International Journal of Cardiology 169 (2013) 288–295

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International Journal of Cardiology

j ourna l homepage: www.e lsev ie r .com/ locate / i j ca rd

Bioluminescence imaging of cardiomyogenic and vascular differentiationof cardiac and subcutaneous adipose tissue-derived progenitor cells in fibrinpatches in a myocardium infarct model

Juli R. Bagó a,b,1, Carolina Soler-Botija c,1, Laura Casaní e, Elisabeth Aguilar a,b, Maria Alieva a,b, Núria Rubio a,b,Antoni Bayes-Genis b,c,d, Jerónimo Blanco a,b,⁎a Institute for Advanced Chemistry of Catalonia, Barcelona 08034, Spainb Networking Biomedical Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spainc Heart Failure and Cardiac Regeneration (ICREC) Research Program, Health Research Institute Germans Trias i Pujol (IGTP), Cardiology Service, Hospital Universitari Germans Trias i Pujol,08916 Badalona, Spaind Department of Medicine, Autonomous University Barcelona, Spaine Cardiovascular Research Center (CSIC-ICCC), 08025 Barcelona, Spain

⁎ Corresponding author at: Instituto deQuímica Avanza26, 08034 Barcelona, Spain. Tel.: +34 93 400 6100x5022.

E-mail address: jeronimo.blanco@iqac.csic.es (J. Blanco1 These two authors contributed equally to this work.

0167-5273/$ – see front matter © 2013 Elsevier Ireland Lhttp://dx.doi.org/10.1016/j.ijcard.2013.09.013

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 26 March 2013Received in revised form 3 September 2013Accepted 27 September 2013Available online 6 October 2013

Keywords:BioluminescenceNon-invasive imagingGene reporterAdipose tissue-derived progenitor cellsAcute myocardial infarction

Background: Adipose tissue-derived progenitor cells (ATDPCs) isolated from human cardiac adipose tissue areuseful for cardiac regeneration in rodent models. These cells do not express cardiac troponin I (cTnI) and onlyexpress low levels of PECAM-1 when cultured under standard conditions. The purpose of the present study wasto evaluate changes in cTnI and PECAM-1 gene expression in cardiac ATDPCs following their delivery through afibrin patch to a murine model of myocardial infarction using a non-invasive bioluminescence imaging procedure.Methods and results: Cardiac and subcutaneous ATDPCs were doubly transduced with lentiviral vectors for theexpression of chimerical bioluminescent–fluorescent reporters driven by constitutively active and tissue-specificpromoters (cardiac and endothelial for cTnI and PECAM-1, respectively). Labeled cells mixed with fibrin wereapplied as a 3-D fibrin patch over the infarcted tissue. Both cell types exhibited de novo expression of cTnI,though the levels were remarkably higher in cardiac ATDPCs. Endothelial differentiation was similar inboth ATDPCs, though cardiac cells induced vascularizationmore effectively. The imaging results were corroborated

by standard techniques, validating the use of bioluminescence imaging for in vivo analysis of tissue repairstrategies. Accordingly, ATDPC treatment translated into detectable functional andmorphological improvementsin heart function.Conclusions: Both ATDPCs differentiate to the endothelial lineage at a similar level, cardiac ATDPCs differentiatedmore readily to the cardiomyogenic lineage than subcutaneous ATDPCs. Non-invasive bioluminescence imagingwas a useful tool for real time monitoring of gene expression changes in implanted ATDPCs that could facilitatethe development of procedures for tissue repair.

© 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

The goal of tissue engineering (TE) is the generation of a functionaltissue replacement using biological and synthetic materials, oftenin combination with cells and other biochemical factors. One of themajor challenges of TE is the restoration of cardiac function after myo-cardial infarction (MI). Cumulative evidence indicates that TE has onlybeen capable of modest improvements to cardiac function. Thus, novelcell sources, scaffolds, and biochemical factors with the potential to

da de Cataluña, Jordi Girona 18-

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td. All rights reserved.

repair injured tissue are needed. In vivo analysis of these elements andtheir interactions requires a great amount of resources and time. Thus,agile analytical procedures for in vivo evaluation have the potential tofacilitate the development of TE strategies [1–3].

We used non-invasive bioluminescence imaging (BLI) to monitorthe behavior of cells seeded in a biomaterial implanted in a mousemodel of MI. Due to the capacity of visible light photons to transverseliving tissues, BLI allows the distribution, proliferation, and differentiationof luciferase-expressing cells to bemonitored in real time in living tissues.By using tissue-specific promoters to regulate the expression of luciferasereporters introduced into living cells, changes in promoter activitytranslate into measurable changes in photon fluxes that correlate withtranscriptional activity. Cardiac (troponin I) and endothelial (PECAM-1)specific promoters were used in the present study to regulate theexpression of chimerical luciferase-fluorescent protein reporters.

