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Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, 75 Francis Street, Boston, Massachusetts, 02115 USA. Correspondenceshould be addressed to V.J.D. (vdzau@partners.org).

Published online 10 August 2003; doi:10.1038/nm912

NATURE MEDICINE VOLUME 9 | NUMBER 9 | SEPTEMBER 2003 1195

Prolonged interruption of myocardial blood flow initiates events thatculminate in cardiac myocyte death1. Proposed endogenous reparativemechanisms include cardiac myocyte hypertrophy2 and hyperplasia3

and the trafficking of bone marrow–derived stem cells (BMCs) to themyocardium for repair and angiogenesis4,5. None of these proposedmechanisms are adequate in restoring lost myocardium or sustainingcardiac function. Harvesting BMCs followed by direct injection intoischemic myocardium results in angiogenesis and myogenesis6–8, butfunctional improvement is incomplete. It is unclear whether trans-plantation of BMCs affects repair of the ischemic myocardium pri-marily by angioblast-mediated vasculogenesis9,10, which preventsapoptosis of native cardiac myocytes, or by direct regeneration of thelost myocytes.

Mesenchymal stem cells (MSCs) are self-renewing, clonal pre-cursors of non-hematopoietic tissues. They are expandable in cultureand multipotent, and can differentiate into osteoblasts11, chondro-cytes11, astrocytes12, neurons13 and skeletal muscle14. Several groupshave reported that putative MSCs derived from bone marrow can dif-ferentiate into cardiac muscle in vitro15 and in vivo16,17. However, it hasbeen observed that transplantation of as many as 6 × 107 of these puta-tive MSCs into infarcted porcine hearts yielded only marginalimprovement in cardiac function17. This is explained at least in part bypoor viability of the transplanted cells. It has been estimated that>99% of MSCs die 4 d after transplantation into uninjured nude-mouse hearts16. Cell transplantation strategies to replace lostmyocardium are limited by the inability to deliver large numbers ofcells that resist peritransplantation graft cell death17–20.

Accordingly, we set out to isolate and expand a highly purified pop-ulation of adult rat bone marrow–derived MSCs, and to engineer these

cells to overexpress Akt, a serine threonine kinase and powerful sur-vival signal in many systems21,22, to test the hypothesis that Akt-engineered MSCs are more resistant to apoptosis and can enhance cardiac repair after transplantation into the ischemic rat heart. Ourresults documented significant retention of Akt-MSCs in the ischemicheart that was associated with inhibition of cardiac remodeling, agreater volume of regenerated myocardium, and near-complete normalization of systolic and diastolic cardiac function.

RESULTSMSCs express markers distinct from hematopoietic stem cellsMSCs proliferated in mixed culture with hematopoietic cells, yielding 5 × 106 cells by day 15 of culture. We separated MSCs from hematopoi-etic cells based on their preferential attachment to polystyrene sur-faces23. By immunocytochemistry, over 99% of MSCs expressed CD29,CD71, CD90, CD106 and CD117 (Fig. 1); 60% expressed Ki67 and 15%expressed the transcription factors Nkx2.5 (Fig. 1) and Gata-4 (datanot shown). MSCs did not express the hematopoietic markers CD34and CD45 (Fig. 1) or the cardiac-specific markers myosin heavy chain(MHC), myosin light chain (MLC), cardiac troponin I (CTnI), α-sarc-omeric-actin (αSA) or MEF-2 (data not shown). In addition, MSCsalso expressed the gap junction protein connexin-43 as verified by RT-PCR (data not shown). We further purified MSCs using negative para-magnetic bead sorting targeting CD34, resulting in a population thatwas >99.9% pure. We were unable to induce these MSCs to differentiateinto megakaryocytes and erythroid cells using described methods24.

Increased Akt activity protects against MSC apoptosis We used retroviruses to transduce MSCs with genes expressing green

Mesenchymal stem cells modified with Akt preventremodeling and restore performance of infarcted heartsAbeel A Mangi, Nicolas Noiseux, Deling Kong, Huamei He, Mojgan Rezvani, Joanne S Ingwall & Victor J Dzau

Transplantation of adult bone marrow–derived mesenchymal stem cells has been proposed as a strategy for cardiac repair followingmyocardial damage. However, poor cell viability associated with transplantation has limited the reparative capacity of these cells in vivo. In this study, we genetically engineered rat mesenchymal stem cells using ex vivo retroviral transduction to overexpress the prosurvival gene Akt1 (encoding the Akt protein). Transplantation of 5 × 106 cells overexpressing Akt into the ischemic ratmyocardium inhibited the process of cardiac remodeling by reducing intramyocardial inflammation, collagen deposition andcardiac myocyte hypertrophy, regenerated 80–90% of lost myocardial volume, and completely normalized systolic and diastoliccardiac function. These observed effects were dose (cell number) dependent. Mesenchymal stem cells transduced with Akt1restored fourfold greater myocardial volume than equal numbers of cells transduced with the reporter gene lacZ. Thus,mesenchymal stem cells genetically enhanced with Akt1 can repair infarcted myocardium, prevent remodeling and nearlynormalize cardiac performance.

