Suncombe Components and Spares

ZhangSwingen, Robert Deans, Arthur H.L. From, Robert J. Bache, Catherine M. Verfaillie and JianyiZhang, Piradeep Suntharalingam, Sherry Boozer, Abner Mhashilkar, Carmelo J. Panetta, Cory Lepeng Zeng, Qingsong Hu, Xiaohong Wang, Abdul Mansoor, Joseph Lee, Julia Feygin, Ge

RemodelingProgenitor Cell Transplantation in Hearts With Postinfarction Left Ventricular

Derived Multipotent−Bioenergetic and Functional Consequences of Bone Marrow

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2007 American Heart Association, Inc. All rights reserved.

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Bioenergetic and Functional Consequences of BoneMarrow–Derived Multipotent Progenitor Cell

Transplantation in Hearts With Postinfarction LeftVentricular Remodeling

Lepeng Zeng, PhD*; Qingsong Hu, MD, MS*; Xiaohong Wang, MD, PhD*; Abdul Mansoor, MD, PhD;Joseph Lee, PhD; Julia Feygin, BS; Ge Zhang, MD, PhD; Piradeep Suntharalingam, BS;

Sherry Boozer, BS; Abner Mhashilkar, PhD; Carmelo J. Panetta, MD; Cory Swingen, PhD;Robert Deans, PhD; Arthur H.L. From, MD; Robert J. Bache, MD;

Catherine M. Verfaillie, MD; Jianyi Zhang, MD, PhD

Background—The present study examined whether transplantation of adherent bone marrow–derived stem cells, termedpMultistem, induces neovascularization and cardiomyocyte regeneration that stabilizes bioenergetic and contractilefunction in the infarct zone and border zone (BZ) after coronary artery occlusion.

Methods and Results—Permanent left anterior descending artery occlusion in swine caused left ventricular remodelingwith a decrease of ejection fraction from 55�5.6% to 30�5.4% (magnetic resonance imaging). Four weeks after leftanterior descending artery occlusion, BZ myocardium demonstrated profound bioenergetic abnormalities, with a markeddecrease in subendocardial phosphocreatine/ATP (31P magnetic resonance spectroscopy; 1.06�0.30 in infarcted hearts[n�9] versus 1.90�0.15 in normal hearts [n�8; P�0.01]). This abnormality was significantly improved bytransplantation of allogeneic pMultistem cells (subendocardial phosphocreatine/ATP to 1.34�0.29; n�7; P�0.05). TheBZ protein expression of creatine kinase–mt and creatine kinase–m isoforms was significantly reduced in infarctedhearts but recovered significantly in response to cell transplantation. MRI demonstrated that the infarct zone systolicthickening fraction improved significantly from systolic “bulging” in untreated animals with myocardial infarction toactive thickening (19.7�9.8%, P�0.01), whereas the left ventricular ejection fraction improved to 42.0�6.5% (P�0.05versus myocardial infarction). Only 0.35�0.05% donor cells could be detected 4 weeks after left anterior descendingartery ligation, independent of cell transplantation with or without immunosuppression with cyclosporine A (withcyclosporine A, n�6; no cyclosporine A, n�7). The fraction of grafted cells that acquired an endothelial orcardiomyocyte phenotype was 3% and �2%, respectively. Patchy spared myocytes in the infarct zone were found onlyin pMultistem transplanted hearts. Vascular density was significantly higher in both BZ and infarct zone of cell-treatedhearts than in untreated myocardial infarction hearts (P�0.05).

Conclusions—Thus, allogeneic pMultistem improved BZ energetics, regional contractile performance, and global leftventricular ejection fraction. These improvements may have resulted from paracrine effects that include increasedvascular density in the BZ and spared myocytes in the infarct zone. (Circulation. 2007;115:1866-1875.)

Key Words: cells � heart failure � hypertrophy � magnetic resonance imaging � metabolism� myocardial contraction � myocardial infarction

Myocardial infarction (MI) often results in left ventricu-lar (LV) remodeling in which an initial period of

relative hemodynamic stability is followed by progressivecontractile dysfunction that can eventuate in overt congestiveheart failure. Using a swine model of post-MI LV remodel-

Clinical Perspective p 1875

ing, we recently reported that myocardial energetic charac-teristics, as represented by high-energy phosphate content andthe phosphocreatine (PCr)/ATP ratio, were markedly abnor-

Received August 31, 2006; accepted February 9, 2007.From the Department of Medicine (L.Z., Q.H., X.W., A. Mansoor, J.L., J.F., G.Z., C.J.P., C.S., A.H.L.F., R.J.B., J.Z.) and Stem Cell Institute (L.Z.,

C.M.V.), University of Minnesota Medical School, Minneapolis, Minn; and Athersys, Inc (P.S., S.B., A. Mhashilkar, R.D.), Cleveland, Ohio.*The first 3 authors contributed equally to this work.The online-only Data Supplement, consisting of Methods and a figure, is available with this article at http://circ.ahajournals.

org/cgi/content/full/CIRCULATIONAHA.106.659730/DC1.Correspondence to Jianyi Zhang, MD, PhD, University of Minnesota Health Science Center, Mayo Mail Code 508, 420 Delaware St SE, Minneapolis,

MN 55455. E-mail [email protected]© 2007 American Heart Association, Inc.

Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.106.659730

1866

Heart Failure

at UNIV OF KANSAS SCH OF MED on May 26, 2014http://circ.ahajournals.org/Downloaded from


mal in the peri-infarct border zone (BZ).1 The abnormalitieswere much greater in the BZ subendocardium (ie, PCr/ATPreduced by �50%) than in myocardium remote from theinfarct. Similarly, LV contractile function was most de-pressed in the BZ of infarcted hearts.2,3 These data are inagreement with the hypothesis that bioenergetic and contrac-tile abnormalities of the BZ myocardium result in progressivedeterioration of overall LV chamber function. A potentially“curative” therapy would be to replace infarcted or dysfunc-tional tissue with cardiomyocytes that could prevent LVremodeling and subsequent congestive heart failure.

Both experimental and clinical studies have demonstratedthat cell transplantation can improve LV contractile perfor-mance in infarcted hearts,4–15 but the underlying mechanismsare not totally clear. Although improved cardiac functioncould be due to differentiation of transplanted cells intocardiomyocytes,4–15 it is generally believed that release ofcytokines that exert trophic effects on host cardiac cells andinduce neovascularization may be of equal or greater impor-tance.7,15 In the present study, we examined the effects ofallogeneic bone marrow–derived adherent stem cells (pMul-tistem) transplanted into the BZ of acutely infarcted swinehearts on BZ and remote zone structure (LV chamber size andinfarct size), contractile performance, and energetic status(PCr/ATP ratio) using 31P magnetic resonance spectroscopy(MRS). pMultistem cells were expanded by Athersys, Inc(Cleveland, Ohio) from a cell line of multipotent adultprogenitor cells generated from a young pig (45 days ofage)16 using clinically suitable expansion methods. pMultis-tem cells were transduced with a �-galactosidase–expressingretrovirus, expanded to generate a master cell bank andworking cell bank, which were cryopreserved. pMultistemcells can differentiate into smooth muscle cells and endothe-lial cells. We hypothesized that the primary beneficial effectsof cell transplantation occur in the infarct zone (IZ) and BZ of

infarcted hearts and that preservation of structure and func-tion is primarily the result of limitation of BZ deterioration.We reasoned that grafting of pMultistem cells into the BZ ofacutely infarcted hearts would attenuate BZ contractile andenergetic deterioration and thereby limit overall LV chamberdilation. To determine whether cell transplantation couldaugment oxygen availability in the BZ, myocardial deoxy-myoglobin (Mb-�) was assessed with 1H MRS.17,18 Theresults were compared with nontransplanted infarcted hearts.We also evaluated the effects of immunosuppression withcyclosporine on the pMultistem engraftment rate.

MethodspMultistem CulturepMultistem cells were derived from 1 of the male swine multipotentadult progenitor cell stocks generated at the University of Minneso-ta.16 Details on the generation of the pMultistem cells, their bankingand cryopreservation, and in vitro experiments characterizing thephenotype and differentiation potential are described in the onlineData Supplement.

Creation of Infarct by Coronary Artery LigationThe investigation conformed to the “Guide for the Care and Use ofLaboratory Animals” published by the National Institutes of Health(NIH publication No. 85-23, revised 1985). Details of the animalmodel of postinfarction LV remodeling have been described previ-ously.1,17 Briefly, young Yorkshire female swine (45 days old, �10kg; Manthei Hog Farm, Elk River, Minn) were anesthetized withpentobarbital (30 mg/kg IV), intubated, and ventilated with arespirator with supplemental oxygen. A left thoracotomy was per-formed, and 0.5 cm of the left anterior descending coronary artery(LAD) distal to the second diagonal branch was dissected free andpermanently occluded with a ligature, which usually results in �10%LV mass damage. Lidocaine (1 mg · kg�1 · min�1 IV for 70 minutes,2 mg/kg IV bolus before LAD occlusion) and nitroglycerine (0.5 �g· kg�1 · min�1 IV for 70 minutes starting 10 minutes before LADocclusion) were given before and during LAD ligation to decreasethe arrhythmia. Animals were observed in the open-chest state for 60minutes. If ventricular fibrillation occurred, electrical defibrillation