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This approach allowed us to analyze the cardio-regenerative potentialof two related cell populations from adipose tissue.

Human adipose tissue-derived progenitor cells of cardiac origin(cardiac ATDPCs) have inherent cardiac and endothelial differentiationpotential, exerting a beneficial histopathological and functional effectupon intramyocardial transplantation in experimental models of MI inrodents [4].

In the present work, we embedded cardiac and subcutaneousATDPCs in a natural fibrin scaffold as a cell support and transplantedthis construct into the ischemic myocardium of infarcted mice inorder to compare their gene expression and proliferative behavior.The implanted cells were permanently labeled by transduction withlentiviral vectors for the expression of chimeric photoproteins withtwo types of activities: bioluminescence for non-invasive monitoringand fluorescence for cell enrichment and histological analysis. Compar-ison of the cardiomyogenic and endothelial differentiation capacity ofATDPC cell types using the in vivo BLI procedure indicates that cardiacATDPCs are better cardiomyogenic precursors. Moreover, postmortemvalidation of BLI results with more standard procedures shows thatthis is a convenient and sensitive strategy for non-invasive monitoringof changes in gene expression associated with myocardium repair.

2. Methods

2.1. Isolation and culture of cardiac and subcutaneous ATDPCs

Cells were isolated from the epicardiac adipose tissue (cardiac) and fat tissue abovethe sternum (subcutaneous) of patients undergoing cardiac surgery. Informed consentwas obtained from all subjects, and the study protocol conformed to the principlesoutlined in the Declaration of Helsinki. Biopsy samples were processed and cells isolatedas described previously [5]. Adhered cells were cultured to subconfluence under standardconditions.

2.2. Flow cytometry

Immunophenotypical characterization of cardiac and subcutaneous ATDPCs was per-formed as described previously [4,6].

2.3. Generation of luciferase-fluorescent protein reporters regulated by specific human cardiactroponin I promoter (hcTnIp) and specific human PECAM-1 promoter (hPECAM-1p)

A pLox:PLuc:GFP lentiviral vector containing a fusion reporter comprising PLuc andgreen fluorescent protein (GFP), was obtained by PCR amplification and standard cloningprocedures using the PLuc and GFP genes from the pGL4.10:PLuc (Promega Corporation,Madison, WI) and pEGFP-N1 plasmids (Clontech Lab., Palo Alto, CA). The 340-bp humancTnIp promoter (hcTnIp) was PCR amplified from genomic DNA using 5′-TCCTTGTGTGAGGGAGTGG-3′ and 5′-GGGTGACCTTCAGGGTCC-3′ as described previously [5] and clonedinto the pCR2.11 vector (Invitrogen, Paisley, UK). The promoter sequence was removedfrom the pCR2.11 vector and cloned into the pLox:PLuc:GFP lentivirus vector to obtainPlox:hcTnIp:PLuc:GFP. Human PECAM-1 promoter (hPECAM-1p) was kindly provided byDr. Carmelo Bernabéu (Centro de Investigaciones Biológicas CSIC, Madrid, Spain) and clonedinto pLox:PLuc:GFP to obtain Plox:hPECAM-1p:PLuc:GFP.

The constitutively expressed reporter, the CMV:RLuc:RFP:ttk lentiviral vector contain-ing a trifunctional chimeric construct comprising the RLuc reporter gene, monomeric redfluorescent protein (RFP), and a truncated version of the herpes simplex virus thymidinekinase gene sr39tk (ttk) under transcriptional control of CMV,was a kind gift from Profes-sor S.S. Gambhir (Dept. Radiology, Stanford University, US).

2.4. Lentiviral particle production and ATDPC transduction

Lentiviral production was performed as described [7–9]. ATDPCs were transducedusing CMV:hRLuc:RFP:ttk concentrated lentiviral stock (2 × 106 transduction units/mL,MOI=21) for 48h. The highest 11% of RFP-expressing cells were selected by FACS. Sortedcells were transduced again with either concentrated lentiviral Plox:hcTnIp:PLuc:GFPor Plox:hPECAM-1p:PLuc:GFP stocks (2×106transduction units/mL,MOI=21) for 48h toobtain double-transduced cells: hcTnIp:PLuc·GFP/CMV:RLuc:RFP:ttk-ATDPCs and hPECAM-1p:PLuc:GFP/CMV:RLuc:RFP:ttk-ATDPCs.

