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Cell therapy for acute myocardial infarction

Kai C Wollert
The infarcted heart heals by scar formation, and large myocardial infarctions typically result in heart failure. Although adult stem cells with the capacity to transform into various cardiac cell types and to secrete cardioprotective cytokines have been identied, endogenous repair mechanisms in the adult heart are not sufcient for meaningful tissue regeneration. These observations, however, suggest that it may be feasible to develop interventions aimed at enhancing these processes, and to promote functional, and eventually, structural recovery of the infarcted heart. Recent randomized clinical trials indicate that intracoronary delivery of bone marrow (stem) cells leads to an improvement in systolic function after acute myocardial infarction, thereby providing the rst evidence that cell therapy may work in patients.
Address Department of Cardiology and Angiology, Hannover University Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany Corresponding author: Wollert, Kai C (wollert.kai@mh-hannover.de)

Current Opinion in Pharmacology 2008, 8:202210 This review comes from a themed issue on Cardiovasular and renal Edited by Antoine Bril and Metin Avkiran Available online 1st February 2008 1471-4892/$ see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2007.12.011

believed to differentiate only within tissue lineage boundaries (e.g. hematopoietic stem cells giving rise to mature hematopoietic cells). The fairly recent concept of adult stem cell plasticity implies that stem cells can transdifferentiate into cell types outside their original lineage [2]. Along this line, it has been reported that hematopoietic stem cells when transplanted into infarcted mouse myocardium transdifferentiate into cardiomyocytes and vascular cells [3]. Another hypothesis that has created some excitement recently, predicts that limited myocardial regeneration can occur after tissue injury through the recruitment of resident cardiac stem cells [4]. Ironically, while these new ideas have already triggered clinical trials, fusion of transplanted stem cells with resident cardiomyocytes has been offered as an alternative explanation for previous claims of transdifferentiation [57,8]. Moreover, it has been proposed that stem cells secrete cytokines and growth factors which may promote angiogenesis, suppress cell death of resident cardiomyocytes, modulate interstitial matrix composition, and maybe even recruit cardiac stem cells [9,10]. Regardless of the mechanisms, it appears that stem cell therapy has the potential to improve perfusion and contractile performance of the injured heart (Figure 1). This article will provide the reader with essential information on donor cells and strategies for cell delivery, and will provide an opinionated review of the current status and future perspectives of cell therapy for patients with acute myocardial infarction.

Potential donor cells Introduction

Modern reperfusion strategies and advances in pharmacological management of acute myocardial infarction (AMI) have resulted in an increasing proportion of patients surviving the acute event. Some of these patients eventually develop adverse left ventricular remodeling and heart failure. None of our current therapies addresses the underlying cause of the remodeling process, that is the damage to the cardiomyocytes and the vasculature in the infarcted area. The alleged transdifferentiation capacity of adult stem cells and the recent discovery of endogenous cardiac repair mechanisms have suggested that cardiac repair (i.e. replacement of necrotic or scarred tissue with viable myocardium) might be achieved in the clinical setting [1]. Stem cells are capable of self-renewal, transformation into dedicated progenitor cells, and differentiation into specialized progeny. Traditionally, adult stem cells were
Current Opinion in Pharmacology 2008, 8:202210

Conceptually, a variety of stem and progenitor cell populations could be used for cardiac repair after AMI. Each cell type has its own prole of advantages, limitations, and practicability issues. Studies comparing the regenerative capacity of distinct cell populations are scarce. Most investigators have therefore chosen a pragmatic approach by using unfractionated nucleated bone marrow cells, which contain different stem and progenitor cell populations, including endothelial progenitor cells and mesenchymal stem cells.
Endothelial progenitor cells

Endothelial progenitor cells (EPCs) have been dened by their cell surface expression of the hematopoietic marker proteins CD133 and CD34 and the endothelial marker vascular endothelial growth factor receptor-2, and their capacity to incorporate into sites of neovascularization and to differentiate into endothelial cells in situ. The cell surface antigen CD133 is expressed on early hematopoietic stem cells and EPCs, both of which collaborate to
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Figure 1

