and (iv) induced pluripotent stem cells,somatic cells that have been genetically reprogrammed to anembryonic stem cell-like state. After systemic or local trans-plantation, stem cells may be able tomigrate and differentiate atpathologic sites in the body to produce a therapeutic effect. In1959, the first human bone marrow transplantation by EdwardThomas operated on the concept that infusing bone marrowcould provide hematological reconstitution in lethally irradiat-ed patients with acute leukemia [6]. Since then, several ad-vancements have been made in the field of regenerative med-icine, with some very promising outcomes. There has been arise in clinical trials involving stem cell therapies in the last3 years. The public clinical trials database, http://www.clinicaltrials.gov, shows 290 trials using mesenchymal stemcells (MSCs) in different disease conditions (accessed 06/18/2013, unknown status excluded).

A. K. Srivastava : J. W. M. Bulte (*)Russell H. Morgan Department of Radiology and RadiologicalScience, Division of MR Research, The Johns Hopkins UniversitySchool of Medicine, 217 Traylor Building, 720 Rutland Avenue,Baltimore, MD 21205-1832, USAe-mail: [email protected]

A. K. Srivastava : J. W. M. BulteCellular Imaging Section, Institute for Cell Engineering, The JohnsHopkins University School of Medicine, Baltimore, MD21205-1832, USA

Stem Cell Rev and RepDOI 10.1007/s12015-013-9468-x

http://www.clinicaltrials.gov/


CartiCel, a cell therapy product to repair articular cartilageinjuries in the knee of adults, was launched byGenzyme, USAand approved by the Food and Drug Administration (FDA) in2010 (www.fda.gov; Submission Tracking Number: BL103661). In Europe, ChondroCelect (TiGenix, Leuven,Belgium) was licensed in 2009 by the European MedicinesAgency as advanced therapeutic medicinal product forcartilage regeneration in the knee (www.emea.europa.eu). Inthe USA, the Cardiovascular Cell Therapy Research Network(CCTRN), established by the National Heart, Lung, andBlood Institute (NHLBI), develops, coordinates, and conductsmultiple collaborative protocols to test the effects of stem celltherapy in cardiovascular diseases. The main goal of theCCTRN is to determine whether stem cell therapies are safefor use in humans and will not lead to any adverse events. Thesuccess of any stem cell-based therapy depends on severalcritical issues, such as type of cell, route and accuracy of celltransplant, functionality of transplanted cells (long-term as-sessment), and, above all, how cells interact with the micro-environment after transplantation (short-term assessment).Long-term assessment of transplanted cells may not be rele-vant if the cells do not initially home and engraft. To addressthese questions, it is imperative to monitor transplanted cellsin real time.

While safety has been consistently demonstrated, sustainedtherapeutic benefits may not always be obtained with stemcell-based therapies. Several risks may arise following thesetherapies, including teratoma formation, the potential for ma-lignant transformation, graft-versus-host disease, graft failure,and organ damage [7, 8]. According to the FDA guidelines onstem cell-based therapies, although in vitro assays may indi-cate how the stem cell-based product is likely to functionin vivo, pre-clinical animal studies are needed to monitor thefunction of the cells after transplantation. Such observationsare particularly important for a stem cell-based product thatcontains cells that are not terminally differentiated at the timeof transplantation. These studies should mimic the route andmethod of administration to be used in subsequent clinicalstudies (http://www.fda.gov). Therefore, in vivo tracking oftransplanted cells over time is a crucial step in determining thesafety and efficacy of cell therapy.

Traditionally, histological investigation has been requiredto determine the fate of transplanted cells. However, it ishighly invasive, requires multiple tissue biopsies, and alsolimits our ability to monitor transplanted cells in real time. Inrecent years, the development of in vivo cell imaging tech-niques has significantly improved our ability to non-invasively track transplanted cells in real time. We can nowmonitor distribution, differentiation, and viability of thesecells at the site of injury or elsewhere in the body. Along withthese benefits, other important information about the action ofengrafted stem cells can also be gained by non-invasively

visualizing the changes in the target tissue. For example, theefficacy of transplanted neural stem cells for new myelinationin a dysmyelinated animal model can be detected by diffusiontensor imaging (DTI) [9]. Similarly, the therapeutic outcomeof stem cell therapy for treatment of myocardial infarct can beevaluated by magnetic resonance imaging (MRI) [10], with-out labeling and visualizing cells. Positron emission tomogra-phy (PET) imaging can be used for monitoring myelin repairin the spinal cord [11].

Cell tracking can be useful for the preclinical assessment ofnew cell therapies, the design of clinical trials, and for mon-itoring these therapies in clinical practice. In this review, weprovide an overview of different methods of cell labeling, aswell as discuss the use of different imaging modalities for celltracking and their key strengths and limitations.

Cell Labeling

Labeling or tagging of cells is necessary to track the migrationand differentiation of cells by imaging after transplantation. Acell can be labeled by direct or indirect methods. Directmethods are simple and do not involve genetic modificationof the cell. Indirect cell labeling methods require geneticalteration of the cell to introduce a reporter gene into it.

Direct Labeling Methods

Direct labeling is a commonly used cell labeling method. Inthis method, a labeling agent is introduced into the cells priorto transplantation. The labeling agent is then imaged as asurrogate for the transplanted cells. Depending on the imagingmodality to be used, cells can be labeled with fluorescentsemiconductor nanocrystals or fluorochromes for optical fluo-rescence imaging, paramagnetic and superparamagnetic ironoxide particles (SPIO) for MRI, and radionuclides for singlephoton emission computed tomography (SPECT) or PET.

Direct Labeling for Optical Imaging

In this method, cells are labeled with optical fluorescent probes(fluorochromes). After the transplantation of labeled cells inthe body, the fluorescent probes are excited with light of adefined wavelength and the emitted photons are registeredwith a highly sensitive charge-coupled device (CCD) camera.Organic fluorochromes are affordable, easy to use, and have afavorable toxicity profile. Past approaches have investigatedseveral fluorochromes, including the most commonly usedfluorochromes, cyanine dyes. Organic fluorochromes areprone to photobleaching, an obstacle when longer observationperiods are required. Another major limitation of these fluoro-chromes is the low tissue penetration of light. The visible

Stem Cell Rev and Rep

http://www.fda.gov/

http://www.emea.europa.eu/

http://www.fda.gov/

spectrum emitted by fluorescence has only a limited tissuepenetration of a few hundredmicrometers; thus, fluorochromesaccumulated a few centimeters from the skin may not bedetected. An alternative to conventional, traditional fluoro-chrome probes is semi-conductive, light-emitting inorganicnanocrystals, also called quantum dots (QDs). These are anew group of agents for cell labeling whose emission spectracan be tuned to any desired wavelength [12]. Contrary totraditional fluorochromes, the emission wavelength of QDsdoes not depend on their chemical structure, but rather, onthe size, shape, and chemical composition. These nanoparticlesare highly photostable and preferred over organic fluoro-chromes for multicolor cell labeling because of their highquantum yield, high molar absorption coefficient, broadexcitation spectra, long fluorescence lifetime, and narrowemission spectra. One of the early applications of QDs forcell trackingwas demonstrated by Lei and colleagues in whichMSCs labeled with QDs were systemically injected intoimmuodeficient NOD/SCID beta2 M null mice [13]. Thefluorescence PEG-encapsulated CdSe/ZnS QDs were usedto quantify the tissue distribution of MSCs in different organs.Ohyabu et al. further showed that QDs have no adverse effectson the differentiation of MSCs in vitro or in vivo [14]. In theabove-mentioned studies, histological evaluations wereperformed to track the cells labeled with QDs. A noninvasivetracking of cells is possible with the use of near-infrared QDs.Sugiyama and colleagues showed that MSCs labeled withnear-infrared QDs (QD800) could be tracked noninvasivelyafter transplantation into the ipsilateral striatum of rats withcerebral stroke [15]. Another study discovered that QDs donot affect growth, differentiation, or proliferation of primarynerve cells, mouse embryonic stem cells, or kidney stemcells, and are suitable for short-term cell tracking [8, 16].Recently developed organic far-red/near-infrared dots withaggregation-induced emission have also been demonstratedto be useful as long-term non-invasive cell tracers [17].

Despite the promise of this labeling technique, the mainlimitation with the use of QDs is heavy metal toxicity. QDs areoften composed of atoms from groups II–VI or III–Velementsin the periodic table, such as CdSe, CdTe, InP, and InAs. Thisraises concerns about metal toxicity, and limits the use of theseQDs in humans.

Alternatively, another near-infrared fluorescent lipophilicdye 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanineiodide (DiR), can be used for direct cell labeling and opticalimaging. DiR has been primarily used for macrophage label-ing [18] and also for embryonic stem cell labeling [19]. DiR issafe and typically provides a rapid and stronger labeling [20].The two long 18-carbon chains of DiR dye can enter throughthe cell membrane, resulting in a stable labeling with negligi-ble dye transfer between cells, which has been a significantproblem when obtaining cell specificity [21].

Direct Labeling for Magnetic Resonance Imaging

In this method, cells are labeled with a suitable contrast agent.MR contrast agents contain metal ions that define their relax-ation enhancement properties. These agents alter the relaxa-tion rate of nearby tissue water protons, making them discern-ible on post-contrast MRI. Contrast agents can be introducedto cells by binding to the external surface of the cell membraneby immuno-magnetic linking to an antibody [22, 23] or byinternalization into the cytoplasm through facilitated trans-membrane uptake [24]. In immunomagnetic labeling, contrastagents in general do not enter the cells, and thus, do not affectcell viability. However, there is the risk of contrast agentdetachment from the cell membrane or the transfer of contrastagent to other cells with this method.

The following types of MR contrast agents are generallyused for cell labeling and tracking: (i) paramagnetic agents;(ii) superparamagnetic agents; and (iii) contrast agents thatcontain nuclei other than hydrogen.

Labeling with Paramagnetic Agents

Gadolinium (III)-Based Contrast Agents Paramagnetic agentsare the most widely used contrast agents in the clinical setting.Gadolinium (III) [Gd (III)] chelates, such as gadopentetatedimeglumine pentaacetic acid (Gd-DTPA), are effective para-magnetic contrast agents owing to their seven unpaired elec-trons. These unpaired electrons create a magnetic moment thatincreases the relaxation rates of the surrounding water protonspins and shortens the longitudinal relaxation rate (T1). Thisincreases the signal by creating “positive” contrast on a T1-weighted scan. The clinical safety of paramagnetic contrastagents is largely dependent on the stability of the chelatein vivo. Major factors that determine safety include thermo-dynamics, solubility, selectivity, and kinetics. Gadolinium is ametal of the lanthanide series, which are rare earth elements.The safety of this class of agents is based on the use ofchelates, firmly holding the gadolinium ion and ensuring rapidand complete excretion. If dechelated, the gadolinium ion canbe deposited in organs. Gadolinium has been used to labeldifferent types of stem cells, such as hematopoietic progenitorcells, monocytic cells, endothelial progenitor cells, and MSCsfor cell tracking [25–27]. However the long-term presence ofgadolinium in engrafted cells may affect cell function andhence the therapeutic benefit of cell therapy. For instance,Modo and colleagues found that in rats with middle cerebralartery occlusion (MCAO), transplantation of the neural stemcell lineMHP36 labeled with Gadolinium-Rhodamine Dextran(GRID) had an actual negative therapeutic effect. Over a periodof 1 year, administration of GRID-labeled cells resulted in aslight increase in lesion size compared to untreated MCAO rats[28], a highly undesirable outcome.


