Sustained Persistence of Transplanted Proangiogenic Cells Contributes to Neovascularization and Cardiac Function After Ischemia

Guillaume Carmona, Carmen Urbich, Andreas M. Zeiher and Stefanie DimmelerFabian Kiessling, Stefan Stein, Manuel Grez, Christian Ihling, Marion Muhly-Reinholz,

Thomas Ziebart, Chang-Hwan Yoon, Thomas Trepels, Astrid Wietelmann, Thomas Braun,Neovascularization and Cardiac Function After Ischemia

Sustained Persistence of Transplanted Proangiogenic Cells Contributes to

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Sustained Persistence of Transplanted Proangiogenic CellsContributes to Neovascularization and Cardiac Function

After IschemiaThomas Ziebart,* Chang-Hwan Yoon,* Thomas Trepels, Astrid Wietelmann, Thomas Braun,

Fabian Kiessling, Stefan Stein, Manuel Grez, Christian Ihling, Marion Muhly-Reinholz,Guillaume Carmona, Carmen Urbich, Andreas M. Zeiher, Stefanie Dimmeler

Abstract—Circulating blood–derived vasculogenic cells improve neovascularization of ischemic tissue by a broadrepertoire of potential therapeutic actions. Whereas initial studies documented that the cells incorporate and differentiateto cardiovascular cells, other studies suggested that short-time paracrine mechanisms mediate the beneficial effects. Thequestion remains to what extent a physical incorporation is contributing to the beneficial effects of cell therapy. By usingthe inducible suicide gene thymidine kinase to deplete transplanted cells, we determined the contribution of physicalincorporation in 3 animal models. After acute myocardial infarction, depletion of cells 14 days after infusion resultedin a reduction of capillary density and a substantial deterioration of heart function. Likewise, neovascularization ofMatrigel plugs and ischemic limbs was significantly suppressed when infused cells were depleted 7 days after infusion.Induction of cell death in the previously transplanted cells reduced perfusion and led to vascular leakage as evidencedby Evans blue extravasation. These results indicate that physical incorporation and persistence of cells contribute tocell-mediated improvement of neovascularization and cardiac function. Long-term paracrine activities and/or cellintrinsic mechanisms may have contributed to the maintenance of functional improvement. (Circ Res. 2008;103:1327-1334.)

Key Words: progenitor cells � neovascularization � cell therapy

Cell therapy is a promising option for treating ischemicdiseases. Adult stem and progenitor cells from various

sources have experimentally been shown to augment thefunctional recovery after ischemia. Clinical trials confirmedthat autologous cell therapy using bone marrow–derived orcirculating blood-derived progenitor cells is safe and providesbeneficial effects1–3 (see also recent metaanalysis4). Endothe-lial progenitor cells (EPCs) were initially isolated from bonemarrow or peripheral blood and were characterized by theexpression of the hematopoietic stem cell marker CD34 orCD133 and endothelial markers such as vascular endothelialgrowth factor receptor 2 (KDR).5,6 Isolated CD34� cellsdifferentiate to endothelial cells in vitro and incorporate intonewly formed vessels during angiogenesis in vivo.5 Thesepioneering studies suggested that bone marrow–derived cellscan participate in vascular repair. Indeed, a single hemato-poietic stem cell was shown to give rise to both blood cellsand vascular endothelium.7–9 The contribution of circulatingcells to the endothelium was further supported by sex-mismatched heart transplantation in humans.10,11 Other inves-tigators used culture assays to enrich for EPC-yielding cells,

which coexpress endothelial and myeloid marker (“earlyEPCs,” proangiogenic cells), or to show a more matureendothelial cell phenotype on further culture and expansion(“late or outgrowing EPCs”).12,13 Although the origin and thephenotype of the cultured EPCs is not entirely clear and mayvary depending on the defined specific culture conditions,14

the therapeutic potential and improved neovascularizationmediated by the cultured cells has been documented exten-sively in ischemic models.12,15,16