Working hypothesis of therapeutic stem cell transplantation for cardiac repair. Conceptually, stem and progenitor cell transplantation may have a favorable impact on tissue perfusion and contractile performance by promoting vascularization and myocyte formation. Depending on the stem cell type and local milieu, the relative contribution of cell incorporation (transdifferentiation and/or fusion) versus paracrine effects may vary. Stem and progenitor cell numbers and functional capacity are influenced by a patients age, gender, cardiovascular risk factors, and underlying disease state.

promote vascularization of ischemic tissues. There is increasing evidence that culture-expanded EPCs contain a CD14+/CD34/CD133-mononuclear cell population with EPC-capacity, which mediates its angiogenic effects by releasing paracrine factors [11].
Mesenchymal stem cells

stem cells can be expanded in vitro, and reportedly have a low immunogenicity, they might be used in an allogeneic setting in the future [12].

Skeletal myoblasts

Mesenchymal stem cells represent a rare population of CD34 and CD133 cells present in bone marrow stroma and other mesenchymal tissues [12]. Mesenchymal stem cells can differentiate into osteocytes, chondrocytes, adipocytes, and cardiomyocyte-like cells under specic culture conditions. When injected into infarct tissue, mesenchymal stem cells may enhance regional wall motion and prevent adverse remodeling. It has been suggested that these effects may be related to paracrine effects rather than differentiation of mesenchymal stem cells into cardiomyocytes [13,14]. Since mesenchymal
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Skeletal myoblasts (satellite cells) are progenitor cells which normally lie in a quiescent state under the basal membrane of mature muscular bers. Myoblasts can be isolated from skeletal muscle biopsies and expanded in vitro. Myoblasts differentiate into myotubes and retain skeletal muscle properties when transplanted into an infarct scar. Although grafted myotubes may contract in response to electrical stimulation, they do not express intercalated disk proteins, indicating that the majority are not electromechanically coupled to their host cardiomyocytes. Nevertheless, myoblast transplantation has been shown to augment systolic and diastolic performance in
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animal models, possibly through the release of pararine factors [15].

Resident cardiac stem cells

Recently, several groups have detected stem and progenitor cells within the heart that are capable of differentiating into cardiomyocytes and/or vascular lineages. It has been suggested that these cells can be clonally expanded and used for cardiac repair in an autologous setting [4]. Clearly, independent conrmation of these provocative ndings is required. If conrmed, cardiac resident stem and progenitor cells hold great promise for clinical applications, although, it is conceivable that the bone marrow contains a pluripotent stem cell population with similar properties [16,17].
Embryonic stem cells

transplanted cells retained in the myocardium for a short period of time (hours). The local milieu is an important determinant of cell retention, as it will inuence shortterm cell survival and, if a transvascular approach is used, cell adhesion, transmigration through the vascular wall, and tissue invasion. Transvascular strategies are especially suited for the treatment of recently infarcted and reperfused myocardium when chemoattractants are highly expressed [22]. Direct injection techniques have been used in patients presenting late in the disease process when an occluded coronary artery precludes transvascular cell delivery (e.g. patients with chronic myocardial ischemia) or when cell homing signals are expressed at low levels in the heart (scar tissue). However, cell delivery by direct injection may not be safe in patients with AMI and friable necrotic tissue. Selective intracoronary application delivers a maximum concentration of cells homogeneously to the site of injury. Unselected bone marrow cells, circulating blood-derived progenitors cells, and mesenchymal stem cells have been delivered via the intracoronary route in patients with AMI. In these studies, cells were delivered through the central lumen of an over-the-wire balloon catheter during transient balloon inations to maximize the contact time of the cells with the microcirculation of the infarct-related artery [23]. In experimental models, intravenous delivery of endothelial progenitors cells and mesenchymal stem cells has been shown to improve cardiac function after AMI. However, cell homing to non-cardiac organs limits the applicability of this approach. Indeed, in a recent clinical study, homing of unselected bone marrow
Figure 2