The low cellular uptake of paramagnetic contrast agents isthe main obstacle in cellular imaging. Different methods, e.g.,transfection using transfection agents (lipofectin andlipofectamine) [29], or coupling of the contrast agent to amembrane translocation peptide (13-mer HIV-tat peptide)[30], can be used to increase the uptake of Gd (III) chelates.Several advancements have been made in the last severalyears to improve the efficacy of paramagnetic contrast agents.For example, Tseng et al. developed a paramagnetic contrastagent, gadolinium hexanedione nanoparticles (GdH-NP), totrack transplanted stem cells [31]. They found that GdH-NPwas nontoxic to human MSCs and had the ability to sponta-neously internalize in the cells. A liposomal-based Gd nano-particle as a MR contrast agent has also been developed. Thisdual-Gd liposomal contrast agent consists of a core-encapsulated Gd liposome and a surface-conjugated Gd lipo-some. This provides the contrast agent with an even greaterability to deliver a higher concentration of Gd to the cell,resulting in greater signal enhancement [32]. Furthermore,the use of PEGylated ultra-small (mean diameter 5 nm) gad-olinium oxide (Gd2O3) nanoparticles has also been reported toenhance signal. Very small particles provide the optimalsurface-to-volume ratios necessary to reach high relaxivitiesand generate strong positive contrast enhancement on T1-weighted MRI [33, 34].

Paramagnetic Chemical Exchange Saturation Transfer(PARACEST) Contrast Agents A new class of paramagneticcontrast agents is the PARACEST (paramagnetic chemicalexchange saturation transfer) agents. Contrary to conventionalGd-based contrast agents that affect T1 relaxation, these agentsenhance image contrast only at a specific radiofrequencies[35]. Therefore, this technique may make it possible to tracktwo different types of cell populations in the same experiment[36]. Ferrauto and colleagues used two PARACEST agents,Yb- and Eu-HPDO3A, to track two cell populations: J774.A1murine macrophages and B16-F10 murine melanoma cellsthat co-localize in tumor phenotypes. They found that trackingof the two cell types is possible using these two agents. BothPARACEST agents have potential for clinical translation asthey share the same stability and in vivo pharmacokineticproperties of clinically approved Gd-HPDO3A (ProHance)[37].

Manganese (Mn)-Based Contract Agents After Gd, manga-nese (Mn) has been the most studied T1 contrast agent. Mn-enhanced MRI (MEMRI) has been used to study neuronalactivity, and monitor the neuronal tract and neuronal connec-tivity in different animal models [38]. MnO nanoparticles area novel T1 contrast agent for cell labeling and tracking. Theycan be used to detect cells with positive contrast in vivo,and can be combined with iron oxide to detect two cellpopulations simultaneously with opposite cell contrast [39].

Novel mesoporous, silica-coated, hollow MnO nanoparticleshave also been developed as positive T1 contrast agents forthe labeling and MRI tracking of MSCs (Fig. 1). Mesoporoussilica material is known for its excellent stability in aqueoussolution and high labeling efficiency. It also allows easyaccess for water molecules to the magnetic center, whichsignificantly improves the longitudinal water proton relaxa-tion. These MnO nanoparticles proved to be a better positiveT1 contrast for MR cell tracking as compared to conventionalMnO nanoparticle-based contrast agents [40].

Labeling with Superparamagnetic Iron Oxide NanoparticlesOne of the major limitations of paramagnetic agents for celltracking is that theMRI signal generated by these agents is notstrong enough to make small numbers of cells visible. SPIOparticles, however, have a stronger effect on MR reflexivitythan paramagnetic agents [41]. SPIOs are based on magnetite(Fe3O4) or maghemite (γ-Fe2O3) cores embedded and stabi-lized with a hydrophilic shell of dextran [42], siloxan [43],citrate [44], or other polystyrene [45]. The SPIO core containsthousands to millions of iron atoms, which enables the detec-tion of small numbers of cells by increasing the iron concen-tration in the cells. These particles create a large dipolarmagnetic field [46] and signal spin-spin dephasing due tothe local field inhomogeneity induced in water molecule nearparticles [47]. This results in negative contrast on T2-weightedMRI. The unique properties of SPIO were initially utilized tolabel and track transplanted cells in the rat brain [48]. Sincethen, several advancements have been made in this field,including the development of dextran-coated monocrystallineiron-oxide nanoparticles (MION) [42, 49] and the detec-tion of the migration of oligodendrocyte progenitors usingMION [50]. In 2001, it was shown that magnetodendrimer-encapsulated superparamagnetic iron oxide could be used tolabel and track stem cells in vivo [51]. In a phase 1/2 open-safety clinical trial SPIOs were used to track the CNS homingof injected MSCs by MRI in multiple sclerosis (MS) andamyotrophic lateral sclerosis (ALS) patients [52] (Fig. 2).

SPIOs were found to have no effect on the pluripotency ordifferentiation capacity of cells [53, 54]. Clinical trials aboutthe safety and feasibility of in vivo mononuclear cell trackingusing SPIOs (http://www.clinicaltrials.gov; unique identifier:NCT00972946, NCT01169935) demonstrated that SPIOlabeling of human peripheral inflammatory cells does notaffect their viability or function [55]. However, in mice,although the labeling of bone marrow-derived dendritic cellswith SPIO did not affect phenotype or cytokine profiles,overall cell migration was reduced. The reduction in cellmigration might be due to nanoparticle size [56]. SPIOs alsodo not affect the therapeutic efficacy of labeled stem cells aftertransplantation [57] To date, two iron oxide-based agentshave been developed clinically and approved for MR imagingof the liver: ferumoxide (Endorem® in Europe, Feridex®



in USA), developed by AMAG Pharma, with a particle size of50 to 180 nm; and ferucarbotran (SHU555 A, Resovist®),developed by Schering AG, with a particle size of about60 nm. Endorem®/Feridex® consists of SPIO nanoparticlescoated with dextran, while Resovist® consists of SPIOnanoparticles coated with lowmolecular weight carboxydextran.With regard to the safety of these agents when injected I.V. forMRI of the liver, the USA package insert for Feridex® reports anoverall incidence of adverse events of 11/2240 (0.5 %) of thepatientswho received Feridex I.V. These events include dyspnea,other respiratory symptoms, angioedema, generalized urticaria,and hypotension, which required treatment. For ferucarbotran,the overall incidence of adverse events was 7.1 % (75/1053subjects), with vasodilatation and paraesthesia themost commonevent reported (< 2 %). Ferucarbotran was also found to have aunique ability to promote the cell growth of bone marrow-derived hMSCs by diminishing intracellular H2O2 [58].Unfortunately, while these SPIO formulations have been usedoff-label for clinical studies, they are no longer manufactured[59].

To facilitate the internalization of SPIO contrast agents incells, several strategies have been used, such as monoclonalantibody conjugation [50, 60], magnetodendrimers [51],transfection agents [61], and electroporation [62]. Limitedin vitro stability and species specificity are major limitationswith the use of monoclonal antibodies. An anaphylactic shockmay also occur as a result of human immune response toforeign immunoglobulin. Magnetodendrimers, however, canincrease the possibility of cell internalization without trigger-ing an immune response. Embedding iron-oxide nanoparticles

Fig. 1 Manganese (Mn)-basedcontract agents for stem celltracking. a Labeling of MSCswith silica-coated, hollow MnOnanoparticles as MR contrastagent. b No hyperintense signal(red arrow) was detected in amouse transplanted withunlabeled MSCs. c Hyperintensesignals (green arrows) weredetected in a mouse transplantedwith HMnO@mSiO2-labeledMSCs and were still visible14 days after injection.(Reproduced from Ref. 40, withpermission)

Fig. 2 MRI after injection of Feridex®-labeled mesenchymal stem cells. aAn axial T2-weighted gradient echo scan through the inferior thoracic cordshows a hypointense pial signal coating the cord similar to that of super-ficial siderosis, characteristic of Feridex®-labeled cells. b Axial T2-weight-ed gradient echo scan through the cervical cord shows hypointensity of thedorsal roots and their entry zone and a similar hypointensity of the ventralroot entry zones, suggesting the presence of Feridex®-labeled cells.(Reproduced from Ref. 52, with permission)


in carboxyl-terminated dendrimers increases the efficiency ofinternalization and accumulation of magnetodendrimers incellular endosomes. These dendrimers did not affect the via-bility, differentiation, or proliferation capabilities of cells [51].

Another popular and currently most widely used method isthe use of transfection agents. Here, poly-L-lysine or prot-amine sulfate are first multiplexed with SPIOs via electrostaticinteractions to provide the formation of SPIO oligomers with ahighly positive surface charge, which induces endocytosis ofthe transfection agent-conjugated SPIO [24, 63]. Because ofparticle clustering of SPIOs within endosomes, T2* effects aremore pronounced, and, thus, the signal is hypointense [64].This method does not affect the viability and proliferation ofcells, but can inhibit the chondrogenic differentiation of hu-man MSCs when poly-L-lysine is used [65].

To avoid the use of any additional agents with SPIOs, andto achieve an instant intracytoplasmatic magnetic labeling ofcells, another method called magnetoelectroporation (MEP)can be used. In MEP, small-pulsed voltage is used to internal-ize SPIO in the cell [62]. This method does not require anytransfection agent, which makes this method more attractivefor clinical use. This method also provides the ability to labelseveral million cells in a few seconds. This is especiallyhelpful in case of tagging of cells that are prone to morpho-logical changes due to adhesion to the plastic surface of tissueculture dishes. MEP does not affect the viability or prolifera-tion of cells in vivo. Feridex®-labeled mouse neuronal stemcells maintained 98±2 % viability right after a single 130 V,17-ms pulse MEP. These magnetoelectroporated cells werealso able to maintain their proliferative capacity in vivo [66].

One of limitation of SPIO-labeling method is the extracel-lular deposition of the iron label following death of engraftedcells [67] or by active exocytosis [68]. When SPIO-labeledneural stem cells and human pluripotent stem cells weretransplanted into mouse brain, rapid exocytosis of SPIO bylive cells was observed as early as 48 h post-transplantation[68]. Exocytosed and deposited iron particles are scavengedby macrophages and may generate a false-positive signal onMRI, particular when these now indirectly labeled macro-phages migrate away from the injection site. Nevertheless,direct labeling of cells SPIO is well suited for real-timeimage-guided cell delivery [59, 69] and for short-term moni-toring of immediate cell engraftment.

Labeling with Micron-Sized Iron Oxide Particles The sizeand number of iron particles greatly affect the MRI contrastenhancement. Micron-sized iron oxide (MPIO) particles havean approximately three-fold higher iron content per particle(≈100 pg) compared to SPIO. The use of MPIO can signifi-cantly increase the possibility of detecting smaller numbers oflabeled cells or cells with fewer particles. Labeling of only oneor a few MPIO particles per cell is sufficient to produce detect-able signal attenuation within one imaging voxel. MPIOs have

been used forMRI detection of single cells in vivo [70]. Despitethe bigger size, internalization of these particles in cells is easy.Shapiro and colleagues reported that murine hepatocytes canreadily endocytoseMPIOs from 0.96μm in diameter to as largeas 5.80 μm without the need for lipofection [71]. Several typesof cells have been labeled with MPIOs, including hematopoi-etic progenitor cells, MSCs [72], T cells [73], glioma cells [74],macrophages [75], adult neural progenitors [76], and dendriticcells [77]. The viability of all cells remained unchanged afterMPIO labeling. The main limitation with the use of MPIOs,however, is that inert, non-biodegradable polymer polystyreneis used to construct MPIOs. Polystyrene is not FDA-approvedfor use in humans. To overcome this limitation Nkansah et al.developed and characterized biodegradable, superparamagneticmicroparticles of poly(lactide-co-glycolide) (PLGA) and cellu-lose for cell labeling. Both polymers, commonly used for drugdelivery and oral tablet formulations, are FDA-approved for usein humans. PLGA- and cellulose-based particles exhibited veryhigh R2 molar relaxivity. Labeling of MSCs and human breastadenocarcinoma cells with these particles did not result in anydetectable cytotoxicity [78]. As in the case of SPIO, the use ofpoly-L-lysine or coating of MPIOs with –NH2 improves thelabeling efficiency of particles [79].