Progenitor cell–mediated improved neovascularization ofischemic tissue might be attributable to various therapeuticactions including a physical incorporation of the infused cellsin the endothelium leading to the formation of new capillar-ies.16–18 Transplanted or infused hematopoietic or endothelialprogenitor cells also were shown to differentiate to cardiacmuscle cells.5,19,20 However, the capacity of hematopoietic orendothelial progenitor cells to acquire a cardiac phenotype isnot well established.21 Bone marrow–derived cells also weredetected in the perivascular region, where they were proposedto provide paracrine factors contributing to vessel growth.22

Indeed, cultured endothelial progenitor cells release proan-

Original received May 31, 2008; revision received September 30, 2008; accepted October 6, 2008.From Molecular Cardiology (T.Z., C.-H.Y., T.T., M.M.-R., G.C., C.U., A.M.Z., S.D.), Department of Internal Medicine III, University Frankfurt; Max

Planck Institute (A.W., T.B.), Bad Nauheim; Junior Group Molecular Imaging (F.K.), German Cancer Research Center, Heidelberg; AngewandteVirologie und Gentherapie (S.S., M.G.), Georg-Speyer-Haus, Frankfurt; and Gemeinschaftspraxis fur Pathologie (C.I.), Frankfurt, Germany.

*Both authors contributed equally to this work.Correspondence to Prof Dr Stefanie Dimmeler Molecular Cardiology, Department of Internal Medicine III University of Frankfurt, Theodor-Stern-Kai

7 60590 Frankfurt am Main, Germany. E-mail [email protected]© 2008 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.108.180463

1327 by guest on June 7, 2013http://circres.ahajournals.org/Downloaded from


giogenic and antiapoptotic cytokines, which may contributeto enhanced angiogenesis and endogenous repair.23,24 Basedon these findings, it was speculated that short-term paracrinemechanisms account for the observed therapeutic benefits ofcell therapy.25–27

Thus, despite ample data providing evidence for functionalimprovements after cell therapy, the mechanisms by whichcells augment the functional recovery after ischemia are notwell defined, and it is not known to what extent the physicalincorporation and persistence of transplanted cells contributesto the beneficial effects of cell therapy.

To determine the role of cell incorporation and persistenceafter cell therapy, we used the suicide gene thymidine kinase(TK), which activates the prodrug gancyclovir (GV),28 toinduce cell death in infused cells at specific time points afterinfusion and assessed the influence on neovascularization andcardiac function after induction of ischemia. Our resultsdemonstrate that depletion of cells weeks after infusionreduces vascularization and causes a deterioration of functionin 3 different animal models.

Materials and Methods

Culture of Proangiogenic CellsHuman peripheral blood mononuclear cells (MNCs) were isolated bydensity gradient centrifugation with Biocoll (Biochrom AG) fromperipheral venous blood according to Vasa et al.29 Immediatelyfollowing isolation, 8�106 MNCs per milliliter of medium wereplated on culture flask coated with human fibronectin (Sigma,Taufkirchen, Germany) and maintained in endothelial basal medium(EBM; LONZA, Verviers, Belgium) supplemented with EGMSingleQuots and 20% FBS. After 3 days in culture, nonadherent cellswere removed by thorough washing with PBS, and adherent cellswere incubated in fresh medium.

Cell CulturePooled human umbilical vein endothelial cells (HUVECs) werepurchased from LONZA (Verviers, Belgium) and were cultured inEBM medium supplemented with EGM SingleQuots and 10% FBS.HEK 293 cells were maintained in DMEM containing 10% FBS andpenicillin–streptomycin.

Preparation of Lentiviral StocksSelf-inactivating lentiviral vectors containing the enhanced greenfluorescent protein (GFP) gene or the viral TK gene of the herpessimplex type 2 virus and a WPRE (woodchuck posttranscriptionalregulatory element) under the control of a spleen focus-forming viruspromoter were generated by transient transfection in 293T cells usingpCMV�R8.91 as packaging plasmid and pMD2.G for vesicularstomatitis virus–G protein (VSV-G) pseudotyping as described.30,31

After 8 hours, the medium was replaced by DMEM/10 mmol/Lsodium butyrate for 20 hours. Lentiviral particles were collected inEBM supplemented with EGM SingleQuots and 20% FBS every 24hours for 2 days, pooled (200 to 250 mL), and filtered through0.22-�m filters.

