Autologous cell transplantation for the treatment of damaged myocardium

Autologous Cell Transplantation for the Treatment of Damaged Myocardium Marc S. Penn, Gary S. Francis, Stephen G. Ellis, James B. Young, Patrick M. McCarthy, and Eric J. Topol

Autologous cell transplantation for the treatment of damaged myocardium after myocardial infarction is becoming an increasingly promising strategy. This form of treatment can be divided into 2 treatment strategies: The first uses differentiated cell types to replace the scarred tissue with living cells, while the second strategy uses stem cells in an attempt to regenerate myocardium. Over the past decade, multiple cell types have been used in animal studies, and clinical trials to determine the safety of injecting and engrafting skeletal myoblasts into damaged myocardium are presently being conducted. Animals studies focused on using stem cells to regenerate damaged myocardium have shown a naturally occurring reparative process that consists of up-regulation of progenitor cell release from the bone marrow after myocardial infarction, homing of these cells to the injured tissue, and differentiation of these progenitor cells into vascular cells and cardiac myocytes within the infarcted tissue. Unfortunately, this process occurs with great infrequency. Strategies to regenerate myocardium with stem cells either extract stem cells from the bone marrow and inject these cells into the damaged area or they attempt to increase the efficiency of the natural reparative process by increasing the mobilization of bone marrow– derived stem cells after myocardial infarction. This review summarizes the field of cell transplantation over the past decade, discusses areas of controversy, and proposes an outline of advancements that need to be made in both the clinical and scientific arenas for autologous cell transplantation to fully reach its clinical potential. Copyright 2002, Elsevier Science (USA). All rights reserved.

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ecent clinical trials of acute myocardial infarction testing enhanced reperfusion strategies have failed to demonstrate a significant improvement in mortality. These recent studies also

demonstrated that no significant progress has been made on encouraging patients to present earlier with their symptoms since the time to presentation for patients enrolled in clinical trials, nearly 3 hours after symptom onset, has not changed over the past decade. These observations demonstrate that need for the development of a new treatment paradigm for the treatment of patients with acute myocardial infarction and the consequences of myocardial necrosis. Strategies are needed to either (1) prevent myocardial cell loss during acute myocardial infarction, (2) optimize the pathological remodeling, or (3) regenerate myocardium after acute myocardial infarction, particularly since now over 10% of the American population over 65 years of age has congestive heart failure. The concept of transplanting autologous cells successfully into a damaged heart has received significant attention. Clinical trials are ongoing that examine the safety and efficacy of autologous skeletal myoblast transplantation in patients initially as an adjunct to coronary artery bypass surgery.1 Furthermore, case studies using autologous bone marrow transplantation after acute myocardial infarction are beginning to appear at national meetings describing experiences with bone marrow cells injected into the myocardium or directly through the infarct-related epicardial artery.2

From the Departments of Cardiovascular Medicine, Cell Biology, and Cardiothoracic Surgery, Cleveland Clinic Foundation, Cleveland, OH. Address reprint requests to Marc S. Penn, MD, PhD, Director, Experimental Animal Laboratory Departments of Cardiovascular Medicine and Cell Biology, 9500 Euclid Avenue, NC10, Cleveland, OH 44195. Copyright 2002, Elsevier Science (USA). All rights reserved. 0033-0620/02/4501-0001$35.00/22/1/123466 doi:10.1053/pcad.2002.123466

Progress in Cardiovascular Diseases, Vol. 45, No. 1, (July/August) 2002: pp 21-32

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22 The scientific issues surrounding autologous cell transplantation are significant. These cells must incorporate themselves into the heart, survive, mature, and electromechanically couple to each other and native cardiac myocytes, or at least not be arrhythmogenic, and ultimately benefit organ function. Potential cells for autologous cell transplantation might be myoblasts grown from skeletal muscle,1,3 smooth muscle cells from blood vessels,4 or hematopoietic or mesenchymal stem cells either mobilized with pharmaceuticals or by biopsy from the bone marrow.5,6 If injected during the peri-infarct period, the transplanted cells should at least promote positive ventricular remodeling by increasing myocardial wall thickness and promoting efficient ventricular function; however, ideally, the donor cells should lead to regeneration of infarcted myocardial tissue including differentiating into blood vessels or promoting local angiogenesis. This review encapsulates the recent burgeoning literature on autologous cell transplantation in the treatment of infarcted myocardium remodeling and discusses the promise and challenges of the field.