Embryonic stem (ES) cells are totipotent stem cells derived from the inner cell mass of blastocysts. Under specic culture conditions, ES cells differentiate into multicellular embryoid bodies containing differentiated cells from all three germ layers including cardiomyocytes. Human ES cell-derived cardiomyocytes display structural and functional properties of early stage cardiomyocytes that couple with host cardiomyocytes when transplanted into normal or infarcted myocardium [18,19]. In theory, innite numbers of cardiomyocytes could be obtained from human ES cell clones. However, unresolved ethical and legal issues, concerns about the tumorigenicity of residual ES cells in ES cell-derived cardiomyocyte preparations, and the need to use allogeneic cells for transplantation currently hamper their use in clinical studies. Eventually, nuclear transfer techniques or somatic cell reprogramming may provide means for generating an unlimited supply of histocompatible stem cells for the treatment of cardiac disease.
Multipotent adult germline stem cells

Recent studies have highlighted the pluripotency and plasticity of spermatogonial stem cells, which are responsible for maintaining spermatogenesis throughout life in the male [20,21]. In culture, adult spermatogonial stem cells acquire ES cell properties. These multipotent adult germline stem cells spontaneously differentiate into derivatives of the three embryonic germ layers in vitro, including cardiomyocytes and vascular cells. Conceptually, establishment of human multipotent adult germline stem cells from testicular biopsies may allow cell-based therapy without the ethical and immunological problems associated with human ES-cells.

Modes of cell delivery in the setting of acute myocardial infarction

The goal of any cell delivery strategy is to transplant sufcient numbers of cells into the myocardial region of interest and to achieve maximum retention of cells within that area. Cell retention may be dened as the fraction of
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Myocardial homing and biodistribution of unfractionated bone marrow cells after intracoronary transfer. Nine days after primary angioplasty and stent implantation for AMI, this patient received an intracoronary infusion of autologous nucleated bone marrow cells into the stented left circumflex coronary artery. A small fraction of the cells was radiolabeled with 18F-FDG just prior to intracoronary transfer. Positron emission tomography imaging was performed 65 min after cell transfer. Left posterior oblique (panel A) and left anterior oblique (panel B) views of the chest and upper abdomen are shown. Approximately 3% of the cells homed to the lateral wall of the heart; most remaining activity is detected in liver and spleen (adapted from Ref. [24]). www.sciencedirect.com

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cells to the infarct region was observed only after intracoronary stop-ow delivery but not after intravenous infusion (Figure 2) [24]. Considering that the acutely infarcted myocardium recruits circulating stem and progenitor cells to the site of injury, mobilization of stem and progenitor cells by cytokines may offer a non-invasive strategy for cardiac regeneration. Indeed, it has been reported that the stem cell mobilizing cytokines stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) improve cardiac function after AMI in mice [25,26]. Notably, G-CSF can accelerate infarct healing directly by enhancing macrophage inltration and matrix metalloproteinase activation, and suppressing cardiomyocyte apoptosis, suggesting that stem-cell independent mechanisms may contribute to the favorable effects of G-CSF post AMI [27,28].