Lentiviral TransductionFor lentiviral transduction, EPCs were transduced on days 3 and 4after isolation. Transduction was carried out by adding viral super-natant to the EBM supplemented with EGM SingleQuots and 20%FBS. After 8 hours, medium was changed and EPCs were transduceda second time. Transduced EPCs were used on days 5 or 6 afterisolation for the following experiments.

Matrigel Plug ModelAll animal experiments were approved by the RefierungspracsidiumDarmstadt, Germany. A total of 500 �L of Matrigel BasementMembrane Matrix (BD Biosciences) without supplements was in-jected subcutaneously into 6- to 8-week-old female athymic nudemice (Harlan) along the abdominal midline. After 7 or 14 days, bloodvessel infiltration in Matrigel plugs was quantified by analysis oflectin and smooth muscle actin-stained sections using microscopy.

Hindlimb Ischemia ModelEight-week-old male Balb/c nude mice (Charles River) were anes-thetized with isoflurane. Right femoral artery and vein were coagu-lated and then cut out to induce critical ischemia. One day afteroperation, EPCs (1 million) were administrated via the tail vein.Laser Doppler perfusion image was taken as indicated and perfusionof each limb was calculated from mean value multiplied by thenumber of pixel of the region below the inguinal ligament. Data wererepresented as ratios of the ischemic limb to the nonischemic limb.Evans blue (30 mg/kg) was injected intravenously at day 9. Micewere euthanized 24 hours after dye injection and cardioperfused withnormal saline. Tissue was harvested and frozen. Evans blue wasextracted from tissue using trichloroacetic acid and ethanol. Absor-bance was measured at 620 nm.

Acute Myocardial Infarction ModelLeft coronary artery occlusion was performed in female nu/nu mice(8 to 9 weeks; Harlan) under mechanical ventilation and anesthesia.Acute myocardial ischemia was induced by ligating the left coronaryartery. Cells (2 million) were intravenously infused 24 hours afteroperation.

Physiological Assessment of LeftVentricular FunctionIn the myocardial infarction study, transthoracic echocardiographywas performed to evaluate left ventricular function by measuringejection fraction 2, 3, and 4 weeks after myocardial ischemia.

MRI ProcedureCardiac MRI was performed under volatile isoflurane (1.5% to2.0%) anesthesia with a Bruker Pharmascan 7.0 T, a custom-builtcircularly polarized birdcage resonator and use of the Early AccessPackage for Self-gated Cardiac Imaging (Intragate).32 This measure-ment is based on the gradient echo method (repetition time�44.4 ms;echo time�6.0 ms; field of view�2.20�2.20 cm; slice thick-ness�1.0 mm; matrix�128�128; repetitions�100). The imagingplane was localized using scout images showing the 4- and2-chamber view of the heart, followed by acquisition in short axisview, orthogonal on the septum in both scouts. Multiple contiguousshort-axis slices consisting of 6 to 8 slices were acquired for completecoverage of the left ventricle. All MRI data were analyzed using Qmassdigital imaging software (Medis, Leiden, The Netherlands).

ImmunostainingsSections were deparaffinized and were incubated with biotinylatedIsolectin B4 (Vector B1201) followed by streptavidin Alexa Fluor488. TUNEL assays (Roche) was performed according to theinstructions of the manufacturer. Confocal microscopic analysis wasperformed using a Zeiss LSM 510 Meta.

Statistical AnalysisData are expressed as means�SEM. Two treatment groups werecompared by Mann–Whitney test, 3 or more treatment groups werecompared by 1-way ANOVA, followed by post hoc analysis adjustedwith a least significant difference correction for multiple compari-sons (SPSS). Results were considered statistically significant whenP�0.05.

An expanded Materials and Methods section is available in theonline data supplement at http://circres.ahajournals.org.