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fluorescent protein (GFP), LacZ or murine Akt, with over 90% effi-ciency. Using PCR for genomic DNA amplification with probesdesigned to detect both endogenous rat Akt1 and exogenous murineAkt1, we observed a threefold increase in the total Akt signal in Akt-

MSCs, suggesting the successful incorporation of copies of the exoge-nous Akt1 gene (data not shown). The amount of murine Akt1 mRNAwas, on average, 7.8-fold higher in Akt-MSCs than the amounts ofendogenous rat Akt1 mRNA in MSCs, GFP-MSCs or LacZ-MSCs

Figure 1 Immunocytochemical characterization of MSCs. Immunostaining was performed with antibodies to c-kit, CD29, CD106, Ki-67, cTnI, MHC, MLC,Nkx2.5, GATA-4, MEF-2C, N-cadherin, CD90, CD34, CD45, connexin-43, GFP, α-SA and CD71. Appropriate fluorochrome-linked secondary monoclonalantibodies were used. Negative controls for cell type included NIH-3T3 fibroblasts and human umbilical vein endothelial cells. Negative controls for antibodytype were performed on MSCs using appropriate blocking peptide when available, or by omitting the primary antibody.

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dFigure 2 Effect of Akt1 transduction in MSC. (a) Real-time RT-PCR showing that 24 h of hypoxia and serum deprivation (+) had no effect on the level of the endogenous Akt1 mRNA expression ascompared with normoxia (–). Note transduction of exogenous murine Akt1 (filled bar) resulted insignificant suppression of the endogenous rat Akt mRNA (open bar) in the Akt-MSC group (*, P <0.05). Hspa1b mRNA abundance was increased after hypoxia in all cells, except the Akt-MSCs (top).(b) Representative western blots showing that at normoxia (–), the abundances of total and phospho-Akt protein were very low in all groups, but high in Akt-MSCs. Phospho-Akt increased in all groupsafter exposure to hypoxia (+). The antibody is known to detect phospho-Akt isoforms44,45. Akt activitywas also higher in the Akt-MSCs at normoxia, and increased significantly in all groups in response tohypoxia. (c) Akt overexpression reduces MSC apoptosis in vitro by 79% as judged by TUNEL assay forapoptosis. (d) Quantification of intramyocardial c-kit+ cells and percentage of TUNEL-positive c-kit+ cells in heart of animals 24 h after Akt-MSC injection ascompared to GFP-MSC injection. The fraction of c-kit+ apoptotic cells was significantly smaller in Akt-MSCs than in GFP-MSCs (19% of 82 ± 6.7 × 104 versus68% of 33 ± 1.5 × 104, *, P < 0.001, after 24 h). After 72 h, the fraction of c-kit+ apoptotic cells was even smaller in Akt-MSCs than in GFP-MSCs (17% of66 ± 3.5 × 104 versus 37% of the 13 ± 7.8 × 104 P < 0.001, after 72 h; data not shown).

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(P < 0.05; Fig. 2a). Overexpression of murine Akt was associated withsuppression of endogenous rat Akt1 mRNA. Twenty-four hours ofexposure to hypoxia (1% ambient O2) and serum deprivation had noeffect on the amount of endogenous Akt1 mRNA. On the other hand,the amount of Hspa1b (formerly Hsp70) mRNA increased significantly,as expected, in response to hypoxia (Fig. 2a). Total Akt protein was onaverage 19.7-fold more abundant in Akt-MSCs than in the other groupsat 21% O2, and these relative abundances were unchanged after expo-sure to hypoxia (Fig. 2b). At normoxia, phosphorylated Akt protein waselevated in Akt-MSCs, but remained very low in the other groups. With

hypoxia, phospho-Akt increased further in all groups, suggesting post-translational regulation. Similarly, Akt activity in Akt-MSCs was higherat normoxia and increased further after 24 h of hypoxia than in the othergroups. In response to hypoxia, endogenous Akt activity also increasedin MSCs, GFP-MSCs and LacZ-MSCs (Fig. 2b).

