transcoronary delivery of bone marrow cells to the infarcted murine myocardium

Christian TemplinDaniel KotlarzFrederik MarquartJoerg FaulhaberVanessa BrendeckeArnd SchaeferDimitrios TsikasTomasz BondaDenise Hilfiker-KleinerLars OhlHassan Y. NaimReinhold FoersterHelmut DrexlerFlorian P. Limbourg

Transcoronary delivery of bone marrowcells to the infarcted murine myocardium

Feasibility, cellular kinetics, and improvementin cardiac function

Received: 1 September 2005Return for 1st revision: 19 September 20051st Revision received: 4 January 2006Accepted: 13 February 2006Published online: 16 May 2006

j Abstract Efficient strategies for labelling and delivery of bone marrowderived stem cells (BMCs) are required to elucidate the cellular kineticsand therapeutic effects after BMC transfer for myocardial infarction (MI).Lineage negative (lin)) BMCs, labelled ex vivo in a simple procedure withthe cell tracker dye tetramethyl-rhodamine (TAMRA), were reliablydetected by fluorescence microscopy with higher specificity thanretroviral enhanced green fluorescence protein (EGFP) marking anddetection. Only few cells entered the ischemic myocardium afterintravenous (i.v.) application, but this number increased more than 18-fold after transcoronary delivery. Time course and kinetic analysis over12 h revealed that myocardial colonization seems to be a biphasic processof first order decay with different elimination half-lifes. Most cells areeliminated rapidly during the first 2 h (t1/2 40 min), but the remainingcells are retained significantly longer in the ischemic heart (t1/2 5.2 h). Incontrast, BMC colonization of the spleen increased rather in a linearfashion. Although transcoronary BMC transfusion did not alter infarctsize, it increased capillary density in the infarct border zone andimproved LV function 4 weeks after MI. In conclusion, BMCs deliveredby transcoronary injection increase capillary density and improve LVfunction after MI although homing to the ischemic heart is only transient.

j Key words ischemia/reperfusion – bone marrow stem cells – celllabelling – homing – transcoronary cell delivery

Introduction

Several recent reports suggest that bone marrow derived stem cells(BMCs) have myogenic potential and are therefore promising candidatesfor various cell-based therapies for myocardial diseases [16, 18, 30, 36]. Anumber of fundamental issues need to be addressed before this thera-peutic approach can be considered for translational studies.

Simple and efficient strategies for cell labelling and detection arecrucial issues in any study addressing the cellular fate and therapeuticeffect of transplanted cells. Reporter genes expressing a fluorescenceprotein like enhanced green fluorescence protein (EGFP) have been usedto follow the fate of transplanted cells [29]. However, attention has to be

ORIGINAL CONTRIBUTIONBasic Res Cardiol 101: 301–310 (2006)DOI 10.1007/s00395-006-0590-7

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C. Templin (&) Æ D. KotlarzF. Marquart Æ V. Brendecke Æ A. SchaeferT. Bonda Æ D. Hilfiker-KleinerH. Drexler Æ F.P. LimbourgDepartment of Cardiology and AngiologyHannover Medical SchoolCarl-Neuberg-Strasse 130625 Hannover, GermanyTel.: +49-511/532-8751Fax: +49-511/532-5412E-Mail:[email protected]

F. Marquart Æ H.Y. NaimDepartment of Physiological ChemistryUniversity of Veterinary MedicineHannover, Hannover, Germany

J. FaulhaberDepartment of Vegetative Physiology andPathophysiologyUniversity of HamburgHamburg, Germany

J. FaulhaberDepartment of DermatologyVenereology and AllergologyUniversity Medical Center MannheimRuprecht-Karls-University of HeidelbergMannheim, Germany

D. TsikasInstitute of Clinical PharmacologyHannover Medical SchoolHannover, Germany

L. Ohl Æ R. FoersterDepartment of ImmunologyHannover Medical SchoolHannover, Germany

paid as dead and dying cardiomyocytes have anautofluorescent spectrum that partially overlaps withthat of EGFP. Consequently, dead or dying host car-diomyocytes can mistakenly be scored as donor cells[8]. In this study, we describe a simple procedure tolabel transplanted cells with a cell tracker dye fordetection by fluorescence microscopy.