1328 Circulation Research November 21, 2008



ResultsInduction of Cell Death by Virally TransducedSuicide GeneTo investigate the physical contribution of proangiogeniccells, peripheral blood–derived mononuclear cells were cul-tivated on fibronectin-coated dishes under conditions favor-ing endothelial commitment as previously described.15,16

Adherent cells at day 4 express endothelial markers (KDR,von Willebrand factor, endothelial nitric oxide synthase), butalso hematopoietic/myeloid markers such as CD31, CD45,and CD14 (see previous publications for cell characteris-tics13,33). To determine the role of cell incorporation andpersistence, cells were transduced using a lentiviral vectorencoding TK. The transfection efficiency of the GFP controlvector was �80%, and control experiments confirmed thatthe viral transduction did not affect cell function (viability,migration, colony forming activity) (data not shown). Addi-tion of GV induced toxicity in the TK-transduced cells butnot in the GFP control vector–expressing cells (Figure 1a).To determine potential indirect effects of GV-induced cyto-toxicity, culture supernatants of the GV treated TK-expressing cells were added to HUVECs or cardiomyocytes.However, conditioned medium did not induce cell death(Figure 1b and data not shown). Moreover, TK-expressingcells were cocultured with GFP-transfected HUVECs beforeadding GV to induce cell death in the TK-expressing cells.The viability of GFP-transfected HUVECs was not reducedby inducing cell death in the TK-expressing cells (99.9% ofcontrol), indicating that the treatment with GV selectivelykills the transduced cells.

Depletion of Infused Cells Impairs FunctionalRecovery After Acute Myocardial InfarctionNext, we infused TK-expressing cells in nude mice afteracute myocardial infarction to determine the effect of cellablation on heart function in vivo (Figure 2a). Myocardialinfarction was induced by permanent ligation of the leftcoronary artery, and TK- or GFP-transduced cells wereinjected 1 day later. After 4 weeks, 1.55�0.2% of cells werepositive for human specific Alu sequence in the border zoneof the infarcts. Human cells were preferentially localized in oraround vessels, whereas only a small number of myocytes

(�0.1%) were detected (Figures I and II in the online datasupplement). Two weeks after acute myocardial infarction,left ventricular ejection fraction was significantly improvedin both cell-treated groups compared with PBS control mice(Figure 2b). In GFP cell-treated mice, the ejection fractionwas not affected by GV treatment, which was initiated 2weeks after cell transplantation, and the beneficial effect ofcell therapy was maintained during the 4-week observationperiod. In contrast, left ventricular ejection fraction signifi-cantly deteriorated after injection of GV in TK cell–treatedmice, indicating that ablation of the cells 2 weeks afterinfusion impaired heart function. To confirm these results,MRI was used as an additional means to corroborate ourresults. Again, left ventricular ejection fraction was signifi-cantly improved in mice treated with TK cells in the absenceof GV (Figure 2c and 2e). Injection of GV after 2 weeks inTK cell–treated mice resulted in a reduced left ventricularejection fraction at 4 weeks compared with TK cell–treatedmice not receiving GV (Figure 2c). Consistent with theseresults, end-systolic volume was significantly reduced inmice injected with TK cells but significantly increased byadditional GV treatment (Figure 2d and 2e).

Because injected cells preferentially incorporated in oraround capillaries, we investigated whether ablation of thecells affected capillary density. We detected a significantreduction of the increased capillary density area in TKcell–treated mice 4 weeks after myocardial infarction, whenGV was injected at 2 weeks (Figure 2f; for higher magnifi-cation, see supplemental Figure IV). To obtain a moredetailed view of the acute effects induced by GV treatment inTK cell–treated mice, TUNEL stainings were performed. Asshown in Figure 2g and supplemental Figure III, GV treat-ment induced cell death in TK cell–treated mice 3 days afteronset of GV treatment (1.7�0.28-fold compared with mice,which had received TK cells without GV treatment, P�0.05).

Depletion of Infused CellsReduces NeovascularizationHaving demonstrated that depletion of transplanted cellsreduced capillary density, we investigated the relevance ofcell incorporation in 2 models of vascularization. First, weinfused TK-expressing cells in nude mice implanted with a

Figure 1. GV induces cell death in TK-expressing cells. a, GV reduces cell viability (MTT assay) dose-dependently in TK-transducedperipheral blood–derived fibronectin-adherent cells after 36 hours of incubation (N�3). b, Incubation with conditioned medium ofGV-treated TK-expressing cell did not affect viability of HUVECs. MTT assay after 24 hours of incubation is shown in comparison with1 mmol/L H2O2 treatment (N�3).