This increase in Akt activity in MSCs translated into a reductionof DNA laddering, a 27% reduction in the pro-apoptotic gene Baxand a 50% upregulation in the apoptotic gene Bcl2 (data notshown). As a result, we observed an 80% reduction in apoptosis ofAkt-MSCs as compared with GFP-MSCs in vitro after 24 h of

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Figure 3 Analysis of myocardial repair 3 weeks after MSC injection intoischemic rat hearts. (a) H&E staining demonstrates the difference inscar size and infiltration of viable, mature cardiac myocytes from theborder zone into the scarred area (arrow), comparing Akt-MSC groupswith the saline (control) group. (b) There was no blue staining of X-galseen in remote areas of the infarcted heart or in saline-injected heart(negative control), but blue staining is seen in what would have beenthe infarct area after injection of LacZ-MSCs into border zone ofischemic myocardium. Examination of the border zone (×10) of LacZ-MSC–injected sections showed cells with phenotypiccharacteristics of cardiac myocytes (large, elongated, centrallymultinucleated cells) with blue nuclei (arrows) in the border zone. (c) Merged images of double staining of sections for GFP (green) andfor cardiac-specific proteins: MHC, CTn1, α-SA and MLC (red)demonstrated the colocalization of the reporter with these cardiac-specific proteins (yellow). (d) Merged images of double staining ofsections for GFP (green) and for connexin-43 (Cx-43) (red) or N-cadherin (red) demonstrated that MSC-derived cardiac myocytesexpress connexin-43 and that the expression abuts native cardiacmyocytes (yellow). This suggests that MSC-derived cardiac myocytes arecapable of electromechanical coupling with native cardiac myocytes.(e) FISH staining for the Y chromosome (green), which is colocalizedwith α-SA (red), confirming the male origin of donor cells.

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Figure 4 Injection of MSCs into ischemic rat heart reduced infarct volume.Area at risk (arbitrary units) for infarction in hearts of all groups of animalswas equivalent (top). Infarct volume was greatest after saline injection andwas reduced in dose-dependent fashion after injection of LacZ-MSCs.Injection of equivalent numbers of Akt-MSCs resulted in a smaller infarctvolume by a factor of 2–5 (*, P < 0.01). Saline, LacZ and Akt representanimals injected with saline, LacZ-MSCs and Akt-MSCs, respectively (2.5 × 105 or 5 × 106 cells).

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hypoxia (from 53 ± 9% apoptotic cells to 9 ± 0.5%, P < 0.01;Fig. 2c).

We assessed the cytoprotective effects of Akt overexpression in vivoby injecting 5 × 106 Akt or GFP-MSCs into the left ventricle (LV) ofadult female rats 60 min after myocardial infarction. At 24 h after injec-tion, we double-stained LV sections for c-kit immunoreactivity and ter-minal deoxynucleotidyl transferase–mediated dUTP nick end-labeling(TUNEL). In the Akt-MSC group, we observed that a greater numberof c-kit+ cells were retained in the myocardium and a smaller percent-age of these cells were TUNEL+ (Fig. 2d). We were unable to detect c-kit+ cells in the myocardium after 2 weeks, suggesting that MSCs didnot persist in the undifferentiated state in the ischemic myocardium.

MSCs develop into cardiac myocyte-like cells after transplantationH&E staining of post–myocardial infarction hearts injected withMSCs showed infiltration of finger-like extensions of organized car-diac myocytes (Fig. 3a) into the myocardial scar. β-Galactosidasestaining of thick sections of whole hearts injected with LacZ-MSCsshowed intense blue coloration in the peri-infarct zone resulting fromthe blue nuclear staining of cells that bore the phenotype of cardiacmyocytes (Fig. 3b). In ischemic heartsinjected with GFP-MSCs, we observed large,multinucleated syncytiae oriented in the samedirection as the native cardiac myocytes in theborder zone (data not shown). Double stain-ing of the sections for cardiac-specific pro-teins showed that GFP colocalized withMHC, CtnI, α-SA and MLC (Fig. 3c). Cardiacmyocytes expressing GFP also expressed con-nexin-43 and N-cadherin (Fig. 3d) at contactpoints with native cardiac myocytes. We veri-fied the male donor origin of these cardiacmyocyte–like cells in female recipient heartsby fluorescent in situ hybridization for the Ychromosome, which colocalized with theabove-mentioned cardiac-specific proteins(Fig. 3e). Three weeks after transplantation,cardiac myocytes expressing GFP and/orstaining positively for the presence of the Ychromosome no longer expressed c-kit orCD90.

We examined the contribution of ischemic myocardium to thelocalization of MSC-derived cardiac myocytes and were unable todetect GFP-containing cardiac myocytes after injection of GFP-MSCs into uninjured myocardium. We also did not detect GFP inendothelium, smooth muscle, hematopoietic elements or remoteareas of the heart after injection of GFP-MSCs into ischemicmyocardium. As a control for cell type, we injected equivalent num-bers of GFP-transduced c-kit– CD34+ cells into ischemicmyocardium and could not detect GFP in cardiac myocytes (datanot shown).

