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P U B L I S H E D B Y E L S E V I E R I N C . h t t p : / / d x . d o i . o r g / 1 0 . 1 0 1 6 / j . j c m g . 2 0 1 2 . 1 1 . 0 1 8

Noninvasive Monitoring of Oxidative Stressin Transplanted Mesenchymal Stromal Cells

Peter J. Psaltis, MBBS, PHD,* Karen M. Peterson, BSC,* Rende Xu, MD,*

Federico Franchi, PHD,* Tyra Witt, CVT,* Ian Y. Chen, MD, PHD,y Amir Lerman, MD,*

Robert D. Simari, MD,* Sanjiv S. Gambhir, MD, PHD,z Martin Rodriguez-Porcel, MD*

Rochester, Minnesota; and Stanford, California

OBJECTIVES The goal of this study was to validate a pathway-specific reporter gene that could be

used to noninvasively image the oxidative status of progenitor cells.

BACKGROUND In cell therapy studies, reporter gene imaging plays a valuable role in the

assessment of cell fate in living subjects. After myocardial injury, noxious stimuli in the host tissue

confer oxidative stress to transplanted cells that may influence their survival and reparative function.

METHODS Rat mesenchymal stromal cells (MSCs) were studied for phenotypic evidence of increased

oxidative stress under in vitro stress. On the basis of their up-regulation of the pro-oxidant enzyme p67phox

subunit of nicotinamide adenine dinucleotide phosphate (NAD[P]H oxidase p67phox), an oxidative stress

sensor was constructed, comprising the firefly luciferase (Fluc) reporter gene driven by the NAD(P)H

p67phox promoter. MSCs cotransfected with NAD(P)H p67phox–Fluc and a cell viability reporter gene

(cytomegalovirus–Renilla luciferase) were studied under in vitro and in vivo pro-oxidant conditions.

RESULTS After in vitro validation of the sensor during low-serum culture, transfected MSCs were

transplanted into a rat model of myocardial ischemia/reperfusion (IR) and monitored by using

bioluminescence imaging. Compared with sham controls (no IR), cardiac Fluc intensity was significantly

higher in IR rats (3.5-fold at 6 h, 2.6-fold at 24 h, 5.4-fold at 48 h; p < 0.01), indicating increased cellular

oxidative stress. This finding was corroborated by ex vivo luminometry after correcting for Renilla

luciferase activity as a measure of viable MSC number (Fluc:Renilla luciferase ratio 0.011 � 0.003 for

sham vs. 0.026 � 0.004 for IR at 48 h; p < 0.05). Furthermore, in IR animals that received MSCs

preconditioned with an antioxidant agent (tempol), Fluc signal was strongly attenuated, substantiating

the specificity of the oxidative stress sensor.

CONCLUSIONS Pathway-specific reporter gene imaging allows assessment of changes in the

oxidative status of MSCs after delivery to ischemic myocardium, providing a template to monitor key

biological interactions between transplanted cells and their host environment in living

subjects. (J Am Coll Cardiol Img 2013;6:795–802) ª 2013 by the American College of Cardiology

Foundation

From the *Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota;

yDivision of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California;

and the zDepartment of Radiology and Molecular Imaging Program at Stanford, Stanford University School of Medicine,

Stanford, California. The luminometer used was obtained through a grant from Turner BioSystems. This work was supported in

part by the National Institutes of Health (HL 88048, Dr. Rodriguez-Porcel) and the Mayo Foundation (Dr. Rodriguez-Porcel).

Dr. Psaltis has received funding from the National Health and Medical Research Council of Australia. All other authors have

reported that they have no relationships relevant to the contents of this paper to disclose.

Manuscript received July 27, 2012; revised manuscript received November 1, 2012, accepted November 9, 2012.

P

A B B R E V I A T I O N S

A N D A C R O N YM S

BLI = bioluminescence ima

CMV = cytomegalovirus

FBS = fetal bovine serum

Fluc = firefly luciferase

IR = ischemia/reperfusion

LV = left ventricular

MSC = mesenchymal strom

NAD(P)H = nicotinamide

adenine dinucleotide phos

PET = positron emission

tomography

ROS = reactive oxygen spe

TSTA = 2-step transcriptio

amplification

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rogenitor cell therapies are being developedas a therapeutic alternative for myocardial

repair in coronary artery disease (1). Al-though studies have shown an overall benefit

with improvements in left ventricular (LV) cardiacfunction after myocardial injury, the beneficial effectof cell-based intervention after myocardial injury hasbeen modest, likely due to poor survival of trans-planted cells (2). Until recently, progenitor cellstudies were limited in their capacity to assess cellsurvival, both in cell culture and living subjects.