Ziebart et al Persistence of Transplanted Progenitor Cells 1329



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Figure 2. Depletion of infused TK-expressing cells by GV impairs functional recovery of hearts after acute myocardial infarction. a andb, PBS-transduced (N�6), GFP-transduced (N�4), or TK-transduced cells (N�8) were injected 1 day after induction of acute myocar-dial infarction. GV (100 mg/kg) was injected at days 14 to 18 daily. Echocardiography was performed to assess heart function at day14 (before GV injection), at day 21 and day 28 as indicated. c through e, Heart function and end-systolic volumes were determined byMRI in N�4 cell-treated mice per group after 28 days. *P�0.05 compared with PBS, P�0.06 compared with TK cells�GV, **P�0.05 vsTK cells�GV. Representative images are shown in e. f, Vessel density was determined by automatic measurement of lectin-positivearea per total area of a cross-section of the left ventricle (N�5). *P�0.05 compared with PBS and TK cells�GV. g, TUNEL staining(red) was performed in sections of mice receiving TK-transduced cells 1 day after induction of acute myocardial infarction. GV wasinjected at day 14 to day 17 daily, and mice were euthanized at day 17. Sections were counterstained with lectin (green) and DAPI(nuclei, blue). Representative images of the border zone are shown.




Matrigel plug to determine the effect of cell ablation onangiogenesis. Infusion of GFP- or TK-expressing cells in-creased the number of invading vessel-like structures in atime-dependent manner (Figure 3a and 3b). To determinewhether persistence of the cells contributes to angiogenesis 7days after infusion of the cells, we started GV infusion at day7 and documented vessel growth at day 14 (Figure 3a and 3b).As shown in Figure 3b, GV treatment at day 7 significantlyreduced Matrigel plaque vascularization in mice treated withTK cells, but not in mice receiving GFP cells. Similarly, inTK cell–infused mice, the number of lectin-positive cells wasdecreased by GV treatment to 38.9�1.4% (P�0.05). How-ever, only a partial inhibition by GV-induced cell depletionwas observed and the vascularization of plaques from micereceiving TK cells and GV was significantly higher comparedwith the PBS controls group (Figure 3b), indicating that inthis angiogenesis model vascularization is mediated by bothimmediate paracrine effects on host-derived endothelial cellsand physical incorporation of the infused cells.

To additionally determine the role of physical incorpora-tion and persistence of infused cells in blood flow recoveryafter ischemia, we used a hindlimb ischemia model (Figure 4aand 4b). Infusion of TK-transduced cells time-dependentlyimproved blood flow recovery after ischemia (Figure 4b). Atday 7 after infusion of TK cells, mice were randomized toreceive either GV or PBS and laser Doppler-derived bloodflow was analyzed at 14 and 21 days (Figure 4b). The TKcell–treated mice receiving PBS showed a further increase inblood flow recovery (Figure 4b, blue line). However, in theTK cell–treated mice receiving GV, perfusion did not furtherincrease and remained at the levels detected at day 7 (Figure4b, red line). Consistently, capillary density was significantlyreduced by GV treatment in TK cell–treated mice (Figure 4cand 4d). Moreover, 3 days after injection of GV, a reducedperfusion was detected by MRI in TK cell–treated micecompared with TK cell–treated mice, which received PBS(supplemental Figure V). However, the extension of theobservation period revealed a recovery of perfusion at day 21in the GV-treated group, indicating that ablation of cells doesnot irreversibly affect the recovery after ischemia. As acontrol, we infused TK-expressing macrophages. Intravenous

infusion of macrophages did not augment laser Doppler–derived blood flow compared with PBS and was not affectedby GV treatment (data not shown).