months). Bone marrow harvest and intracoronary cell delivery are not associated with bleeding complications or ischemic damage to the myocardium. No increased rates of in-stent restenosis have been observed. Clinical surveillance, Holter monitoring, and data from an electrophysiological study indicate that bone marrow cell transfer is not associated with an increased propensity to ventricular arrhythmias; moreover, intramyocardial calcications or tumor formation were not observed after intracoronary bone marrow cell delivery [29,30,31,32,33]. In the bone marrow transfer to enhance ST-elevation infarct regeneration (BOOST) trial, intracoronary transfer of nucleated bone marrow cells resulted in an improvement of global left ventricular ejection fraction of 6 percentage points after six months as compared to the control group. This improvement of ejection fraction was due mostly to improved regional wall motion in the infarct border zone [29]. For comparison, improvements of 34 percentage points are achieved by primary angioplasty and stent implantation in patients with AMI, suggesting that the improvement of ejection fraction by cell therapy may be clinically meaningful. The benecial effects of intracoronary bone marrow cell transfer were conrmed in the reinfusion of enriched progenitor cells and infarct remodeling in acute myocardial infarction (REPAIRAMI) trial. In this study, mononucleated bone marrow cell transfer promoted an increase in left ventricular ejection fraction of 2.5 percentage points after four months as compared to a control group that also underwent bone marrow aspiration but received an intracoronary infusion of a placebo [30]. In contrast to BOOST and REPAIR-AMI, two other randomized studies did not report signicant improvements of left ventricular ejection fraction after intracoronary cell transfer [31,32]. While the exact reasons for the differing results remain elusive, it is worthwhile to

Current status of cell therapy in patients with acute Myocardial infarction

Inspired by experimental data suggesting that functional recovery after AMI can be augmented by stem cell transfer, clinical trials were initiated to assess the therapeutic potential of cell therapy in patients post AMI. All clinical studies have included patients, who had undergone primary angioplasty and stent implantation to reopen the infarct-related artery and who received optimal medical treatment during the acute phase and follow-up, and cells were infused intracoronarily by using the stop-ow balloon-catheter approach. Current trials may be categorized into studies using unselected bone marrow cells or selected stem cell populations.
Unselected bone marrow cells

Following initial safety and feasibility studies, four larger randomized trials of bone marrow cell therapy after AMI have now been completed (Table 1) [2932]. The combined experience from these trials indicates that intracoronary delivery of unselected bone marrow cells is feasible and safe in the short- and mid-term (up to 18
Table 1

Randomized bone marrow cell trials in patients with acute myocardial infarction Study Design n Cell type Dose (mL) Time of delivery post AMI 6 1 Days 36 Days 1 Day 6 1 Days Outcomes Improved Global LVEF Global LVEF No change LVEDV LVEDV Global LVEF LVEDV Global LVEF LVEDV

BOOST (Ref. [29]) REPAIR-AMI (Ref. [30]) Janssens (Ref. [31]) ASTAMI (Ref. [32])

Randomizedcontrolled Placebocontrolled Placebocontrolled Randomizedcontrolled

30 30 95 92 32 34 47 50

treated controls treated controls treated controls treated controls

Nucleated BMCs Mononucleated BMCs Mononucleated BMCs Lymphocytic BMCs

128 50 130 50

BMC denotes bone marrow cell; n, number of patients; AMI, acute myocardial infarction; LVEF, left ventricular ejection fraction; LVEDV, left ventricular end-diastolic volume. In BOOST, cells were prepared by gelatine-polysuccinate density gradient sedimentation which retrieves all nucleated cell types from the bone marrow; REPAIR-AMI and Janssens employed a Ficoll gradient, which recovers the mononuclear cell fraction. In ASTAMI, lymphocytic bone marrow cells were enriched. Dose refers to the average amount of bone marrow that was harvested.