Figure 1. In vitro differentiation of pMultistemto cells with phenotypic and functional char-acteristics of endothelial cells. pMultistem(�90 population doublings) were plated at50 000 cells/cm2 in fibronectin-coated wellsin basal medium with 100 ng/mL VEGF for10 days. Cultures were fixed with 4% para-formaldehyde on day 10 and double stainedwith (A) anti–von Willebrand factor (vWF)labeled with Cy3, (B) anti-CD31 labeled withCy2, (C) overlay of (A) and (B), (D) anti-vWFlabeled with Cy3, (E) anti–VE-cadherinlabeled with Cy2, and (F) overlay of (D) and(E); nuclei were stained by DAPI. G, Vasculartube formation by pMultistem-derived endo-thelium-like cells. pMultistem-derived endo-thelial cells were replated in ECMatrix. After6 hours, typical vascular tubes could beseen. H, Quantitative polymerase chain reac-tion analysis with endothelium-specific prim-ers vWF, VE-cadherin, CD31(PECAM; plate-let and endothelial cell adhesion molecule),and Tie-1 also confirmed the differentiationof pMultistem into an endothelial phenotype.Lane 1, ladder marker; lane2, negative con-trol; lane3, before differentiation; lane 4,induced differentiation at day 10; and lane 5,positive control. Magnification: A–F, �40;G, �10.

Zeng et al Stem Cells in Postinfarction LV Remodeling 1867



was performed immediately and was usually successful. Sixtyminutes after LAD ligation, the surviving pigs were randomized toligation only (n�7), MI plus cell transplantation group (n�7), andMI plus cell transplantation plus cyclosporine A (CsA) group (n�6).The ischemic myocardial region became cyanotic in response to theLAD occlusion. pMultistem cells were injected directly into 5regions of the peri-infarct BZ (10 million cells per location; 50million total diluted in 2 mL of saline). Injection sites were markedwith a stitch to allow identification of the areas for histologicalstudies. Animals in the control group (MI) received 2 mL of salinein 5 injections at similar BZ injection sites. The chest was thenclosed. Animals received standard postoperative care, includinganalgesia, until they ate normally and became active. pMultistem-treated animals were randomized to receive or not receive CsA (15mg · kg�1 · d�1 mixed with food). Animals returned to the laboratory3.5 and 4 weeks later for magnetic resonance imaging (MRI) and 31PMRS studies, respectively.

Tagged and Cine MRI ProtocolMRI was performed �25 days after surgery on a 1.5-T clinicalscanner (Siemens Sontata, Siemens Medical Systems, Islen, NJ) witha phased-array, 4-channel surface coil and ECG gating. Details onmethods of MRI and MRS are described in the Data Supplement.

Spatially Localized 31P-MRS and1H-MRS TechniqueSpatially localized 31P nuclear magnetic resonance spectroscopy wasperformed with the rotating frame experiment using adiaboticplane-rotation pulses for phase modulation–imaging selected in vivo

spectroscopy/Fourier series window method.17,19–21 There is abso-lutely no overlap between the 135° voxel (corresponding to thesubepicardium) and the 45° voxel (corresponding to the subendocar-dium).17,19–21 The 31P-MRS and 1H-MRS techniques are describedbriefly in the Data Supplement.

Surgical Preparation for Open-Chest MRS StudyDetailed surgical preparations for the MRS study have been pub-lished previously and are described briefly in the Data Supplement.

Hemodynamic MeasurementsAortic and LV pressures were measured with pressure transducerspositioned at the midchest level19–21 and recorded on an 8-channelrecorder.

Infarct SizeAt the completion of the open-chest MRS and hemodynamicmeasurements, animals were euthanized by an overdose of pento-barbital, and the heart was explanted. The LV was opened at thelateral wall from base to apex, and a photograph was taken for infarctsize measurement. Infarct size was expressed as a percent of LVsurface area by an image-analysis system (NIH Image J program,available at http://rsb.info.nih.gov/ij).22

ImmunohistochemistryDetailed immunohistochemistry methods are described in the DataSupplement.

Figure 2. In vitro differentiation of pMulti-stem to cells with phenotypic character-istics of smooth muscle (Sm) cells. pMul-tistem (�90 population doublings) wereplated at 3000 cells/cm2 in fibronectin-coated wells in basal medium with 10ng/mL platelet-derived growth factor and5 ng/mL transforming growth factor-� for12 days. Cultures were fixed with �20°Cmethanol on day 12 and double stainedwith (A) anti-smooth muscle-�-actinlabeled with Cy3, (B) anti-calponinlabeled with Cy2, (C) overlay of (A) and(B), (D) anti-caldesmon labeled with Cy3,(E) anti-Sm22 labeled with Cy2, and (F)overlay of (D) and (E); nuclei werestained by DAPI. G, Smooth muscle dif-ferentiation was evaluated by reverse-transcription polymerase chain reactionfor myocardin, calponin, and smoothmuscle-�-actin. Lane 1, Negative control;lane 2, before differentiation; lane 3,induced differentiation at day 14; andlane 4, positive control. Magnification:A–F, �20.