Depletion of Infused Cells InduceVascular LeakagePrevious studies documented the incorporation of trans-planted human EPCs in 10 to 20% of the capillaries in theischemic hindlimb muscle.16,18 Therefore, we investigatedwhether ablation of the TK-transduced cells might inducecapillary damage and leakage leading to edema formation andtransient perfusion defects in the hindlimb ischemia model.Injections of Evans blue in TK cell–treated mice 3 days afteronset of GV treatment (Figure 4e; GV treatment started at day7) revealed multiple spots of Evans blue staining in the thighmuscle of GV-injected mice (305�78% increase quantifiedby dye extraction of muscle tissue compared with PBScontrols, P�0.05), whereas no staining was evident in PBS-injected mice (Figure 4e), suggesting that ablation of thepreviously administered cells by GV injection induces vas-cular damage and increases permeability.

DiscussionOur data demonstrate that ablation of transplanted cells 1week after infusion reduced vessel growth and perfusionrecovery in 2 different models of neovascularization. More-over, ablation of infused cells 2 weeks after administrationinduced a rapid decline of ejection fraction and capillarydensity in a model of acute myocardial infarction. These datademonstrate that infused cells are physically incorporatedinto host tissue and persist weeks after administration. Thus,it appears unlikely that the beneficial effects of cell therapyusing EPCs can be solely explained by short-time paracrineeffects. However, the present study does not exclude that theincorporated cells may provide long term paracrine effects.Of note, the functional consequences of cell depletion weredifferent in the animal models used. In the angiogenesisMatrigel plug assay, vessel growth was only partially inhib-ited, indicating that a paracrine mechanism promoting host-derived angiogenesis within the first days after infusionsignificantly contributed to the vascularization of the plugs.

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Figure 3. Depletion of infused TK-expressing cells by GV impairs vessel-growth in Matrigel plugs (a and b). GFP- or TK-expressingcells were injected IV in mice with subcutaneously implanted Matrigel plugs at the same day (day 0). GV (100 mg/kg) or PBS wasinjected daily at days 7 to 11. Matrigel plugs were explanted at days 7 (N�4) or 14 (N�4), and vessels were counted in sectionsstained with smooth muscle actin antibodies.




After hindlimb ischemia, a profound increase in vascularleakage and decreased perfusion was detected after cellablation. However, this defect was transient and mice treatedwith GV partially recovered at day 21, indicating thathost-derived endogenous repair compensated for the loss ofcells at later time points. The extent to which early paracrinemechanisms may have contributed to the rapid recoverydetected later is difficult to evaluate.

In contrast to the partial inhibitory or transient effectsdetected in the Matrigel and hindlimb ischemia model,depletion of the cells after acute myocardial infarction in-duced a profound and sustained deterioration of cardiacfunction and capillary density was still significantly reducedin the GV treated group at 4 weeks. These findings might beexplained by the crucial role of angiogenesis in the heart.Two recently published experimental models demonstratedthat disruption of angiogenesis in the heart induced a declinein heart function,34 resulting in heart failure during pressure

overload or induction of hypertrophy.35 In addition, theinduction of cell death in cardiac myocytes derived from theinjected cells might have contributed to a reduction of heartfunction. However, although we detected human cardiacmyocytes, the number was below 0.1%, suggesting that this isunlikely to account for the deterioration of function. How-ever, we cannot exclude that the activated prodrug GV can betransported by gap junctions to physically connected neigh-boring cells,36 although we excluded unspecific cytotoxicityof GV in vivo (supplemental Figure VI) and “kiss of death”effects imposed by TK-transduced cells on neighboringendothelial cells in culture in vitro. Because cultivatedvascular progenitor cells were previously shown to connect tocardiac myocytes via gap junctions in vitro,20 and trans-planted c-kit� bone marrow–derived cells were shown toform interactions with host cells via gap junctions,37 suchbystander effects might have amplified the detrimental effectscaused by depletion of TK-expressing cells on cardiac func-