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take a closer look at the design of these studies (Table 1). In one of the negative studies, cells were delivered already 24 hours after coronary reperfusion [31]. In BOOST and REPAIR-AMI, cells were transplanted several days later. Subgroup analyses in REPAIR-AMI indicate that the timing of cell delivery may be important, and that the benecial effects on ejection fraction are lost when the cells are delivered too early [30]. The autologous stem cell transplantation in acute myocardial infarction (ASTAMI) trial also did not nd a signicant effect of bone marrow cell transfer on left ventricular ejection fraction recovery [32]. While the cells were delivered late after reperfusion in ASTAMI (after 46 days), a particular cell preparation method that leads to an enrichment of lymphocytic bone marrow cells was employed. It is not clear whether this method actually recovers the bone marrow cell populations that are required to achieve functional improvements after AMI [34]. Together, these data remind us that procedural issues, such as the cell preparation method and timing of cell transfer need to be further rened. It should be noted that none of the trials so far has revealed a signicant effect of bone marrow cell transfer on left ventricular end-diastolic volumes, an index of left ventricular remodeling. However, larger studies may be required to settle this issue. Moreover, little data are available regarding the long-term effects of bone marrow cell transfer after AMI. Follow-up data from the BOOST trial indicate that the improvements of left ventricular ejection fraction are maintained 18 months after cell transfer; [33] however, ejection fraction also increased somewhat in the control group during long-term followup [33], which would be expected in AMI patients on chronic angiotensin converting enzyme inhibitor and bblocker therapy. Tissue Doppler echocardiography analyses in BOOST indicate that bone marrow cell transfer may prevent the development of diastolic dysfunction during long-term follow-up. Signicant effects of cell transfer on E/A ratio and tissue Doppler Ea/Aa ratio, but not on isovolumic relaxation time suggest that cell therapy positively affects left ventricular stiffness but not active relaxation [35].
Selected bone marrow cell populations

CHF) trial, patients received an intracoronary infusion of unselected mononucleated bone marrow cells or circulating blood-derived progenitor cells months or even years after an AMI. Notably, a signicant improvement in left ventricular ejection fraction was observed only in patients receiving mononucleated bone marrow cells in this setting [37]. The effects of culture-expanded mesenchymal stem cells after AMI have been investigated in one randomized clinical trial [38]. While no serious side effects were reported, it is not known whether intracoronary mesenchymal stem cell delivery promoted ischemic damage to the myocardium, a complication that has occurred after intracoronary mesenchymal stem cell infusions in dogs [39]. Six months after cell transfer, regional wall motion and global left ventricular ejection fraction were improved and left ventricular enddiastolic volumes were decreased [38]. These striking effects need to be conrmed by additional studies. In another trial, CD133+ enriched bone marrow cells were infused into the infarct-related artery in post AMI patients. Higher luminal losses within the stented segment and the distal, non-stented segments of the infarctrelated artery were observed, raising the concern that CD133+ cells may promote in-stent restenosis and atherosclerosis progression [40]. As these data were obtained from retrospective analysis and lacked randomized controls, future studies need to dene the risk and mechanisms of such adverse effects on the epicardial coronary circulation (which were not observed in any of the trials using unfractionated nucleated bone marrow cells).
Stem and progenitor cell mobilization

The transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI) trial compared mononucleated bone marrow cells with circulating blood-derived progenitor cells (mostly EPCs) in post AMI patients a few days after reperfusion. Both cell types appeared to have similar safety and efcacy proles [36]. However, since TOPCARE-AMI was not randomized, rm conclusions regarding the efcacy of endothelial progenitor cells post AMI cannot be drawn at the present time. In the randomized transplantation of progenitor cells and regeneration enhancement in chronic heart failure (TOPCARECurrent Opinion in Pharmacology 2008, 8:202210

The front-integrated revascularization and stem cell liberation in evolving acute myocardial infarction by use of granulocyte-colony-stimulating factor (FIRSTLINE-AMI) trial randomized 50 patients with AMI to a control group or a six day open-label course of G-CSF that was initiated within 12 hours after primary angioplasty and stenting [41]. G-CSF therapy after stent implantation was not associated with an enhanced rate of in-stent restenosis or other serious adverse events, and promoted signicant improvements in left ventricular ejection fraction and metabolic activity in the infarct territory [41]. Critics have pointed out that the benecial effects of G-CSF in FIRSTLINE-AMI were magnied by an unexpected decrease in ejection fraction in the control group [42]. The favorable safety prole of G-CSF post AMI was conrmed two other recent trials which, however, did not observe a benecial effect on left ventricular ejection fraction [43,44]. It remains to be seen whether differences in study design, for example later start of G-CSF injections in the negative trials, account for the discrepant results.

