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Journal of the American College of Cardiology Vol. 55, No. 13, 2010© 2010 by the American College of Cardiology Foundation ISSN 0735-1097/10/$36.00P

CELL THERAPY

Red Blood Cell Contamination of theFinal Cell Product Impairs the Efficacy ofAutologous Bone Marrow Mononuclear Cell Therapy

Birgit Assmus, MD,* Torsten Tonn, MD,‡ Florian H. Seeger, MD,*† Chang-Hwan Yoon, MD,†David Leistner, MD,* Jens Klotsche, MSC,§ Volker Schächinger, MD,* Erhard Seifried, MD,‡Andreas M. Zeiher, MD,* Stefanie Dimmeler, PHD†

Frankfurt and Dresden, Germany

Objectives The aim of this study was to identify an association between the quality and functional activity of bone marrow-derived progenitor cells (BMCs) used for cardiovascular regenerative therapies and contractile recovery in pa-tients with acute myocardial infarction included in the placebo-controlled REPAIR-AMI (Reinfusion of EnrichedProgenitor cells And Infarct Remodeling in Acute Myocardial Infarction) trial.

Background Isolation procedures of autologous BMCs might affect cell functionality and therapeutic efficacy.

Methods Quality of cell isolation was assessed by measuring the total number of isolated BMCs, CD34� and CD133�

cells, their colony-forming unit (CFU) and invasion capacity, cell viability, and contamination of the final BMCpreparation with thrombocytes and red blood cells (RBCs).

Results The number of RBCs contaminating the final cell product significantly correlated with reduced recovery of left ventric-ular ejection fraction 4 months after BMC therapy (p � 0.007). Higher numbers of RBCs in the BMC preparation wereassociated with reduced BMC viability (r � �0.23, p � 0.001), CFU capacity (r � �0.16, p � 0.03), and invasion ca-pacity (r � �0.27, p � 0.001). To assess a causal role for RBC contamination, we coincubated isolated BMCs withRBCs for 24 h in vitro. The addition of RBCs dose-dependently abrogated migratory capacity (p � 0.003) and reducedCFU capacity (p � 0.05) of isolated BMCs. Neovascularization capacity was significantly impaired after infusion ofBMCs contaminated with RBCs, compared with BMCs alone (p � 0.05). Mechanistically, the addition of RBCs wasassociated with a profound reduction in mitochondrial membrane potential of BMCs.

Conclusions Contaminating RBCs affects the functionality of isolated BMCs and determines the extent of left ventricular ejec-tion fraction recovery after intracoronary BMC infusion in patients with acute myocardial infarction. These resultssuggest a bioactivity response relationship very much like a dose–response relationship in drug trials. (Reinfu-sion of Enriched Progenitor cells and Infarct Remodeling in Acute Myocardial Infarction [REPAIR-AMI];NCT00279175) (J Am Coll Cardiol 2010;55:1385–94) © 2010 by the American College of CardiologyFoundation

ublished by Elsevier Inc. doi:10.1016/j.jacc.2009.10.059

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lthough reperfusion therapy has significantly improvedrognosis of patients with acute myocardial infarctionAMI), the development of post-infarction heart failure

rom *Cardiology, Department of Medicine III, and the †Institute for Cardiovascularegeneration, CMM, Goethe University, Frankfurt, Germany; ‡Institute for Transfusionedicine and Immunohematology, Red Cross Blood Donor Service Baden-Württemberg-essen, Frankfurt, Germany; and the §Institute of Clinical Psychology and Psychotherapy,entre of Clinical Epidemiology and Longitudinal Studies, Faculty of Mathematics andatural Sciences, Technical University, Dresden, Germany. This study was supported by theFG (FOR 501-1: WA 146/2-1) and by the Foundation Leducq Transatlantic Network

f Excellence for Cardiac Regeneration. Drs. Dimmeler and Zeiher are cofounders of2cure, a for-profit company focused on regenerative therapies for cardiovascular disease,nd serve as scientific advisors and are shareholders. Dr. Schächinger has received grantupport from Guidant. Drs. Assmus and Schächinger report being scientific advisors of2cure. Drs. Assmus, Tonn, and Seeger contributed equally to this work.

tManuscript received March 9, 2009; revised manuscript received September 30,

009, accepted October 14, 2009.

emains a major challenge, particularly in patients witharge AMIs (1,2). Numerous experimental and severallinical studies suggest that cell therapy might provide anttractive novel option to beneficially interfere with leftentricular (LV) remodeling processes, and thereby at-enuate the development of post-infarction heart failure.n experimental studies, infusion of various types ofells—including circulating endothelial progenitor cells,one marrow mononuclear progenitor cells (BMCs),D34� cells, mesenchymal stem cells, adipose-tissueerived cells, and cardiac stem cells—leads to improve-ent of neovascularization and cardiac function (3– 6).