Intramyocardial MSC injection reduces infarct volumeThe area at risk of infarction after coronary artery ligation was equiva-lent in all groups. Three weeks after coronary ligation, the volume ofleft ventricular infarct (Vinfarct) varied based on the type and numberof cells injected (Fig. 4). Vinfarct was maximal after saline injection.Injection of 2.5 × 105 LacZ-MSCs yielded a 9.8% smaller Vinfarct andinjection of 5 × 106 LacZ-MSCs yielded a 12.9% smaller Vinfarct.Genetic modification with Akt exerted a powerful inhibitory effect onVinfarct: Vinfarct was 44.8% smaller after injection of 2.5 × 105 Akt-MSCs, whereas 5 × 106 Akt-MSCs resulted in almost complete aboli-tion of Vinfarct.

MSC transplantation normalizes cardiac functionTo avoid the confounding effects of preload, afterload, in vivo sympa-thetic activity and anesthesia, we measured ventricular performanceusing isolated, perfused, isovolumetrically contracting hearts 2 weeksafter MSC transplantation. LV systolic performance in post-infarct,saline-injected control hearts decreased to 58% of sham-operatedhearts. Transplantation of 2.5 × 105 or 5 × 106 LacZ-MSCs did notimprove LV systolic performance, but transplantation of 2.5 × 105 Akt-MSCs resulted in a 37% increase in baseline LV systolic pressure ascompared with controls. Transplantation of 5 × 106 Akt-MSCsresulted in a further increase in systolic performance to a level that wasindistinguishable from sham-operated animals (Fig. 5a). These differ-ences persisted during inotropic challenge with dobutamine (Fig. 5b).Diastolic function, as assessed by –dp/dt, also deteriorated in thepost–myocardial infarction saline control group as compared withsham, but improved in the Akt-MSC group at baseline and with dobu-tamine (Fig. 5c).

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Figure 5 MSC injection improved cardiac function. (a) Left ventricular systolic pressure (LVSP) pergram of viable tissue was lowest in control infarct animals, and did not change after injection of 2.5 ×105 LacZ-MSCs or 5 × 106 LacZ-MSCs. Injection of 2.5 × 105 or 5 × 106 Akt-MSCs increased LVSP ina dose-dependent fashion (*, P < 0.001 as compared with corresponding LacZ-MSCs). LVSP wasnormalized after injection of 5 × 106 Akt-MSCs. (b) Further improvements of LVSP were seenconsistently after infusion of the inotrope dobutamine (*, P < 0.001 as compared with correspondingLacZ-MSCs). (c) Left ventricular –dp/dt (in the presence of dobutamine) deteriorated in the salinegroup as compared with sham (*, P < 0.05), suggesting that diastolic relaxation in saline-injectedhearts is impaired. This improved with transplantation of 5 × 106 Akt-MSCs (†, P < 0.05 as comparedwith saline group), suggesting that cell implantation rescued diastolic relaxation.

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MSC transplantation prevents remodelingAt 5 d after induction of myocardial infarction and injection of 5 × 106

Akt-MSCs, we observed that CD45+ infiltration of the myocardiumwas significantly lower than that of the post-myocardial infarctionsaline control (36 ± 1.1 versus 226.7 ± 3 cell per high-power field;P < 0.01) and was at the level seen in sham-operated animals. Twoweeks after transplantation of 5 × 106 Akt-MSCs, the increases inwhole heart collagen-area fraction (Fig. 6a,b) and cardiac myocytediameter in histologic cross-sections (Fig. 6c) seen in saline-injectedmyocardial infarction controls were completely inhibited, to levelsobserved in sham-operated animals. Transplantation of 5 × 106 LacZ-MSCs did not result in the same degree of reduction. Three weeks aftermyocardial infarction, we observed that saline-injected controls hadlarger LV than did sham-operated animals (LV mass, excludinginfarcted tissue, increased from 537 ± 39 to 672 ± 4.2 mg; P < 0.05).Transplantation of 5 × 106 Akt-MSCs prevented ventricular enlarge-ment, whereas LacZ-MSCs had a more moderate effect (533 ± 77 and597 ± 50 mg, respectively).

DISCUSSIONWe have shown here that CD117+ CD90+ CD34– MSCs can be isolatedbased on adhesion to polystyrene surfaces and highly purified byimmunoselection, and are amenable to genetic engineering with retro-viruses. MSCs injected into the border zone migrate specifically towardthe ischemic myocardium and develop into cardiac myocyte–like cellsthat form connections with native myocytes. These cellular responsesseem to be unique to the microenvironment of the ischemicmyocardium because they are not activated after transplantation ofMSCs into normal uninjured myocardium, nor are MSCs discovered inremote, uninjured parts of coronary ligated hearts. Finally, in this set-ting, MSCs do not seem to have the potential to differentiate into newblood vessels or to form new capillaries from existing arterioles.

The mechanism by which MSCs develop into cardiac myocyte–likecells remains controversial. Although several investigators haveclaimed that these cells differentiate into cardiac myocytes, previous in vitro studies have shown that stem cells may fuse with existingnative cells25,26, theoretically improving function by contributingtheir own genetic and cellular materials. Our experiments were notdesigned to address fusion versus differentiation. Future experiments

defining the exact mechanism of therapeutic cardiac repair by MSCsare clearly necessary.