TABLE 1. Anatomic Data Obtained 4 Weeks After MI

BW, kg LVW, g RVW, gLVW/BW,

g/kgRVW/BW,

g/kg SSA/LVSA, %

Normal (n�8) 29�2 79�15 28�7 2.71�0.64 0.96�0.25 0

MI (n�9) 29�7 103�25* 33�8 3.58�0.27* 1.16�0.08 13�7

MI�pMultistem (n�7) 26�5 90�18 29�6 3.51�0.34* 1.14�0.20* 11�5

MI�pMultistem�CsA (n�6) 30�5 94�13 31�10 3.19�0.26 1.02�0.16 10�7

Values are mean�SD. BW indicates body weight; LVW, LV weight; RVW, right ventricular weight; SSA, scar surfacearea; LVSA, LV surface area; and n, number of pigs.

*P�0.05 vs normal.

1868 Circulation April 10, 2007



Data AnalysisHemodynamic data were measured from the strip chart record-ings.18–20 Data were analyzed with 1-way ANOVA. A value ofP�0.05 was considered significant. When ANOVA demonstrated asignificant effect, post hoc analysis was performed with the 2-tailedt test with Bonferroni correction. A least-squares linear regressionmodel was used for fitting the regression lines in the evaluation ofthe relationships between PCr/ATP and myocardial systolic shorten-ing fraction.

The authors had full access to and take full responsibility for theintegrity of the data. All authors have read and agree to themanuscript as written.

ResultsPhenotypic Characteristics of pMultistemMethods for pMultistem generation, expansion, banking, andquantification are described in detail in the online DataSupplement. Figures 1 and 2 illustrate the results of in vitrodifferentiation of pMultistem cells to endothelial and smoothmuscle cells, respectively. These data demonstrate that withappropriate inducers, pMultistem cells differentiate intosmooth muscle–like and endothelium-like cells and are con-sistent with the in vivo cell fate detected in these experiments.

Animal Model and Anatomic DataSeven of the 27 pigs with LAD ligation died within the first60 minutes after coronary occlusion. The surviving 20 pigswere randomized to ligation only (MI; n�7), ligation pluscell transplantation (n�7), and ligation plus cell transplanta-tion plus CsA (n�6). In addition, 8 size-matched normal pigsand 2 ligation-only pigs were subjected to the identical studyprotocol before randomization. These 10 pigs have beenincluded in the respective study groups. Table 1 summarizesanatomic data from 13 swine with LAD ligation into whichpMultistem cells were grafted with or without CsA, comparedwith 9 swine with LAD ligation with no cells grafted and noCsA treatment, as well as 8 normal pigs. The LV weight to

body weight ratio and right ventricular weight to body weightratio were 32% and 21% greater in hearts after LADocclusion than in hearts of size-matched normal swine,respectively (P�0.05; Table 1). LAD occlusion resulted in10% to 13% LV infarct, expressed as the ratio of scar surfacearea to LV surface area (Table 1). Infarct size, LV weight tobody weight ratio, and right ventricular weight to bodyweight ratio were not significantly affected by pMultistemtransplantation in the presence or absence of CsA (Table 1).

Hemodynamics and LV FunctionHemodynamic and LV ejection fraction data are summarizedin Table 2. One month after LAD ligation, hemodynamicvariables were not significantly different between the heartswith or without pMultistem treatment (Table 2). The cineMRI–measured LV ejection fraction decreased from 55%(normal hearts) to 30% in hearts with postinfarction LVremodeling without pMultistem treatment; LV ejection frac-tion was significantly greater in animals with pMultistemtransplantation irrespective of CsA treatment (P�0.05; Table 2).

LV end-systolic wall thickness and thickening fractionmeasured by MRI are summarized in Table 3. The LV IZ walltended to be thinner than other regions of the LV (P�0.01;Table 3), which suggests higher wall stress in this region.

Myocardial High-Energy Phosphate and Pi LevelsFigure 3 illustrates typical transmurally differentiated 31Pnuclear magnetic resonance spectra for a normal heart (Figure3) compared with the BZ of an infarcted heart not treated withpMultistem (Figure 3) and the BZ of a heart with pMultistemtransplantation with CsA (Figure 3). The voxel labeled EPIwas positioned over the outer edge of the LV wall, whereasthe voxel most distant from the coil (labeled ENDO) waspositioned over the subendocardium. Myocardial high-energyphosphate and Pi levels are summarized in Table 4. PCr/ATPwas most severely decreased in the inner layer of the BZ,

TABLE 2. Hemodynamic Data Obtained 4 Weeks After MI

Heart Rate,bpm

Ao-S,mm Hg

Ao-D,mm Hg

Mean AoP,mm Hg

LVSP,mm Hg

LVEDP,mm Hg RPP, �103 EF, %

Normal (n�8) 120�23 113�12 77�13 89�12 113�16 7.2�7.4 13.42�2.82 55.0�5.6

MI (n�9) 108�23 110�15 79�15 89�15 110�15 11.2�7.9 11.77�2.46 30.4�5.4†

MI�pMultistem (n�7) 116�10 109�15 84�13 93�13 114�13 6.6�2.1 13.27�1.90 42.0�6.5*†