o far, most of the clinical trials used autologous BMCsor cell therapy of AMI (7). In contrast to an allogeneic
herapy with an “off the shelf ” cell product, autologous
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1386 Assmus et al. JACC Vol. 55, No. 13, 2010RBC Contamination Impairs Bone Marrow Cell Therapy March 30, 2010:1385–94

cell therapy requires isolation ofcells from each individual pa-tient, and thus the applied cellcomposit ion and functionmight vary individually. Indeed,risk factors for atherosclerosissuch as diabetes and age wereshown to impair cell function-ality (8 –11). Previous experi-mental studies and clinical pilottrials showed that a reductionof functional activity of the in-fused cells defined as impairedmigratory capacity was associ-ated with reduced therapeutic ef-fects (12,13). However, the im-pact of the composition of thecell product and the potentialeffect of contaminating cells areunclear.

Therefore, we determined whether total cell number, theumber of selected subpopulations of progenitor cells aseasured by fluorescent-activated cell sorting (FACS, Bectonickinson, Franklin Lakes, New Jersey), in vitro assays toeasure cell activity, or contamination of the cell preparation

y platelets or erythrocytes affects the recovery of left ventric-lar ejection fraction (LVEF) in the multicenter, double-blind,andomized, placebo-controlled REPAIR-AMI (Reinfusionf Enriched Progenitor cells And Infarct Remodeling in Acuteyocardial Infarction) trial including 204 patients with AMI,

f whom 101 patients have been treated with BMCs. At-month follow-up, BMC-treated patients showed signifi-antly improved LVEF compared with the placebo group, andhe treatment was associated with a reduced number ofomposite clinical end points at 1 year (14,15). Here we reporthat, among the various parameters tested, the contaminationf the isolated BMCs with red blood cells (RBCs) was aignificant independent predictor of reduced recovery of LVEFn the BMC group. Experimental studies confirmed thesendings by demonstrating that the addition of RBCs impairsMC cell function in vitro and in vivo.

ethods

tudy population and protocol. Patients with an acuteT-segment elevation myocardial infarction successfullyeperfused with stent implantation with a residual signifi-ant LV regional wall motion abnormality (LVEF �45% byisual estimate at the time of reperfusion therapy) werencluded in the REPAIR-AMI trial. A total of 204 patientsere randomized to receive either intracoronary infusion oflacebo medium or BMCs. In 187 patients (92 in thelacebo and 95 in the BMC group), paired analysis of LVngiograms at baseline immediately before BMC/placebodministration (4.3 � 1 day after AMI reperfusion) and

Abbreviationsand Acronyms

AMI � acute myocardialinfarction

BMC � bone marrowmononuclear progenitor cell

CFU � colony-forming unit

FACS � fluorescent-activated cell sorting

LV � left ventricle/ventricular

LVEF � left ventricularejection fraction

NO � nitric oxide

RBC � red blood cell(erythrocyte)

SDF � stromal cell-derived factor

-month follow-up were available (for detailed baseline w

haracteristics see Schächinger et al. [14]). Cell processingnd quality controls were performed in a central coreaboratory, and patients were randomized to receive intra-oronary infusion of placebo medium or BMCs into thenfarct-related artery during low-pressure balloon occlusion.he BMCs were infused 17 � 11 h after bone marrow

ollection and were stored in X-vivo 10 medium (Lonza,alkersville, Maryland) � 20% autologous serum. In the

lacebo group, bone marrow was aspirated, processed, andunctionally tested as in the BMC group, but patients receivednly X-Vivo 10 medium supplemented with 20% autologouserum, never having contained any cells. Further details of cellreparation and administration have been described previously16,17). The ethics review board of each individual participat-ng center approved the protocol, and the study was conductedn accordance with the Declaration of Helsinki.

The BMCs for the experimental in vitro and in vivoalidation and confirmation experiments were obtainedrom patients with ischemic cardiomyopathy undergoingntracoronary infusion of BMCs within an ongoing registry,aving the same inclusion and exclusion criteria as thereviously published randomized TOPCARE-CHDTransplantation of Progenitor Cells and Recovery of LVunction in Patients with Chronic Ischemic Heart Disease)

rial (18,19). The Ethics Review Board of the Hospital ofhe Johann Wolfgang Goethe University of Frankfurt,ermany, approved the protocol, and the study was con-

ucted in accordance with the Declaration of Helsinki.ritten informed consent was obtained from each patient.