Generating functional cardiac myocytes from autologous MSCsseems to be a superior strategy to the intramyocardial implantation ofskeletal myoblasts27–31, which lack the capacity for electromechanicalcoupling31 and have the potential to proliferate in an uncontrolledfashion32; and to the transplantation of adult, fetal33 or neonatal car-diac myocytes34 or of embryonic stem cell–derived cardiacmyocytes35, all of which are difficult to obtain in clinically meaningfulnumbers and are susceptible to immunologic rejection. Our data alsosuggest that MSC transplantation (especially transplantation of Akt-MSCs) exerts a marked inhibitory effect on pathological myocardialremodeling that may be important in mediating the therapeutic bene-fit. Whether the effect on remodeling is due to mechanical improve-ment resulting from cell transplantation or to paracrine mediatorsreleased by MSCs requires further investigation.

Our approach to myocardial repair is enhanced by the ability togenetically engineer stem cells addressing, in this case, the problem ofcell death. The causes of cell death in this setting are multifactorial andare influenced by the ischemic environment, which is devoid of nutri-ents and oxygen19, coupled with the loss of survival signals from matrixattachments and cell–cell interactions. Akt is activated by hypoxia and avariety of other stimuli, including cytokines22. It is a general mediatorof survival signals and is both necessary and sufficient for cell sur-vival22. Akt achieves this by targeting apoptotic family members Ced-9/Bcl-2 and Ced-3/caspases, forkhead transcription factors, IKK-α andIKK-β. In addition, Akt also has a role in modulating intracellular glu-cose metabolism22, thereby enhancing energy production duringhypoxia. Thus, Akt is an excellent therapeutic gene for preserving MSCviability in the early post-transplant period. As shown in our study, useof wild-type Akt whose gene product was not constitutively active36,but was activated in response to hypoxia, protected cells from apoptosiswhile avoiding the potential detrimental effects of constitutive acti-vated Akt expression37. As a result, intracardiac retention and engraft-ment of MSCs genetically enhanced to overexpress Akt are superior tothat of control MSCs expressing reporter genes alone.

We have described genetic modification of mesenchymal stem cellswith a therapeutic gene before transplantation as a strategy for regen-erative medicine. We speculate that future therapy for acute myocar-

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Figure 6 Injection of MSCs inhibits ventricular remodeling. (a,b) Masson’strichrome staining showing that collagen deposition within the whole heartwas reduced 2 weeks after injection of 5 × 106 Akt-MSCs to a level that wasnot significantly different from that of sham-operated heart. Collagendeposition was higher in LacZ-MSCs than in Akt-MSC–treated hearts (*, P < 0.001). (c) Cardiac myocyte diameter as measured in histologiccross-section was significantly lower in hearts injected with 5 × 106

Akt-MSCs (†, P < 0.01) and was not significantly different from that insham-operated hearts. Cardiac myocyte diameter was higher in heartstreated with LacZ-MSCs as opposed to Akt-MSCs (*, P < 0.01).

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dial infarction may involve transplantation of MSCs overexpressingAkt to facilitate the repair of the damaged heart. This novel, cell-based,gene-therapy approach has the potential to address cell availability andclinical scalability, and to make cell-based therapy an effective treat-ment for human cardiac disease.

METHODSAnimals and surgical procedures. We used 250-g Sprague-Dawley rats withapproval of the Harvard Medical Area Standing Committee on Animals. Weused 17 animals for MSC isolation and characterization. Eight animals pergroup (vehicle, c-kit– CD34+ cells, LacZ-MSCs, Akt-MSCs, sham ligation)were used for morphologic analysis at 2 weeks. Additional animals were pre-pared (n = 8 per group) for studies requiring different time points. For func-tional studies, we used four animals per group for echocardiography at 2 weeks, followed by isolated Langendorff preparations to assess ventricularperformance. Each experiment was performed independently, in triplicate.

Sixty minutes after suture ligation of the left anterior descending artery35 offemale rat hearts, MSCs suspended in saline were injected into five sites in theborder zone of the ischemic LV. Controls underwent coronary ligation and onlysaline injection. Sham animals underwent placement of the suture without liga-tion.

Retroviral design and transduction. IRES-GFP (Clontech) was cloned into theMurine Stem Cell Virus vector (pMSCV; Clontech) after digestion with XhoIand BamHI. We PCR-amplified cDNA for murine Akt38 (forward, 5′-GCAA-GATCTGATACCATGAACGACGTAGCC-3′; reverse, 5′-CGGTCACCGT-GTCGGACTCCTAGGATC-3′) and cloned it into pMSCV using BglII andBamH1. Nuclear-localized LacZ expression plasmid was obtained from theHarvard Gene Therapy Initiative. We exposed MSCs three times to 1 × 108 par-ticles of high-titer VSV-G pseudotyped retrovirus with 6 µg/ml polybrene(Sigma-Aldrich) for 6 h.