Conclusions and future directions

Taken together, it appears that bone marrow cell therapy has the potential to improve left ventricular systolic
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Cell therapy for AMI Wollert 207

Table 2 Ongoing randomized cell therapy trials in patients with acute myocardial infarction Acronym REGENT HEBE Country Poland The Netherlands Switzerland Design Multicenter, controlled Multicenter, controlled Multicenter, controlled n 200 200 Groups MN-BMCs vs. CD34+/CXCR4+ cells Standard therapy vs. MN-BMCs vs. MN blood cells Standard therapy vs. MN-BMCs (57 days vs. 34 weeks after PCI) Placebo vs. MN-BMCs (after thrombolysis for AMI) Primary endpoints LVEF, LV volumes by MRI Regional function in dysfunctional segments by MRI LVEF by MRI Principal investigators Michal Tendera Alexander Hirsch, Jan J. Piek Roberto Corti

SWISS-AMI

>60

Finnland

Placebocontrolled

80

BOOST-2

Germany, Norway, Bulgaria

Multicenter, placebo-controlled, factorial design

200

Placebo vs. nucleated BMCs (low-dose vs. high-dose, pre-treated vs. not pre-treated)

Arrhythmia risk variables, LV function by echo and angio, restenosis by IVUS LVEF by MRI

Heikki V. Huikuri

Kai C. Wollert, Gerd P. Meyer, Helmut Drexler

n denotes number of patients (expected total enrollment); MN, mononucleated; BMCs, bone marrow cells; LVEF, left ventricular ejection fraction; LV, left ventricular; MRI, magnetic resonance imaging; echo, echocardiography; angio, angiography; IVUS, intravascular ultrasound.

function in patients with AMI when the right cell type and delivery strategies are employed. While two metaanalyses support this conclusion [45,46], further research is clearly needed. A recently established task force of the European Society of Cardiology advocates to no longer perform studies involving small numbers of patients, but rather to conduct larger, double-blind, randomized-controlled clinical trials to rmly establish the effects of cell therapy on surrogate markers, like left ventricular ejection fraction and remodeling, myocardial perfusion, or exercise capacity [47]. Most importantly, upcoming trials should address procedural issues such as the optimal cell type, cell dosage, and timing of cell transfer. While these trials may also look at combined morbidity and mortality endpoints, they may be too small to be conclusive in this regard. Some of the ongoing trials of intracoronary bone marrow cell transfer after AMI are summarized in Table 2. Ultimately, outcome trials and cost-benet analyses will be required. Notably, the absolute number of transplanted bone marrow cells did not correlate with subsequent improvements in ejection fraction in previous post AMI studies. This may be because the cell numbers infused were within a narrow range, or because differences in the functional capacity of the cells, such as the ability to home to and engraft in the infarcted area, to undergo transdifferentiation and/or to produce paracrine factors, may override differences in cell numbers. Intriguingly, cell labeling studies indicate that less than 5% of unselected nucleated bone marrow cells are retained in the infarcted area after intracoronary delivery in patients (Figure 2) [24]. Although this rate of cell retention was sufcient to improve left ventricular systolic and diastolic function in the BOOST trial, it is conceivable that pharmacological
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strategies might be used to enhance the homing capacity or other functional parameters of the cells. Experimental studies are already pointing in this direction [48]. Patient subgroups that derive the greatest benet from cell transfer need to be identied in future trials (e.g. patients presenting late after symptom onset in whom little myocardial salvage can be expected from reperfusion therapy). In this regard, data from REPAIR-AMI indicate that the effects of bone marrow cell transfer may be more pronounced in patients with more severely depressed baseline left ventricular ejection fraction [30]. Cytokines with stem-cell mobilizing and/or direct cardioprotective properties should be further evaluated as stand alone therapy or in combination with cell transfer after AMI. Eventually, cytokines may emerge as a non-invasive alternative or as an adjunct to cell therapy. Meanwhile, fundamental questions need to be addressed experimentally. What is the fate of the injected cells after transplantation? Genetic and transgenic markers should be employed to determine lineage commitment of engrafted cells. Cell labeling and imaging techniques need to be developed to track stem cell fate in patients and correlate cell retention and engraftment with functional outcomes. Can the regenerative capacity of transplanted stem cells be enhanced by drugs, cytokines, or gene therapy approaches? Pharmacological and genetic strategies may help to enhance stem cell retention, engraftment, differentiation, and paracrine capability [49,50]. For the time being, cardiac repair remains the holy grail of cell therapy. While unselected bone marrow cells may have a favorable impact on systolic function, they probably do not make new myocardium. This should
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stimulate further basic research into the prospects of cell types with transdifferentiation capacity, such as ES-cells, multipotent adult germline stem cells, and, possibly, multipotent bone marrow-derived stem cells and resident cardiac stem cells.