V angiography. Left ventricular angiograms were ob-ained in identical standard projections at the baselinerocedure (immediately before intracoronary cell infusion)nd at 4-month follow-up. Quantitative analysis of pairedV angiograms was performed by an experienced investi-ator (BA) in a central core laboratory, blinded to thereatment modality of the individual patients with theoftware CMS version 6.0 (Medis, Leiden, the Nether-ands), as previously described (14). The LVEF was calcu-ated with use of the area-length method.solation of BMCs and viability assessment. Briefly, inhe REPAIR-AMI trial, bone marrow aspirates were di-uted with 0.9% sodium chloride (1:5) and filtrated (100m), and mononuclear cells were isolated by density gradi-nt centrifugation with Lymphocyte Separation MediumLonza, 800 g, 20 min, without brake). Mononuclear cellsere washed 3 times with 50 ml phosphate-buffered saline

800 g) and counted; viability was assessed by a dyexclusion stain with trypan blue. Cells were resuspended in-vivo 10 medium (Lonza), supplemented with 20% autol-gous serum, and released for immediate use. Trial centersutside of the greater area of Frankfurt received the cellreparations the next day.solation of erythrocytes (RBCs). With a leukocyte de-letion filter for whole blood donations (Leucoflex LCR5,acopharma, Langen, Germany), erythrocytes were eluted

ith 10 ml X-vivo 10 medium (Cambrex) from 5-ml bone

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1387JACC Vol. 55, No. 13, 2010 Assmus et al.March 30, 2010:1385–94 RBC Contamination Impairs Bone Marrow Cell Therapy

arrow aspirate obtained from patients with ischemic heartailure. Enumeration of RBCs and the depleted total nu-leated cells was performed with an automated differentiallood count (Sysmex XT 1800i [Sysmex, Mundelein, Illi-ois], linearity of analysis: total nucleated cells: 0 to 100.0 �03/�l � 300; RBC: 0 to 8.00 � 106/�l � 30.000; HCT:.0% to 60 � 1%).For all coincubation experiments, the defined amount of

BCs was added to the bone marrow preparation in X-vivo0 medium containing 20% autologous serum and wastored for 24 h at room temperature to mimic the settingssed in the REPAIR-AMI trial (16). The RBCs andMCs used were from the same patient.low cytometry analysis of BMCs. After Ficoll densityradient centrifugation, BMCs were analyzed by FACS.or the enumeration of bone marrow cell populations, wesed directly conjugated antibodies against human CD45FITC-labeled, BD Pharmingen, San Diego, California),D34 (PE-labeled, BD Pharmingen), CD133 (APC-

abeled, Miltenyi, Bergisch Gladbach, Germany), and KDRPE-labeled, R&D Systems, Minneapolis, Minnesota).ating was performed according to the International Society

f Hematotherapy and Graft Engineering guideline (20).For analysis of the mitochondrial membrane potential, BMCs

ere stained with the lipophilic cation 5,5=,6,6=tetrachloro-,1=,3,3=-tetraethylbenzimidazol-carbocyanine iodide (JC-1, In-itrogen, Carlsbad, California) according to the manufacturer’snstruction. Briefly, 24 h after storage, JC-1 was added to the cellsnd incubated for 20 min at 37°C. After washing with phosphate-uffered saline, FACS analysis was immediately performedFACS Canto II, Becton Dickinson).

ssessment of hematopoietic colonies. The BMCs (1 �05/dish) were seeded in methylcellulose plates (MethocultF H4534, StemCell, Vancouver, British Columbia, Can-

da). Plates were studied under phase-contrast microscopy,nd colony-forming units (CFUs) (colonies �50 cells) wereounted after 14 days of incubation at 37°C by an indepen-ent investigator. CFUs were examined in duplicates.ssessment of invasion capacity of BMCs. A total of 1 �06 BMCs were resuspended in 250 �l X-vivo 10 mediumnd placed in the upper chamber of a modified Boydenhamber filled with Matrigel (BioCoat invasion assay, 8 �more size, Becton Dickinson). Then, the chamber waslaced in a 24-well culture dish containing 500 �l endothe-

ial basal medium. For stimulation of invasion, 100 ng/mltromal cell-derived factor (SDF)-1 was added in the lowerhamber. After incubation for 24 h at 37°C, transmigratedells were counted by independent investigators. Invasionssays were run in duplicates.ell-matrix adhesion. Cell-matrix adhesion was per-

ormed as previously described (21). Ninety-six-well platesere coated overnight at 4°C with 2.5 �g/ml soluble

ecombinant human ICAM-1 (R&D Systems) in coatinguffer (150 mmol/l sodium chloride, 20 mmol/l Tris hydro-hloride, 1 mmol/l magnesium chloride, pH 9.0) and then
locked for 1 h at room temperature with 3% (w/v) v
eat-inactivated (2 h, 56°C) bovine serum albumin (Sigma,t. Louis, Missouri). Human BMCs were stained with=,7=-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-ctoxymethyl-ester (BCECF, Invitrogen; 2.5 �M) andtored overnight with different concentrations of RBCs asndicated in X-vivo medium containing 20% serum. Then,ells were seeded at 1.1 � 105 cells/well in 100 �l in theoated wells for 10 min at 37°C. After washing with warmPMI 1640, adherent cells were quantified in triplicatesith a fluorescence plate reader (Synergy HT, Biotek, Badriedrichshall, Germany).indlimb ischemia model. The neovascularization capac-