MI�pMultistem�CsA (n�6) 115�23 116�15 85�10 95�11 117�16 8.5�2.6 13.49�4.18 41.2�5.0*†

Values are mean�SD. Ao-S indicates aortic systolic pressure; Ao-D, aortic diastolic pressure; AoP, aortic pressure; LVSP, LV systolic pressure;LVEDP, LV end-diastolic pressure; RPP, rate pressure product (mm Hg; HR�LV systolic pressure); EF, ejection fraction; and n, number of pigs.

*P�0.05 vs MI; †P�0.01 vs normal.

TABLE 3. LV Wall Thickness and Systolic Thickening Fraction 25 Days After MI Measured by MRI

IZ BZ Remote Zone

ES Wall Thickness,mm TF, %



MI (n�9) 4.5�0.6 �1.1�4.0 6.6�1.5‡ 21.1�7.2 6.9�0.9‡ 27.8�8.1

MI�pMultistem (n�7) 6.0�1.1† 19.7�9.8* 7.1�0.8 23.4�10.1 7.2�1.1 29.1�12.4

MI�pMultistem�CsA (n�6) 5.7�0.9† 18.5�13.0* 7.1�1.5 22.2�9.8 7.5�0.7‡ 34.9�14.2

Values are mean�SD. ES indicates LV end-systolic; TF, thickening fraction; and n, number of pigs.*P�0.01 vs MI; †P�0.05 vs MI; ‡P�0.05 vs IZ.




being �56% of that in normal hearts (Figure 3; Table 4). TheBZ PCr/ATP ratio was significantly improved in response topMultistem transplantation (P�0.03, Table 4). The BZ PCr/ATP ratio was not significantly different in animals that didor did not receive CsA (Table 4). The combined pMultistem-treated hearts showed a significant increase of PCr/ATPacross the LV wall compared with the hearts with postinfarctionLV remodeling without cell treatment (P�0.05; Table 4).

Figure 4 illustrates relationships between BZ myocardialPCr/ATP and tagged MRI measuring LV systolic shorteningfraction from the combined IZ and BZ. Regional LV contrac-tile function calculated for segments 1 through 6 represents

the anterior papillary, anterior, septal, posterior, posteriorpapillary, and lateral LV segments. LAD ligation resulted ina thin-walled infarct that mainly involved the anterior wallwithin segment 2, whereas the septal and anterior papillarysegments (segments 1 and 3, respectively) represent the BZ.The remaining segments (4, 5, and 6) represent the remotezone. pMultistem treatment significantly increased IZ LVwall contractile function irrespective of CsA. When eachPCr/ATP ratio was plotted against the respective IZ/BZsystolic shortening fraction for each heart, significant corre-lations were observed (Figure 4). These data suggest thatpMultistem transplantation significantly improved IZ con-tractile performance, which in turn was associated withremarkably improved BZ bioenergetics.

In principle, the deeper voxels (ie, more distant from theouter LV wall) contain contributions from LV cavity bloodbecause of partial volume effects (ie, they can be occupiedboth by LV wall and LV chamber), recognizable by thepresence of 2,3-diphosphoglycerate resonances in the �3ppm region of the spectra.1,23 The presence of both blood andcardiac muscle in the same voxel has the potential to distortATP levels and PCr/ATP ratios, because blood contains ATPbut not PCr. The ATP contribution from blood to thesubendocardial spectrum PCr/ATP has been examined previ-ously1,23 and found to be trivial. In the present study, thecontribution of blood in the nuclear magnetic resonanceregion of interest might be greater because of the thinner wall

Figure 3. Transmurally differentiated 31P-nuclear magnetic resonance spectra from a normal heart, a heart with MI, and a heart with MIand pMultistem cell transplantation. Spectra were obtained from the peri-infarct BZ area near apex. Each transmural data set consistsof a track of 5 spectra corresponding to voxels centered around phase angles 45° (60°, 90°, and 120°, not shown) and 135°. The 135°voxel (corresponding to the subepicardium) and the 45° voxel (corresponding to the subendocardium) are the outermost and innermostvoxels relative to the surface coil. ENDO indicates the subendocardial voxel; EPI, the subepicardial voxel. Resonance peaks are the 2,3diphosphoglycerate (2,3 DPG) from the erythrocytes; PCr, the creatine phosphates; and 3 resonances from ATP. The PCr/ATP ratiosare substantially decreased in MI hearts, which is most severe in ENDO. pMultistem is accompanied by a significant improvement inmyocardial energy efficiency.