2.5. RNA extraction and real-time PCR

Total RNA was extracted from each mouse heart 3weeks after MI using the RNAeasymini kit (Qiagen, Dusseldorf, Germany) according to themanufacturer's instructions. Onemicrogram of total RNA was reverse transcribed using the Revertaid First Strand cDNASynthesis Kit (Fermentas, St. Leon Rot, Germany). FAM-labeled primers/probes were

human specific. Details of the quantitative real-time RT-PCR protocol are provided in theSupplemental Material online.

2.6. Myocardial infarction model

The study was performed using 97 female SCID mice (20 to 25 g; Charles RiverLaboratories Inc.). Myocardial infarction was induced as described previously [4].Briefly, the animals were intubated and anesthetized with a mixture of O2/isofluraneand mechanically ventilated. The heart was exposed and the left anterior descending(LAD) coronary artery permanently occluded with an intramural stitch (7–0 silk suture).Sham-operated animals were prepared in a similar manner except without occluding theLAD coronary artery. Mice were then randomly distributed into six experimental groups:control MI, control MI with implanted fibrin patch, sham implanted cell-loaded fibrinpatch (cardiac or subcutaneous ATDPCs), andMI implanted cell-loaded fibrin patch (cardiacor subcutaneous ATDPCs).

All procedures have the approval of the Animal Care Committee of the ResearchCentre and the Government of Catalonia.

2.7. Development and delivery of fibrin patch

To produce thefibrinpatch, Tissucol duo Baxterfibrin adhesivewas used. Eightmicro-liters of Tissucol solution (fibrinogen 90 mg/mL) were mixed with 1.5 × 106 transducedcells or culture medium. Then, 8 μL of thrombin solution (500 IU/mL) were added forjellification. Fibrin patches loaded with cells were cultured under standard conditionswith supplemented α-MEM (10% FBS, 1% P/S, 1% L-glutamine) for 24 h. Cell-loaded andnon-loaded fibrin patches were then implanted after MI induction, covering the injuredtissue using synthetic surgical glue (Glubran®2) in the healthy myocardium. Fibrinpatches were also implanted in sham-operated animals.

Three weeks after implantation, the hearts were arrested in diastole using arrestsolution [4], excised, fixed, cryopreserved in 30% sucrose in PBS, embedded in OCT(Sakura), and snap-frozen in liquid nitrogen-cooled isopentane.

2.8. Non-invasive BLI of luciferase activity

For in vivo BLI, anesthetized mice bearing a fibrin patch seeded with luciferasereporter-expressing cells were intraperitoneally injected with 150 μL of luciferin(16.7 mg/mL in physiological serum) (Caliper, Hopkinton, MA) to image PLuc activity.The mice were injected intravenously (tail vein) with 25 μL of benzyl coelenterazine(hCTZ) (1 mg/mL in 50/50 propylene glycol/ethanol) (Nanolight Technology, Pinetop,AZ) diluted in 125 μL of water to image RLuc activity. PLuc and RLuc activities were mea-sured in consecutive days. Mice were monitored during a 3-week period at the indicatedtimes. Photons recorded in images were quantified and analyzed using theWasabi imageanalysis software (Hamamatsu Photonics).

2.9. Echocardiography

Mouse cardiac function was assessed by transthoracic echocardiography. Anultrasound system (iE33 Echocardiography System, Philips) equipped with a 15-7io MHz linear-array transducer was used to take measurements at baseline(1 day pre-infarction), 1 day post-infarction, and 2 and 3week post-cell transplan-tation. The investigators were blinded to the treatment groups. Images wereobtained in B-Mode and M-Mode in the parasternal long-axis view. Functional parameterswere measured over five consecutive cardiac cycles and calculated using standard methods[10,11]. Left ventricular end-diastolic diameter (LVDd), LV end-systolic diameter (LVDs), LVend-diastolic volume (LVEDV), and LV end-systolic volume (LVESV) were quantified. Leftventricular ejection fraction (LVEF) was calculated as (LVEDV− LVESV)/LVEDV]×100 [12].

2.10. Morphometry

Mouse hearts were transversally sliced in two segments: apex and base. Eight serialcryosections (spaced 100-μm apart) from the apex segment were stained with Masson'strichrome and morphometric parameters determined using image analysis software(ImageJ, NIH). The infarct size was calculated as: percentage of mean scar area/total LVwall surface × 100. To evaluate infarct thickness, wall thickness was measured at thethinnest and border zones of the infarction. Three measurements per sectionwere performed to determine posterior wall thickness. The mean value of six 200-μmsections was calculated for the thickness parameters. All sections were photographedusing a Leica Stereoscope (Leica TL RCI) and a blind analysis performed.