Labeling with 19F-Rich Perfluorocarbon Particles In additionto 1H nuclei, several other nuclei, such as 31P, 23Na, 13C, and19F, also exhibit MR phenomena. However, 31P, 23Na, 13C donot qualify for cell tracking studies because of their lower ratioof magnetic dipole moment to angular moment (gyromagneticratio) and abundance in the body. A natural absence of 19F atomsin the body and the mere 6 % difference in gyromagnetic ratiowith 1H fluorine makes 19F MRI a promising tool for selectivedetection of labeled cells with MRI [80]. Perfluorocarbon (PFC)nanoparticles have been widely employed within the field ofMRI and have been used for 19F MRI cell tracking of labeledprogenitor, neuronal stem, immune, and dendritic cells [81–86].These nanoparticles have little effect on viability or proliferationand differentiation of labeled cells. However, large particles(560 nm) have been found to alter the immunological propertiesof dendritic cells [87].

PFCs are the most biologically inert and stable C-F polymers.There are no known enzymes that metabolize PFCs in vivo, andPFCs do not degrade at typical lysosomal pH values. PFCs arewater insoluble and do not attach to the cells very easily becauseof their “non-stick” property. For effective labeling of cells, PFCsmust be formulated into a biocompatible nanoemulsion. PFCnanoemulsions can label a wide range of cell types, causing notoxicity or changes in phenotype, morphology, or function. Cellscan be labeled by two methods using PFCs: (i) ex vivo labelingwith PFC nanoemulsions. The labeled cells are tracked using 19FMRI post-transplantation as shown in Fig. 3. This method iscalled ‘in vivo cytometry’ [88] as the fluorine signal is linear toits concentration, allowing quantification of cell numbers. With


this method, a wide variety of cells can be tracked in a cell-specific manner. (ii) Direct injection of PFC-nanoemulsions. ThePFCs are taken up by cells of the reticuloendothelial system,including monocytes and macrophages, post-injection. This ap-proach is mainly helpful in noninvasive visualization of inflam-mation [89] in different disease conditions and to monitor in-flammatory responses associated with graft rejection.

A major disadvantage of 19FMRI is its low sensitivity. Celllabeling with PFCs generally results in a cell loading of 1011–1013 19F atoms per cell, with the levels higher in phagocyticthan in non-phagocytic cells [80, 88, 90]. This uptake of 19Fatoms per cell results in a minimum detection sensitivity ofaround 2,000 cells/voxel at 7 T; however, the detection limit ofSPIO-labeled cells was shown to be 1,000 cells/mm3 at 3 Tand 500 cells/mm3 at 7 T [91]. Another disadvantage of thistechnique is that imaging with PFCs can be about 10-foldslower than with other contrast agents [92]. A combinationof pulse sequences with high SNR per image acquisitiontime may reduce the total imaging acquisition time. Further

studies are required to improve image acquisition time in19F MRI.

Labeling with MRI-Detectable pH Nanosensors The assess-ment of cell death following in vivo transplantation of stemcells is challenging. A new technique for non-invasive mon-itoring of in vivo cell death in capsule hydrogels has beendeveloped usingMRI. In this technique, L-arginine, which is amolecule with multiple exchangeable NH protons, is used as apH-sensitive chemical exchange saturation transfer (CEST)contrast agent. The proton exchange rate with water stronglydepends on the pH, and when pH decreases due to cell death,the exchange rate decreases for base-catalyzed guanidyl NHexchangeable protons in L-arginine, leading to decreases inCEST contrast [93]. Arginine-rich LipoCEST microcapsuleswere synthesized by incorporating an L-arginine-filled lipo-some inside the capsule and using protamine sulfate as anarginine-rich cross-linker in the capsule coating. It wasdemonstrated that apoptotic encapsulated hepatocytes can

Fig. 3 Ex vivo 19F and diffusion MR imaging. A middle cerebral arteryocclusion induces a hyperintense signal on a T2-weighted MR imagecompared to a normal control. In the absence of 19F-labeled cells, nosignal is detected on the 19F scan. The apparent diffusion coefficient(ADC) image shows an increase in diffusion in the area of stroke infarc-tion. Injection of extracellular matrix (ECM) derived from decellularizedtissue affects both the hyperintense T2-weighted signal and the diffusionscan. On the T2-weighted image, the lesion cavity almost completely

disappeared, although the ADC image still exhibits areas of high diffu-sivity. Only 19F-labeled cells can be detected using the 19F channel. It isevident here that injection of the ECM and 19F F-labeled cells extensivelycover the lesion cavity. Transplantation of unlabeled cells with the ECMdid not significantly affect the ADC compared to injection of just ECM,indicating that the ECMwas primarily responsible for significant changesin the lesion cavity’s characteristics. (Reproduced from Ref. 86, withpermission)


be detected with CEST MRI in vivo (Fig. 4). As thesenanosensors are clinical-grade, this technique should betranslatable to the clinic.

Direct Labeling for PET and SPECT

The nuclear imaging modalities PET and SPECT provideimages of the in vivo distribution of administered radionu-clides. These modalities have better clinical potential in that

they have superior tissue penetration capability and are morehighly quantitative than optical imaging [94]. PET andSPECT mainly differ in the radionuclide used and the mech-anism for signal generation and detection. For cell labelingand tracking, different radionuclides, such as 64Cu-PTSMand 18F-FDG (for PET), and 111In-oxine, 99mTc- HMPAO(for SPECT), are widely used [94–96]. Due to their nanomolarsensitivity (PET; 10−11–10−12 M and SPECT; 10−10 M), theseradionuclides are able to measure biological processes at non-

Fig. 4 Use of LipoCEST microcapsules for the detection of apoptoticencapsulated hepatocytes by MRI. a The L-arginine (R) moieties (red)entrapped in liposomes (1) and in protamine (2) have exchangeableprotons that provide CEST contrast. b A mixture of cells, alginate andL-arginine liposomes is passed through a needle using a nanoinjectorpump. The single-layered charged alginate droplets are collected, washedand resuspended in a crosslinker solution (protamine sulfate), followed bya coating with a second layer of alginate. c Phase-contrast image (top)and fluorescent image (bottom ; with NBD-PE (1,2-distearoyl-sn -glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl))-

labelled liposomes) showing a uniform distribution of cells and liposomeswithin the microcapsule. Scale bar=200 μm. d BALB/c mice weresubcutaneously transplanted with 2,500 empty LipoCEST capsules(−Cells), with LipoCEST capsules containing Luc-transfected hepato-cytes while receiving immunosuppression (+Cells/+IS), and with cap-sules containing cells but no immunosuppression (+Cells/−IS). CEST/MTw (magnetization transfer-weighted) overlays using a frequency offsetof 2 ppm at days 0, 1, 7, and 14 after transplantation. (Reproduced fromRef. 93, with permission)


pharmacological doses. For cell labeling, these radionuclidesare introduced into the cells by a lipophilic chelator andsubsequently bind to intracellular proteins, whereas the che-lator leaves the cells. Cells are simply incubated with radio-isotopes in vitro, washed, and transplanted. Labeling efficien-cy varies from 60 to 90 %, depending upon the labelingcondition and cell type [97, 98]. 111In and 99mTc are used asthe “gold-standard” clinical procedure for the detection ofsites of occult infections and inflammation by imaging theleukocyte distribution [99, 100]. 111In-oxine was approved bythe FDA in 1985 for white blood cell (WBC) scintigraphy, andamong the several options, has been used most extensively forstem cell labeling due to its relatively long-decay half-life of2.8 days [101]. In a study in patients with advanced cirrhosis,intravenously administered MSCs labeled with 111In-oxinecould be detected up to day 10-post injection [102]. Thisradionuclide usually does not have any adverse effect on cellviability or function; however, human MSCs have shownmoderate functional impairment after labeling [103]. But,111In-oxine labeling does not affect the stemness of those cells[104]. 99mTc-HMPAO is another clinically used tracer that hasa different biodistribution than 111In-oxine 99. 99mTc-HMPAO-labeled, bonemarrow-derivedMSCswere visualized up to 4 hafter cell transplantation in a rat model of myocardial infarc-tion [105]. However, the trade-off between half-life and long-term exposure to ionizing radiation and a possible transfer ofradiometal to other non-stem cells are the major limitations ofusing 99mTc-HMPAO for cell tracking. Similarly 18F-FDGmay also be used for cell labeling, but the disadvantage ofthe short half-life of 18F (only 2 h) makes this tracer less thanideal for follow-up studies [106].

The detachment and efflux of radioisotopes into the extra-cellular space are obvious disadvantages with this method. Forexample, 111In-oxine showed a significant efflux within 24 hafter labeling [107]. This may cause an inaccurate estimationof the number of labeled cells. However, detached radiotracersare not retained in body for a long period, so pose no apparenthealth risk [108].

Labeling by Microencapsulation

Prevention of transplanted cells from host immune attack andavoidance of immunosuppression by encapsulating cells withbiomaterials, such as an alginate hydrogel, are the primarygoals of microencapsulation. Later, it was realized that differ-ent contrast agents could also be incorporated in the micro-capsule [109, 110]. MRI-visible ‘magnetocapsules’ weremade by modifying the alginate–poly-L-lysine–alginate mi-croencapsulation method to include SPIO. Infusion of these‘magnetocapsules’ through the portal vein of the liver enabledMR–guided, real-time, targeted delivery and imaging of pan-creatic islet cells [69], as well as MRI visualization of encap-sulated hepatocytes engrafted within the abdomen [111].

Simultaneous immunoprotection and trimodal, non-invasivemonitoring of cell engraftment could be achieved by co-encapsulating gold nanoparticles functionalized with Gd-DTDTPA with pancreatic islet cells using protamine sulfate asa clinical-grade alginate cross-linker. Encapsulated cells werereadily visualized with high-field MRI, micro-computed to-mography (μ-CT), and ultrasound imaging, both in vitro andin vivo [112]. Other examples include incorporation of bariumor bismuth sulfate for X-ray imaging [113], perfluorocarbonsfor 19F MRI [114], and trimodal capsule-in capsules [109].

Labeling with Gold and Multimodal Gold and SilicaNanoparticles

An alternative approach for non-invasive stem cell tracking,with high spatial and temporal resolution at sufficient depths,is the use of gold and silica nanoparticles. These nanoparticlesalso provide an advantage over radionuclides, as they can beused for long-term imaging and allow for repeated imagingover time. Gold nanoparticles or nanotracers possess opticallytunable properties and inert characteristics. Nam and col-leagues used gold nanotracers to trace MSCs in vivo withCT and ultrasound-guided photoacoustic (US/PA) imaging[115]. Gold nanoparticles provide sufficient radiopacity tobe detected, and MSCs have been successfully labeled anddetected by CT follow their ex vivo labeling. Loading MSCswith gold nanotracers was found to have no adverse effects oncell function [116]. However, due to the lack of sensitivity andlow contrast-to-noise, CT imaging of stem cells has beenpursued in a limited fashion. This has also provided an alter-native way for their detection by US/PA imaging [115].

Similarly, silica nanoparticles (SiNPs) can also be used asan alternative to MRI-based stem cell imaging. The use ofSiNPs for cell tracking is particularly attractive because theseparticles can be detected by fluorescent, MR, and ultrasoundimaging. Jokerst et al. reported that silica-based nanoparticlescan be used for cell sorting (fluorescence), real-time guidedcell implantation with ultrasound, and high-resolution, long-term monitoring by MRI. They found that SiNPs labelingcould increase the ultrasound and MRI contrast of labeledhMSCs 700 % and 200 % versus unlabeled cells, respectively,and allowed cell imaging in animal models for 13 days afterimplantation. The SiNPs were found to have no effect on theproliferation or pluripotency of hMSCs [117].