TABLE 4. Myocardial PCr/ATP Measured by 31P MRS 4 WeeksAfter MI

PCr/ATP

EPI ENDO Whole LV Wall

Normal (n�8) 2.11�0.19 1.90�0.15 2.03�0.24

MI (n�9) 1.26�0.27‡ 1.06�0.30‡ 1.20�0.29‡

MI�pMultistem (n�7) 1.55�0.35*† 1.34�0.29*† 1.52�0.35*†

MI�pMultistem�CsA (n�6) 1.53�0.44† 1.36�0.43† 1.51�0.42†

MI�pMultistem(combined) (n�13)

1.54�0.37*† 1.34�0.34*† 1.52�0.37*†

Values are mean�SD. EPI indicates subepicardial layers; ENDO, subendo-cardial layers; and n, number of pigs.

*P�0.05 vs MI, †P�0.05 vs normal; ‡P�0.01 vs normal.




in the peri-infarct area. To assess this possibility, the bloodATP contribution was examined with a phantom filled withfresh heparinized blood using the identical spectrometersetup. Prominent resonance peaks of 2,3-diphosphoglycerateappeared at �3 ppm. No ATP resonance was detected. Thesedata demonstrate that the contribution of LV cavity bloodATP to the subendocardial PCr/ATP ratio was negligible.

Myocardial Oxygenation Evaluated by 1H-MRSNo deoxymyoglobin resonance peak was detected either innormal hearts or in the peri-infarct or remote regions ofinfarcted hearts (spectra not shown). On the basis of thesignal to noise ratio of Mb-� during partial and completeLAD occlusions,16 the resonance peak should be recognizedwhen there is �10% myoglobin desaturation.17 These dataindicate that the BZ bioenergetic abnormality in hearts withpostinfarction LV remodeling (Figure 3; Table 4) was not theresult of persistent myocardial ischemia.

Transplanted pMultistem Engraftment andDifferentiation In VivoFour weeks after transplantation, 0.35�0.05% of a total 50million injected LacZ pMultistem cells (CsA: n�6, no CsA:n�7) could be detected. Approximately 70% of these cellswere found at areas near the injection sites in the BZ, whereas�30% migrated into the IZ. In hearts that received stem celltransplantation, patchy spared myocytes were observed in theIZ (Figure 5A), and this was only observed in the hearts withcell transplantation. Among these cells, a small number(shown by the arrows) were costaining positive for X-Gal andcardiac-specific troponin T (�2%; Figure 5A). LacZ-labeledpMultistem cells were also detected in coronary vessels,where they expressed von Willebrand factor, which suggestsdifferentiation into endothelial cells (�3%; Figure 5B).

Vascular DensityMeasurements of vascular density are illustrated in Figure 6.Transplantation of pMultistem cells was associated with a

Figure 4. Scatterplots showing the improvement in LV systolic shortening fraction (SF%) plotted against BZ myocardial PCr/ATP ratiofrom spectra of whole LV wall (left) and from subepicardial (EPI) and subendocardial (ENDO) layers in the periscar BZ myocardium of15 pigs. The filled circles with brackets are from normal pigs. The improved BZ myocardial energetic efficiency secondary to pMultis-tem transplantation is associated with IZ contractile function.




significant increase of vascular density (per mm2) in both IZand BZ (P�0.05). The vascular density was not significantlydifferent between hearts with or without cell transplantationin the remote zone (Figure 6).

Myocardial CK Isoform Protein ExpressionFigure 7 illustrates Western blots examining CK isoform pro-teins in the BZ. The protein levels of CK-M and CK-mito weredecreased significantly in hearts with MI and recovered signif-icantly in response to cell transplantation (Figure 7). In contrast,CK-B isoform protein expression did not change significantly.

DiscussionIn the present study, injection of banked cryopreserved allogeneicpMultistem cells at the time of acute coronary occlusion attenuatedthe structural, contractile, and energetic abnormalities that occurredin the IZ and peri-infarct BZ at 4 weeks after occlusion, withresultant preservation of global LV function. The beneficial effectsof cell transplantation occurred despite the fact that (1) cell trans-plantation did not reduce infarct size, (2) the engraftment rate 4weeks after transplantation was very low and mainly confined to theBZ and IZ, and (3) differentiation of pMultistem cells into cardio-myocytes and endothelial cells was minimal. The results support thehypothesis that protection is mediated by paracrine effects ofpMultistem on IZ and BZ cardiomyocytes and neovascularization.Interestingly, immunosuppression with CsA did not increase theengraftment rate of the allogeneic pMultistem cells.