A critical step for the clinical success of any cell-based therapy is an efficient method for cell delivery.There are various ways to implant cells into themyocardium: multiple experimental studies injecteddifferent types of donor cells directly into the myo-cardium [17, 24, 29, 35, 37]. On the one hand thismethod enables a selective transport into specificareas of the myocardium. On the other hand the di-rect injection is invasive and therefore combined withdifferent risks particularly for patients with diffusecardiomyopathy. Intravenous (i.v.) or intraarterial(i.a.) delivery of BMCs to patients recovering frommyocardial infarction (MI) is an attractive non-inva-sive strategy that allows administration of largenumbers of cells. There are insufficient data to sup-port systemic delivery of BMCs as a strategy formyocardial repair.

Clinically, the most practical route for BMCadministration is the intravascular delivery, but it isunknown whether BMCs injected into the coronarycirculation can cross the vessel wall, engraft to theinfarcted region, and initiate effective myocardialregeneration. Furthermore, the kinetics of cell colo-nization and decolonization as well as the fate of thetransfused cells are still unknown or controversial[25].

In the present study, we delineate the feasibilityand efficacy of proximally injecting BMCs into theascending aorta in a murine model of myocardialischemia/reperfusion (I/R) to simulate the clinicalprocedure of coronary delivery by coronary arterycatheterization. We then analyzed the cellular kineticsof BMCs in myocardium and spleen.

The goal of this study was to address these issuesthrough a thorough comprehensive investigation in aclinically relevant mouse model designed to mimicthe most common clinical condition in acute MI.

Methods

j Animals, ischemia/reperfusion, and experimental protocol

Ischemia/reperfusion injury was performed in 12-week-old C57BL/6 mice as described in prior experiments [9]. In brief, mice wereanesthetized and mechanically ventilated with isoflurane (1–2%). Aleft thoracotomy was performed after which the left anteriordescending artery (LAD) was ligated with 8–0 Prolone with aslipknot, which was removed 60 min later. Control mice underwenta thoracotomy and an incision of the pericard solely (sham). The

thoracic wall was closed in layers. After extubation, all animals wereallowed to recover from surgery in an incubator (37�C). At varioustime points (see below), mice were sacrificed, left ventricles (LVs)were removed, embedded in OCT for later immunohistochemistry,and frozen in liquid N2.

All animal experiments were approved by the local committeeon animal research. The investigation conforms with the ‘‘Guide forthe Care and Use of Laboratory Animals’’ published by the USNational Institutes of Health (NIH Publication No. 85–23, revised1985).

j Cell isolation

Whole BMCs were harvested from femurs and tibias of maleC57BL/6 mice. After intensive resuspension cells were filteredthrough nylon meshes and suspended in PBS containing 2% fetalcalf serum. The cells were incubated in a cocktail of biotin-conjugated monoclonal antibodies against mouse CD5, CD45R(B220), CD11b, Ly-6G (Gr-1) 7–4, and Ter-119 (Miltenyi Biotech)and subsequently labelled with monoclonal anti-biotin antibodiesconjugated to microbeads for sorting. These cells were loaded onan LS column (Miltenyi Biotech), and magnetically labelled cellswere retained in the magnetic field of a MACS separator. Lin)

depleted cells present in the flow-through were collected bycentrifugation at 250 g for 5 min and resuspended in 200 ll PBS.

j Cell tracker dye labelling

TAMRA [5-(and 6)-Carboxy-Tetramethylrhodamine Succinimidy-lester] was obtained from Molecular Probes. Cells were incubatedin IMDM medium for 20 min at 37�C and finally labelled for10 min at 37�C with 200 lM TAMRA per 107 cells. Washed cellswere suspended in 250 ll PBS and injected into the tail vein or theaortic root of recipient mice.

j Retroviral gene transfer

For retroviral gene transfer we used the vector SFb-91-eGFP-wPRE.Recombinant vesicular stomatitis virus-G (VSV-G) pseudotypedretroviruses were generated using transient transfection into thepackaging cell line Phoenix-gp. For retroviral gene transfer, lin)

progenitor cells were stimulated for 48 h in the presence of a stemcell cytokine cocktail of murine IL-3 (10 ng/ml), human IL-11(50 ng/ml), human Flt-3 (50 ng/ml) and murine SCF (50 ng/ml)and transduced at a multiplicity of infection (MOI) of 10 in thepresence of 8 lg/ml polybrene (Sigma). In brief, cells were exposedto recombinant retrovirus for 1 h at 37�C, followed by centrifuga-tion for 2 h at ·700 g and further incubation at 37�C in 5% CO2.Subsequently, cells were washed, cultured for additional 48 h in thepresence of the stem cell cocktail and used for in vitro and in vivoexperiments. The average transduction efficiency was 60–70%.