Purification of mesenchymal stem cells. Whole bone marrow was flushed fromthe tibia and femur of adult male rats. MSCs preferentially attached to the poly-styrene surface23 and were further purified by incubating the mixed culturewith CD34 antibody (Santa Cruz Biotechnology) linked to biotin (Sigma-Aldrich) and collecting the negative fraction after exposure to avidin-coatedmagnetic beads (Beckman Coulter) in the presence of a magnet. This CD34-negative fraction was further propagated in alpha minimum essential medium(αMEM), with Glutamax (Invitrogen) plus 10% fetal bovine serum(Invitrogen) and antibiotics (Invitrogen).

Assays for Akt mRNA, protein, activity and cell death. MSCs were cultured inserum-free medium in 1% O2 at 37 °C for 24 h. Ten minutes after normoxia incomplete medium, protein extracts were prepared for western blot analysis.Membranes were incubated according to manufacturer’s protocols with spe-cific antibodies to Akt and phospho-Akt (Ser-473; Cell Signaling) and to actin(Santa Cruz Biotechnology). The Akt kinase activity was assayed, using GSK-3α/β fusion protein as substrate, by the Akt Kinase Assay Kit (Cell Signaling) onimmunoprecipitated Akt.

RNA was extracted with TRIzol reagent (Invitrogen). Real-time RT-PCRwas carried out using SuperScript One-Step RT-PCR with Platinum Taqkits (Invitrogen). The primers were designed to match both rat and mouseAkt (total Akt) (forward, 5′-AACGGACTTCGGGCTGTG-3′ ; reverse,5′-TTGTCCTCCAGCACCTCAGG-3′), but the probes were designed to bespecific for rat (5′-CGTTCTGCGGGACACCCGAGTACC-3′) and mouse(5′-AAGACATTCTGCGGAACGCCGGAGTA-3′). The fluorogenic probescontained a 5′-FAM report dye and a 3′-BHQ1 quencher dye. TaqMan 18SRibosomal RNA (Applied Biosystems) was used as housekeeping gene.RT-PCR reactions were carried out in iCycler IQ Real-Time DetectionSystems (Bio-Rad). Conventional RT-PCR was carried out using theSuperscript first-strand synthesis system (Invitrogen) to transcribe cDNAthat was ultimately used for PCR amplification of Hspa1b and Gapd usingthe following primer sets: Hspa1b, forward, 5′-TGCTGACCAAGATGAAG-3′ , reverse, 5′-AGAGTCGATCTCCAGGC-3′ ; Gapd, forward, 5?-GGTGAT-GCTGGTGCTGAGTATGTC-3′ , reverse, 5′-CACCAGTGGATGCAGGG-ATGA-3′ .

TUNEL for detection of apoptotic nuclei was performed according to themanufacturer’s protocol (Roche). DNA was isolated by glass-fiber column elu-tion (Roche) and electrophoresed.

Genomic DNA PCR amplification. Genomic DNA was isolated using Easy-DNA Kit (Invitrogen) and PCR amplified using primer sets for Akt1(forward, 5′-GTGCTGGAGGACAACGACT-3′; reverse, 5′-GTGTAGGGTC-CTTCTTGAGCA-3′), α-actin (forward, 5′-GTTTGCCGGAATCAATTTTC-3′;reverse, 5′-AGCCAGAGCTGTGATCTCCTT-3′) and murine Akt from plasmidpMSCV-Akt (forward, 5′-CTCGATCCTCCCTTTATCCAG-3′; reverse, 5′-TGTGCCACTGAGAAGTTGTTG-3′).

Morphometry. Area at risk was estimated by Evans blue retrograde perfusion39.LV volume was calculated by dividing wet weight by density (1.06 g/ml)40. OnMasson’s Trichrome staining, all blue staining was quantified morphometri-cally and divided by all nonwhite area. This yielded the ‘area of infarct’ per 5-µm section. This area of infarct was then added up for all 15 sections. To con-vert this to the ‘volume of infarct’ for the thick slice from which the sectionswere obtained, we multiplied it by the appropriate multiplier based on thethickness of the slice. We then added up the volume of infarct (mm3) of thewhole heart. Collagen area fraction was measured as described41. Cardiacmyocyte diameter in histologic cross-section was measured using morphomet-ric techniques as described42.

Analysis of cardiac function. Isolated rat hearts were perfused in theLangendorff model43. Indices of isovolumic contractile and relaxation per-formance were measured by placing a polyvinylchloride balloon in the LV con-nected to a data acquisition system at baseline and in the presence of 300 nMdobutamine.

Statistics. Student’s t-test was used for two-group comparison and ANOVA fol-lowed by an unpaired Student’s t-test with Bonferroni’s correction was used formultiple group comparisons.