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Acknowledgement
This work was supported by the German Research Foundation (KFO136, WO 552/4-1).

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. 2. 3.  Wollert KC, Drexler H: Clinical applications of stem cells for the heart. Circ Res 2005, 96:151-163. Wagers AJ, Weissman IL: Plasticity of adult stem cells. Cell 2004, 116:639-648.

Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM et al.: Bone marrow cells regenerate infarcted myocardium. Nature 2001, 410:701-705. This study provided the initial evidence that hematopoietic stem cells from the bone marrow promote an improvement of systolic function when injected into the acutely infarcted mouse myocardium. The proposed mechanism of this effect transdifferentiation of stem cells into cardiac cell types has been questioned in subsequent publications from other groups. 4. Torella D, Ellison GM, Mendez-Ferrer S, Ibanez B, Nadal-Ginard B: Resident human cardiac stem cells: role in cardiac cellular homeostasis and potential for myocardial regeneration. Nat Clin Pract Cardiovasc Med 2006, 3(Suppl. 1):S8-S13. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K, Lois C, Morrison SJ, Alvarez-Buylla A: Fusion of bonemarrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 2003, 425:968-973. Nygren JM, Jovinge S, Breitbach M, Sawen P, Roll W, Hescheler J, Taneera J, Fleischmann BK, Jacobsen SE: Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation. Nat Med 2004, 10:494-501. Murry CE, Soonpaa MH, Reinecke H, Nakajima H, Nakajima HO, Rubart M, Pasumarthi KB, Virag JI, Bartelmez SH, Poppa V et al.: Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 2004, 428:664668.

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Balsam LB, Wagers AJ, Christensen JL, Kodis T, Weissman IL, Robbins RC: Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 2004, 428:668-673. This study conrmed that hematopoietic stem cells from the bone marrow promote an improvement of systolic function when injected into the acutely infarcted mouse myocardium. However, transdifferentiation of stem cells into cardiac cell types was not observed in this work using sophisticated genetic labeling strategies.

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Kamihata H, Matsubara H, Nishiue T, Fujiyama S, Tsutsumi Y, Ozono R, Masaki H, Mori Y, Iba O, Tateishi E et al.: Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation 2001, 104:1046-1052. Using a pig model of acute myocardial infarction, this study shows that intramyocardial injection of unfractionated bone marrow cells promotes an improvement in systolic function. Secretion of cardioactive cytokines is offered as a potential mechanism of this effect. 10. Kinnaird T, Stabile E, Burnett MS, Lee CW, Barr S, Fuchs S, Epstein SE: Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and