Figure 4. a and b, Depletion of infused TK-expressing cells after hindlimb ischemia by GV reduces perfusion and induces vascularleakage. TK-expressing cells or PBS was injected 1 day after induction of hindlimb ischemia. GV (100 mg/kg) or PBS was injected dailyat days 7 to 11, and perfusion was assessed at days 1, 7, 14, and 21 using laser Doppler (N�11 to 18 per group). P�0.05 comparedwith TK cells�GV and PBS. c and d, Capillary density was determined in muscle sections by lectin staining (n�5 to 7 mice per group).*P�0.05 vs PBS and TK cells�GV. e, TK-expressing cells were injected 1 day after inducing hindlimb ischemia. GV was injected atdays 7 to 9 daily. At day 9, Evans blue was injected to determine vessel leakage. A representative image of thigh muscles is shown.Arrows indicate Evans blue uptake.




tion in the myocardial infarction model. Nevertheless, even ifbystander effects on physically connected neighboring cellsmight have contributed to the observed abrogation of neovas-cularization recovery and deterioration of cardiac functionfollowing ablation of the administered cells, such mecha-nisms would require physical incorporation of the cells intothe host tissue. Thus, sustained persistence of the adminis-tered cells in the host tissue contributes to the beneficialeffects of cell therapy on neovascularization and recovery ofcardiac function. The contribution of specific cell lineages tothe maintenance of functional improvement warrants furtherinvestigations.

AcknowledgmentsWe thank Tino Roexe and Ariane Fischer for experttechnical assistance.

Sources of FundingThis study was supported by Deutsche Forschungsgemeinschaftgrants Di600/6-3 (to S.D.) and TR-SFB23 (to F.K.) and ExcellenceCluster Cardiopulmonary Systems grant Exc 147/1.

DisclosuresA.M.Z. and S.D. are cofounders and advisors to t2cure GmbH.

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Supplementary files:

Supplementary methods: page 1-2 Supplementary figures: page 3-8 Supplementary figure legends: page 9



Supplementary Methods

Magnetic Resonance Imaging (MRI) Dynamic contrast enhanced (DCE) MRI was performed using a 1.5 Tesla whole-body MR-

scanner (Siemens Symphony, Erlangen, Germany) in combination with a custom-made

radio-frequency-coil for excitation and signal reception. Contrast agent kinetics in hind limbs

were recorded using a T1-weighted inversion-recovery Turbo FLASH (IRTF) sequence (TR =

13 ms, TE = 5.3 ms, TI = 300 ms, Slice Thickness = 2 mm, FOV = 60 x 22.5 mm2, Matrix 128

x 48). In total, 120 dynamic scans were acquired from two sections within 14:55 min. Five

seconds after starting the DCE-MRI measurement, 100 µl of the paramagnetic contrast agent

Gadomer (0.1 mmol/kg body weight; Bayer-Schering Pharma, Berlin, Germany) were

injected manually within 5 s into the tail vein. Data were analysed using the pharmacokinetic

two compartment model of Brix (1) providing the parameters amplitude (related to relative

blood volume) and exchange rate constant kep (surrogate marker of vessel permeability and

perfusion). A and kep were determined pixelwise, color coded and overlaid on the

morphological MR-images. In addition, region of interest (ROI) analyses of both upper thighs

for each animal were performed. All postprocessing steps were conducted on the computer-

based workstation Dyna Lab (Mevis Research, Germany). Dynamic MR-measurements were

supplemented with morphological precontrast T2-weighted (TSE, TR = 1510 ms, TE = 59 ms,

Slice Thickness = 1 mm, FOV = 50 x 38 mm2, Matrix 128 x 96) and postcontrast T1-weighted

(SE, TR = 600 ms, TE = 1 ms, Slice Thickness = 1 mm, FOV = 61 x 38 mm², Matrix 512 x

320) sequences.

Stainings Paraffin embedded sections were deparaffinized and were incubated with proteinase K (15

min, 37 °C) and was hybridised with human specific fluorescein-labeled Alu probe (PR-1001-

01, InnoGenex) at 60 °C (overnight). Sections were then incubated with goat anti-fluorescein

followed by donkey FITC-conjugated anti-goat antibodies. Rabbit polyclonal anti-von

Willbrand factor antibodies (DAKO) followed by anti-rabbit Alexa Fluor 555 antibodies were

used for detecting vessel structures. Cardiomyocytes were detected by α-sarcomeric actin

(Sigma) and anti-mouse IgM rhodamine (Jackson). Nuclei were couterstained with ToPro

(Molecular Probes). Confocal microscopic analysis was performed using a Zeiss LSM 510

Meta.