Autologous Cell Transplantation into Normal Myocardium Some of the first studies directed at investigating the potential role of autologous cell transplantation for the regeneration of myocardial tissue involved normal hearts. These studies performed by Field et al3,7,8 demonstrated engraftment of either skeletal muscle or cardiac myocytes. In particular, C2C12 cells, a skeletal myoblast cell line,3 or AT-1 cardiomyocytes, a T-antigen immortalized cell line,8 were injected into syngeneic normal hearts. Cell engraftment was present through at least 3 months in the case of transplantation of the C2C12 cells3 and 4 months for AT-1 cardiomyocytes.8 Further, electron microscopy of engrafted nonimmortalized fetal cardiac myocytes demonstrated that the transplanted cells could form intercalated disks connecting them to the host myocardium.9 These early observations offered the potential that transplanted cells could lead to formation of functional myocardium. Field et al went on to demonstrate that the use of autologous cell transplantation could be expanded to include local drug delivery. Skeletal myoblast cell line stably transfected with an in-

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ducible promoter-driving TGF-␤1 expression transplanted into normal myocardium demonstrated increased endothelial cell DNA synthesis as well as histological evidence of neovascularization7 compared with control subjects (non-stably transfected C2C12 cells). These early studies demonstrated the potential efficacy of engrafting autologous cells of either skeletal or cardiac myocyte origin into normal myocardium. Critically important to the future of the field was the observation that engraftment of skeletal myoblasts3 or cardiomyocytes8 into normal myocardium did not lead to the development of ventricular arrhythmias. Although these studies were pivotal toward establishing the potential of autologous cell transplantation for the treatment of cardiac dysfunction, the potential efficacy of engrafting normal (nonimmortalized) skeletal myoblasts or cardiac myocytes into ischemic myocardium remained to be demonstrated.

Autologous Transplantation of Differentiated Cells into Ischemic Myocardium A number of investigators have used multiple animal models to study the efficacy of transplantation of differentiated cells into ischemic or infarcted myocardium. Myocardial injury was usually induced either by left anterior descending (LAD) artery ligation5,6 or direct cryoinjury to the epicardial surface.4,10,11 Although the physiological relevance of cryoinjury is unclear, we chose to include these studies in this review owing to the important findings that have come from this model.

Differentiated Cell Transplantation A schematic diagram of the general protocol of transplantation of differentiated cells into infarcted myocardium is depicted in Fig 1. Differentiated cells include those that may be fully differentiated cells, such as smooth muscle cells, fibroblasts, or skeletal myocytes, as well as cells committed to differentiation along a specific pathway, such as skeletal myoblasts. The advantages of using differentiated cells are (1) the relative accessibility of biopsies to obtain cells of interest; (2) it allows for expansion of autologous cells in vitro before cell injection;

CELL TRANSPLANTATION AND MYOCARDIAL REGENERATION

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Fig 1. Schematic representation of protocol for autologous cell transplantation of skeletal myoblast or heart cells. Step 1 is the isolation of tissue containing the differen-tiated cell type of interest; step 2 is expansion of the differentiated cell type of interest in culture; and step 3 is the injection of the differentiated cells type into the peri-infarct or infarct zone of the myocadum. During step 2, cells can potentially be transfected with vectors that encode for genes of interest.