Mechanism of Postinfarction LV RemodelingLV remodeling after MI has been attributed to several factors,including neurohormonal activation and increased systolic wall

stress in the BZ, which is mechanically tethered to the dysfunctionalregion of infarcted myocardium.1–3 Increased biomechanical strainhas been found to trigger stretch-activated signaling pathways thatlead to cardiomyocyte hypertrophy (with associated changes ingene expression patterns) and apoptosis.1 We recently reported thathigh-energy phosphate levels, PCr/ATP values, and the expressionof several proteins crucial to oxidative ATP production are signif-icantly decreased in the preserved myocardium of infarcted swinehearts and that these abnormalities are most severe in the BZ(especially the BZ subendocardium).1 The profound depression ofhigh-energy phosphate levels and PCr/ATP in the BZ myocardiumwas in marked contrast to remote-zone bioenergetic characteristics,which remained relatively normal.1,17,27,28 These data are compati-ble with the concept that impaired energetic capacity in the BZcontributed to contractile dysfunction.

Mechanisms of pMultistem Effects on IZContractile Function and BZ EnergeticsThere are several possible mechanisms by which cellengraftment might protect myocardial energetics. First, theengrafted cells, via differentiation, could make directcontributions to IZ and BZ cardiomyocyte regenerationand/or neovascularization. This possibility is contradictedby the small fraction of the grafted cell population detected4 weeks after transplantation and the even smaller numberof engrafted cells that acquired cardiomyocyte or endothe-lial cell features. Second, recruitment of endogenouscardiac progenitors to the injury site might provide aprotective effect.9,22 Third, engrafted cells could influenceIZ and BZ structure and function by exerting trophiceffects to enhance native cardiomyocyte survival and

Figure 5. Engraftment and differentiation of pMultistem in vivo. Infarcted swine hearts with LacZ-labeled pMultistem injection were harvestedand dissected into 10-�m sections. Dissected samples were fixed in zinc fixative, stained for both LacZ (blue) and troponin T or von Wille-brand factor (VWF). Nuclei were stained by DAPI. A, IZ of a Lac-Z� pMultistem-treated heart expressing cardiac myocyte phenotype troponinT. The left 2 pictures are phase-contrast images with magnifications of �10 and �40, respectively. The right 2 pictures are fluorescenceimages with magnifications of �10 and �40, respectively. Patches of spared myocytes were observed only in the pMultistem-treated hearts,not in untreated hearts (shown in B). C, Lac-Z� pMultistem cells in vascular structures that coexpress VWF. The left 2 pictures are phase-contrast images with magnifications of �10 and �40, respectively. The right 2 pictures are fluorescence images with magnifications of �10and �40, respectively. The arrows indicate LacZ-positive cells stained with troponin T and VWF markers.




function and promote neovascularization,29,30 which islikely the mechanism underlying the observed effects onBZ energetics and function. Gnecchi et al29 recentlyreported that injection of concentrated conditioned me-dium harvested from cultured Akt-overexpressing mesen-

chymal stem cells into infarcted heart was highly protec-tive, decreasing infarct size and improving contractilefunction in a rat ischemia-reperfusion model. That studydemonstrated high concentrations of a number of cytokinesand growth factors in the concentrated culture medium,

Figure 6. Cardiac sections from IZ, BZ, and remote-zone myocardium stained with von Willebrand factor and CD31. Vascular densitywas significantly higher in both the IZ and BZ of the pMultistem-treated hearts.

Figure 7. Representative Western blotsfrom normal hearts (Normal), hearts withpostinfarction LV remodeling (MI), and aheart with MI that received cell trans-plantation (MI�pMultistem) showing pro-tein levels of myocardial CK isoforms (A)and the respective normalized data (toGAPDH) in B, C, and D. Values aremean�SD. *P�0.05. The same blotsreprobed with GAPDH antibody wereused as controls for equal loading.




namely, vascular endothelial growth factor (VEGF),insulin-like growth factor, hepatocyte growth factor, andfibroblast growth factor.29

pMultistem-Induced NeovascularizationThe IZ and BZ of pMultistem-treated hearts demonstratedsignificant neovascularization compared with the untreatedhearts (Figure 6). This could enhance delivery of oxygen andcarbon substrate to the BZ and might thereby facilitate ATPproduction.15 The lack of myoglobin desaturation in the BZargues against limitation of oxygen and carbon substrate deliv-ery to the BZ, and previous reports31,32 have suggested that BZperfusion is not compromised in single-vessel coronary arteryligation models. However, the myoglobin saturation data indi-cate only that ischemia was not present during basal conditions,so the possibility of ischemia during increased periods ofincreased cardiac work is not excluded, especially becauseoxygen demands are likely increased in the BZ due to theincreased systolic wall stress. Increased BZ vascularity15 couldpotentially prevent recurrent episodes of demand-inducedischemia.