Labeling with Microbubbles

Another approach for non-invasive cell tracking is the use ofgas-filled microbubbles (MBs)-based contrast agents. Thesecontrast agents are clinically approved and have been success-fully used to label and track stem cells by ultrasound (US)imaging [118]. Because of the strong signal induced by MBs,a large acoustic impedance mismatch with tissues, a single


MB and thus a single cell can be detected with this imagingmodality [118]. However, the use of US imaging is still in itsinfancy, and further studies will have to demonstrate its overallapplicability in cell tracking.

Indirect Labeling Methods

In this method, target cells are genetically manipulated tointroduce a reporter gene. The reporter gene is translated intoan enzyme, a receptor, or a fluorescent or bioluminescentprotein. The interaction between the substrate and the encodedreporter protein will result in a signal that can be detected non-invasively. Indirect labeling methods have a clear advantageover direct labeling methods in terms of assessment of cellviability and division. The biggest limitation of any directlabeling method is that it does not provide accurate informa-tion about cell viability. In addition, cell numbers increaseafter every cell division, but the numbers of labeled probesremain the same. This results in “dilution” of the signal overtime. Since reporter genes can pass on to the progenies aftercell division, imaging of dividing cells is possible. In addition,in contrast to direct methods, where a probe is present anddetectable even after cell death, a reporter gene does notcontinue to be expressed after cell death, and thus, producesno signal. However, epigenetic gene silencing may affect theexpression of a reporter gene and suppress the signal. Thislimitation can be overcome by treating target cells with DNAmethyltransferase inhibitor.

Reporter genes are currently mainly used for ex vivo stud-ies (beta-galactosidase and alkaline phosphatase) to assess thetrans-gene expression in cells, tissues, and organs [119, 120]by histology. The use of firefly luciferase (for bioluminescentimaging) and green fluorescent protein (for fluorescent imag-ing) genes are initial evidence that reporter genes can be usedto monitor trans-gene expression in living organisms [121,122]. Just like direct labeling methods, indirect labelingmethods can also be used with several different imagingmodalities.

Reporter Genes for Optical Imaging

Optical imaging techniques using reporter genes can be sep-arated into two types: (i) fluorescence and (ii) biolumines-cence. Several reporter gene strategies are based on fluoro-chromes, e.g., green fluorescent protein (GFP- the first fluo-rescent protein gene isolated from Aequorea victoria jellyfish), mutants of the GFP gene emitting blue, cyan, or yellowlight, and fluorochromes with red and far-red spectra isolatedfrom different species [123, 124], have been in use for manyyears. However, a number of issues have been identified within vivo optical fluorescent imaging. As mentioned earlier inthis review, excitation and emission wavelengths of fluoro-chromes have limited penetration in tissues and a poor signal-

to-noise ratio limits the use of fluorochromes in vivo, partic-ularly in deep tissues. Novel technologies, such as diffuseoptical tomography and optical coherence tomography, mayovercome these problems; however, their current use is limit-ed to small animal studies, and further development is neededto transfer these technologies to clinical settings.

In contrast to fluorescence imaging, where an external lightsource excites the fluorochrome, bioluminescence imaging(BLI) is based on the emission of photons in reactions cata-lyzed by luciferase enzymes. Luciferases emit photons duringthe oxidation of a substrate, such as D-luciferin, in the pres-ence of oxygen and ATP. The most commonly utilized lucif-erases for in vivo imaging are Firefly (isolated from Photinuspyralis , the North American firefly) and Renilla (isolatedfrom Renilla reniformis , the click beetle) luciferases. TheFirefly and Renilla luciferase reporter systems, in combinationwith their corresponding luminescent substrates, luciferin andcoelenterazine, have several advantages for small animal im-aging. There is no cross-reactivity between luciferin andcoelenterazine substrates, so both luciferases can be imagedsimultaneously. BLI has been extensively used for the non-invasive in vivo tracking of transplanted stem cells, includingembryonic [125, 126] and neural stem cells [127], in smallanimals. Target cells can be stably transfected with luciferasesgene and be tracked for very long time. This gives an edge tothis technique for long-term sequential studies. For example, aregenerative cell population derived from human subcutane-ous adipose tissue (hASCs), implanted into the peri-infarctregion of a mouse model of myocardial infarction, could betracked over a period of 10 weeks [128]. This is because BLsignal generation is ATP-dependent and BLI can be used tomonitor cell survival post-transplantation. Transplantationsite-dependent survival of neuronal progenitor allografts inthe mouse brain [129] and neuronal progenitor cell survivalwithin hydrogels can be determined in vivo by BLI (Fig. 5)[130]. Recently, BLI has also been used for the non-invasiveimaging of disease progression in muscular dystrophy.Maguire and colleagues monitored luciferase activity in miceexpressing an inducible luciferase reporter gene in musclestem cells. These stem cells proliferated in response to muscledegeneration, thus increasing the level of luciferase expressionin dystrophic muscle [131].

One of the major limitations of in vivo BLI is light absor-bance from hemoglobin, particularly with a high blood-to-tissue ratio wheremaximal emission overlaps with themaximalabsorption of hemoglobin, and selectively transports the sub-strate back to the blood system. Multi-layer anatomical barriersalso limit the emission. The photon emission per cell can bemaximized by using a strong promoter or by using luciferasewith a red-shifted spectrum to overcome some of the limitations[132]. Physiological parameters, such as anesthesia [133]and the route of substrate delivery (i.p. vs. subcutaneous)[134], also affect signal intensity. Volatile anesthetics, such


as isoflurane, sevoflurane and desflurane, have an inhibitoryeffect on the luciferase activity in rodents. Pentobarbital wasfound to have a less inhibitory effect. The possible reasons forthis inhibitory effect might be attributable to the hemodynam-ic effects of anesthetics [135] and the binding of anesthetics tothe domain that regulates the opening and closing of theenzymatic pocket, and to the inhibition of the binding of D-luciferin to luciferase [136]. Concentrations of serum proteinsmay also have negative effects on BLI signal intensity. Forexample, a hypoalbuminemic condition, where levels of albu-min in blood serum are abnormally low, is associated with ahigher BLI signal intensity [137].

Reporter Genes for MRI

High spatial resolution and the ability to gather accurateanatomical and physiological information simultaneously aretwo of the biggest advantages of MRI reporter gene imaging.In addition, unlike the optical reporter gene imaging approachwhere there is a limit on light tissue penetration, there is nolimit on the size of the subject to be imaged as long as it fitsinto the magnet. In the past decade, several MRI reportergenes have been developed and used in neurological, cardiac,and cancer research [138–141]. For MRI reporter gene imag-ing, cells are genetically modified to either increase theiraffinity for a contrast agent, or produce iron-containing pro-teins, or provide an endogenous contrast agent. Based onthe mode of action, MRI reporter gene imaging can be mainly

divided into the following types: enzyme-based; iron-based;and chemical exchange saturation transfer (CEST)-based.

Enzyme-Based MRI Reporter Genes Louie and colleaguespioneered the enzyme-based MRI approach by developing agadolinium-based substrate (EgadMe) that contains a galac-tose group. In the presence of lacZ-transfected cells express-ing β-galactosidase, the galactopyranose moiety is cleaved,which allows increased water molecule diffusion to the gad-olinium, thus increasing the T1 signal [142]. More recently,lacZ-transfected tumor cells, combined with 3,4-cyclo-hexenoesculetinb-D-galactopyranoside and iron, resultedin T2* relaxation on MRI [143]. Another example of theenzyme-based approach is genetically manipulated cellsoverexpressing tyrosinase. Tyrosinase is a rate-limiting en-zyme that controls the production of melanin. Melanin bindsparamagnetic iron ions to produce metallomelanin, and thus,cells overexpressing tyrosinase exhibit high signal intensityon T1-weighted MRI [144]. Some of the potential pitfalls ofthis approach are tissue delivery barriers, falseMR signals dueto the presence of “leftover” galactose, or persistence ofmetallomelanin in the cells, even when the reporter gene isnot activated [145].

Iron-Based MRI Reporter Genes Genetically engineered ironbinding proteins, other than metallomelanin, have also exten-sively been studied for reporter gene MRI. The engineeredtransferrin receptor (ETR) generates contrast by the receptor-

Fig. 5 Detection of in vivo survival of neuronal progenitor cell by BLI(a). BLI of representative animals transplanted with 1×105 neuronalprogenitor cell (ReNcells) with or without helper cells into Rag2−/−

immunodeficient mice show strong BLI signal on the same day aftertransplantation, which decreases progressively over the following days. b

Quantification of BLI signal for each group (n =5 each), with the intensityexpressed as photon/s/cm2/sr. c Estimation of percent donor cell survivalplotted as % signal activity (normalized to day 0) over the 5-day periodfollowing transplantation. (Reproduced from Ref. 130, with permission)


mediated internalization of iron-bound transferrin [146].However, overexpression of etR could lead to iron-catalyzedfree radical formation via the Fenton reaction, and could betoxic to cells [147]. Overexpression of the ubiquitous proteinferritin, which stores and releases the iron to maintain ironhomeostasis, traps excess intracellular iron and causes signif-icant shortening of the T2 relaxation time [148]. Recent stud-ies showed that a ferritin reporter gene could be used to trackstem/progenitor cells in vivo [149, 150]. Differences in tissuetype, molecular aggregation, strength of the MR field, andquantification methods may affect the ferritin-related relaxa-tion rate [151].

Intracellular particles of biogenic magnetite (Fe3O4), syn-thesized by theMagA gene (a putative iron transporter), foundin some fresh-water magnetotactic bacteria of the genusMagnetospirillum sp., also have properties similar to that ofSPIO nanoparticles and can also be used as MRI reportergenes. It was observed that MagA-positive cells show a sig-nificant signal drop on T2*-weighted MRI [152, 153].

Chemical-Exchange Saturation Transfer (CEST)-Based MRIReporter Genes This class of MR reporter genes utilize aprocess called chemical-exchange saturation transfer (CEST).In CEST, applying a saturation radiofrequency (RF) pulse atthe exchangeable proton resonance frequency for a long dura-tion saturates the proton’s magnetization, creating a chemicalexchange. Since these protons constantly exchange with bulkwater protons, they can be detected as a reduction in the waterproton MR signal. Our group has designed a non-metallic,biodegradable, lysine-rich protein (LRP) reporter (containinghigh-density amide protons), which can be successfully used asa MR CEST reporter [138]. The major advantages of CESTcontrast agents are: (i) the CEST contrast is switchable. Thiscontrast is only detectable when a saturation pulse is applied ata specific frequency characteristic of an agent’s exchangeableprotons. This unique feature allows the CEST contrast to beundeletable when a saturation pulse is switched off. Thus, theCEST contrast does not interfere with other MRI contrasts. (ii)The ability to create multiple colors by using a saturation pulse

Fig. 6 Radionuclide imagingusing reporter genes. a Lethallyirradiated C57BL/6 (CD45.2)mice were transplanted withretrovirally transduced 5-FU–enriched HSCs (CD45.1).Animals were monitored forhematopoietic reconstitution overtheir total lifespan. MicroPETscans shown from Left, coronal;Right Upper, sagittal; Center,coronal; and Lower, transverse. b[18F]-L-FMAU at 4 week post-transplant. Reporter signalobserved in hdCK3mut animalsin spleen (S), thymus (T), andbone marrow (BM). Probemetabolism in both cohorts seenin gastrointestinal (GI), bladder(Bl), and kidney (K). c [18F]-FDGMicroPET scan at 4 week post-transplant. Signal unrelated to thereporter was sen in both cohorts inheart (H), spleen (S),gastrointestinal (GI), brain (Br),with metabolism in kidneys (K)and bladder (Bl). d In vivoaccumulation of [18F]-L-FMAUin sorted hematopoietic cells fromhdCK3mut animals. Reporterpositive: CD45.1+, YFP+ andreporter negative: CD45.1+,YFP−. (P<0.05). (Reproducedfrom Ref. 165, with permission)


at different frequencies. This may allow simultaneous MRimaging of more than two target cells [139, 154].