ACKNOWLEDGMENTSWe thank D.G. Phinney, D.J. Prockop and R. Pratt for helpful discussions and assistance in establishing culture conditions for propagation of bonemarrow–derived MSCs; M.A. Perrella for sharing Akt1 constructs and for helpfuldiscussions on Akt biology; and S. Colgan for the use of a hypoxia chamber. Thiswork was supported by grants HL35610 (V.J.D.), HL058516 (V.J.D.), HL072010(V.J.D.), HL073219 (V.J.D.) and HL52320 (J.S.I.) from the National Heart, Lungand Blood Institute, US National Institutes of Health. A.A.M. is the recipient of aNational Research Service Award (1 F32 NHL 10503-01) from the National Heart,Lung and Blood Institute, National Institutes of Health; and the Robert R. LintonResearch Fellowship from the Department of Surgery, Massachusetts GeneralHospital. N.N. is a recipient of a scholarship from the Canadian Institutes ofHealth Research. M.R. is the recipient of an American Heart Association Research Award (0120195T).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 29 May; accepted 17 July 2003Published online at http://www.nature.com/naturemedicine/

1. Williams, R.S. & Benjamin, I.J. Protective responses in the ischemic myocardium.J. Clin. Invest. 106, 813–818 (2000).

2. Pfeffer, J.M., Pfeffer, M.A., Fletcher, P.J. & Braunwald, E. Progressive ventricularremodeling in rat with myocardial infarction. Am. J. Physiol. 260, H1406–1414(1991).

3. Beltrami, A.P. et al. Evidence that human cardiac myocytes divide after myocar-dial infarction. N. Engl. J. Med. 344, 1750–1757 (2001).

4. Jackson, K.A. et al. Regeneration of ischemic cardiac muscle and vascularendothelium by adult stem cells. J. Clin. Invest. 107, 1395–1402 (2001).

5. Quaini, F. et al. Chimerism of the transplanted heart. N. Engl. J. Med. 346, 5–15(2002).

6. Tomita, S. et al. Autologous transplantation of bone marrow cells improves dam-aged heart function. Circulation 100, II247–256 (1999).

7. Wang, J.S. et al. Marrow stromal cells for cellular cardiomyoplasty: feasibility andpotential clinical advantages. J. Thorac. Cardiovasc. Surg. 120, 999–1005(2000).

8. Orlic, D. et al. Bone marrow cells regenerate infarcted myocardium. Nature 410,701–705 (2001).

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9. Kocher, A.A. et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodel-ing and improves cardiac function. Nat. Med. 7, 430–436 (2001).

10. Kawamoto, A. et al. Therapeutic potential of ex vivo expanded endothelial progen-itor cells for myocardial ischemia. Circulation 103, 634–637 (2001).

11. Pereira, R.F. et al. Cultured adherent cells from marrow can serve as long-lastingprecursor cells for bone, cartilage, and lung in irradiated mice. Proc. Natl. Acad.Sci. USA 92, 4857–4861 (1995).

12. Azizi, S.A. et al. Engraftment and migration of human bone marrow stromal cellsimplanted in the brains of albino rats—similarities to astrocyte grafts. Proc. Natl.Acad. Sci. USA 95, 3908–3913 (1998).

13. Chen, J. et al. Therapeutic benefit of intracerebral transplantation of bone marrowstromal cells after cerebral ischemia in rats. J. Neurol. Sci. 189, 49–57 (2001).

14. Ferrari, G. et al. Muscle regeneration by bone marrow-derived myogenic progeni-tors. Science 279, 1528–1530 (1998).

15. Makino, S. et al. Cardiomyocytes can be generated from marrow stromal cells invitro. J. Clin. Invest. 103, 697–705 (1999).

16. Toma, C. et al. Human mesenchymal stem cells differentiate to a cardiomyocytephenotype in the adult murine heart. Circulation 105, 93–98 (2002).

17. Shake, J.G. et al. Mesenchymal stem cell implantation in a swine myocardialinfarct model: engraftment and functional effects. Ann. Thorac. Surg. 73,1919–1925, 1926 (2002).

18. Reinecke, H., Zhang, M., Bartosek, T. & Murry, C.E. Survival, integration, and dif-ferentiation of cardiomyocyte grafts: a study in normal and injured rat hearts.Circulation 100, 193–202 (1999).

19. Zhang, M. et al. Cardiomyocyte grafting for cardiac repair: graft cell death andanti-death strategies. J. Mol. Cell. Cardiol. 33, 907–921 (2001).

20. Muller-Ehmsen, J. et al. Survival and development of neonatal rat cardiomyocytestransplanted into adult myocardium. J. Mol. Cell. Cardiol. 34, 107–116 (2002).