See page 803

Assessment of the fate and biology of cells aftertransfer to recipient myocardium relied on tradi-tional ex vivo assays and molecular techniques (e.g.,

ging

al cell

phate

cies

nal

histology, western blotting), which areboth invasive and restricted in their ca-pacity to monitor temporal changes in agiven subject. However, developments inmolecular imaging techniques, such as re-porter gene technology, have increasinglyenabled the noninvasive surveillance of cellfate after cardiovascular application (3).Bioluminescent imaging (BLI) has pro-vided helpful information in small animalmodels regarding the kinetics of cellviability by detecting transgene expressionunder constitutional promoters in livingcells (4). Recent studies have demonstratedthat reporter gene imaging can also be usedto monitor biological processes, in additionto cellular viability, by using conditional

transgenes whose expression is regulated bypathway-specific promoters (5,6).In the setting of cell delivery after myocardial

infarction or ischemia/reperfusion (IR), the recipientmicroenvironment may confer a heightened state ofoxidative stress to transplanted cells. Depending onboth cell type and degree of oxidative stress, this mayhave adaptive or maladaptive effects on cell survival andbiological function, ultimately influencing capacity forrepair and/or regeneration (7–9). The ability to assessthe biological interactions of transplanted cells withtheir host milieu in vivo, including their pro-oxidantstatus, could provide invaluable insights to assist withefforts to optimize stem cell functionality and thera-peutic efficacy (10). Here we report on the novel use ofconditional reporter gene labeling and imaging tomonitor oxidative stress in mesenchymal stromal cells

(MSCs) after their in vivo delivery to a rodent model ofmyocardial IR.

METHODS

Detailed methods are provided in the OnlineAppendix. Bone marrow MSCs from rats werephenotypically characterized under in vitro condi-tions of increased oxidative stress. DNA reportergene plasmids were designed, constructed, and usedto transfect MSCs to enable assessment of oxidativestress signal. This involved using firefly luciferase(Fluc) driven by the promoter for the p67phox sub-unit of nicotinamide adenine dinucleotide phos-phate (NAD[P]H p67phox–Fluc). To overcome thelow expression levels typically inherent with tran-scriptional targeting of reporter genes, signalamplification was achieved by a recently validated2-step transcriptional amplification (TSTA) strat-egy (11). The oxidant signal was normalized to thenumber of viable cells, assessed by using Renillaluciferase under the regulation of a constitutivecytomegalovirus promoter (CMV-Rluc). Trans-fected cells were transplanted to animals after IR(n ¼ 7) and compared with sham controls (n ¼ 8).Furthermore, the oxidative stress sensor was alsoevaluated in MSCs that had been pretreated with anantioxidant (n ¼ 5).

Statistical comparisons were performed withparametric or nonparametric 2-sample Student ttests or 1-way analysis of variance (with Bonferroniposttest comparison), as appropriate. Results areexpressed as mean � SEM of multiple experiments.In all cases, statistical significance was established at2-tailed p < 0.05.

RESULTS

Reporter gene detection of cellular oxidative stressin vitro. Initially, MSCs were assessed for theirsusceptibility to increased oxidative stress underin vitro stressors. After exposure to hypoxia (12) orlow-serum culture for 24 h (2% fetal bovine serum[FBS]), MSCs displayed increased oxidativestress conversion of 2070-dichlorodihydrofluoresceindiacetate (H2DCFDA) to 20,70-dichlorofluorescein,compared with control conditions (10% FBS)(2.9-fold difference; p < 0.05) (Fig. 1A). Bothconditions stimulated differences in MSC expres-sion of various proteins involved in producing orscavenging reactive oxygen species (ROS), includingup-regulation of the p67phox regulatory subunit of