2.11. Immunohistochemistry

Mouse heart cryosections were incubated with primary antibodies against CD31(0.8 μg/mL) (Abcam), cTnI (2 μg/mL) (Abcam), or phospho-histone 3 (phospho-H3)(2 μg/mL) (CellSignaling). Sections were also incubated with antibodies against RFP andGFP (5μg/mL) (Abcam) to enhance thedetection of transduced cells. Secondary antibodiesconjugated with Cy2, DyLight 549, and Cy5 (1 μg/mL) (Jackson ImmunoResearch) wereused for detection. Nuclei were counterstained with Hoechst 33342 and the resultsanalyzed using a Leica TCS SP2 laser confocal microscope.

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2.12. Vessel density

To determine vessel density at the border and distal zones from the infarction, mouseheart sections were stained with biotinylated GSLI B4 isolectin (vector Labs), and AlexaFluor 647-conjugated streptavidin was used as a detection system. Images were taken inat least 20 randomly selected fields (10 border areas + 10 distal areas) and analyzedusing image analysis software (ImageJ, NIH). The results were expressed as a percentageof the mean isolectin-positive area per area of tissue surface.

2.13. Fluorescence angiography

FITC-dextran (Sigma, St. Louis, USA)was used as a fluorescent tracer of microvascularstructures in fibrin patches and the heart. FITC-dextran (200 μL; 10mg/mL) was injectedthrough the lateral tail vein of anesthetizedmice 3weeks afterMI. Tenminutes after inoc-ulation, the mice were sacrificed, and the heart retrieved, fixed in 10% formalin solution(Sigma), and sectioned for microscopy. Laser confocal microscopy was used to analyzemicrovascular structures connected to the host vascular system in fibrin patches andtheir relationship with implanted ATDPCs.

2.14. Statistical analysis

The significance of the bioluminescence signal and echocardiographic parameterswasevaluated by analysis of variance (ANOVA) using the factors time and treatment, and aGreenhouse correctionwas applied. Vessel density, survival rate, andmorphometric com-parisons between control and cell-treated groups were performed using parametric one-way ANOVA. Pairwise comparisons between groups were made by post-hoc analysis(Tukey method) for multiple comparisons. All of the results were presented as mean ±SEM. P-values b0.05 were considered significant. All analyses were performed usingSPSS Statistics (version 19).

3. Results

3.1. Cardiac and subcutaneous ATDPCs differentiate into the cardiomyogeniclineage in a mouse model of MI

To monitor survival and cardiomyogenic lineage differentiation,cardiac and subcutaneous ATDPCs were transduced using lentiviralvector CMV:RLuc:RFP:ttk for expression of the RLuc:RFP reporter underthe regulation of the CMV promoter, a cell number reporter. Positivelytransduced cells expressing RLuc-RFP were selected by FACS and trans-duced with lentiviral vector pLox:hcTnIp:PLuc:GFP to express the PLuc:GFP reporter under the regulation of hcTnIp, a reporter of cardiomyogenicdifferentiation. Transduced cells were then loaded into a fibrin patch anddelivered to the infarcted area in a mouse model of MI.

Quantification of photon counts acquired weekly for 3 weeks frommice implanted with each cell type showed an increase in the ratiobetween hcTnIp-regulated PLuc and CMV-regulated RLuc activities ininfarcted and sham-infarcted animals (Fig. 1A and B). These resultsindicate that de novo gene expression of cTnI was already induced1week post-implantation. Interestingly, BLI revealed that animals treat-ed with cardiac ATDPCs expressed higher levels of cTnI compared tosubcutaneous ATDPC-treated animals (p = 0.009). Although the cTnIlevels in cardiac ATDPCs tended to decrease over time, they were greaterthan those of subcutaneous ATDPCs at all time points analyzed (Fig. 1B).

As an independent procedure, RT-PCR was used to validate the BLIdata usingmRNA extracted from the cell-loaded fibrin patches implantedin the infarcted animals. Changes in cardiac gene expression levels werecompared to those from cardiac and subcutaneous ATDPCs culturedin vitro during the same time period. The relative gene expression indicat-ed an increase in the cardiac markers in the implanted cells compared tothose fromcultured cells (Fig. 1C). A comparisonof the cardiac and subcu-taneous cell types revealed higher expression of Tbx5, Mef2, Nkx2.5,GATA4, sarcomeric α-actinin, SERCA2, and Cx43 in implanted cardiacATDPCs. Importantly, no amplification of mouse mRNAs was measuredwith any of the human FAM-labeled primers, except for cTnI, as analysisof this marker could not be performed (data not shown).