ty of the BMC was investigated in a murine model ofindlimb ischemia by use of 8-week-old athymic Balb/Cude mice (Jackson Laboratory, Bar Harbor, Maine) weigh-

ng 18 to 22 g. The proximal portion of the femoral arteryncluding the superficial and the deep branch as well as theroximal portion of the saphenous artery were obliterated andemoved with an electrical coagulator. The overlying skin waslosed with 7-0 silk suture. After 24 h, 1 � 106 BMC/mouseontaining the indicated number of RBCs were injectedntravenously. Injection of 5 � 106 RBCs obtained aftereukocyte depletion of the bone marrow served as control.

imb perfusion measurements. After 21 days, we mea-ured ischemic (right)/normal (left) limb perfusion ratioith a laser Doppler blood flowmeter (Laser Dopplererfusion Imager System, moorLDI-Mark 2, Moor Instru-ents, Wilmington, Delaware). The perfusions of the

schemic and nonischemic limb were calculated from meanalue multiplied by the number of pixels of the region belowhe inguinal ligament. To minimize variables includingmbient light and temperature, calculated perfusion wasxpressed as the ratio of ischemic to nonischemic hind-imb perfusion.tatistical analysis. If not stated otherwise, data are showns mean � SEM. Statistical comparisons were made by theonparametric Wilcoxon 2-sample or Friedman=s testspaired analyses) and the nonparametric Mann-Whitney Ur Kruskal-Wallis tests for between-group analyses. Fornivariate analysis, nonparametric Spearman correlation waspplied. The multivariable analysis was done by a parametricinear regression model. Because this approach assumes theormality of the dependent variable, we applied the Box-ox transformation for the dependent variable in theultivariate analysis (22). The stepwise linear regressionodel was performed with the transformed approximately

ormal distributed dependent variable. Statistical signifi-ance was assumed, if p � 0.05. All reported p values are-sided. All statistical analysis was performed with SPSSversion 17.0, SPSS, Inc., Chicago, Illinois).

esults

mpact of cell characteristics on functional recovery ofatients treated with BMCs. To analyze the effect of
arious cell quality and functional parameters on LV con-

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ractile recovery measured as absolute change in LVEF, weerformed a univariate analysis (Table 1). None of the cellharacteristics, which include the total number of cells andhe subpopulations of hematopoietic progenitor cells, cor-elated with the contractile recovery in the BMC-treatedatients. Because in the placebo group BMCs were isolatedccordingly but were not infused in the patients, analysis ofhe placebo group offered the unique opportunity to excludehe possibility that the contamination of the final cellroduct with RBCs is affected by patient-specific factorsnfluencing LVEF recovery.

Moreover, we quantified the cell intrinsic function ofrogenitor cells by measuring the hematopoietic CFUapacity. Likewise, we determined both the basal as well asDF-1–induced migratory capacity of BMCs, which washown to correlate with cell recruitment and homing after

aseline Cell Characteristics of the Final Cellreparation and Impact on Contractile Recoveryithin the REPAIR-AMI TrialTable 1

Baseline Cell Characteristics of the Final CellPreparation and Impact on Contractile RecoveryWithin the REPAIR-AMI Trial

Median (25th/75th Percentile)

Placebo Patients(n � 103)

BMC Patients(n � 101)

Total mononuclear cellsisolated (�106)

210 (110/336) 198 (130/284)

Viability (%) 99 (98/99) 99 (98/99)

Invasion basal 82 (49/125) 81 (46/139)

SDF-1–induced invasion 161 (90/210) 163 (98/209)

CFU capacity 29 (20/43) 27 (19/40)

CD34� CD45� (%) 1.43 (1.01/2.04) 1.35 (0.96/1.91)

CD133� CD45� (%) 1.10 (0.80/1.50) 1.00 (0.69/1.40)

CD45� KDR� (%) 0.06 (0.30/0.80) 0.06 (0.03/0.11)

RBC (�109) 0.30 (0.10/0.40) 0.20 (0.10/0.30)

Hematocrit (%) 0.3 (0.2/0.5) 0.2 (0.2/0.4)

Platelets (�106) 150 (100/330) 140 (100/200)

Neutrophils (%) 38 (32/47) 36 (27/49)

Lymphocytic cells (%) 43 (36/50) 42 (34/53)

Univariate Analysis for �LVEF

Placebo (n � 92) BMC (n � 95)