Reporter Genes for Radionuclide Imaging

Radionuclide reporter genes encode for receptors or trans-porters that promote the uptake or accumulation of radiolabeledtracers in target cells. Reporter genes are transferred to thetarget cells via viral or non-viral methods. Herpes simplex virusthymidine kinase type 1 (HSV1-tk) is the most commonly usedradionuclide reporter gene. Thymidine kinase (TK) adds anegative charge to the cell surface by phosphorylatingradiolabeled nucleoside substrates, and thereby preventsthe radiolabeled tracer from exiting the cell. Thus, the traceraccumulates in the cell [155]. HSV1-tk has been used to tracktumor-specific lymphocytes [156], T-cell activation [157],bone marrow MSCs [158], and hESCs [159]. However, asHSV1-tk is a non-human gene, it poses the threat of generat-ing an immune response against the cells. This immunogenic-ity has prevented the routine use of PET reporter genes clin-ically [160]. To avoid immunogenicity, the human nucleosidekinases deoxycytidine kinase (dCK) and thymidine kinase 2(TK2) have been used. Both human kinases have a substratespecificity similar to HSV1-tk [161]. These reporter geneshave been successfully used in mouse models [162, 163]and a tumor patient [164]. A mutant of dCK (hdCK3mut)with three amino acid substitutions within the active site hasbeen used with the PET probe 18F-L-FMAU to track mouseand human hematopoietic stem cells after transplantation forlong-term observation (Fig. 6) [165]. Another disadvantage ofradionuclide imaging is the inability of certain tracers to crossthe blood–brain barrier. This limits the ability to track cellstransplanted intracerebrally. The use of a somatostatin recep-tor, with its ligand, 111In-octreotide [47], a sodium-iodinesymporter [166], or dopamine receptors [167], can be usedimprove the uptake of tracers. These reporter genes have beenused to track neural stem cells [168] and cardiac stem cells[169], and also to monitor cellular differentiation [170]. Aninteresting novel approach has transformed HSV1-tk into anMRI reporter gene. To this end, nucleoside analogues weresynthesized that are rich in imino protons. Upon accumulationof this substrate, that acts as a CESTcontrast agent, implantedglioma cells could be tracked in vivo by MRI [171].

Conclusions

Stem cell-based therapies promises a new mode of care thathas the potential to greatly reduce the burden of disease.Imaging technologies have become a driving force in stemcell research. Extraordinary efforts are currently beingmade to

improve cellular imaging techniques. The biocompatibility ofmolecular probes, temporal resolution, detection thresholds,need for real-time image-guided injection, and cost effective-ness are a few important criteria when selecting the appropri-ate imaging modality for cellular imaging. While optical im-aging techniques have demonstrated a clear advantage inimaging small animals and are firmly established in preclinicalstudies, the use of these techniques is not practically feasiblein bigger animals and humans, given the poor tissue penetra-tion and low spatial resolution of signals. The use of MRIand PET/SPECT in cell tracking has an advantage over opticalimaging in terms of higher resolution and sensitivity. However,these techniques have their own advantages and disadvantages.The first clinical MRI cell-tracking study on humans demon-strated the key advantages of this technique, being that ana-tomical information is provided [172], dictating the need formonitoring the accuracy of cell injections, preferable in real-time [59]. PET, however, may possess more sensitivity, butsuffers from low spatial resolution and lack of anatomicalinformation. Therefore, employing multimodal imaging tech-niques with double- or triple-reporter systems for optical, MR,and PET imaging will greatly facilitate the transition of cellularimaging techniques from the bench to the bed.

Conflict of interest The authors declare no conflict of interest.

References

1. Jacobson, L. O., Simmons, E. L., & Bethard, W. F. (1950). Studieson hematopoietic recovery from radiation injury. The Journal ofClinical Investigation, 29(6), 825.

2. Simmons, E. L., Jacobson, L. O., Marks, E. K., & Gaston, E. O.(1959). Long-term survival of irradiated mice treated with homolo-gous tissue suspensions. Nature, 183(4660), 556.

3. Jacobson, L. O., & Simmons, E. L. (1960). Comparison of theeffects of isologous, homologous, and heterologous hematopoietictissues on post-irradiation survival. Radiology, 75, 6–10.

4. Barnes, D. W., & Loutit, J. F. (1953). Protective effects of implantsof splenic tissue. Proceedings of the Royal Society of Medicine,46(4), 251–252.

5. Main, J. M., & Prehn, R. T. (1957). Fate of skin homografts in x-irradiated mice treated with homologous marrow. Journal of theNational Cancer Institute, 19(6), 1053–1064.

6. Thomas, E. D., Lochte, H. L., Jr., Cannon, J. H., Sahler, O. D., &Ferrebee, J. W. (1959). Supralethal whole body irradiation andisologous marrow transplantation in man. The Journal of ClinicalInvestigation, 38 , 1709–1716.

7. Hatzistergos, K. E., Blum, A., Ince, T., Grichnik, J. M., & Hare, J.M. (2011).What is the oncologic risk of stem cell treatment for heartdisease? Circulation Research, 108(11), 1300–1303.

8. Rocha, V., Wagner, J. E., Jr., Sobocinski, K. A., et al. (2000). Graft-versus-host disease in children who have received a cord-blood orbone marrow transplant from an HLA-identical sibling. Eurocordand International Bone Marrow Transplant Registry Working


Committee on Alternative Donor and Stem Cell Sources. The NewEngland Journal of Medicine, 342(25), 1846–1854.

9. Nair, G., Tanahashi, Y., Low, H. P., Billings-Gagliardi, S., Schwartz,W. J., & Duong, T. Q. (2005). Myelination and long diffusion timesalter diffusion-tensor-imaging contrast in myelin-deficient shiverermice. NeuroImage, 28(1), 165–174.

10. Rickers, C., Gallegos, R., Seethamraju, R. T., et al. (2004).Applications of magnetic resonance imaging for cardiac stem celltherapy. Journal of Interventional Cardiology, 17(1), 37–46.

11. Wu, C., Zhu, J., Baeslack, J., et al. (2013). Longitudinal PETimaging for monitoring myelin repair in the spinal cord. Annals ofNeurology. doi:10.1002/ana.23965.

12. Voura, E. B., Jaiswal, J. K., Mattoussi, H., & Simon, S. M. (2004).Tracking metastatic tumor cell extravasation with quantum dotnanocrystals and fluorescence emission-scanning microscopy.Nature Medicine, 10(9), 993–998.

13. Lei, Y., Tang, H., Yao, L., Yu, R., Feng, M., & Zou, B. (2008).Applications of mesenchymal stem cells labeled with Tat peptideconjugated quantum dots to cell tracking in mouse body.Bioconjugate Chemistry, 19(2), 421–427.

14. Ohyabu, Y., Kaul, Z., Yoshioka, T., et al. (2009). Stable andnondisruptive in vitro/in vivo labeling of mesenchymal stem cells byinternalizing quantum dots. Human Gene Therapy, 20(3), 217–224.

15. Sugiyama, T., Kuroda, S., Osanai, T., et al. (2011). Near-infraredfluorescence labeling allows noninvasive tracking of bone marrowstromal cells transplanted into rat infarct brain.Neurosurgery, 68(4),1036–1047.

16. Rak-Raszewska, A., Marcello, M., Kenny, S., Edgar, D., See, V., &Murray, P. (2012). Quantum dots do not affect the behaviour ofmouse embryonic stem cells and kidney stem cells and are suitablefor short-term tracking. PLoS One, 7(3), e32650.

17. Li, K., Qin, W., Ding, D., et al. (2013). Photostable fluorescentorganic dots with aggregation-induced emission (AIE dots) fornoninvasive long-term cell tracing. Scientific Reports, 3 , 1150.

18. Eisenblatter, M., Ehrchen, J., Varga, G., et al. (2009). In vivo opticalimaging of cellular inflammatory response in granuloma formationusing fluorescence-labeled macrophages. Journal of NuclearMedicine, 50(10), 1676–1682.

19. Ruan, J., Song, H., Li, C., et al. (2012). DiR-labeled embryonic stemcells for targeted imaging of in vivo gastric cancer cells.Theranostics, 2(6), 618–628.

20. Shan, L. (2004). Near-infrared fluorescence 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR)-labeled mac-rophages for cell imaging. Molecular Imaging and Contrast AgentDatabase (MICAD) (Internet). Bethsda (MD): National Centre ForBiotechnology Information (US); 2004–2013.

21. Frangioni, J. V. (2003). In vivo near-infrared fluorescence imaging.Current Opinion in Chemical Biology, 7(1), 626–634.

22. Sykova, E., & Jendelova, P. (2007). Migration, fate and in vivoimaging of adult stem cells in the CNS. Cell Death andDifferentiation, 14(7), 1336–1342.

23. Bulte, J. W., Hoekstra, Y., Kamman, R. L., et al. (2003). Clinicallyapplicable labeling of mammalian and stem cells by combiningsuperparamagnetic iron oxides and transfection agents. Radiology,228(2), 480–487.

24. Frank, J. A., Miller, B. R., Arbab, A. S., et al. (2003). Clinicallyapplicable labeling of mammalian and stem cells by combiningsuperparamagnetic iron oxides and transfection agents. Radiology,228(2), 480–487.

25. Hedlund, A., Ahren, M., Gustafsson, H., et al. (2011). Gd(2)O(3)nanoparticles in hematopoietic cells for MRI contrast enhancement.International Journal of Nanomedicine, 6 , 3233–3240.

26. Agudelo, C. A., Tachibana, Y., Hurtado, A. F., Ose, T., Iida, H., &Yamaoka, T. (2012). The use of magnetic resonance cell tracking tomonitor endothelial progenitor cells in a rat hindlimb ischemicmodel. Biomaterials, 33(8), 2439–2448.

27. Guenoun, J., Koning, G. A., Doeswijk, G., et al. (2012). CationicGd-DTPA liposomes for highly efficient labeling of mesenchymalstem cells and cell tracking with MRI. Cell Transplantation, 21(1),191–205.

28. Modo, M., Beech, J. S., Meade, T. J., Williams, S. C., & Price, J.(2009). A chronic 1 year assessment of MRI contrast agent-labelledneural stem cell transplants in stroke. NeuroImage, 47(Suppl 2),T133–T142.

29. Rudelius, M., Daldrup-Link, H. E., Heinzmann, U., et al. (2003).Highly efficient paramagnetic labelling of embryonic and neuronalstem cells. European Journal of Nuclear Medicine and MolecularImaging, 30(7), 1038–1044.

30. Bhorade, R., Weissleder, R., Nakakoshi, T., Moore, A., & Tung, C.H. (2000). Macrocyclic chelators with paramagnetic cations areinternalized into mammalian cells via a HIV-tat derived membranetranslocation peptide. Bioconjugate Chemistry, 11(3), 301–305.

31. Tseng, C. L., Shih, I. L., Stobinski, L., & Lin, F. H. (2010).Gadolinium hexanedione nanoparticles for stem cell labeling andtracking via magnetic resonance imaging. Biomaterials, 31(20),5427–5435.