Excised heart cross-sections were analyzed to determine the histo-logical distribution and differentiation of the implanted human cells.Immunostaining for RFP revealed the presence of cardiac and subcuta-neous ATDPCs in the fibrin patch 30 day post-implantation in all cases(Fig. 1E and I). However, migration of the ATDPCs into themyocardium

was observed rarely in infarcted or sham animals (Supplemental Fig. 1).To demonstrate that cTnI gene expression correlated with the presenceof the protein, we used an anti-cTnI antibody to immunostain cells inthe implanted fibrin patch. The results (Fig. 1D–K) confirmed the pres-ence of the cTnI protein in RFP- and GFP-expressing cells, validating theBLI data and providing further evidence of their differentiation to thecardiac lineage.

3.2. Cardiac and subcutaneous ATDPCs differentiate into the endotheliallineage when transplanted into a mouse model of MI

Tomonitor in vivo differentiation of cardiac and subcutaneous ATDPCsto the endothelial lineage, cells were first transducedwith the CMV:RLuc:RFP:ttk photoprotein reporter, and the positively transduced cells weretransduced a second time with pLox:hPECAM-1p:PLuc:GFP. Doublytransduced cells were mixed with fibrin and implanted as a fibrin patchover experimental infarcts and sham controls in SCID mice. Results fromboth cell types (Fig. 2A and B) revealed an important increase in thePLuc/RLuc ratio 2week post-implantation, relative to the ratio at implan-tation time, showing an up-regulation of PECAM-1 gene expression. Incardiac ATDPC-treated hearts from the MI and sham groups, thePLuc/RLuc ratio increased during the first week post-implantation,followed by a decrease by week 3 (Fig. 2B). Interestingly, infarcted ani-mals treated with subcutaneous ATDPCs exhibited an up-regulation ofPECAM-1 similar to that of cardiac ATDPCs between the first and secondweek post-implantation, but this level was maintained throughout theexperiment (3weeks). Sham-operated animals treatedwith subcutane-ous ATDPCs presented with a gradual increase in PECAM-1 expression,but the levels were lower compared to those of infarcted animals.ANOVA did not demonstrate significant differences (p = 0.330) inthese changes for both cell types and experimental groups.

A comparison of changes in gene expression in ATDPCs from fibrinpatches in excised hearts and in vitro culture confirmed the up-regulation of human PECAM-1 gene expression in both subcutaneousand cardiac cell types (Fig. 2C). In addition, other human endothelialmarkers, including CD34 and EGR3, also exhibited increased geneexpression. Thus, in vivo changes in PLuc activity regulated by the pro-moter of a gene expressed in endothelial cell lineages (PECAM-1) corre-lated with changes in mRNA expression.

Histological examination of hearts bearing fibrin implants re-vealed the presence of human cells (RFP-positive) expressing thehPECAM-1p-regulated PLuc-GFP reporter (GFP-positive) that were alsopositive for the expression of PECAM-1 protein. Importantly, some ofthe PECAM-1-positive cells exhibited endothelial cell morphology andwere associatedwith vessel structures, further supporting the endotheliallineage differentiation of cardiac and subcutaneous ATDPCs in themyocardium (Fig. 2D–K).

3.3. Cardiac ATDPCs and fibrin increase vessel density in subjacentmyocardial tissue

To investigate whether ATDPCs promote angiogenesis whenadministered in a fibrin patch, wemeasured vessel density in the infarctregion subjacent to the patch. By using fluorescent isolectin to stain en-dothelial cells, we found that the vessel density was 32.9% greater in thesubjacentmyocardium tissue sections fromhearts that received cardiacATDPCs than from equivalent controls without a patch (p b 0.01)(Fig. 3A–C). Interestingly, the administration of fibrin glue withoutcells also significantly increased vascularization by 22.8% (p b 0.01).Nevertheless, differences between MI-fibrin and cell treated groupsreached statistical significance (cardiac ATDPCs p=0.036; subcutane-ous ATDPCs p=0.016). Although subcutaneous ATDPC-treated animalsalso presented a 5.7% greater vessel density compared to controls, thedifferences were not significant. Taken together, these results suggestthat fibrin glue may promote new vessel formation in the ischemicmyocardium and that this effect is enhanced by cardiac ATDPCs.