pMultistem Paracrine Effects on BZ andIZ CardiomyocytesAs previously discussed, there is strong evidence that stretch canactivate signaling pathways that can trigger pathological hyper-trophy and apoptosis of cardiomyocytes.33 The resultant alter-ations can include genes involved in carbon substrate metabo-lism and ATP generation.1 There is similarly strong evidencethat other marrow-derived (or other tissue-derived) progenitorcells secrete cytokines involved in stimulation of cell growth andsuppression of apoptosis.7,15,29 We have shown that transplanta-tion of mesenchymal stem cells overexpressing VEGF intoswine myocardium at the time of imposition of an acute pressureoverload (ascending aortic banding) limited the development ofLV hypertrophy, contractile dysfunction, and bioenergetic ab-normalities.15 Transplantation of cells not overexpressing VEGFproduced significant but smaller beneficial effects.15 In anexperiment in which mesenchymal stem cells overexpressingVEGF were co-cultured with neonatal myocytes, the VEGF-modified mesenchymal stem cells inhibited myocyte apopto-sis.15 Similarly, Dzau and colleagues have shown that paracrinefactors released by mesenchymal stem cells induce neovascular-ization and protect surviving cardiomyocytes in infarcted mousehearts.7,29 In other studies, we have shown that mouse multipo-tent adult progenitor cells secrete a number of cytokines andchemokines, including VEGF, platelet-derived growth factor,and monocyte chemoattractant protein-1.34 Taken together, thesedata suggest that pMultistem, like mouse multipotent adultprogenitor cells, may secrete soluble factors that exert paracrineeffects that inhibit activation of pathological signaling pathwaysinduced by the increased mechanical and oxidative stresses inthe infarct BZ, and that limiting the activation of these pathwaysmay restrict the development of gene expression patterns that areassociated with contractile and energetic dysfunction. The re-sultant preservation of cardiomyocyte contractile and energy-generating systems could preserve BZ function and thereby limitglobal LV remodeling. It is likely that these paracrine effects are

crucial to the protective effects of cell transplantation on nativecardiomyocytes in the acutely infarcted heart.

BZ Myocardial CK Isoform Protein ExpressionThe BZ bioenergetic improvement produced by cell trans-plantation was accompanied by increased expression ofCK-M and CK-mito isoform proteins but unchanged CK-Bisoform (Figure 7), which occurred independent of changes inmitochondrial density, which suggests again that alterationsin protein expression secondary to mechanical or oxidativestress were prevented by a paracrine effect of stem celltransplantation. These observations also indicate an urgentneed for studies of BZ signaling pathways that are activatedand/or inhibited by cell transplantation.

Does Immune Rejection Contribute to the LowpMultistem Engraftment Rate?In the present study, immunosuppression with CsA did not increasethe rate of engraftment of pMultistem. This suggests that eitherintrinsic rejection processes are limited over the 4-week observationperiod or that, as suggested by Thum et al,35 apoptosis of trans-planted cells induces immunosuppression. Other possible mecha-nisms for the low engraftment rate include early loss of pMultistemfrom the injection sites and apoptotic loss of cells over time. Thelatter is supported by our recent observation in a murine infarctmodel that the number of engrafted cells fell sequentially over a4-week period after transplantation.22 It remains to be determinedwhether higher engraftment rates would be associated with greaterprotective effects.

ConclusionsTransplantation of pMultistem at the time of coronary arteryligation resulted in improved IZ contractile function and pre-vented BZ bioenergetic deterioration. It is likely that the LVchamber response to cell transplantation resulted from thebeneficial effects of sparing myocytes and increasing revascu-larization in both IZ and BZ. A direct structural contribution ofthe engrafted cells to cardiomyocyte regeneration or neovascu-larization appears unlikely. It remains to be determined whetherthe primary benefits of pMultistem transplantation are a conse-quence of (1) increased blood flow reserve resulting fromincreased BZ capillarity, (2) paracrine effects emanating frompMultistem, or (3) a combination of these effects. The resultsimply that progenitor cell transplantation can exert beneficialeffects at the time of an acute myocardial infarct.

Sources of FundingThis work was supported by US Public Health Service grantsHL50470, HL61353, HL 67828, HL71970, and HL21872.

DisclosuresP. Suntharalingam, S. Boozer, Dr Mhashilkar, and Dr Deans wereemployees of Athersys, Inc. None of these 4 coauthors were involvedin data collection or analysis. Dr Verfaillie has received researchfunding from Athersys, Inc, for work with multipotent adult progen-itor cells unrelated to the studies in this report. The remainingauthors report no conflicts.

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CLINICAL PERSPECTIVECell transplantation into border-zone myocardium at the time of percutaneous intervention for acute myocardial infarction is apotentially feasible therapeutic strategy, because banked frozen bone marrow–derived stem cells (pMultistem) can be made readilyavailable, and the methodology for percutaneous cell injection already exists. It must first be shown, however, that the observedshort-term therapeutic effects translate into long-term benefit. It is possible that strategies to enhance the pMultistem engraftment rateor in vivo proliferation and differentiation could also contribute to the long-term efficacy of this therapeutic approach.




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