and (3) a theoretical decrease in the potential for tumor genesis. The disadvantages of using differentiated cells are (1) the time required (on the order of weeks) to expand the cells in culture and (2) the inability of differentiated cells to regenerate myocardium. The general goal of these studies has been to demonstrate engraftment of the differentiated cell type of interest into infarcted myocardium; however, some of these studies have attempted to demonstrate efficacy of cell transplantation. Measures of left ventricular function have varied from quantifying dP/dT12 to the more clinically relevant ejection fraction by two-dimensional echocardiography.5,13,14

Cell Types and Species Studied Differentiated cell populations that have been studied include “heart cells” grown directly from right heart biopsies,15 skeletal myoblasts isolated from muscle biopsies,13 and fetal or transformed cardiac myocytes16 (Table 1).9,17 Transplantation of autologous differentiated cells into infarcted tissue has proven successful in sheep,13 dog,10 rabbit,11,18 porcine,15,16 and rat4,14,19 models of myocardial infarction. Cell engraftment has been successful as early as immediately after transmural myocardial injury (induced by placing a cryoprobe on the epicardial surface) to as late as 5 weeks after infarction

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Table 1. Published Studies Designed to Investigate the Safety and Efficacy of Transplanting Differentiated Autologous Cells into Infarcted Myocardium Study

Animal Model

Time after Infarct

Effect on LV Function

Marelli et al10

Adult

Immediate (cryoinjury)

N/A

Skeletal myoblasts

Neonatal

Immediate (cryoinjury)

N/A

Rat

Ventricle patches

Fetal

1 to 2 weeks

N/A

Watanabe et al16

Porcine

Cardiomyocyte

HL-1 and fetal

5 weeks

N/A

Taylor et al11

Rabbit

Adult

Cryoinjury

Improved

Neonatal

10 days

N/A

Cell Type

Source

Dog

Skeletal myoblasts

Murry et al21

Rat

Leor et al19

Matsushita et al

Rat

Skeletal myoblasts Cardiomyocyte

Li et al4

Rat

SMC

Fetal stomach

4 weeks (cryoinjury)

Atkins et al18

Rabbit

Skeletal myoblasts

Adult

1 week

Scorsin et al44

Rat

Skeletal myoblasts Cardiomyocyte

Neonatal Fetal

1 week 1 week

Improved in Langendorf Prep Improved diastolic performance Improved EF Improved EF

Li et al15

Porcine

Heart cells

Right heart biopsy

4 weeks

N/A

Pouzet et al14

Rat

Skeletal myoblasts

Adult

1 week

Improved EF

Jain et al12

Rat

Skeletal myoblasts

Adult

1 week

Rajnoch et al13

Sheep

Adult

3 weeks

Etzion et al17

Rat

Skeletal myoblasts Cardiomyocte

Improved exercise capacity Improved EF

Embryonic

1 week

Improved EF

43

Comments Variable persistence of myoblasts as a function time after transplantation Sketetal myoblasts contracted with external stimulus Fetal cardiomyocytes were engrafted at least out to 65 days Engraftment seen only in normal, not ischemic, myocardium

Transplanated cells formed gap junctions with native myocardium Increased angiogenesis in transplanted scar

Animals were immunosuppressed Fetal cardiomyocytes were as efficacious as neonatal skeletal myoblasts Increased perfusion and wall thickness of infarct zone Improvement correlated with cell number injected Attenuated LV dilation in treated animals Engrafted cell survival at least 2 months Embryonic phenotype maintained in cells isolated by scar from native myocardium

Abbreviations: LV, left ventricular; SMC, smooth muscle cell; EF, ejection fraction.