Figure 1. Detection of Oxidative Stress in MSCs In Vitro

(A) Conversion of 2070-dichlorodihydrofluorescein diacetate (H2DCFDA) to 20 ,70-dichlorofluorescein (DCF) (measure of oxidative stress) inmesenchymal stromal cells (MSCs) after culture in normal (10% fetal bovine serum [FBS]) or low (2% FBS) serum media for 24 h. RFU ¼ relativefluorescence units. (B) Densitometric analysis from immunoblotting of MSC lysates for the p67phox subunit of nicotinamide adeninedinucleotide phosphate (NAD[P]H p67phox), corrected for beta-actin. (C) Oxidative stress plasmid vector NAD(P)H p67phox–firefly luciferase(Fluc). Gal4-VP2 ¼ fusion gene combining yeast Gal4 and 2 tandem repeats of herpes simplex virus VP16; E4 ¼ adenovirus E4 minimalpromoter; PA ¼ SV40 poly(A) tail; TSTA ¼ 2-step transcription amplification. (D) Bioluminescence imaging and (E) luminometry of Fluc activity(oxidative stress signal) corrected for Renilla luciferase (Rluc; viable cell number) in MSCs under different culture conditions. Values are mean� SEM. *p < 0.05, zp < 0.001. (F) There was strong correlation (R2 ¼ 0.95, p < 0.01) between Fluc activity and oxidative stress (by H2DCFDAconversion) for MSCs cultured at varying serum concentrations (0.5%, 1%, 2%, 5%, and 10%). RLU ¼ relative light units.

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NAD(P)H oxidase (Fig. 1B) (12). On this basis, atransfection plasmid was designed for an oxidativestress sensor that comprised the NAD(P)H p67phox

promoter (353bp, PubMed nucleotide DQ662934)to regulate expression of the Fluc reporter gene(Fig. 1C). Cells were cotransfected with theCMV-Rluc and NAD(P)H p67phox–Fluc plasmids.After 24 hours of culture in low-serum media,MSC Fluc signal, corrected for cell number andtransfection efficiency, was significantly increasedcompared with control conditions, as shown inde-pendently by BLI using a cooled charge-coupleddevice camera (1.6-fold difference; p < 0.05)(Fig. 1D) and luminometry (1.8-fold difference;p < 0.05) (Fig. 1E). In further experiments, MSCFluc activity correlated strongly with oxidative stressreadout in response to varying concentrations of

FBS (R2 ¼ 0.95, p < 0.01) (Fig. 1F). Importantly,MSCs pretreated with tempol, a superoxidedismutase mimetic (4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl; 5 mmol/l) displayed a markedlylower Fluc:Rluc signal ratio after low-serum culture,compared with untreated cells (9.5 � 2.1 vs. 49.6 �6.4, respectively; p < 0.001) (Fig. 1E). Thesein vitro results demonstrated that reporter genes,under the regulation of the NAD(P)H p67phox

promoter, can be used to assess the oxidant status ofMSCs across a range of oxidative stress levels.In vivo monitoring of oxidative stress in MSCs. Wethen tested the oxidative stress sensor in a ratmodel ofanterior wall IR (30-min ischemia). Over the firstweek after IR surgery, ischemic myocardium dis-played histological changes of progressive inflam-matory cell infiltration and collagen deposition

Figure 2. Injurious Changes in Rat Myocardium After IR

(A) Hematoxylin-eosin, (B)Masson’s trichrome, and (C) immunohistochemical staining for 8-hydroxy-2’-deoxyguanosine (OHG) of the ischemicterritory in rats at different intervals after ischemia/reperfusion (IR) surgery (�20 magnification). Yellow arrows, inflammatory cell infiltrates;arrowhead, collagen; black arrows,OHG. (D) Percent area of OHG staining in ischemic myocardium at different intervals after IR; n$ 3 for eachtime. (E) Expression bands of inflammatory, survival, and oxidative stress proteins from lysates of ischemic (or sham) myocardium at differentintervals after IR. For quantitative analysis, refer to Online Figure 1A. NF-kb ¼ nuclear factor kappa-light-chain-enhancer of activated B cells;Bcl-2¼ B-cell lymphoma 2; Bax¼ bcl-2 associated X-protein; XDH¼ xanthine dehydrogenase; HO-1¼ heme oxygenase-1; SOD1¼ copper/zincsuperoxide dismutase; SOD2 ¼ manganese superoxide dismutase. (F) Echocardiographic results for end-diastolic volume (EDV), end-systolicvolume (ESV), and ejection fraction (EF) 2 days after IR or sham surgery; n¼ 4/group. Values are mean� SEM. *p< 0.05, yp< 0.01 versus sham.