32. Ghaghada, K. B., Ravoori, M., Sabapathy, D., Bankson, J., Kundra,V., & Annapragada, A. (2009). New dual mode gadolinium nano-particle contrast agent for magnetic resonance imaging. PLoS One,4(10), e7628.

33. Klasson, A., Ahren, M., Hellqvist, E., et al. (2008). Positive MRIcontrast enhancement in THP-1 cells with Gd2O3 nanoparticles.Contrast Media & Molecular Imaging, 3(3), 106–111.

34. Faucher, L., Tremblay, M., Lagueux, J., Gossuin, Y., & Fortin, M.A. (2012). Rapid synthesis of PEGylated ultrasmall gadoliniumoxide nanoparticles for cell labeling and tracking with MRI. ACSApplied Materials & Interfaces, 4(9), 4506–4515.

35. Ward, K. M., Aletras, A. H., & Balaban, R. S. (2000). A new classof contrast agents for MRI based on proton chemical exchangedependent saturation transfer (CEST). Journal of MagneticResonance, 143(1), 79–87.

36. Aime, S., Carrera, C., Delli Castelli, D., Geninatti Crich, S., Terreno,E. (2005). Tunable imaging of cells labeled with MRI-PARACESTagents. Angewandte Chemie International Edition England, 44(12):1813–1815.

37. Ferrauto, G., Castelli, D. D., Terreno, E., & Aime, S. (2013). In vivoMRI visualization of different cell populations labeled withPARACEST agents. Magnetic Resonance in Medicine, 69 (6),1703–1711.

38. Silva, A. C., & Bock, N. A. (2008). Manganese-enhanced MRI: anexceptional tool in translational neuroimaging. SchizophreniaBulletin, 34(4), 595–604.

39. Gilad, A. A., Walczak, P., McMahon, M. T., et al. (2008). MR trackingof transplanted cells with "positive contrast" using manganese oxidenanoparticles.Magnetic Resonance in Medicine, 60(1), 1–7.

40. Kim, T., Momin, E., Choi, J., et al. (2011). Mesoporous silica-coated hollow manganese oxide nanoparticles as positive T1 con-trast agents for labeling and MRI tracking of adipose-derived mes-enchymal stem cells. Journal of the American Chemical Society,133(9), 2955–2961.

41. Josephson, L., Lewis, J., Jacobs, P., Hahn, P. F., & Stark, D. D.(1988). The effects of iron oxides on proton relaxivity. MagneticResonance Imaging, 6(6), 647–6453.

42. Shen, T.,Weissleder, R., Papisov,M., Bogdanov, A., Jr., & Brady, T.J. (1993). Monocrystalline iron oxide nanocompounds (MION):physicochemical properties. Magnetic Resonance in Medicine,29(5), 599–604.

43. Jung, C. W. (1995). Surface properties of superparamagnetic ironoxide MR contrast agents: ferumoxides, ferumoxtran, ferumoxsil.Magnetic Resonance Imaging, 13(5), 675–691.

44. Wagner, S., Schnorr, J., Pilgrimm, H., Hamm, B., & Taupitz, M.(2002). Monomer-coated very small superparamagnetic iron oxide


http://dx.doi.org/10.1002/ana.23965

particles as contrast medium for magnetic resonance imaging: pre-clinical in vivo characterization. Investigative Radiology, 37(4),167–177.

45. Shapiro, E. M., Skrtic, S., Sharer, K., Hill, J. M., Dunbar, C. E., &Koretsky, A. P. (2004). MRI detection of single particles for cellularimaging. Proceedings of the National Academy of Sciences of theUnited States of America, 101(30), 10901–10906.

46. Hao, R., Xing, R., Xu, Z., Hou, Y., Gao, S., & Sun, S. (2010).Synthesis, functionalization, and biomedical applications of multi-functional magnetic nanoparticles. Advanced Materials, 22(25),2729–2742.

47. Rogers, W. J., Meyer, C. H., & Kramer, C. M. (2006). Technologyinsight: in vivo cell tracking by use ofMRI. Nature clinical practice.Cardiovascular Medicine, 3(10), 554–562.

48. Norman, A. B., Thomas, S. R., Pratt, R. G., Lu, S. Y., &Norgren, R.B. (1992). Magnetic resonance imaging of neural transplants in ratbrain using a superparamagnetic contrast agent. Brain Research,594(2), 279–283.

49. Bulte, J. W., Brooks, R. A., Moskowitz, B. M., Bryant, L. H., Jr., &Frank, J. A. (1998). T1 and T2 relaxometry of monocrystalline ironoxide nanoparticles (MION-46L): theory and experiment.Academic Radiology, 5(Suppl 1), S137–S140.

50. Bulte, J. W., Zhang, S., van Gelderen, P., et al. (1999).Neurotransplantation of magnetically labeled oligodendrocyte pro-genitors: magnetic resonance tracking of cell migration andmyelination. Proceedings of the National Academy of Sciences ofthe United States of America, 96(26), 15256–15261.

51. Bulte, J.W., Douglas, T.,Witwer, B., et al. (2001). Magnetodendrimersallow endosomal magnetic labeling and in vivo tracking of stem cells.Nature Biotechnology, 19(12), 1141–1147.

52. Karussis, D., Karageorgiou, C., Vaknin-Dembinsky, A. et al. (2010)Safety and immunological effects of mesenchymal stem cell trans-plantation in patients with multiple sclerosis and amyotrophic lateralsclerosis. Archives of Neurology, 67(10): 1187–1194.

53. Wang, Y., Wang, L., Che, Y., Li, Z., & Kong, D. (2011). Preparationand evaluation of magnetic nanoparticles for cell labeling. Journalof Nanoscience and Nanotechnology, 11(5), 3749–3756.

54. Nejadnik, H., Henning, T. D., Castaneda, R. T., et al. (2012).Somatic differentiation and MR imaging of magnetically labeledhuman embryonic stem cells. Cell Transplantation, 21(12), 2555–2567.

55. Richards, J. M., Shaw, C. A., Lang, N. N., et al. (2012). In vivomononuclear cell tracking using superparamagnetic particles of ironoxide: feasibility and safety in humans.Circulation. CardiovascularImaging, 5(4), 509–517.

56. de Chickera, S. N., Snir, J., Willert, C., et al. (2011). Labellingdendritic cells with SPIO has implications for their subsequentin vivo migration as assessed with cellular MRI. Contrast Media& Molecular Imaging, 6(4), 314–327.

57. Dunning, M. D., Lakatos, A., Loizou, L., et al. (2004).Superparamagnetic iron oxide-labeled Schwann cells and olfactoryensheathing cells can be traced in vivo by magnetic resonanceimaging and retain functional properties after transplantation intothe CNS. Journal of Neuroscience, 24(44), 9799–9810.

58. Huang, D. M., Hsiao, J. K., Chen, Y. C., et al. (2009). The promo-tion of human mesenchymal stem cell proliferation bysuperparamagnetic iron oxide nanoparticles. Biomaterials, 30(22),3645–3651.

59. Bulte, J. W. (2009). In vivo MRI cell tracking: clinical studies.American Journal of Roentgenology, 193(2), 314–325.

60. Ahrens, E. T., Feili-Hariri, M., Xu, H., Genove, G., & Morel, P. A.(2003). Receptor-mediated endocytosis of iron-oxide particles pro-vides efficient labeling of dendritic cells for in vivo MR imaging.Magnetic Resonance in Medicine, 49(6), 1006–1013.

61. Modo,M., Hoehn, M., & Bulte, J. W. (2005). CellularMR imaging.Molecular Imaging, 4(3), 143–164.

62. Walczak, P., Kedziorek, D. A., Gilad, A. A., Lin, S., & Bulte, J. W.(2005). InstantMR labeling of stem cells usingmagnetoelectroporation.Magnetic Resonance in Medicine, 54 (4), 769–774.

63. Frank, J. A., Zywicke, H., Jordan, E. K., et al. (2002). Magneticintracellular labeling of mammalian cells by combining (FDA-ap-proved) superparamagnetic iron oxide MR contrast agents andcommonly used transfection agents. Academic Radiology, 9(Suppl2), S484–S487.

64. Kraitchman, D. L., & Bulte, J. W. (2008). Imaging of stem cellsusing MRI. Basic Research in Cardiology, 103(2), 105–113.

65. Bulte, J. W., Kraitchman, D. L., Mackay, A. M., & Pittenger, M. F.(2004). Chondrogenic differentiation of mesenchymal stem cells isinhibited after magnetic labeling with ferumoxides. Blood, 104(10),3410–3412.

66. Walczak, P., Ruiz-Cabello, J., Kedziorek, D. A., et al. (2006).Magnetoelectroporation: improved labeling of neural stem cellsand leukocytes for cellular magnetic resonance imaging using asingle FDA-approved agent. Nanomedicine : Nanotechnology,Biology, and Medicine, 2(2), 89–94.

67. Terrovitis, J., Stuber, M., Youssef, A., et al. (2008). Magnetic reso-nance imaging overestimates ferumoxide-labeled stem cell survivalafter transplantation in the heart. Circulation, 117(12), 1555–1562.

68. Cromer Berman, S. M., Kshitiz, Wang, C. J., et al. (2013). Cellmotility of neural stem cells is reduced after SPIO-labeling, which ismitigated after exocytosis.Magnetic Resonance in Medicine, 69(1),255–262.

69. Barnett, B. P., Arepally, A., Karmarkar, P. V., et al. (2007). Magneticresonance-guided, real-time targeted delivery and imaging ofmagnetocapsules immunoprotecting pancreatic islet cells. NatureMedicine, 13(8), 986–991.

70. Shapiro, E. M., Sharer, K., Skrtic, S., & Koretsky, A. P. (2006). Invivo detection of single cells by MRI. Magnetic Resonance inMedicine, 55(2), 242–249.

71. Shapiro, E. M., Skrtic, S., & Koretsky, A. P. (2005). Sizing it up:cellular MRI using micron-sized iron oxide particles. MagneticResonance in Medicine, 53(2), 329–338.

72. Hinds, K. A., Hill, J. M., Shapiro, E. M., et al. (2003). Highlyefficient endosomal labeling of progenitor and stem cells with largemagnetic particles allows magnetic resonance imaging of singlecells. Blood, 102(3), 867–872.

73. Shapiro, E.M., Medford-Davis, L. N., Fahmy, T. M., Dunbar, C. E.,& Koretsky, A. P. (2007). Antibody-mediated cell labeling of pe-ripheral T cells with micron-sized iron oxide particles (MPIOs)allows single cell detection by MRI. Contrast Media & MolecularImaging, 2(3), 147–153.

74. Bernas, L. M., Foster, P. J., & Rutt, B. K. (2007). Magnetic reso-nance imaging of in vitro glioma cell invasion. Journal ofNeurosurgery, 106(2), 306–313.

75. Foley, L. M., Hitchens, T. K., Ho, C., et al. (2009). Magneticresonance imaging assessment of macrophage accumulation inmouse brain after experimental traumatic brain injury. Journal ofNeurotrauma, 26(9), 1509–1519.

76. Sumner, J. P., Shapiro, E.M.,Maric, D., Conroy, R., & Koretsky, A.P. (2009). In vivo labeling of adult neural progenitors for MRI withmicron sized particles of iron oxide: quantification of labeled cellphenotype. NeuroImage, 44(3), 671–678.

77. Rohani, R., de Chickera, S. N., Willert, C., Chen, Y., Dekaban, G.A., & Foster, P. J. (2011). In vivo cellular MRI of dendritic cellmigration using micrometer-sized iron oxide (MPIO) particles.Molecular Imaging and Biology, 13(4), 679–694.

78. Nkansah, M. K., Thakral, D., & Shapiro, E. M. (2011). Magneticpoly(lactide-co-glycolide) and cellulose particles for MRI-basedcell tracking.Magnetic Resonance in Medicine, 65(6), 1776–1785.