j Transcoronary infusion of BMCs

We compared two strategies of cell delivery. In the first group,

BMCs (1·107) were infused via the tail vein (200 ll volume). Ani-mals of the second group received a light anesthesia (ketamine,100 mg·kg)1; xylazine, 1.25 mg·kg)1, atropine, 0.6 mg·kg)1; i.p.).An incision was made in the middle of the neck to expose the leftcarotid artery and the internal jugular vein. A homemade catheterwas placed into the aortic root via the left carotid artery and BMCs(1·107) were infused (200 ll volume).

302 Research in Cardiology, Vol. 101, No. 4 (2006)� Steinkopff Verlag 2006

j Echocardiographic measurements

Echocardiograms were obtained 28 days after I/R. The investiga-tor was blinded to the experimental groups. After intraperitonealinjection of the combination of ketamin (100 mg/kg) and xylazine(1.25 mg/kg), the sedated mice were fixed supine in a left lateralposition on a heated table and shaved at the precordium.Transthoracic echocardiography was performed with a commer-cially available ultrasound machine equipped with a linear15 MHz transducer (ATL HDI 5000 CV) [32]. Short axis two-dimensional images were taken at the papillary muscle level. Thefollowing parameters were measured and averaged from threecardiac cycles: left ventricular enddiastolic diameter (LVEDD), leftventricular endsystolic diameter (LVESD), systolic und diastolicanterior and posterior wall thickness were measured to thenearest 0.1 mm. Fractional shortening (FS) was calculated usingthe following equitation: FS (%)=(LVEDD)LVESD)/LVEDD·100.LV ejection fraction (EF) was calculated as [(LVEDA)LVESA)/LVEDA]·100, where LVEDA and LVESA denote LV end-diastolicand end-systolic areas, respectively [41].

j Specific detection of transplanted BMCs in the murine myocardiumafter labelling with the cell tracker dye TAMRA

At different time points after cell transfer (see below), myocardialcryosections were prepared and analyzed by immunofluorescenceand confocal laser microscopy (LSM510meta). This technologycombines laser scanning microscopy and fluorescence correlationspectrometers. The analysis of the characteristic emission spectrumof TAMRA allowed a specific detection of BMCs. We calculated thenumber of TAMRA+-cells present in the heart and in the spleen at2–5 min, 2 h and 12 h after infusion of BMCs. Cell counts weremade on 6 lm sections and surface area of the myocardium orspleen on the section was measured by planimetry. The cell numberwas referred to a certain tissue volume (mm3). The investigator wasblinded to the experimental group.

j Analysis of capillary density and immunostaining

Capillary density was determined in LV sections with transversely-sectioned cardiomyocytes immunostained against Isolectin B4(Vector) and counterstained with WGA and Hoechst 33258 (Sigma)as described previously [14]. In brief, the ratio of Isolectin B4 po-sitive cells to the total number of nuclei (Hoechst stain) per fieldwas calculated. Capillaries per high power fields were determined inLV sections double stained for Isolectin B4 and WGA. For eachmouse, high power fields (400·; 200·200 lm; eight fields per sec-tion of LV basis, middle part and apex; n=4 per group) withtransversely sectioned cardiomyocytes were digitally recorded tocalculate the number of capillaries per high power field. Immu-nostaining was performed with antibodies recognizing sarcomerica-actinin (Sigma).

j Histomorphometric analysis

LV tissue slices were embedded in OCT, cut into 6 lm sections, andstained with hematoxylin- and eosin. Tissue morphometry wasperformed in a blinded fashion using the Quantiment 500MCdigital image analyzer. Infarction size was determined as describedpreviously [40].

j Statistical analysis

Data are presented as mean±SD. Differences between groups wereanalyzed by the unpaired Student‘s t-test or one-way ANOVA

followed by Bonferroni test. A P<0.05 was considered to indicatestatistical significance.