21. Franke, T.F., Kaplan, D.R. & Cantley, L.C. PI3K: downstream AKTion blocks apop-tosis. Cell 88, 435–437 (1997).

22. Datta, S.R., Brunet, A. & Greenberg, M.E. Cellular survival: a play in three Akts.Genes Dev. 13, 2905–2927 (1999).

23. Colter, D.C., Class, R., DiGirolamo, C.M. & Prockop, D.J. Rapid expansion of recy-cling stem cells in cultures of plastic-adherent cells from human bone marrow.Proc. Natl. Acad. Sci. USA 97, 3213–3218 (2000).

24. Hunnestad, J.A., Steen, R., Tjonnfjord, G.E. & Egeland, T. Thrombopoietin com-bined with early-acting growth factors effectively expands human hematopoieticprogenitor cells in vitro. Stem Cells 17, 31–38 (1999).

25. Terada, N. et al. Bone marrow cells adopt the phenotype of other cells by sponta-neous cell fusion. Nature 416, 542–545 (2002).

26. Ying, Q.L., Nichols, J., Evans, E.P. & Smith, A.G. Changing potency by sponta-neous fusion. Nature 416, 545–548 (2002).

27. Taylor, D.A. et al. Regenerating functional myocardium: improved performanceafter skeletal myoblast transplantation. Nat. Med. 4, 929–933 (1998).

28. Murry, C.E., Wiseman, R.W., Schwartz, S.M. & Hauschka, S.D. Skeletal myoblasttransplantation for repair of myocardial necrosis. J. Clin. Invest. 98, 2512–2523(1996).

29. Atkins, B.Z. et al. Cellular cardiomyoplasty improves diastolic properties ofinjured heart. J. Surg. Res. 85, 234–242 (1999).

30. Menasche, P. et al. Myoblast transplantation for heart failure. Lancet 357,279–280 (2001).

31. Reinecke, H., MacDonald, G.H., Hauschka, S.D. & Murry, C.E. Electromechanicalcoupling between skeletal and cardiac muscle. Implications for infarct repair. J.Cell. Biol. 149, 731–740 (2000).

32. Reinecke, H. & Murry, C.E. Transmural replacement of myocardium after skeletalmyoblast grafting into the heart. Too much of a good thing? Cardiovasc. Pathol. 9,337– (2000).

33. Li, R.K. et al. Natural history of fetal rat cardiomyocytes transplanted into adultrat myocardial scar tissue. Circulation 96, II-179–186, 186–177 (1997).

34. Watanabe, E. et al. Cardiomyocyte transplantation in a porcine myocardial infarc-tion model. Cell Transplant. 7, 239–246 (1998).

35. Min, J.Y. et al. Transplantation of embryonic stem cells improves cardiac functionin postinfarcted rats. J. Appl. Physiol. 92, 288–296 (2002).

36. Fujio, Y. et al. Akt promotes survival of cardiomyocytes in vitro and protectsagainst ischemia-reperfusion injury in mouse heart. Circulation 101, 660–667(2000).

37. Matsui, T. et al. Phenotypic spectrum caused by transgenic overexpression of acti-vated Akt in the heart. J. Biol. Chem. 277, 22896–22901 (2002).

38. Fukumoto, S. et al. Akt participation in the Wnt signaling pathway throughDishevelled. J. Biol. Chem. 276, 17479–17483 (2001).

39. Melo, L.G. et al. Gene therapy strategy for long-term myocardial protection usingadeno-associated virus-mediated delivery of heme oxygenase gene. Circulation105, 602–607 (2002).

40. Orlic, D. et al. Mobilized bone marrow cells repair the infarcted heart, improvingfunction and survival. Proc. Natl. Acad. Sci. USA 98, 10344–10349 (2001).

41. Nwogu, J. et al. Inhibition of collagen synthesis with prolyl 4-hydroxylase inhibitorimproves left ventricular function and alters the pattern of left ventricular dilata-tion after myocardial infarction. Circulation 104, 2216–2221 (2001).

42. Choukroun, G. et al. Regulation of cardiac hypertrophy in vivo by the stress-acti-vated protein kinases/c-Jun NH2-terminal kinases. J. Clin. Invest. 104, 391–398(1999).

43. Spindler, M. et al. Diastolic dysfunction and altered energetics in the αMHC403/+mouse model of familial hypertrophic cardiomyopathy. J. Clin. Invest. 101,1775–1783 (1998).

44. Okano, J. et al. Akt/protein kinase B isoforms are differentially regulated by epi-dermal growth factor stimulation. J. Biol. Chem. 275, 30934–30942 (2000).

45. Teruel, T., Hernandez, R. & Lorenzo, M. Ceramide mediates insulin resistance bytumor necrosis factor-α in brown adipocytes by maintaining Akt in an inactivedephosphorylated state. Diabetes 50, 2563–2571 (2001).

NATURE MEDICINE VOLUME 9 | NUMBER 9 | SEPTEMBER 2003 1201

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