Total mononuclear cellsisolated (�106)

r � �0.05; p � 0.7 r � �0.08; p � 0.5

Viability (%) r � �0.17; p � 0.1 r � 0.15; p � 0.2

Invasion basal r � 0.07; p � 0.5 r � �0.05; p � 0.6

SDF-1–induced invasion r � 0.06; p � 0.6 r � 0.02; p � 0.8

CFU capacity r � �0.01; p � 1.0 r � �0.09; p � 0.4

CD34� CD45� (%) r � �0.09; p � 0.4 r � �0.10; p � 0.3

CD133� CD45� (%) r � �0.09, p � 0.4 r � �0.09, p � 0.4

CD45� KDR� (%) r � �0.15, p � 0.2 r � �0.04, p � 0.7

RBC (�109) r � 0.13, p � 0.2 r � �0.25, p � 0.02

Hematocrit (%) r � 0.10, p � 0.3 r � �0.22, p � 0.04

Platelets (�106) r � 0.02, p � 0.8 r � �0.23, p � 0.03

Neutrophils (%) r � 0.12, p � 0.3 r � 0.06, p � 0.6

Lymphocytic cells (%) r � 0.03, p � 0.7 r � �0.10, p � 0.4

ell characteristics of the placebo group are shown for comparative reasons only; these patientsid not receive the cell preparation but a cell-free placebo preparation.BMC � bone marrow mononuclear progenitor cell; CFU � colony-forming unit; LVEF � left

entricular ejection fraction; RBC � red blood cell; REPAIR-AMI � Reinfusion of Enriched Progen-tor Cells and Infarct Remodeling in Acute Myocardial Infarction; SDF � stromal cell-derived factor.

ntravascular administration of BMCs (13). However, noneS

f these functional properties of the administered BMCsas associated with the extent of LV contractile recovery aseasured by changes in LVEF (Table 1). Only the number

f contaminating RBCs and the hematocrit of the final cellroduct were significantly and inversely correlated with theecovery of LVEF, whereas no significant association wasetected for the number of contaminating neutrophils and

ymphocytic cells. In addition, the number of contaminatinglatelets also showed a significant inverse correlation withVEF recovery by univariate analysis (Table 1). To identifyhether RBC contamination is an independent predictor ofVEF recovery at 4 months, we performed a multivariatenalysis including classical determinants of LV contractileecovery after AMI, like end-systolic volume, peak creati-ine kinase levels, time of symptom onset to treatment, age,nd the presence of diabetes as well as the previouslydentified predictors of LVEF recovery, namely a reducedVEF at baseline and the time to treatment (14). As shown

n Table 2, the number of contaminating RBCs togetherith a reduced baseline LVEF and maximum creatinineinase levels remained as the only independent predictorsor the recovery of LVEF. In contrast, the contaminatinglatelets were not an independent predictor of LVEFecovery (Online Tables 1A and 1B). Thus, univariatend multivariate analysis revealed that the contaminationf the final cell product with RBCs is an independentredictor of reduced recovery of LVEF in BMC-treatedatients with AMI. Of note, the negative impact ofBCs on the recovery of LVEF was only detected in theMC group but not in the placebo control group, whichid not receive the cell product (Fig. 1A). Thus, the

mpact of RBC contamination is confined to patientseceiving BMC administration but does not reflect aatient-intrinsic association. Moreover, the number ofBCs in the bone marrow aspirate (before densityradient centrifugation) did not correlate with the recov-ry of LVEF in the BMC group (p � 0.73), indicatinghat the composition of the bone marrow aspirate beforerocessing did not affect functional recovery. These datauggest that the number of contaminating RBCs in the

ultivariate Analysis for LVEFmprovement After 4 Months in the BMC GroupTable 2 Multivariate Analysis for LVEFImprovement After 4 Months in the BMC Group

p ValueStandardizedCoefficient

Age 0.57 0.06

Diabetes 0.22 0.13

Maximum creatinine kinase levels 0.02 0.27

Time symptom to stent 0.22 �0.13

Baseline end-systolic volume 0.39 0.12

Baseline LVEF 0.001 0.48

Time to BMC administration 0.76 �0.03

RBC (�109) contamination of the final cell product 0.01 0.27

ignificance (analysis of variance) � 0.003.Abbreviations as in Table 1.

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urified BMC population might have influenced theunctionality of the cells used for cell therapy.

Therefore, in the next step, we analyzed the effect of RBContamination on functional parameters of isolated BMCsn the REPAIR-AMI trial. Importantly, increasing num-ers of contaminating RBCs were not only associated withreduced viability of the isolated cells, as measured by

rypan blue exclusion assay, but also correlated with aignificant impairment of invasion capacity and CFU ca-acity of the isolated BMCs (Table 3, Fig. 1B), indicatinghat RBCs directly affect cell viability and cell functionality.