Results

j Specific detection of transplanted BMCsin myocardium after labelling with the cell trackerdye tetramethyl-rhodamin (TAMRA)

To determine a simple method for direct visualizationof transplanted cells after myocardial infarction wecompared two strategies of fluorescent cell labellingand detection. Highly purified lin) BMCs were eithertransduced with a retrovirus-encoding EGFP or la-belled with the cell tracker dye TAMRA, which hasbeen successfully used in leukocyte tracking studies[27]. TAMRA-labelling stained 100% of lin) BMCswithout loss of cell viability (data not shown). Micewere subjected to myocardial I/R by transient LADligation followed by tail vein injection of 1·107 EGFP+-or TAMRA+-BMCs or an equal volume of PBS 24 hlater. Analysis of heart sections by direct fluorescencemicroscopy for EGFP+-cells demonstrated multiplebright spots in ischemic and non-ischemic regionsfrom EGFP+-BMC treated animals, but also fromcontrol treated animals which had not received cells(Fig. 1a). Spectral analysis of these signals revealed anemission wavelength distribution characteristic ofmyocardium autofluorescence, which preventeddetection of specific cell signals (Fig. 1c, left panel). Incontrast, TAMRA+-cells could reliably and unequivo-cally be detected by fluorescence microscopy in theheart and the spleen (Fig. 1b). Each TAMRA+-BMChad a distinct nucleus and gave a specific signal in thecorresponding spectral analysis (Fig. 1c, right panel).These data demonstrate specificity and feasibility ofTAMRA labelling for cell detection.

j Transcoronary delivery increases BMC numbersin the infarcted myocardium

To determine whether the route of cell administrationinfluences colonization to the infarcted myocardiumwe quantified the number of TAMRA+-BMCs in theheart 2 h after i.v. or intraarterial, transcoronary (i.a.)cell delivery following I/R injury. While only few cellscould be detected after i.v. application, the number ofretained cells increased more than 18-fold after i.a.delivery (cells/mm3: i.v. 3±3.5, n=8; i.a. 55±26.32,n=9, P<0.001; Fig. 2a).

To characterize the time course and kinetics ofBMC colonization in ischemic myocardium wequantified the number of TAMRA+-cells at three timepoints after i.a. cell transfusion and analyzed the re-

C. Templin et al. 303Transcoronary delivery of BMCs for myocardial infarction

sults by standard kinetic modelling and integraltransformation. Immediately after transfusion(2 min) we observed 488±105 TAMRA+-BMCs/mm3

myocardium (Fig. 2b). This corresponds to a totalnumber of 0.5–1.0·104 BMCs/heart and represents0.5–1.0 % of total number of BMCs transfused. BMCsat this time point were mostly intravascular, in con-tact with the endothelium, and distributed equallyamong all areas of the heart (Fig. 2b, lower left panel,and data not shown). However, cellular retention wasnot sustained since the number of TAMRA+-BMCsdecreased over the following 12 h (Fig. 2b;2 h=61±29/mm3, 12 h=13±9/mm3, n=8 each, P<0.001vs. 2 min). Residual cells at subsequent time pointshad mostly transmigrated into ischemic myocardium(Fig. 2b, lower right panel). In contrast, colonizationin the spleen showed inverse behaviour, with thenumber of TAMRA+-BMCs steadily increasing over aperiod of 12 h (Fig. 2c, see also Fig. 1b; 2 min=164±74/mm3, 2 h=464±95/mm3, 12 h=1288±299/mm3,n=8 each, P<0.05 2 min vs. 2 h and P<0.001 2 min vs.12 h and 2 h vs. 12 h).