79. Tang, K. S., & Shapiro, E. M. (2011). Enhanced magnetic celllabeling efficiency using -NH2 coated MPIOs. MagneticResonance in Medicine, 65(6), 1564–1569.


80. Ahrens, E. T., Flores, R., Xu, H., & Morel, P. A. (2005). In vivoimaging platform for tracking immunotherapeutic cells. NatureBiotechnology, 23(8), 983–987.

81. Partlow, K. C., Chen, J., Brant, J. A., et al. (2007). 19F magneticresonance imaging for stem/progenitor cell tracking with multipleunique perfluorocarbon nanobeacons. FASEB Journal, 21 (8),1647–1654.

82. Ruiz-Cabello, J., Walczak, P., Kedziorek, D. A., et al. (2008). Invivo “hot spot” MR imaging of neural stem cells using fluorinatednanoparticles.Magnetic Resonance inMedicine, 60(6), 1506–1511.

83. Waiczies, H., Guenther,M., Skodowski, J., et al. (2013).Monitoringdendritic cell migration using 19F/1H Magnetic ResonanceImaging. Journal of Visulaized Experiments, 73, e50251.

84. Boehm-Sturm, P., Mengler, L., Wecker, S., Hoehn, M., & Kallur, T.(2011). In vivo tracking of human neural stem cells with 19Fmagnetic resonance imaging. PLoS One, 6(12), e29040.

85. Hitchens, T. K., Ye, Q., Eytan, D. F., Janjic, J. M., Ahrens, E. T., &Ho, C. (2011). 19F MRI detection of acute allograft rejection within vivo perfluorocarbon labeling of immune cells. MagneticResonance in Medicine, 65(4), 1144–1153.

86. Bible, E., Dell'Acqua, F., Solanky, B., et al. (2012). Non-invasiveimaging of transplanted human neural stem cells and ECM scaffoldremodeling in the stroke-damaged rat brain by (19)F- and diffusion-MRI. Biomaterials, 33(10), 2858–2871.

87. Waiczies, H., Lepore, S., Janitzek, N., et al. (2011). Perfluorocarbonparticle size influences magnetic resonance signal and immunolog-ical properties of dendritic cells. PLoS One, 6(7), e21981.

88. Srinivas, M., Morel, P. A., Ernst, L. A., Laidlaw, D. H., & Ahrens,E. T. (2007). Fluorine-19 MRI for visualization and quantificationof cell migration in a diabetes model. Magnetic Resonance inMedicine, 58(4), 725–734.

89. Kadayakkara, D. K., Ranganathan, S., Young, W. B., & Ahrens, E.T. (2012). Assaying macrophage activity in a murine model ofinflammatory bowel disease using fluorine-19 MRI. LaboratoryInvestigation, 92(4), 636–645.

90. Janjic, J. M., Srinivas, M., Kadayakkara, D. K., & Ahrens, E. T.(2008). Self-delivering nanoemulsions for dual fluorine-19 MRIand fluorescence detection. Journal of the American ChemicalSociety, 130(9), 2832–2841.

91. Verdijk, P., Scheenen, T. W., Lesterhuis, W. J., et al. (2007).Sensitivity of magnetic resonance imaging of dendritic cells forin vivo tracking of cellular cancer vaccines. International Journalof Cancer, 120(5), 978–984.

92. Srinivas, M., Heerschap, A., Ahrens, E. T., Figdor, C. G., & deVries, I. J. (2010). (19)F MRI for quantitative in vivo cell tracking.Trends in Biotechnology, 28(7), 363–370.

93. Chan, K. W., Liu, G., Song, X., et al. (2013). MRI-detectable pHnanosensors incorporated into hydrogels for in vivo sensing oftransplanted-cell viability. Nature Materials, 12(3), 268–275.

94. Massoud, T. F., & Gambhir, S. S. (2003). Molecular imaging inliving subjects: seeing fundamental biological processes in a newlight. Genes & Development, 17(5), 545–580.

95. Adonai, N., Nguyen, K. N., Walsh, J., et al. (2002). Ex vivo celllabeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone)for imaging cell trafficking inmicewith positron-emission tomography.Proceedings of the National Academy of Sciences of the United Statesof America, 99(5), 3030–3035.

96. Zanzonico, P., Koehne, G., Gallardo, H. F., et al. (2006).[131I]FIAU labeling of genetically transduced, tumor-reactive lym-phocytes: cell-level dosimetry and dose-dependent toxicity.European Journal of Nuclear Medicine and Molecular Imaging,33(9), 988–997.

97. Bhargava, K. K., Gupta, R. K., Nichols, K. J., & Palestro, C. J.(2009). In vitro human leukocyte labeling with (64)Cu: anintraindividual comparison with (111)In-oxine and (18)F-FDG.Nuclear Medicine and Biology, 36(5), 545–549.

98. Brenner, W., Aicher, A., Eckey, T., et al. (2004). 111In-labeledCD34+ hematopoietic progenitor cells in a rat myocardial infarctionmodel. Journal of Nuclear Medicine, 45(3), 512–518.

99. Rennen, H. J., Boerman, O. C., Oyen, W. J., & Corstens, F. H.(2001). Imaging infection/inflammation in the new millennium.European Journal of Nuclear Medicine, 28(2), 241–252.

100. Becker, W., & Meller, J. (2001). The role of nuclear medicine ininfection and inflammation. The Lancet Infectious Diseases, 1(5),326–333.

101. Jin, Y., Kong, H., Stodilka, R. Z., et al. (2005). Determiningthe minimum number of detectable cardiac-transplanted111In-tropolone-labelled bone-marrow-derived mesenchymalstem cells by SPECT. Physics in Medicine and Biology,50 (19), 4445–4455.

102. Gholamrezanezhad, A., Mirpour, S., Bagheri, M., et al. (2011). Invivo tracking of 111In-oxine labeled mesenchymal stem cells fol-lowing infusion in patients with advanced cirrhosis. NuclearMedicine and Biology, 38(7), 961–967.

103. Gholamrezanezhad, A., Mirpour, S., Ardekani, J. M., et al. (2009).Cytotoxicity of 111In-oxine on mesenchymal stem cells: a time-dependent adverse effect. Nuclear Medicine Communications, 30(3),210–216.

104. Monteiro-Riviere, N. A., Inman, A. O., & Zhang, L. W. (2009).Limitations and relative utility of screening assays to assessengineered nanoparticle toxicity in a human cell line. Toxicologyand Applied Pharmacology, 234(2), 222–235.

105. Barbash, I. M., Chouraqui, P., Baron, J., et al. (2003). Systemicdelivery of bone marrow-derived mesenchymal stem cells to theinfarcted myocardium: feasibility, cell migration, and body distri-bution. Circulation, 108(7), 863–868.

106. Kang, W. J., Kang, H. J., Kim, H. S., Chung, J. K., Lee, M. C., &Lee, D. S. (2006). Tissue distribution of 18F-FDG-labeled periph-eral hematopoietic stem cells after intracoronary administration inpatients with myocardial infarction. Journal of Nuclear Medicine,47(8), 1295–1301.

107. Zhou, R., Thomas, D. H., Qiao, H., et al. (2005). In vivo detection ofstem cells grafted in infarcted rat myocardium. Journal of NuclearMedicine, 46(5), 816–822.

108. Swirski, F. K., Pittet, M. J., Kircher, M. F., et al. (2006).Monocyte accumulation in mouse atherogenesis is progressiveand proportional to extent of disease. Proceedings of the NationalAcademy of Sciences of the United States of America, 103(27),10340–10345.

109. Kim, J., Arifin, D. R., Muja, N., et al. (2011). Multifunctionalcapsule-in-capsules for immunoprotection and trimodal imaging.Angewandte Chemie International Edition England, 50 (10),2317–2321.

110. Arifin, D. R., Kedziorek, D. A., Fu, Y., et al. (2012).Microencapsulated cell tracking. NMR in Biomedicine . doi:10.1002/nbm.2894.

111. Link, T. W., Arifin, D. R., Long, C. M., et al. (2012). Use ofmagnetocapsules for in vivo visualization and enhanced survivalof xenogeneic HepG2 cell transplants. Cell Medicine, 4(2), 77–784.

112. Arifin, D. R., Long, C. M., Gilad, A. A., et al. (2011). Trimodalgadolinium-gold microcapsules containing pancreatic islet cells re-store normoglycemia in diabetic mice and can be tracked by usingUS, CT, and positive-contrast MR imaging. Radiology, 260(3),790–798.

113. Barnett, B. P., Kraitchman, D. L., Lauzon, C., et al. (2006). Radiopaquealginate microcapsules for X-ray visualization and immunoprotectionof cellular therapeutics. Molecular Pharmaceutics, 3 (5), 531–538.

114. Barnett, B. P., Ruiz-Cabello, J., Hota, P., et al. (2011).Fluorocapsules for improved function, immunoprotection, and vi-sualization of cellular therapeutics with MR, US, and CT imaging.Radiology, 258(1), 182–191.


http://dx.doi.org/10.1002/nbm.2894

http://dx.doi.org/10.1002/nbm.2894

115. Nam, S. Y., Ricles, L. M., Suggs, L. J., & Emelianov, S. Y. (2012).In vivo ultrasound and photoacoustic monitoring of mesenchymalstem cells labeled with gold nanotracers. PLoS One, 7(5), e37267.

116. Ricles, L. M., Nam, S. Y., Sokolov, K., Emelianov, S. Y., & Suggs,L. J. (2011). Function of mesenchymal stem cells following loadingof gold nanotracers. International Journal of Nanomedicine, 6 ,407–416.

117. Jokerst, J. V., Khademi, C., & Gambhir, S. S. (2013). Intracellularaggregation of multimodal silica nanoparticles for ultrasound-guided stem cell implantation. Science Translational Medicine,5(177), 177ra35.

118. Cui, W., Tavri, S., Benchimol, M. J., et al. (2013). Neural progenitorcells labeling with microbubble contrast agent for ultrasound imag-ing in vivo. Biomaterials, 34(21), 4926–4935.

119. Forss-Petter, S., Danielson, P. E., Catsicas, S., et al. (1990).Transgenic mice expressing beta-galactosidase in mature neuronsunder neuron-specific enolase promoter control.Neuron, 5(2), 187–197.

120. Himes, S. R., & Shannon, M. F. (2000). Assays for transcriptionalactivity based on the luciferase reporter gene.Methods in MolecularBiology, 130 , 165–174.

121. Contag, C. H., Jenkins, D., Contag, P. R., & Negrin, R. S. (2000).Use of reporter genes for optical measurements of neoplastic diseasein vivo. Neoplasia, 2(1–2), 41–52.

122. Zhuo, L., Sun, B., Zhang, C. L., Fine, A., Chiu, S. Y., &Messing, A.(1997). Live astrocytes visualized by green fluorescent protein intransgenic mice. Developmental Biology, 187(1), 36–42.

123. Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N.,Palmer, A. E., & Tsien, R. Y. (2004). Improved monomeric red,orange and yellow fluorescent proteins derived from Discosoma sp.red fluorescent protein. Nature Biotechnology, 22(12), 1567–1572.

124. Kremers, G. J., Gilbert, S. G., Cranfill, P. J., Davidson, M. W., &Piston, D. W. (2011). Fluorescent proteins at a glance. Journal ofCell Science, 124(2), 157–160.

125. Priddle, H., Grabowska, A., Morris, T., et al. (2009).Bioluminescence imaging of human embryonic stem cellstransplanted in vivo in murine and chick models. Cloning andStem Cells, 11(2), 259–267.