BC contamination impairs BMC function in vitro andn vivo. To prospectively test the hypothesis that contamina-

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(A) Association between left ventricular ejection fraction (LVEF) improvement at 4derived progenitor cell (BMC) group within the REPAIR-AMI (Reinfusion of Enrichedshows quartiles of RBC contamination. Statistical comparison between groups waviability within the REPAIR-AMI trial. Statistical comparison between groups was pe

ion with RBCs influences the functionality of BMCs used for v

ell therapy, we coincubated isolated BMCs with increasingumbers of RBCs for 24 h in vitro. The highest number used

n the experiments reflects the median concentration of con-aminating RBCs observed in the clinical trial. The RBCsose-dependently reduced the viability of BMCs (Fig. 2A),onsistent with the statistical evaluation of the clinical trial.urthermore, the invasion capacity of BMCs at baseline andfter stimulation with SDF-1 was significantly reduced byBC addition. In fact, the highest dose of RBC contami-ation completely abolished the migratory capacity of iso-

ated BMCs toward SDF-1 (Fig. 2B). Moreover, directncubation of RBCs with freshly isolated BMCs in the

igration assay significantly inhibited SDF-1-induced in-

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s and red blood cell (RBC) contamination for the placebo and the bone marrow-itor cells And Infarct Remodeling in Acute Myocardial Infarction) trial. The x-axisrmed with the Kruskal-Wallis test. (B) The RBC contamination influences BMCd with the Kruskal-Wallis test.

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BCs for 24 h further reduced the formation ofranulocyte/macrophage-CFUs, a prototypical marker forunctionally competent hematopoietic progenitor cells (Fig.C), but did not affect the adhesion of the BMCs (Fig. 2D).The integrity and function of mitochondria is essential

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(A) After isolation, RBCs were added to 1 � 106 BMCs in the indicated concentrawas assessed with a trypan blue dye exclusion assay. Data are mean � SEM of nindicated concentrations of RBCs for 24 h under basal conditions as well as towachambers. Data are mean � SEM of n � 4; �p � 0.05 versus basal migration. (Ccated numbers of RBCs. Data are mean � SEM of n � 4; �p for trend �0.05 verslis, Minnesota) after storage with 5 � 106 RBC/1 � 106 BMCs for 24 h. Data are

nfluence of RBC Contamination on Cellharacteristics Within the REPAIR-AMI TrialTable 3 Influence of RBC Contamination on CellCharacteristics Within the REPAIR-AMI Trial

Univariate Analysis (RBC/MNC)

Viability (%) r � �0.23; p � 0.001

Invasion basal r � �0.21; p � 0.003

SDF-1–induced invasion r � �0.27; p � 0.001

CFU capacity r � �0.16; p � 0.03

CD34� CD45� (%) r � 0.13; p � 0.06

CD133� CD45� (%) r � �0.04, p � 0.5

CD45� KDR� (%) r � �0.06, p � 0.4

NC � mononuclear cell; other abbreviations as in Table 1.

ells (24), and cell survival (25). Therefore, we testedhether the addition of RBCs might influence mitochon-rial function of BMCs by measuring the mitochondrialembrane potential with JC-1 staining. As demonstrated inigure 3, contamination with RBCs dose-dependently re-uced the quantitative mitochondrial membrane potentialf BMCs, indicating that RBCs impair the mitochondrialunction of BMCs.

To test whether RBCs affect the functional benefit of cellherapy in vivo, we injected BMCs that had been co-ncubated with or without RBCs for 24 h into nude micefter induction of hindlimb ischemia. Indeed, laser Doppler-erived blood flow recovery was significantly higher in micereated with BMCs compared with the group that receivedMCs after co-incubation with RBCs (Fig. 4). Taken to-ether, these data confirm that RBC contamination dose-ependently impairs the in vitro and in vivo functions of

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BMC + RBC

basal SDF-1

p=0.59

p=0.11

adhe

sion

capa

city

(% c

ontr

ol)

D

and cells were stored according to the REPAIR-AMI protocol. After 24 h, viability�p � 0.05 versus control. (B) Invasion capacity of BMCs after storage with theromal cell-derived factor (SDF)-1 gradient was assessed with modified Boydenber of colony-forming units of 1 � 105 BMCs after 24 h storage with the indi-Cs alone. (D) Adhesion capacity of BMCs on ICAM-1 (R&D Systems, Minneapo-

SD of n � 3 experiments measured in triplicates. Abbreviations as in Figure 1.

Inva

ded

cells

(% c

ontr

ol)

B

tions,� 4;

rd a st) Numus BM

mean �

RnihfhcaiutimitiecHm

psi

D

PcicoivTcLait


BCs impair BMC function via a secreted factor. Fi-ally, we performed further experiments to gain mechanistic

nsights into RBC action on BMC function. Becauseemolysis of RBCs is well-known to result in high levels ofree heme, causing cellular injury (26), we tested theypothesis of whether the release of hemoglobin mightause BMC dysfunction. However, supernatants of RBCsfter hypotonic lysis did not affect BMC invasion (Fig. 5A)ndicating that the release of hemoglobin by RBCs isnlikely to induce migratory defects of BMCs. Next, weested whether a secreted RBC-derived factor is sufficient tompair BMCs. Therefore, RBCs were incubated in X-vivo

edium for 24 h, and the supernatant was subsequentlyncubated with BMCs. As shown in Figure 5B, superna-ants of intact RBCs indeed inhibited SDF-1–inducednvasion of BMCs. Furthermore, we addressed whether theffect might be caused by shed microparticles and, therefore,entrifuged the RBC supernatant to pellet microparticles.owever, RBC-derived microparticles did not affect BMC

controunstained

Lymphocytes

Monocytes

10

20

30

40

Lymphocytesp <0.05

PE

-po

siti

ve c

ells

(%

)

contro

l0.