j Biphasic cardiac decolonization of BMCsafter transcoronary delivery

To characterize the BMC behaviour in more detail weanalyzed the number of cardiac BMCs over time. Asmice were sacrificed 2 min, 2 h and 12 h after BMCdelivery, the kinetics of the decrease of cell density inthe myocardium and of the colonization in the spleencould not be satisfactorily modelled. Nevertheless, weattempted to analyze the results by means of standardkinetic models. The density of the BMCs initiallycolonizing the myocardium decreased exponentiallyover time (Fig. 2b, upper left panel). Analysis of thedata from all mice by the integral method, i.e. plottingof the natural logarithm of the ratio of the meanconcentration of the BMCs at time 2 min (i.e. Co) tothe mean concentration at time 2 h or 12 h (i.e. C), i.e.ln[Co/C], versus time (t) did not reveal a straight line(Fig. 2b, upper right panel). This finding suggests thatthe decolonization cannot be described by the one-compartment model and first order kinetic over thewhole observation time interval. The curve in Fig. 2b,(upper left panel) which was obtained by fitting thedata using exponential decay of first order, shows agood agreement with the experimental data only forthe first time interval of up to 2 h (i.e. a-phase), andcan be described by the function C=505·e)1.059t,where 505 is the cell density at 2 min and 1.059 h)1 isthe rate constant (ka). Thus, the ‘‘decolonization’’mean half-life (t½a) of the BMCs in the myocardium isdetermined to be 0.655 h. Almost the same values forka and t½a were observed by the integral method for

the early phase of the decolonization process, i.e.1.115 h)1 and 0.622 h, respectively. In analogy,application of the integral method to the later decol-onization phase from 2 to 12 h (i.e. the b phase)revealed kb and t½b values of 0.134 h)1 and 5.17 h,respectively. The data agree with the data obtained byapplying the integral method separately to each ani-mal and by calculating the mean and standard error.These values were ka=1.146±0.117 h)1 andt½a=0.665±0.059 h for the a phase, and kb=0.152±0.022 h)1 and t½b=5.216±0.778 h for the b-phase.These results suggest that the decolonization processof the myocardium is biphasic and could be describedby the one-compartment model of first order kineticsin each phase, but with clearly different rate constantsk. While the mean decolonization half-life of BMCsfrom the myocardium in the initial (a) phase was0.66 h (40 min), it increased to 5.2 h in the following(b) phase. In other words, while most cells areeliminated rapidly during the first 2 h, probablyduring the first pass, the remaining cells are retainedsignificantly longer in the ischemic heart, whichsuggest biological interaction during the second phaseof delayed decolonization.

The colonization of BMCs in the spleen, on theother hand, is also a biphasic process with meanrates of colonization (slopes of straight lines inFig. 2c) of 152 cells ·mm)3·h)1 in the a-phase andapproximately half of that, i.e. 82 cells·mm)3·h)1, inthe b-phase.

In summary, these data suggest that BMCs areretained differentially in ischemic myocardium andspleen. While most BMCs pass through the ischemicheart without significant retention, some cells areretained and stay for a prolonged period, whichsuggests interaction with the parenchym.

j Transcoronary delivery of BMCs after I/Rincreases myocardial capillaries and improvescardiac function

We next characterized the long-term consequences ofi.a. BMC injection 4 weeks after myocardial infarc-tion. Within the whole study period there was nodifference in mortality between BMC treated animalsand control treated animals (BMC 3/12, con 2/10,sham 0/10). Of note, intraoperative mortality due tothe procedure of transcoronary cell delivery was 15%(3/20). There was also no difference in infarct size:transient LAD ligation resulted in non-transmuralmyocardial infarction with comparable average in-farct sizes between BMC and control groups (BMC10.6±0.58%, n=9 vs. con 9.1±0.24%, n=8; P=n.s.).

However, BMC treatment resulted in an increasednumber of myocardial capillaries after I/R. As shown


in Fig. 3a, capillary density in the infarct borderzone was 94.57±9.36 capillaries/high power field inBMC treated mice versus 79.09±13.46 capillaries/cardiomyocytes high power field in control mice(P=0.02). In the remote myocardium, there was nodifference between treatment groups (90.67±11.69 vs.89.50±11.69; P=n.s.).

We performed echocardiography 28 days after MIto obtain data on cardiac performance. I/R injury leadto a significant decrease in fractional shortening and

ejection fraction, which could be partially reversed bytransfusion of BMCs (Fig. 3b; fractional shortening,BMC 16.6±3.3%, n=9 vs. control 11.2±3.6%, n=8;P<0.05; ejection fraction, BMC 24.2±5.8%, n=9 vs.control 16.8±5.5%, n=8; P<0.05). There was also atrend towards improved left ventricular systolicdiameters, although not significant, while end dia-stolic diameters remained unchanged between treat-ment groups (Fig. 3b). The heart rate (bpm) in bothgroups was similar.