Fig. 1. Cardiac differentiation of cardiac and subcutaneous ATDPCs in a mouse model of myocardial infarction. Representative bioluminescent images from dual-labeled cardiac and sub-cutaneous ATDPCswithin an implantedfibrin patch. A cell number reporter (CMV:hRLuc:RFP:ttk) and cell differentiation reporter (PLuc:GFP)were regulated byhcTnIp. Luciferase imagesare superimposed on black and white dorsal images of recipient animal (A). Color bars illustrate the relative light intensities from PLuc and RLuc: low, blue and black; high, red and blue.(B) Histograms showing the PLuc/RLuc ratio calculated from photon fluxes recorded in bioluminescent images. (C) Real-time RT-PCR analysis of cardiac gene expression from cardiac andsubcutaneous ATDPCs in fibrin patches 3week post-transplantation. Valueswere normalized to GAPDH expression and are shown asmean±SEM. (D–K) Immunofluorescent staining oftransplanted cardiac (D–E) and subcutaneous (H–K) ATDPCs. Transplanted cells were detected by immunostaining with an anti-RFP antibody (red, E and I). Troponin-I promoter-regulated expression of GFP and troponin-I protein expression were detected with anti-GFP (green, D and H) and anti-troponin-I protein (white, F and J) antibodies, respectively.(I and K) show merged images. Nuclei were counterstained with Hoechst 33342. Scale bars= 25 μm.

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To determine if vessel connections were present between thefibrin patch and myocardium, FITC-dextran was injected in the tailvein previous to sacrifice. Fluorescence confocal microscope imagesrevealed that the fibrin patches were vascularized in all groups ana-lyzed, regardless of whether infarction had been induced. The FITC-dextran test indicated the presence of functional vessels connected

to the mouse circulatory system, in the fibrin patch, myocardium,and myocardium-fibrin patch interphase (Fig. 3D–G). Moreover,integration of cardiac and subcutaneous ATDPCs into the vesselstructures was also observed inside the fibrin patches (Fig. 3D).FITC-positive vessels were also found in MI fibrin controls withoutATDCPs (Fig. 3G).

Fig. 2. Endothelial differentiation of cardiac and subcutaneous ATDPCs in a mouse model of myocardial infarction. Representative bioluminescent images from dual-labeled cardiac andsubcutaneous ATDPCswithin an implanted fibrin patch. (A) A cell proliferation reporter (CMV:hRLuc:RFP:ttk) and cell differentiation reporter (PLuc:GFP) were regulated by hPECAM-1p.Luciferase images are superimposed on black and white dorsal images of the recipient animal. Color bars illustrate relative light intensities from PLuc and RLuc: low, blue and black; high,red and blue. (B) Histograms showing the PLuc/RLuc ratio calculated fromphoton fluxes recorded in bioluminescent images. (C) Real-time RT-PCR analysis of endothelial genes in cardiacand subcutaneous ATDPCs in fibrin patches 3weeks after transplantation. Values were normalized to GAPDH expression and are shown as mean±SEM. Immunofluorescent staining oftransplanted cardiac (D–G) and subcutaneous (H–K)ATDPCs. Transplanted cellswere detected by immunostainingwith an anti-RFP antibody (red, E and I). PECAM-1promoter-regulatedexpression of GFP and PECAM-1 protein was detected with anti-GFP (green, D and H) and anti-PECAM-1 (white, F and J) antibodies. (G and K) are merged images. Nuclei were counter-stained with Hoechst 33342. Scale bars=25 μm.

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3.4. Cardiac and subcutaneous ATDPCs did not proliferate in the fibrinimplants

Although we were able to detect the presence of cells in the fibrinpatch 30days post-implantation, their survival rate reduced dramatical-ly during the first week post-implantation and continued to decrease

during the experiment (Supplemental Fig. 2). No differences in thisbehavior were observed between cardiac and subcutaneous ATDPCs orbetween MI and sham-operated animals were observed when ANOVAwas applied (p=0.535), suggesting that injury-generated signals werenot capable of modulating cell survival. To confirm the BLI results, heartcross-sections were double immunostained for phospho-H3 and RFP.

Fig. 3.Vascular analysis. (A) Vessel density expressed as the percentage of the area occupied by isolectin-positive cells in controls and ATDPC-treated groups. *pb0.01. Values aremean±SEM. (B and C) GSLI B4 isolectin staining inMI control (B) and cardiac ATDPC-treated animals (C) showing qualitative differences in vessel density. (D–G) Functional vessel detectionwithFITC-dextran (green) and GSLI B4 isolectin (white, D, E, and G) inmice treated with cardiac ATDPCs (red, D–F). (D) Arrowheads indicate the integration of transplanted cells into vessels.(E) Section showing the interface between themyocardium and fibrin patch. Arrowheads indicate vessels transversing between the mouse myocardium and fibrin patch in a cell-treatedanimal (FITC-dextran, green; cardiac ATDPCs, red). Transplanted cells were detected by RFP immunostaining (red, D–F). Nuclei were counterstained with Hoechst 33342. Scale bars=50 μm.

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The images showed no co-localization of either marker, demonstratingthat the implanted cells did not proliferate within the fibrin patch (datanot shown).