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26. Ohtsuka M, Takano H, Zou Y, Toko H, Akazawa H, Qin Y, Suzuki M, Hasegawa H, Nakaya H, Komuro I: Cytokine therapy prevents left ventricular remodeling and dysfunction after myocardial infarction through neovascularization. FASEB J 2004, 18:851853. 27. Minatoguchi S, Takemura G, Chen XH, Wang N, Uno Y, Koda M, Arai M, Misao Y, Lu C, Suzuki K et al.: Acceleration of the healing process and myocardial regeneration may be important as a mechanism of improvement of cardiac function and remodeling by postinfarction granulocyte colony-stimulating factor treatment. Circulation 2004, 109:2572-2580. 28. Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M, Matsuura K, Sano M, Nishi J et al.: G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat Med 2005, 11:305-311. 29. Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P,  Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger D et al.: Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 2004, 364:141-148. The rst randomized-controlled clinical trial of intracoronary bone marrow cell transfer in patients with acute myocardial infarction. Cell transfer led to a signicant increase in left ventricular ejection fraction after six months as compared to the control group. 30. Schachinger V, Erbs S, Elsasser A, Haberbosch W, Hambrecht R,  Holschermann H, Yu J, Corti R, Mathey DG, Hamm CW et al.: Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med 2006, 355: 1210-1221. Placebo-controlled clinical trial of intracoronary bone marrow cell transfer in patients with acute myocardial infarction. Cell transfer led to a signicant increase in left ventricular ejection fraction after four months as compared to the control group. 31. Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C,  Desmet W, Kalantzi M, Herbots L, Sinnaeve P, Dens J et al.: Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled trial. Lancet 2006, 367:113-121. This placebo-controlled clinical trial did not conrm that intracoronary bone marrow cell transfer leads to an improvement in left ventricular ejection fraction in patients with acute myocardial infarction. It has been argued that cells were delivered too early after coronary reperfusion in this study. 32. Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M,  Egeland T, Endresen K, Ilebekk A, Mangschau A, Fjeld JG et al.: Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction. N Engl J Med 2006, 355:1199-1209. This randomized open study did not observe a signicant improvement in left ventricular systolic function after intracoronary cell transfer in patients with acute myocardial infarction. The reasons for these negative results are poorly understood but may relate to the cell preparation methods used in this trial. 33. Meyer GP, Wollert KC, Lotz J, Steffens J, Lippolt P, Fichtner S, Hecker H, Schaefer A, Arseniev L, Hertenstein B et al.: Intracoronary bone marrow cell transfer after myocardial infarction: eighteen months follow-up data from the randomized, controlled BOOST (bone marrow transfer to enhance ST-elevation infarct regeneration) trial. Circulation 2006, 113:1287-1294. 34. Seeger FH, Tonn T, Krzossok N, Zeiher AM, Dimmeler S: Cell isolation procedures matter: a comparison of different isolation protocols of bone marrow mononuclear cells used for cell therapy in patients with acute myocardial infarction. Eur Heart J 2007, 28:766-772. 35. Schaefer A, Meyer GP, Fuchs M, Klein G, Kaplan M, Wollert KC,  Drexler H: Impact of intracoronary bone marrow cell transfer on diastolic function in patients after acute myocardial infarction: results from the BOOST trial. Eur Heart J 2006, 27:929-935. This analysis from the BOOST study indicates that intracoronary bone marrow cell transfer prevents the decline in diastolic function that is observed in patients on standard pharmacological therapy after acute myocardial infarction. www.sciencedirect.com

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of Cardiology concerning the clinical investigation of the use of autologous adult stem cells for repair of the heart. Eur Heart J 2006, 27:1338-1340. 48. Sasaki K, Heeschen C, Aicher A, Ziebart T, Honold J, Urbich C, Rossig L, Koehl U, Koyanagi M, Mohamed A et al.: Ex vivo pretreatment of bone marrow mononuclear cells with endothelial NO synthase enhancer AVE9488 enhances their functional activity for cell therapy. Proc Natl Acad Sci U S A 2006, 103:14537-14541.

49. Kawamoto A, Murayama T, Kusano K, Ii M, Tkebuchava T, Shintani S, Iwakura A, Johnson I, von Samson P, Hanley A et al.: Synergistic effect of bone marrow mobilization and vascular endothelial growth factor-2 gene therapy in myocardial ischemia. Circulation 2004, 110:1398-1405. 50. Gnecchi M, He H, Liang OD, Melo LG, Morello F, Mu H, Noiseux N, Zhang L, Pratt RE, Ingwall JS et al.: Paracrine action accounts for marked protection of ischemic heart by Akt-modied mesenchymal stem cells. Nat Med 2005, 11:367-368.

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