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Fig. 1 Specific detection of TAMRA+ BMCs afterI/R. (A) Direct fluorescence microscopy of ischemicand non-ischemic heart sections after transfer ofcontrol or EGFP-transduced BMCs. (B) Directfluorescence microscopy of heart and spleensections after transfer of TAMRA+-BMCs. Blue:nuclei (Hoechst), red: TAMRA+-BMCs. (C) Spectralanalysis of heart sections after transfer of EGFP+ orTAMRA+-BMCs


Discussion

Simple and efficient strategies for cell labelling,detection and delivery are crucial issues in any studyaddressing the cellular fate and therapeutic effect oftransplanted cells.

We showed that lin) BMCs, labelled in a simpleprocedure with the cell tracker dye TAMRA beforecell transfer, can be reliably detected by fluorescence

microscopy with little confounding tissue artefacts.This technique is superior to retroviral EGFPmarking of BMCs and detection by EGFP fluores-cence for two reasons: first, labelling efficiency byretroviral transduction is inherently low, whileTAMRA labelling has high efficiency. Second, hearttissue displays high autofluorescence in the EGFPspectrum, but low background in the TAMRAspectrum, providing good signal to background ra-tios. Quantification can be further enhanced by flu-

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Fig. 2 Transcoronary delivery increases BMCnumbers in the infarcted myocardium. (A)TAMRA+-BMCs in ischemic hearts 2 h after i.v. ortranscoronary (i.a.) application (left panel, i.v. n=8,i.a. n=9), representative direct fluorescencemicroscopy images of ischemic heart sections 2 hafter BMC transfer (right panel). (B) BMC kineticsafter transcoronary transfer. Upper left panel:quantification of BMCs at different time pointsafter transfer (exponential decay). The curve is thefitting of the measured data on the basis of anexponential decay of first order. Upper right panel:Analysis of the numerical data by means of theintegral method. Values are shown as mean±SD(n=8 for each time point). Lower panel:Intramyocardial localization of TAMRA+-BMCs byfluorescence microscopy. Left: 2 min. after i.a.infusion. Blue: nuclei (Hoechst), red: TAMRA+-BMCs. Right: 2 h after i.a. infusion. Blue: nuclei(Hoechst), red: TAMRA+-BMCs, green: sarcomerica-actinin. (C) Quantification of spleen homing byBMCs after i.a. application. Values are shown asmean±SD (n=8 for each time point)


orescence spectral analysis. Compared to detectionof EGFP by immunofluorescence, which requiresfurther tissue manipulation and is dependent onantibody binding characteristics and specificity,visualization of TAMRA+-cells by fluorescencemicroscopy is direct, providing an ideal tool fortracking different cell populations of various species.Furthermore, TAMRA labelling did not seem tointerfere with cellular function, since homing andcardiac improvements were still observed.

Labelling studies after myocardial infarction usingradiolabelling have been performed in animals with(111)In-oxine [3] and in humans with 100 MBq 2-[18F]-fluoro-2 deoxy-D-glucose (18F-FDG) [15]. Thereare three advantages of the approach described in ourcurrent study: first, cell tracker dye labelling withTAMRA is more stable than labelling with 18F-FDG,which is limited by the short half life of 18F-FDG(about 110 min) and therefore does not allow detec-tion. It is impossible to detect BMCs after a longertime period after administration. In comparison tothis, cell tracker dye labelling has been used for effi-cient detection of bone marrow-derived dendriticcells and T cells 96 h after transplantation in vivo [12,27]. Additionally, Ciulla and coworkers detected redfluorescent cell dye labelled bone marrow mononu-clear cells seven days after myocardial infarction inthe injured area in Fisher rats [6], while we detectedfluorescent cells for at least 12 h post transfer. Fur-thermore, the resolution of the injected BMCs in theorgans is completely different. In live imaging studiescell kinetics can only be examined on an organ levelwhereas with fluorescent labelling detection of cellson a cellular level is possible. Additionally, Brennerand coworker described that after radiolabelling ofCD34+ haematopoietic progenitor cells (HPC) with(111)In-oxine the labelling efficiency of HPC was only32%+/)11% and the viability of radiolabelled cellswas impaired by 30% after 48 and 96 h in comparisonwith unlabelled cells, whereas proliferation and dif-ferentiation of HPCs was nullified after 7 days, asassessed by colony-forming assays. Their findingsdemonstrated a negative effect of (111)In-oxine oncellular function, which resulted in complete impair-ment of haematopoietic progenitor cell proliferationand differentiation [3]. The labelling efficiency forTAMRA, on the other hand, is >99%, withoutimpairment of viability or differentiation capacity(data not shown and [12, 27]).