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(Figs. 2A and 2B), with incremental expression of8-hydroxy-2’-deoxyguanosine, a marker of oxidativestress, that was prominent by 48 h (p< 0.05 vs. sham)(Figs. 2C and 2D). Immunoblotting of myocardiallysates revealed up-regulation of markers/mediators

of inflammatory (nuclear factor kappa-light-chain-enhancer of activated B cells), apoptotic (bcl-2 asso-ciated X-protein), and oxidative stress pathways(NAD[P]H p67phox, xanthine dehydrogenase, hemeoxygenase) in the ischemic and peri-ischemic tissue

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compared with remote and sham myocardium, espe-cially from day 2 after surgery (Fig. 2E,Online Fig. 1).At this time point, there was also echocardiographicevidence of reduced systolic function (ejectionfraction 49.3� 4.9% for IR vs. 75.4� 1.1% for sham;p < 0.05) (Fig. 2F). Therefore, this model of IRproduced early injurious changes that would be ex-pected to create a stressful milieu for cells trans-planted into the anterior LV myocardium.

Next, allogeneic MSCs (7.5 � 105) cotransfectedwith CMV-Rluc and NAD(P)H p67phox–Fluc wereadministered transepicardially into the ischemicterritory of rats, 10 min after reperfusion, or shamsurgery. Bioluminescence imaging was performedat 6, 24, and 48 h to detect Fluc activity afteradministration of its substrate, D-luciferin. Flucsignal was present at low levels over the heart andlungs in sham controls at 6 h, indicating low-gradestress experienced by MSCs early after injectioninto nonischemic myocardium and prompt redistri-bution of cells to the lungs (Online Fig. 2A). Bycomparison, an intense Fluc signal was detected

Figure 3. Reporter Gene Detection of Oxidative Stress in MSCs In V

(A) Representative bioluminescence images for Fluc signal, 2 days after Mradiance of cardiac Fluc signal as determined by bioluminescence at dluminometry, corrected for Rluc activity. Values are mean � SEM. IR, n*p < 0.05 and yp < 0.01 versus IR animals treated with NAD(P)H p67phox

p67phox (red) by green fluorescent protein (GFP) MSCs (green) retainedbottom left: higher magnification of inset box; bottom middle: mergcontrol antibodies. Scale bars: white, 10 mm; yellow, 5 mm. Abbreviat

above background noise in the region of the heart,but not in other organs, throughout the first 48 h inischemic animals and was significantly higher thanfor sham rats at all time-points (p < 0.01) (OnlineFig. 2B). Although absolute values for total Flucradiance diminished over time (IR group: 71.1 �29.0 � 104 photons/s at 6 h, 19.3 � 6.0 � 104

photons/s at 24 h, 11.9 � 2.7 � 104 photons/s at 48h; p < 0.001), the relative difference in cardiac Flucsignal was highest between the ischemic and shamgroups at 48 h (11.9� 2.7� 104 photons/s vs. 2.2�0.5 � 104 photons/s; p < 0.01) (Figs. 3A and 3B,Online Fig. 2B). This increase in Fluc in the IRsetting was confirmed after the signal was correctedfor number of cells with Rluc, which was measuredby using ex vivo luminometry of LV homogenates,because an in vivo signal could not be reliably imageddue to tissue attenuation (Fluc:Rluc ratio 0.026 �0.004 for IR vs. 0.011 � 0.003 for sham at 48 h;p < 0.05) (Fig. 3C, Online Fig. 2B). Tissue im-munofluorescent staining was also used to verify thattransplanted MSCs (transfected to constitutively

ivo

SC transfer in the different experimental groups. (B) Results for totalay 2. (C) Day 2 results for left ventricular Fluc activity by ex vivo¼ 7; sham controls, n ¼ 8; IR Tempol, n ¼ 5; IR null vector, n ¼ 3.–Fluc MSCs. (D) Immunofluorescence showing expression of NAD(P)Hin IR myocardium 2 days after delivery. Top row: low magnification;ed image for sham myocardium; bottom right: immunoglobulin Gions as in Figures 1 and 2.