(induced by LAD ligation). In general, all studies have been within 4 weeks of myocardial infarction (Table 1). From these studies, the following generalizations can be made: 1. Multiple autologous cell types of mesenchymal origin (muscle or fibroblasts) can engraft into infarcted myocardium. 2. Engraftment of differentiated autologous cells

does not lead to significant ventricular arrhythmias in short-term follow-up. 3. Only engraftment of cardiomyocytes reproducibly leads to connection of engrafted cells with native myocardium through intercalated discs. 4. There is no evidence to date that transplantation of fetal myoblasts offers improved efficacy or safety compared with adult skeletal myoblasts.

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Potential Mechanisms of Efficacy The mechanism for improved left ventricular function after transplantation of autologous differentiated cells is unclear. The 2 potential mechanisms believed to modulate left ventricular function after cell transplantation are (1) the establishment of additional muscle cells that contract within the infarct zone or (2) improved postinfarct remodeling of the left ventricular cavity leading to improved wall stress and function. Establishing the mechanism responsible for improved left ventricular function after cell transplantation is actually quite important and will affect the eventual clinical utility of the technique. For example, if improved left ventricular function is because of the establishment of additional contractile tissue, then one could hypothesize that at any time point after myocardial infarction that engraftment can take place, improved function should be demonstrable. However, if improved function is because of improved left ventricular post-infarct remodeling, then cell transplantation needs to be performed within a specific period of time after myocardial infarction. If timing after infarction is critical, rapid identification and treatment initiation may be necessary so that cell expansion could begin as soon as possible. To date, the literature strongly supports that the potential benefit of post-infarct skeletal myoblast transplantation is owing to improved left ventricular remodeling rather than increased contractility.17,18,20,21 Murry et al21 have demonstrated that transplanted skeletal myoblasts could perform cardiac-like duty cycles, but only when externally stimulated, suggesting that the lack of integration of the transplanted skeletal myoblasts with the native myocardium limits their potential contribution to myocardial contraction. In the porcine model, the thickness and end-systolic elastance of the anterior wall after LAD ligation in those animals that received autologous “heart cells” were greater than in untreated control subjects.15 Similarly, rats treated with transplantation of autologous skeletal myoblasts 1 week after 1 hour of ischemia induced by LAD ligation had thicker infarct zones, smaller left ventricular cavities, and lower left ventricular end-diastolic pressures.12 These changes are all consistent with favorable changes in left ventricular wall stress and relaxation. Further, a quick review of the studies in-

cluded in Table 1 shows that all of these studies were conducted soon (within 4 to 5 weeks, the majority within 1 week) after myocardial infarction. This observation could lead one to postulate that a significant benefit of the transplantation of autologous differentiated cells may be to prevent or augment ventricular remodeling after myocardial infarction. The above studies have proven the safety and potential efficacy of differentiated cell transplantation for the treatment of left ventricular dysfunction and have led to the introduction of this technique as an experimental adjunct to coronary artery bypass surgery. Unfortunately, these studies have also demonstrated that the engraftment of these cells is not equivalent to the regeneration of myocardial tissue, and significant improvement in systolic left ventricular function has not been observed. This had lead to studies of the potential regeneration of myocardial tissue using stem cells.

Regeneration of Infarcted Myocardium by Stem Cells The use of stem cells to regenerate damaged organs has received significant attention over the last few years, and it now appears that a normal response to acute myocardial infarction in mammals is to increase release of certain progenitor cells from the bone marrow that then engraft into the damaged myocardium.22,23 Unfortunately, regeneration of damaged myocardium by this pathway is a low-frequency event. The goals of the studies discussed below are to exploit the potential of this naturally occurring system in an attempt to regenerate a sufficient amount of myocardium to cause recovery of cardiac function. Totipotent stem cells are capable of differentiating into any cell type, including adult cardiomyocytes,24 and likely only exist in embryos. There exist well-publicized ethical and political controversies as to whether research into the therapeutic potential of embryonic stems should be supported. Fortunately, for patients afflicted with congestive heart failure, a number of studies have demonstrated that pluripotent adult stem cells can lead to regeneration of myocardium after myocardial infarction.5,6,23,25 Conceptually, regeneration of infarcted myocardium with pluripotent stem cells can be accomplished from 2 approaches, each of which is