Culture of human macrophages CD14-positive monocytes were purified from MNCs by positive selection with anti-CD14

microbeads (Miltenyi Biotec) using a magnetic cell sorter device (Miltenyi Biotec). CD14-



positive monocytes were incubated in RPMI with 10% FCS in the presence of M-CSF (50

ng/mL) to induce macrophage differentiation.

References 1. Brix, G., et al. Pharmacokinetic parameters in CNS Gd-DTPA enhanced MR imaging.

J Comput Assist Tomogr 15, 621-628. (1991).





human cells (Alu)Nuclei

Online figure I

vWF Merge

vWF Nuclei human Alu 20 µm

Nuclei human Alu20 µm



Online figure II

Sarc Actin Nuclei AluNuclei Alu Sarc Actin Nuclei Alu

Nuclei Alu Sarc actin Nuclei Alu

Sarc actin Merge

Sarc actinin Merge

Sarc actin Nuclei AluNuclei Alu

Sarc actin Merge



Online figure III

TK ll

LectinNuclei

TK-cells

TUNEL TUNEL Nuclei Lectin

TK-cells

Nuclei Lectin

+ GV

TUNEL TUNEL Nuclei Lectin



Online figure IV

TK-cells + PBSPBS

TK-cells + GV



Online figure V



Online figure VI

Kid S lKidney Spleen

Liver

IntestineLung



Online figure legends

Online figure I: Human Alu-positive cells (green spots) were detected in TK-cell treated

mice 4 weeks after induction of myocardial infarction. Sections were additionally stained with

von Willebrandt antibodies (vWF; red colour) and ToPro (blue colour) (for study design see

figure 3a)

Online figure II: Human Alu-positive cells (green spots) were detected in TK-cell treated

mice 4 weeks after induction of myocardial infarction. Sections were additionally stained with

α-sarcomeric actin (red colour) and ToPro (blue colour) (for study design see figure 3a)

Online figure III: Lectin staining (green) was performed in mice treated with TK-cells (upper

panel) or TK-cells plus GV treatment (start at 14 days; lower panel) 4 weeks after induction

of myocardial infarction. Dying cells were detected by TUNEL staining (red) and nuclei were

counterstained with DAPI (blue). Representative TUNEL positive cells are indicated by white

arrows (for study design see figure 3a)

Online figure IV: Higher magnification of Lectin stainings quantified in figure 3f. Mice were

treated with PBS (upper left panel), TK-cells with PBS (upper right panel), or with TK-cells

plus GV (GV injections were started at day 14; lower panel) and sections were obtained 4

weeks after the induction of acute myocardial infarction (for study design see figure 3a)

Online Figure V: Color coded parameter maps of the exchange rate constant (kep) in the

upper thighs of mice exposed to hind limb ischemia, which were treated with TK-cells plus

gancyclovir (GV) at day 7 (A) or without GV (B). Images were taken at day 10 after hind limb

ischemia, three days after onset of GV treatment. Corresponding T2-weighted morphological

images are shown in C (TK-cells plus GV) and D (TK-cells without GV). Ischemia was

induced in the right legs of the animals. On morphologic T2-weighted images the oedemas in

the ischemic legs are clearly visibly by enhanced signal intensities as compared with the

contralateral side. When observing the parameter maps higher kep values are found in the

animal that was treated with TK-cells without GV (kep= 0.38/min) as compared with the

animal that was administered GV to kill the infused cells (kep= 0.77/min). Please note that

kep values of the non ischemic upper thighs of both animals were comparable (kep =

0.62/min vs. kep = 0.67/min).



Online figure VI: H&E staining of tissue sections obtained from mice treated with TK-cells

plus GV (injections started after 14 days after induction of myocardial infarction). Animals

were sacrified at day 18 after induction of acute myocardial infarction (for study design see

figure 3a)



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