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express green fluorescent protein) did in fact expressNAD(P)H p67phox 48 h after administration toischemic (but not sham) myocardium (Fig. 3D).MSCs transfected with a Null-Fluc vector dis-

played very low levels of Fluc activity (1.5 � 0.3 �104 photons/s by BLI at 48 h) (Figs. 3A to 3C),underscoring the specificity of the detected oxidativestress signal. Furthermore, treatment of MSCs with5 mmol/l of tempol for 24 h before delivery to IRanimals markedly attenuated the Fluc signal tolevels similar to those in shams (3.7 � 0.7 � 104

photons/s at 48 h). This finding corroborated thein vitro observation that expression of Fluc could beused not only to detect different levels of oxidativestress but also to monitor changes in the biologyof transplanted cells (e.g., after an antioxidantintervention).

D I SCUSS ION

In this proof-of-principle study, we validated apathway-specific reporter gene approach fordetecting oxidative stress in cells after their transferto ischemic myocardium. Using the NAD(P)Hp67phox promoter expressing Fluc, we were able todemonstrate Fluc signal in MSCs transplanted toischemic myocardium. Moreover, this study pro-vides preliminary evidence that this novel oxidativestress sensor may be used to monitor the effect ofadjuvant interventions targeted to improve thebiology of transplanted progenitor cells.A recurring obstacle for cell therapy studies con-

tinues tobe the significant loss of viable cells from targetmyocardium in the early aftermath of transplantation,ultimately limiting the scope of therapeutic benefit(13). Although the immediate retention of cells isadversely influenced by mechanical forces during thedelivery process, ongoing attrition and failure ofengraftment are also contributed to by noxious stimuliin the foreign environment of the recipient heart. Thishas been shown to apply for cell delivery into healthy,intact myocardium but is further accentuated in thesetting of ischemia/infarction, in which a combinationof reduced tissue perfusion and reperfusion injury, in-flammatory cell infiltration, and pro-oxidant factorsincite local release of oxidative free radicals and pro-duction of ROS, imparting increased oxidative stress torecently administered cells (9,14).Although the preciseeffects of increased oxidative stress on cell survival,proliferation, differentiation,maturation, andparacrinefunction are largely undetermined, they are thought todepend on variables such as cell type, myocardial dis-tribution of cell delivery, and severity of the myocardialpro-oxidant state (15–17). Recently, transcriptomic

analysis has revealed that the overwhelming influenceof ischemic and inflamedmyocardiumonbonemarrowmononuclear cells early after transplantation is totrigger survival responses (9). In the case of MSCs,exposure to ROS (likely depending on level of ROS)may cause either deleterious or favorable effectson viability, senescence, and biological function(7,8,12,18). Furthermore, the importance of theoxidative status of MSCs has also been highlighted byshowing that its manipulation (e.g. by gene trans-fection, hypoxic preconditioning, pharmacologicalintervention) may protect against apoptosis andenhance reparative capacity (10,12,19–22).

With our goal of monitoring oxidative stress inMSCs, we elected to use the Fluc reporter genesystem, which has been a cornerstone of BLI, toevaluate cell fate in small animal studies (4,15).Previously, the implementation of reporter genelabeling has mainly been in the context of usingconstitutional promoters, to provide surrogate in-formation about the number of viable cells retainedin myocardium over different time intervals. Recentdevelopments in pathway-specific reporter geneimaging have begun to allow surveillance of otherbiological processes in transplanted cells in vivo,notably their differentiation along cardiomyocyteor endothelial lineages by using conditional pro-moters, such as cardiac sodium–calcium exchanger-1(Ncx-1) or Tie2, respectively (5,6).