3.5. Transplantation of an ATDPC-seeded fibrin patch improves cardiacfunction and diminishes scar size after MI

To determine whether ATDPCs have an effect on the restoration ofcardiac function following infarct, echocardiographic analyses wereperformed. Evaluation of the LVEF revealed that infarcted animals treat-ed with fibrin patches containing cardiac ATDPCs presented with animportant initial improvement (20% recovery), and the values weremaintained throughout the experiment (21.5% recovery pre-sacrifice),whereas MI control animals without patches exhibited only a 2.5% im-provement. Interestingly, infarcted animals treated with subcutaneousATDPCs and MI fibrin controls without cells presented similar LVEFvalues 2 week post-implantation (Fig. 4A), as well as a comparablerecovery (10% and 11%, respectively).

Morphometric analyses of heart cross-sections demonstrated areduced scar size in animals treated with cardiac (30%, p = 0.030) orsubcutaneous (33%, p b 0.005) ATDPCs, or with fibrin patches (27%, p=0.036) compared toMI controls. Differences in scar size were concordantwith LV posterior wall thickness; infarcted animals treated with cells hadthe greatest values compared to non-treated animals (p=0.010 for car-diac ATDPCs and p b 0.01 for subcutaneous ATDPCs). No differences inposterior wall thickness were measured between MI controls and MIfibrin controls (p=0.882; Fig. 4D–I).

4. Discussion

During the past decade, a great deal of attention has been paid tothe use of cell therapy for cardiac regeneration [13–19]. We showedpreviously that the injection of ATDPCs at the infarct border exerts abeneficial effect on cardiac function and reduced scar size in amurine model of MI [4,20]. These cells differentiated into cardiacand endothelial cells in vivo when analyzed at the time of sacrifice

1 month post-implantation. However, few cells and poor interactionwith the host tissue were found, probably due to the adverse effect ofmechanical stress and hypoxic conditions [21].

These results emphasizing the importance of understanding cellbehavior in real time and in vivo suggested the current experimentsbased on the use of non-invasive BLI to evaluate changes in cell differen-tiation and survival in a live mouse model of acute MI. We used a fibrinpatch as a realistic scaffoldmodel for the implantation of cells to evaluatetheir cardiomyogenic and angiogenic differentiation potential forMI therapy.

In our study, the cTnI promoter was used to regulate the expres-sion of a luciferase-fluorescent protein chimerical reporter to ana-lyze cardiac differentiation. In the same cells, a Cytomegalovirus(CMV) constitutive promoter was used to regulate the expressionof a different luciferase-fluorescent protein chimera to report cellnumber [22]. Using this approach, evaluation of the ratio of lightfluxes from both reporters allowed us to measure cell differentiationindependent of cell number.

Bioluminescent quantification indicated that relative to implanta-tion day, de novo expression of cTnI in cardiac ATDPCs was alreadyinduced 1 week post-implantation. Even though cTnI levels tended toslightly decrease over time, they were significantly different comparedto the initial conditions. However, subcutaneous ATDPCs also expressedcTnI in this experimental model, but the levels were remarkably lowerin both infarcted and sham controls, suggesting that cardiac ATDPCshave a greater cardiac differentiation potential. The BLI results werevalidated using an independent procedure (RT-PCR) that showed theinduction of additional cardiac markers in both cell types. Furthervalidation of the induction of cTnI expressionwas provided by immuno-fluorescence detection of cTnI protein in implanted cardiac and subcu-taneous ATDPCs that also expressed the RFP and GFP reporters.

We used a similar approach to evaluate the pro-angiogenic potentialof ATDPCs. However, we used the PECAM-1 gene promoter in place ofthe cTnI promoter to regulate expression of the PLuc-GFP reporter.The BLI results showed up-regulation of PECAM-1 activity 1 weekpost-implantation in both infarcted and sham animals treated with

Fig. 4. Functional and morphometric analysis. (A–C) Echocardiographic functional parameters in the mouse. (A) Quantification of the ejection fraction at baseline, 1 day (MI) and 2weekpost-infarction and at sacrifice. RepresentativeM-mode echocardiograms of MI-control showing LV anterior wall absence of motility (B) andMI-cardiac ATDPC-treated animals showingLV anteriorwall recovery ofmotility (C) before sacrifice.Masson's trichrome staining of cross-sections of the infarctedmousemyocardium3weeks after surgery (D,MI; E,MI-Fibrin; F,MI-cardiac ATDPCs; G, MI-sub ATDPCs). Percentage of the left ventricular (LV) infarct surface (H), posterior LV wall thickness (I), and LV wall thickness (J). Scale bar = 1 mm. Values aremean± SEM. * p b 0.05.