The goal of any cell delivery strategy is to transplantsufficiently high numbers of cells into the injuredmyocardium to exert protective effects. Compared toi.v. injection, transcoronary delivery significantly im-proved BMCs numbers in the heart and improved LVfunction 4 weeks after MI. This approach also betterresembles the clinical setting of cell therapy by tran-

scoronary infusion in patients with acute myocardialinfarction and could therefore serve as a good modelsystem for optimizing treatment strategies for thesepatients [1, 4, 5, 10, 22, 31, 34, 39].

Labelling studies in patients after MI have shownthat less than 3% of unselected BMCs are retained inthe heart after intracoronary delivery [15]. Althoughthis rate of retention was sufficient to improve leftventricular systolic function in one trial [39], thekinetic of the cells and the fate of the transfused cellsare still unknown or controversial [25]. We dem-onstrated that after transcoronary application, highnumbers of mostly intravascular BMCs in contactwith the endothelial lining are observed immediatelyafter transfusion, suggestive of BMC-endothelialinteraction observed in the initial phase of coloni-zation (rolling, attachment). Yet, cell numbers dropcontinuously during the observation period of 12 h,at which time cells are found in the myocardialparenchym.

We showed that transcoronary infusion of BMCsresults in significantly higher numbers of intramyo-cardial BMCs than i.v. infusion. Yet, even with opti-mal delivery, only few BMCs are retained in themyocardium for a prolonged period. Our study sug-gests that the kinetics of myocardial retention can bedescribed by a function of exponential first orderdecay with half-lifes of approximately 0.66 h in theearly a-phase (0.033–2 h) and 5.2 h in the later b-phase (2–12 h). Parallel to the decay in the myocar-dium, the spleen seems to be colonized by BMCsaccording to a biphasic process, too, but presumablyin a linear fashion. We acknowledge that precisemodelling of the decolonization of the myocardiumand colonization of the spleen and eventually of othertarget organs require additional data from at least twotime points, e.g. one time point from the a-phase andone data point from the b-phase. Nevertheless, de-spite the limited time points used, our study suggeststhat the mathematical approach used to describe thedecolonization of the myocardium on a quantitativebasis may be useful in modelling the behaviour ofBMC in animal models.

It is known that injured tissue expresses specificreceptors and soluble factors to facilitate trafficking,adhesion, and infiltration of BMCs to the region ofinjury [26, 28]. Furthermore, establishing rate con-stants and elimination half-lifes for a given cell typeor treatment protocol by kinetic modelling and inte-gral transformation could facilitate comparison ofcolonization characteristics between different studiesand therefore help optimize treatment strategies.

Although lin) BMCs stayed only transiently in theinjured myocardium there was a significant improve-ment in myocardial function, accompanied by an in-crease in infarct related capillary density. These


observations suggest a paracrine mechanism of car-diac protection. It has been proposed that stem cellsrelease angiogenetic ligands, protect cardiomyocytesfrom apoptotic cell death, induce proliferation ofendogenous cardiomyocytes, and may recruit residentcardiac stem cells [7, 11, 13, 19, 23, 33, 38]. Regardlessof the mechanisms, there appears to be generalagreement that stem cell therapy has the potential to

improve perfusion and contractile performance of theinjured heart [2, 19, 21, 29, 33, 38]. Clearly, furtherstudies are necessary to improve application schemesand beneficial mechanisms by BMCs.

Acknowledgments This work was supported by an early career grantfrom Hannover Medical School to C.T. (HiLF Program) and by theHerbert Quandt Foundation of the VARTA AG to D.K.

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]

P = n.s.

B Echocardiographic data

Fig. 3 Transcoronary delivery of BMCs after I/Rincreases myocardial capillaries and improvescardiac function. (A) Myocardial capillary density ininfarct border zone and remote mycardium4 weeks after I/R (n=8 each group). (B)Echocardiographic data 4 weeks after I/R (n=8–9each group). LVESD: left ventricular endsystolicdiameter, LVEDD: left ventricular enddiastolicdiameter


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transcoronary delivery of bone marrow cells to the infarcted murine myocardium

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