In selecting an appropriate promoter that wouldtrigger Fluc expression in “stressed” MSCs, ourchoice of NAD(P)H p67phox was based on itsreliable up-regulation when MSCs were exposed tohypoxic (12) or low-serum conditions, accompaniedby increased production of ROS. Although the useof NAD(P)H p67phox to regulate reporter geneexpression may not necessarily reflect activation ofalternative ROS pathways (e.g., xanthine oxidase,mitochondrial P450 cytochromes), its validity wassupported here by the ability to detect increased Flucsignal in cells exposed to pro-oxidant conditions bothin vitro and in vivo. Compared with IR, Fluc in-tensity was considerably lower in sham animals, inkeeping with the expectation that their myocardiumwould provide a less hostile milieu for transplantedcells, based on lower tissue expression of pro-oxidant,pro-inflammatory, and apoptotic markers. Impor-tantly, immunostaining revealed that MSCs retainedwithin ischemic myocardium did indeed expressNAD(P)H p67phox, confirming the biologicalchanges of MSCs in the in vivo setting of IR.Furthermore, the correction of Fluc signal for viablecell number by using a second reporter gene (CMV-Rluc) clearly demonstrated that the up-regulation of

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NAD(P)H p67phox–Fluc in IR animals comparedwith sham animals was not merely due to retention ofhigher cell numbers in ischemic tissue. It is worthnoting that measurement of Rluc did require ex vivoluminometry, as in vivo detection by BLI wascomplicated by the fact that Rluc emits blue wave-length photons (lpeak ¼ 480 nm) when coelenter-azine is used as its substrate, and these can be stronglyattenuated by the subject’s chest wall. Novel de-velopments of red-shifted Rluc mutants may allowbetter in vivo signal detection in future studies (23).

One of the main challenges for pathway-specificreporter gene labeling is the relatively weak activityinherent in transcriptional targeting with mostconditional promoters. Although the use of anamplification strategy (TSTA) necessitated complexvector design and testing due to the inclusion ofmultiple elements in addition to the promoter andreporter gene (Fig. 1C), it was a critical componentin this study to achieve detectable Fluc signal in ratmyocardium. Although previous work has demon-strated that TSTA vectors may achieve profoundamplification of weak promoters, including thosethat are cardiac specific (e.g. troponin T, alpha-myosin heavy chain) (11,24), the current studyprovides early in vivo evidence of their utility forimaging transfected cells in recipient myocardium.Study limitations. One of the potentially importantapplications for a new imaging sensor for cellularoxidative stress will be to enable surveillance of itsfluctuation over time. This could contribute insightsregarding the optimal therapeutic window for celldelivery after myocardial injury and reveal criticalintervals during which adjuvant interventions mayhelp to modify the oxidative status of retained cells.Unfortunately, a major confounder of the ability tointerpret temporal tends in absolute biological signalis the rapid and progressive diminution of cell numberfrom recipient myocardium (3,15). We have alsoobserved this to be the case in our model systemduring separate experiments, in which CMV-FlucMSCs were used to follow the time course of theirretention (data not shown). This finding, along withthe transient nature of the plasmid transfectionstrategy used here, is likely to have contributed to thesharp decline in absolute NAD(P)H p67phox–Flucsignal observed after cell injection in both the IR andsham groups (Online Fig. 2). Although we found thatreporter gene expression is maintained in vitro for atleast 2 weeks after transfection, peak signal typicallyoccurs within the first 4 days. In future studies, theuse of stable genomic integration (e.g., by retroviralvectors, TALE nucleases) of theNAD(P)H p67phox–Fluc sensor in transplanted cells will be warranted.

It should also be recognized that alternativeoxidative stress pathways may be more relevant thanNAD(P)H oxidase for other progenitor cell typescurrently under investigation in cardiovascularstudies. Further evaluation and refinement ofoxidative stress imaging will therefore need todetermine optimal stress-related promoters for in-dividual cell types, as well as for different diseasecontexts, including chronic myocardial ischemia andnonischemic cardiac disease.Implications and clinical translation. Previous rodentstudies have used constitutive reporter gene imagingby bioluminescence to address key questions per-taining to optimal cell type (15), timing interval(17), and site of administration (16). Their findingshave provided novel insights to assist in the plan-ning and design of clinical stem cell studies. Simi-larly, in the case of an oxidative stress sensor, weanticipate that BLI will enable rapid and highthroughput interrogation of biological interactionsbetween transplanted cells and their recipient envi-ronment to help screen and hone cell and tissueengineering strategies in preclinical models, beforeguiding their translation to the clinical realm. Inthe context of MSCs, such optimization strategiesmay include the use of immunoenriched, genet-ically engineered, preconditioned, or cardiopoiesis-directed MSCs (10). Furthermore, conditionalpromoters, such as NAD(P)H p67phox, may even beimplemented in a “theranostics approach” (11),whereby imaging and therapeutic applications arecombined by using bidirectional systems to regulatethe selective expression of linked transgenes (e.g.,reporter and therapeutic genes) in transfected cells.