1x10

6 RBC

1.0x

106 RBC

5.0x

106 RBC

A

B

Figure 3 Membrane Potential of BMCs

(A) Representative pictures showing histograms of JC-1–stained (Invitrogen, Caof RBCs for 24 h. The fluorescence PE-channel depicts JC-1 dimers at higher mPE-positive BMCs in the lymphocyte or monocyte gate after incubation with RBCobtained when the PE/FITC ratio was analyzed (p � 0.05). Abbreviations as in Fig

igration, whereas the microparticles-free supernatant still a

revented BMC migration (Fig. 5B), indicating that aecreted factor but not microparticles mediate the migrationmpairment of BMCs induced by RBCs.

iscussion

ost-hoc analysis of the REPAIR-AMI trial indicates thatontamination of the autologous cell product with RBCsmpairs functional improvement of LVEF in patients afterell therapy. The number of contaminating RBCs was notnly associated with reduced recovery of LVEF by univar-ate and multivariate analysis but also correlated with theiability, invasion, and CFU capacity of the applied BMCs.his retrospective statistical analysis, indicating that RBC

ontamination negatively influences the improvement ofVEF after cell therapy, was further prospectively validatednd confirmed by experimental studies showing that ex vivoncubation of BMCs with RBCs dose-dependently reducedhe viability and functional activity of the BMCs in vitro

0.1x106 RBC 1x106 RBC 5x106 RBC

20

40

60

80

Monocytesp <0.05

PE

-po

siti

ve c

ells

(%

)

contro

l0.

1x10

6 RBC

1.0x

106 RBC

5.0x

106 RBC

, California) BMCs after incubation of BMCs with the indicated concentrationne potential in the lymphocyte or monocyte gate. (B and C) Quantification of

indicated. Data are mean � SEM of n � 4 experiments. Similar results were

l

C

rlsbadembras as

ure 1.

nd impaired the therapeutic benefit of the applied cells in

adr

dw

(titbp


n animal model in vivo. Thus, RBCs indeed dose-ependently impair BMC functions involved in functionalecovery after ischemia.

It is well-established that risk factors for coronary arteryisease impair the functionality of patient-derived BMCsith respect to their capacity to induce neovascularization

A

BMC

RBC

BB

BMC+RBC

Figure 4 Impairment of In Vivo Functions of BMCs

Representative pictures (A) and quantitative analysis (B) of relative perfusion in aBMCs stored 24 h according to the indicated conditions. Data are mean � SD of

Figure 5 Mechanistic Insights Into the BMC Impairment Media

(A) RBCs (5 � 106 RBC/1 � 106 BMC) were pelleted and lysed with 50 �l of watfuged for 10 min at 800 g. The resulting supernatants or the pellet (resolved in 40invasion was determined by using a modified Boyden chamber assay. Data are me(B) RBCs were incubated in X-vivo-10 medium for 24 h at room temperature, and the20,500 g for 45 min. After centrifugation, the supernatant or the resolved pellet wasassays with SDF-1 as stimulus. Data are mean � SEM of n � 2 experiments measur

9). Moreover, previous clinical pilot trials suggested thathe measurement of cell functionality by detecting thenvasive and migrating capacity defines the benefit of cellherapy (12,18). Whereas these parameters did not seem toe significant univariate determinants in the present study inatients with AMI, the invasive capacity in response to the

BMC

BMC+RBC

RBC* *

BMC

RBC* *BMC+RBC

mb ischemia model 21 days after injection of RBCs ormice. �p � 0.05 versus BMCs alone. Abbreviations as in Figure 1.

y RBCs

25 s. After the addition of 350 ml X-vivo-10 medium, the sample was centri-edium) were added to the BMCs in the invasion chamber. The SDF-1–induced

EM of n � 2 experiments measured in duplicates. �p � 0.05 versus control.oned medium (“supernatant”) was either directly added to BMCs or centrifuged atto BMCs, and BMC invasion was determined by using modified Boyden chamberuplicates. �p � 0.05 versus control. Abbreviations as in Figures 1 and 2.