126. Love, Z., Wang, F., Dennis, J., et al. (2007). Imaging of mesenchy-mal stem cell transplant by bioluminescence and PET. Journal ofNuclear Medicine, 48(12), 2011–2020.

127. Tang, Y., Shah, K., Messerli, S. M., Snyder, E., Breakefield, X., &Weissleder, R. (2003). In vivo tracking of neural progenitor cell migra-tion to glioblastomas. Human Gene Therapy, 14(13), 1247–1254.

128. Bai, X., Yan, Y., Coleman, M., et al. (2011). Tracking long-termsurvival of intramyocardially delivered human adipose tissue-derived stem cells using bioluminescence imaging. MolecularImaging and Biology, 13(4), 633–645.

129. Janowski, M., Engels, C., Gorelik, M., et al. (2013). Survival ofneural progenitors allografted into the CNS of immunocompetentrecipients is highly dependent on transplantation site. CellTransplant, doi: http://dx.doi.org/10.3727/096368912X661328.

130. Liang, Y., Agren, L., Lyczek, A., Walczak, P., & Bulte, J. W. (2013).Neural progenitor cell survival in mouse brain can be improved byco-transplantation of helper cells expressing bFGF under doxycy-cline control. Experimental Neurology, 247C, 73–79.

131. Maguire, K. K., Lim, L., Speedy, S., & Rando, T. A. (2013).Assessment of disease activity in muscular dystrophies by nonin-vasive imaging. The Journal of Clincal Investigation, 123 (5),2298–2305.

132. Liang, Y., Walczak, P., & Bulte, J. W. (2013). Comparison of red-shifted firefly luciferase Ppy RE9 and conventional Luc2 as biolu-minescence imaging reporter genes for in vivo imaging of stemcells. Journal of Biomedical Optics, 17(1), 016004.

133. Ueda, I., Kamaya, H., & Eyring, H. (1976). Molecular mechanismof inhibition of firefly luminescence by local anesthetics.

Proceedings of the National Academy of Sciences of the UnitedStates of America, 73(2), 481–485.

134. Inoue, Y., Kiryu, S., Izawa, K., Watanabe, M., Tojo, A., & Ohtomo,K. (2009). Comparison of subcutaneous and intraperitoneal injec-tion of D-luciferin for in vivo bioluminescence imaging. EuropeanJournal of Nuclear Medicine and Molecular Imaging, 36(5), 771–779.

135. Keyaerts, M., Remory, I., Caveliers, V., et al. (2012). Inhibition offirefly luciferase by general anesthetics: effect on in vitro and in vivobioluminescence imaging. PLoS One, 7(1), e30061.

136. Szarecka, A., Xu, Y., & Tang, P. (2007). Dynamics of fireflyluciferase inhibition by general anesthetics: Gaussian and anisotrop-ic network analyses. Biophysical Journal, 93(6), 1895–1905.

137. Keyaerts, M., Heneweer, C., Gainkam, L. O., et al. (2011). Plasmaprotein binding of luciferase substrates influences sensitivity andaccuracy of bioluminescence imaging. Molecular Imaging andBiology, 13(1), 59–66.

138. Gilad, A. A., McMahon, M. T., Walczak, P., et al. (2007). Artificialreporter gene providing MRI contrast based on proton exchange.Nature Biotechnology, 25(2), 217–219.

139. Gilad, A. A., Ziv, K., McMahon,M. T., van Zijl, P. C., Neeman, M.,& Bulte, J. W. (2008). MRI reporter genes. Journal of NuclearMedicine, 49(12), 1905–1908.

140. Berman, S. C., Galpoththawela, C., Gilad, A. A., Bulte, J. W., &Walczak, P. (2011). Long-termMR cell tracking of neural stem cellsgrafted in immunocompetent versus immunodeficient mice revealsdistinct differences in contrast between live and dead cells.Magnetic Resonance in Medicine, 65(2), 564–574.

141. Liu, G., Bulte, J. W., & Gilad, A. A. (2011). CEST MRI reportergenes. Methods in Molecular Biology, 711 , 271–280.

142. Louie, A. Y., Huber, M. M., Ahrens, E. T., et al. (2000). In vivovisualization of gene expression using magnetic resonance imaging.Nature Biotechnology, 18(3), 321–325.

143. Cui,W., Liu, L., Kodibagkar, V. D., &Mason, R. P. (2010). S-Gal, anovel 1HMRI reporter for beta-galactosidase.Magnetic Resonancein Medicine, 64(1), 65–71.

144. Alfke, H., Stoppler, H., Nocken, F., et al. (2003). In vitroMR imaging of regulated gene expression. Radiology, 228(2),488–492.

145. Gilad, A. A., Winnard, P. T., Jr., van Zijl, P. C., & Bulte, J. W.(2007). Developing MR reporter genes: promises and pitfalls. NMRin Biomedicine, 20(3), 275–290.

146. Weissleder, R., Moore, A., Mahmood, U., et al. (2000). In vivomagnetic resonance imaging of transgene expression. NatureMedicine, 6(3), 351–355.

147. Kotamraju, S., Chitambar, C. R., Kalivendi, S. V., Joseph, J., &Kalyanaraman, B. (2002). Transferrin receptor-dependent iron up-take is responsible for doxorubicin-mediated apoptosis in endothe-lial cells: role of oxidant-induced iron signaling in apoptosis. TheJournal of Biological Chemistry, 277(19), 17179–17187.

148. Cohen, B., Dafni, H., Meir, G., Harmelin, A., & Neeman, M.(2005). Ferritin as an endogenous MRI reporter for noninvasiveimaging of gene expression in C6 glioma tumors. Neoplasia, 7(2),109–117.

149. Naumova, A. V., Reinecke, H., Yarnykh, V., Deem, J., Yuan, C., &Murry, C. E. (2010). Ferritin overexpression for noninvasive mag-netic resonance imaging-based tracking of stem cells transplantedinto the heart. Molecular Imaging, 9(4), 201–210.

150. Campan, M., Lionetti, V., Aquaro, G. D., et al. (2011). Ferritin as areporter gene for in vivo tracking of stem cells by 1.5-TcardiacMRIin a rat model of myocardial infarction. American Journal ofPhysiology. Heart and Circulatory Physiology, 300(6), H2238–H2250.

151. Gossuin, Y., Muller, R. N., & Gillis, P. (2009). Magnetic resonanceimaging of cells overexpressing MagA, an endogenous contrastagent for live cell imaging. Molecular Imaging, 8(3), 129–139.


http://dx.doi.org/10.3727/096368912X661328

152. Zurkiya, O., Chan, A. W., & Hu, X. (2008). MagA is sufficient forproducing magnetic nanoparticles in mammalian cells, making it anMRI reporter.Magnetic Resonance in Medicine, 59(6), 1225–1231.

153. Goldhawk, D. E., Lemaire, C., McCreary, C. R., et al. (2009).Magnetic resonance imaging of cells overexpressing MagA, anendogenous contrast agent for live cell imaging. MolecularImaging, 8(3), 129–139.

154. McMahon, M. T., Gilad, A. A., DeLiso, M. A., Berman, S. M.,Bulte, J. W., & van Zijl, P. C. (2008). New “multicolor” polypeptidediamagnetic chemical exchange saturation transfer (DIACEST)contrast agents for MRI. Magnetic Resonance in Medicine, 60(4),803–812.

155. Alauddin, M. M., Shahinian, A., Gordon, E. M., & Conti, P. S.(2004). Direct comparison of radiolabeled probes FMAU, FHBG,and FHPG as PET imaging agents for HSV1-tk expression in ahuman breast cancer model. Molecular Imaging, 3(2), 76–84.

156. Koehne, G., Doubrovin, M., Doubrovina, E., et al. (2003). Serialin vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes. Nature Biotechnology,21(4), 405–413.

157. Ponomarev, V., Doubrovin, M., Lyddane, C., et al. (2001). ImagingTCR-dependent NFAT-mediated T-cell activation with positronemission tomography in vivo. Neoplasia, 3(6), 480–488.

158. Pei, Z., Lan, X., Cheng, Z., et al. (2012). A multimodality reportergene for monitoring transplanted stem cells. Nuclear Medicine andBiology, 39(6), 813–820.

159. Pomper, M. G., Hammond, H., Yu, X., et al. (2009). Serial imagingof human embryonic stem-cell engraftment and teratoma formationin live mouse models. Cell Research, 19(3), 370–379.

160. Mercier-Letondal, P., Deschamps,M., Sauce, D., et al. (2008). Earlyimmune response against retrovirally transduced herpes simplexvirus thymidine kinase-expressing gene-modified T cells coinfusedwith a T cell-depleted marrow graft: an altered immune response?Human Gene Therapy, 19(9), 937–950.

161. Serganova, I., Ponomarev, V., & Blasberg, R. (2007). Human re-porter genes: potential use in clinical studies. Nuclear Medicine andBiology, 34(7), 791–807.

162. Campbell, D. O., Yaghoubi, S. S., Su, Y., et al. (2012). Structure-guided engineering of human thymidine kinase 2 as a positronemission tomography reporter gene for enhanced phosphorylationof non-natural thymidine analog reporter probe. The Journal ofBiological Chemistry, 287(1), 446–454.

163. Ponomarev, V., Doubrovin, M., Shavrin, A., et al. (2007). A human-derived reporter gene for noninvasive imaging in humans: mito-chondrial thymidine kinase type 2. Journal of Nuclear Medicine,48(5), 819–826.

164. Yaghoubi, S. S., Jensen, M. C., Satyamurthy, N., et al. (2009).Noninvasive detection of therapeutic cytolytic T cells with 18F-FHBG PET in a patient with glioma. Nature Clinical PracticeOncology, 6(1), 53–58.

165. McCracken, M. N., Gschweng, E. H., Nair-Gill, E., et al. (2013).Long-term in vivo monitoring of mouse and human hematopoieticstem cell engraftment with a human positron emission tomographyreporter gene. Proceedings of the National Academy of Sciences ofthe United States of America, 110(5), 1857–1862.

166. Huang, M., Batra, R. K., Kogai, T., et al. (2001). Ectopic expressionof the thyroperoxidase gene augments radioiodide uptake and re-tention mediated by the sodium iodide symporter in non-small celllung cancer. Cancer Gene Therapy, 8(8), 612–618.

167. Auricchio, A., Acton, P. D., Hildinger, M., et al. (2003). In vivoquantitative noninvasive imaging of gene transfer by single-photonemission computerized tomography. Human Gene Therapy, 14(3),255–261.

168. Kim, Y. H., Lee, D. S., Kang, J. H., et al. (2005). Reversing thesilencing of reporter sodium/iodide symporter transgene for stemcell tracking. Journal of Nuclear Medicine, 46(2), 305–311.

169. Terrovitis, J., Kwok, K. F., Lautamaki, R., et al. (2008). Ectopicexpression of the sodium-iodide symporter enables imaging oftransplanted cardiac stem cells in vivo by single-photon emis-sion computed tomography or positron emission tomography.Journal of the American College of Cardiology, 52 (20), 1652–1660.

170. Hwang do, W., Kang, J. H., Jeong, J. M., et al. (2008). Noninvasivein vivo monitoring of neuronal differentiation using reporter drivenby a neuronal promoter. European Journal of NuclearMedicine andMolecular Imaging, 35(1), 135–145.

171. Bar-Shir, A., Liu, G., Liang, Y., et al. (2013). Transforming thymi-dine into a magnetic resonance imaging probe for monitoring geneexpression. Journal of the American Chemical Society, 135(4),1617–1624.

172. de Vries, I. J., Lesterhuis, W. J., Barentsz, J. O., et al. (2005).Magnetic resonance tracking of dendritic cells in melanoma patientsfor monitoring of cellular therapy. Nature Biotechnology, 23(11),1407–1413.


seeing stem cells at work in vivo

Documents