Despite its many advantages for cell tracking inrodents, the lack of spatial resolution and tissue depthpenetration ofBLIprecludes its use in human subjects.Thus, the direct implementation of oxidative stressreporter gene imaging in large animal and clinicalstudies will necessitate the replacement of Fluc withreporter genes specifically designed for clinical imagingmodalities (3). Owing to its superior cell detectionsensitivity (femtomolar range) and radiochemicalflexibility, it is likely that positron emission tomogra-phy (PET) will be better placed for initial clinicaltranslation than either single-photon emission com-puter tomography or magnetic resonance imaging,although hybrid PET–magnetic resonance imagingor PET–computed tomography will be useful forachieving optimal balance of oxidative stress detectionand anatomic resolution. To this end, porcine studieshave already demonstrated the feasibility and validityof PET-based (e.g., 18F-FHBG PET) detection ofconstitutive reporter gene expression (e.g., herpes

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simplex thymidine kinase) in retrovirally transfectedMSCs after myocardial delivery (25–27).

CONCLUS IONS

This study demonstrates the feasibility of usingpathway-specific reporter gene labeling to monitorthe oxidative stress of transplanted progenitor cells,

providing a novel platform to help optimize the useof cell therapies.

Reprint requests and correspondence: Dr. MartinRodriguez-Porcel, Division of Cardiovascular Diseases,Department of Internal Medicine, Mayo Clinic, 200First Street SW, Rochester, Minnesota 55905. E-mail:rodriguez.m@mayo.edu.

R E F E R E N C E S

1. Gersh BJ, Simari RD, Behfar A,Terzic CM, Terzic A. Cardiac cellrepair therapy: a clinical perspective.Mayo Clin Proc 2009;84:876–92.

2. Terrovitis JV, Smith RR, Marban E.Assessment and optimization of cellengraftment after transplantation intothe heart. Circ Res 2010;106:479–94.

3. Psaltis PJ, Simari RD, Rodriguez-PorcelM. Emerging roles for integrated im-aging modalities in cardiovascular cell-based therapeutics: a clinical per-spective. Eur J Nucl Med Mol Imaging2011;39:165–81.

4. Wu JC, Chen IY, Sundaresan G, et al.Molecular imaging of cardiac celltransplantation in living animals usingoptical bioluminescence and positronemission tomography. Circulation2003;108:1302–5.

5. Kammili RK, Taylor DG, Xia J, et al.Generation of novel reporter stem cellsand their application for molecularimaging of cardiac-differentiated stemcells in vivo. Stem Cells Dev 2010;19:1437–48.

6. Wang J, Najjar A, Zhang S, et al.Molecular imaging of mesenchymalstem cell: mechanistic insight intocardiac repair after experimentalmyocardial infarction. Circ CardiovascImaging 2011;5:94–101.

7. OskowitzA,McFerrinH,GutschowM,Carter ML, Pochampally R. Serum-deprived human multipotent mesen-chymal stromal cells (MSCs) are highlyangiogenic. Stem Cell Res 2011;6:215–25.

8. Song H, Cha MJ, Song BW, et al.Reactive oxygen species inhibit adhe-sion of mesenchymal stem cellsimplanted into ischemic myocardiumvia interference of focal adhesioncomplex. Stem Cells 2010;28:555–63.

9. Sheikh AY, Huber BC, Narsinh KH,et al. In vivo functional and tran-scriptional profiling of bone marrowstem cells after transplantation intoischemic myocardium. ArteriosclerThromb Vasc Biol 2012;32:92–102.

10. Richardson JD, Nelson AJ, ZannettinoAC, Gronthos S, Worthley SG, PsaltisPJ. Optimization of the cardiovasculartherapeutic properties of mesenchymal

stromal/stem cellsdtaking the next step.Stem Cell Rev 2013;9:281–302.