Rel

ativ

e pe

rfus

ion

(%le

ft lim

b)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Rel

ativ

e pe

rfus

ion

(%le

ft lim

b)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

hindlin � 8

ted b

er for0 �l m

an � Sconditiaddeded in d

pivSvriBpbt

dcicnMcnB

RBicwlarmthstmahmAaempt

mifhiomitb

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R


hysiologically important cytokine SDF-1 (27) was signif-cantly associated with improved LVEF recovery in multi-ariate statistical analysis. Importantly, normalization ofDF-1–induced invasion by the contaminating RBCs pro-ided a highly significant predictor of global LV contractileecovery (Online Table 2, Online Fig. 1). Thus, combiningntrinsic patient-determined functional parameters ofMCs with extrinsic effects imposed by the cell isolationrocedure indeed discloses a cause-and-effect relationshipetween the cell product and LVEF recovery after cellherapy in patients with AMI.

Interestingly, in the REPAIR-AMI trial, we could notetect an association between the applied cell numbers andontractile recovery. This might be because 85% of the patientsn the BMC group received BMC numbers exceeding 108

ells, which was identified as the minimum cell numberecessary for beneficial effects in a recent meta-analysis byartin-Rendon et al. (28). Thus, to detect a potential direct

ell number-related dose–response relationship, it seems to beecessary to prospectively administer predefined numbers ofMCs varying at least by a factor of 100-fold difference.Mechanistically, our results suggest that addition of

BCs affects the mitochondrial membrane potential ofMCs. Mitochondria act as important signaling organelles

n endothelial cells (29) and are required for the migratoryapacity of endothelial progenitor cells (22). Moreover, it isell-established that mitochondrial function and metabo-

ism are important determinants of stem cell self-renewalnd differentiation (30,31). The mitochondrial marker dyehodamine 123, which measures mitochondrial activity andembrane polarity, shows an age-related relationship be-

ween the mitochondrial dye efflux and the ability of humanematopoietic stem cells to repopulate the hematopoieticystem of irradiated animals (32,33). Importantly, adminis-ration of progenitor cells with a reduced mitochondrialembrane potential induced by hypoxia was associated withsignificantly impaired neovascularization capacity in the

indlimb ischemia model (34). By contrast, increasing theitochondrial membrane potential of progenitor cells via anMP-activated protein kinase-dependent mechanism was

ssociated with enhanced migration and functional re-ndothelialization (24). Thus, the functional integrity of theitochondria seems to be essential not only for stem and

rogenitor cell function and survival but also for regulatinghe vascular repair capacity of administered cells.

The question remains: by which signaling mechanismight RBCs affect viability and mitochondrial function of

solated BMCs? Hemolysis of RBCs results in high levels ofree heme causing cellular injury (26). Moreover, freeemoglobin was shown not only to directly induce apoptosis

n cultured endothelial cells (35) but also to scavenge nitricxide (NO). Nitric oxide plays a crucial role for stem cellaintenance, differentiation, and neovascularization capac-

ty (36–39) and increases mitochondrial function in endo-helial cells (29), thus raising the hypothesis that reduced
ioactive NO levels in the BMC suspension might have
ontributed to impaired benefit of BMCs associated withitochondrial dysfunction. However, supernatants of lysedBCs containing free hemoglobin did not affect BMC

unctions, and the addition of NO donors did not rescue thempaired migratory capacity of BMCs induced by RBCsdata not shown). In contrast, supernatants of intact RBCsere shown to significantly reduce BMC invasion, indicat-

ng that an actively secreted factor mediates BMC impair-ent. Further subfractionation of these RBC supernatants

xcluded a role of microparticles. On the basis of theseesults, one might speculate that secreted lipids or proteinsight mediate the effects of RBCs on BMCs.In summary, the results of the present study demonstrate

or the first time an association between cell quality andontractile recovery within a large randomized, multicenterlinical trial, suggesting a bioactivity response relationshipery much like a dose–response relationship in drug trials.herefore, future studies are warranted to establish rapid

nd reliable tests for quality control of the cell product whensing autologous BMCs. On the basis of the presented data,esting for contaminating RBCs might be used as an ad hocuality control parameter for future clinical trials.

cknowledgmentshe authors greatly appreciate the enthusiastic support of the

taff of our catheterization laboratories and by our technicianseate Mantz, Isabel Geweyer, Heike Wagner, and Lianeiska (study nurses), Tina Rasper, Tino Röxe, Nicola Schüly,

nd Nadine Sorg (biological technicians). In addition, theuthors would like to thank all of the REPAIR-AMI studyenters and investigators for ongoing support.

eprint requests and correspondence: Dr. Andreas M. ZeiherCardiology), Department of Medicine III, Goethe University,heodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail:

[email protected].

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ey Words: myocardial infarction y progenitor cells y REPAIR-AMIrial.

APPENDIX

or supplementary tables and a supplementary
gure, please see the online version of this article.

red blood cell contamination of the final cell product...

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