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cardiac ATDPCs, with a strong increase in the PLuc/RLuc ratio at2 weeks. Previous studies showed that subcutaneous ATDPCs werenegative for PECAM-1 expression [23,24]. However, a fraction of thesecells resided in a perivascular location and were able to differentiateinto endothelial cells that integrated into newly formed blood vesselsduring angiogenesis and neovasculogenesis [25,26]. BLI revealed anincrease in PECAM-1 activity in subcutaneous ATDPCs implanted ininfarcted animals. In contrast to cardiac ATDPCs, PECAM-1 expressionlevels were maintained over time in subcutaneous ATDPCs. Analysisby RT-PCR confirmed not only the up-regulation of PECAM-1 expression,but also that of additional endothelial markers in implanted cells,compared to those maintained in in vitro cultures; thus, validatingthe BLI results. When immunofluorescence was used to detect PECAM-1 in order to localize human cells in the host tissue, we found that mostof the PECAM-1-expressing implanted cells were located in vesselstructures adopting endothelial cell morphology. Local endothelial

differentiation and incorporation into new blood vessels was reportedpreviously in stem cells from subcutaneous adipose tissue [27,28]. Moreimportantly, these vessels were functional, as they contained the tailvein-injected FITC-dextran in their lumen. Comparable results werefound by Lyliana et al. [29] when also implanted mesenchymal stemcells from subaminion embedded in fibrin glue and covered by anomental flap in the rat model of MI.

To determinewhether the observedATDPC behavior correlatedwithfunctional changes, we also evaluated vessel density in the myocardialtissue subjacent to the fibrin patches and found that, though both celltypes were capable of inducing vascularization, cardiac ATDPCs weremore effective than those from subcutaneous fat. Interestingly, whenthe fibrin control group without cells was analyzed, functional vesselswere also found to traverse the myocardium-fibrin patch interphase,showing that the fibrin glue itself was a good vascular inducer as previ-ously reported [30].

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In accordance with the pro-angiogenic effect of ATDPCs and fibringlue, cardiac functional and morphometric analysis revealed that bothcells types exerted a beneficial influence; cardiac ATDPCs were moreeffective than subcutaneous ATDPCs. Infarcted animals treated withcardiac or subcutaneous ATDPCs presented a smaller infarct scar thannon-treated controls, probably due to the capacity of these cells to in-duce an increase in vessel density and preserve tissue from ischemicdamage [1,4,26,31]. In addition to reducing infarct size, both cell typesalso inhibited the extension of the infarct to the anterior area, asevidenced by the lack of significant difference in the LV posterior wallthickness of cell-treated groups compared to sham controls. However,fibrin glue alone also improved cardiac function and reduced infarctsize, as previously reported [32]. Thus, fibrin glue may prevent LV re-modeling by increasing the mechanical strength of the infarct beforethe scar is fully developed, being its effect as significant as the effect ofcardiac ATDPCs already reported in a previous work [4].

Thus, although the current study is directed to the analysis of geneexpression and proliferation of implanted cells and not the effect ofthe scaffold used for implantation, our functional analysis also revealedan interesting vascular induction effect of fibrin, strong enough to over-shadow the effect of ATDPCs in functional studies.

In summary, we evaluated the capacity of cardiac and subcutane-ous ATDPCs for cardiomiogenic and endothelial differentiation in vivousing a BLI procedure to monitor changes in gene expression. We findthat cardiac fat ATDPCs are more effective than adipose fat ATDPCsfor cardiomyogenic differentiation. However, although both cell typeshave similar endothelial differentiation capacity, cardiac fat ATDPCswere more potent inducers of vascularization of subjacent tissue.BLI results were validated using standard RT-PCR, immunologicalcriteria and in agreement with functional studies.

The fibrin glue used as a scaffold in these experiments had a clearvascularization promoting activity, even in the absence of ATDPCs.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.ijcard.2013.09.013.

Funding sources

This work was supported by the Ministerio de Ciencia e Innovación(SAF 2008-05144-C02-01 and SAF2009-07102), European Com-mission 7th Framework Programme (RECATABI, NMP3-SL-2009-229239), Fundació La Marató de TV3 (080330), Red de TerapiaCelular-TerCel (RD12/0019/0029), Red Cardio-vascular (RD12/0042/0047) and Fundació Privada Daniel Bravo Andreu.

Disclosures

None.

Acknowledgments

The authors of this manuscript have certified that they complywith the Principles of Ethical Publishing in the International Journalof Cardiology. The authors also wish to thank the patients whomade this study possible and the members of the Department ofCardiac Surgery for their collaboration in obtaining human samples.We greatly appreciate the help of Carolina Gálvez-Montón in the statis-tical analysis.

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