11. Chen IY, Gheysens O, Ray S, et al.Indirect imaging of cardiac-specifictransgene expression using a bidirec-tional two-step transcriptional ampli-fication strategy. Gene Ther 2010;17:827–38.

12. Peterson KM, Aly A, Lerman A,Lerman LO, Rodriguez-Porcel M.Improved survival of mesenchymalstromal cell after hypoxia pre-conditioning: role of oxidative stress.Life Sci 2011;88:65–73.

13. Segers VF, Lee RT. Stem-cell therapyfor cardiac disease. Nature 2008;451:937–42.

14. Rodriguez-Porcel M, Gheysens O,Paulmurugan R, et al. Antioxidantsimprove early survival of cardiomyo-blasts after transplantation to themyocardium. Mol Imaging Biol 2010;12:325–34.

15. van der Bogt KE, Sheikh AY,Schrepfer S, et al. Comparison ofdifferent adult stem cell types fortreatment of myocardial ischemia.Circulation 2008;118:S121–9.

16. Hung TC, Suzuki Y, Urashima T, et al.Multimodality evaluation of the viabil-ity of stem cells delivered into differentzones of myocardial infarction. CircCardiovasc Imaging 2008;1:6–13.

17. Swijnenburg RJ, Govaert JA, van derBogt KE, et al. Timing of bonemarrow cell delivery has minimal ef-fects on cell viability and cardiac re-covery after myocardial infarction. CircCardiovasc Imaging 2010;3:77–85.

18. Brandl A, Meyer M, Bechmann V,Nerlich M, Angele P. Oxidative stressinduces senescence in human mesen-chymal stem cells. Exp Cell Res 2011;317:1541–7.

19. Sanchez C, Oskowitz A, PochampallyRR. Epigenetic reprogramming of IGF1and leptin genes by serum deprivation inmultipotential mesenchymal stromalcells. Stem Cells 2009;27:375–82.

20. Wang X, Zhao T, Huang W, et al.Hsp20-engineered mesenchymal stemcells are resistant to oxidative stress viaenhanced activation of Akt andincreased secretion of growth factors.Stem Cells 2009;27:3021–31.

21. Wang ZJ, Zhang FM, Wang LS, YaoYW, Zhao Q, Gao X. Lipopolysac-charides can protect mesenchymalstem cells (MSCs) from oxidativestress-induced apoptosis and enhanceproliferation of MSCs via Toll-likereceptor(TLR)-4 and PI3K/Akt. CellBiol Int 2009;33:665–74.

22. Tsubokawa T, Yagi K, Nakanishi C,et al. Impact of anti-apoptotic and anti-oxidative effects of bone marrowmesenchymal stem cells with transientoverexpression of heme oxygenase-1 onmyocardial ischemia. Am J PhysiolHeart Circ Physiol 2010;298:H1320–9.

23. Loening AM, Wu AM, Gambhir SS.Red-shifted Renilla reniformis lucif-erase variants for imaging in livingsubjects. Nat Methods 2007;4:641–3.

24. Iyer M, Wu L, Carey M, Wang Y,Smallwood A, Gambhir SS. Two-steptranscriptional amplification as amethodfor imaging reporter gene expressionusing weak promoters. Proc Natl AcadSci U S A 2001;98:14595–600.

25. GyongyosiM,Blanco J,MarianT, et al.Serial noninvasive in vivo positronemission tomographic tracking ofpercutaneously intramyocardially injec-ted autologous porcine mesenchymalstem cells modified for transgenereporter gene expression. Circ Car-diovasc Imaging 2008;1:94–103.

26. Willmann JK, Paulmurugan R,Rodriguez-Porcel M, et al. Imaginggene expression in human mesen-chymal stem cells: from small to largeanimals. Radiology 2009;252:117–27.

27. Perin EC, Tian M, Marini FC 3rd,et al. Imaging long-term fate ofintramyocardially implanted mesen-chymal stem cells in a porcinemyocardial infarction model. PLoSOne 2011;6:e22949.

Key Words: bioluminescence -

mesenchymal stem cells -

NAD(P)H oxidase - oxidativestress - reporter gene.

A P P E N D I X

For the full protocol and supplemental figures,please see the online version of this article.