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Fig 2. Schematic representation of protocol for 2 potential methods, either intrinsic or extrinsic, for autologous stem cell transplantation. For intrinsic stem cell transplantation, cells are not removed from the body; rather, stem cells expansion and release are induced from native bone marrow. Potential strategies that have been shown to increase stem cell expansion and release include VEGF, statin therapy, and G-CSF. These released cells then home to and engraft into the infarct zone by mechanisms that are still under investigation. In extrinsic stem cell transplantation, stem cells are removed from the bone marrow and injected into the peri-infarct or infarct zones of the myocardium. Before injection, specific stem cells of interest can be identified and enriched using fluorescence-activated cell sorting (FACS).

outlined in Fig 2. The first approach is autologous stem cell transplantation and involves isolating autologous stem cells from bone marrow or other tissue and reinjecting them directly into the myocardium or blood stream sometime after myocardial infarction.5,6,25 The second technique is stem cell mobilization, which in concept involves stimulating the expansion of specific populations of stem cells within the bone marrow,22,23,26,27 and then directing released stem cells or progenitor cells to the infarct zone without ever removing them from the patient. Rather, in concept, this form of transplantation involves developing the pharmacological or genetic means to stimulate specific populations of stem cells and direct their engraftment to damaged tissue.

Autologous Stem Cell Transplantation The ability of stem cell transplantation to regenerate infarcted myocardium has been shown to have considerable therapeutic potential by multiple groups.6,23,25 Autologous stem cell transplantation offers several advantages, including the ability to use flow cytometry to isolate specific cell populations for transplantation as well as control over the timing and number of cells transplanted. Studies using autologous stem cell transplanta-

tion are summarized in Table 2. Orlic et al demonstrated that the injection of Lin⫺ c-kitPOS bone marrow– derived stem cells into the peri-infarct myocardium 3 to 5 hours after LAD ligation resulted in near complete regeneration of the infarcted anterior wall.6 These stem cells lead to regeneration of cardiac myocytes as well as vascular structures, including endothelial and smooth muscle cells. Hearts that received stem cell transplantation did not demonstrate normal function but did exhibit improved hemodynamics with lower left ventricular and diastolic pressure (LVEDP) and increased contractility, 9 days after myocardial infarction compared with saline control subjects.6 The transplantation of human CD34⫹ hematopoietic stem cells, a portion of which are angioblasts, into nude rats 2 days after LAD ligation resulted in neovascularization of the infarct zone with a significant decrease in the amount of scar tissue in the left ventricle and improvement in left ventricular ejection fraction and cardiac index.5 This study using CD34⫹ cells also demonstrated that stem cells can home to the infarct zone because the human CD34⫹ cells in this study were injected into the tail vein of the rats. The pathways involved in the homing of stem cells to damaged myocardium are yet to be identified; although the

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Table 2. Published Studies Designed to Investigate Regenerating Damaged Myocardium by Stem Cells

Study

Animal Model

Cell Type

Source

Effect on LV Function

Time After Infarct

Comments

Myocardial regeneration by stem cell transplantation Tomita et al25

Rat

Bone marrow cells

Autologous

3 weeks (Cryoinjury)

Improved

Kocher et al5

Rat

CD34⫹ Bone marrow cells

Human

48 h

Improved

Orlic et al6

Mouse

Autologous

3 to 5 h

Improved

Malouf et al45

Rat

Lin c-kitpos Bone marrow cells Liver stem cell line WB-F344

Autologous

N/A

N/A

Improved function only in cells grown in 5-azacytidine to promote cardiomyocytes. Nude rats due to use of human BMC. CD34⫹ cell reintroduced i.v. Cells injected into peri-infarct myocardium. Mature cardiac myocytes identified 6 weeks after transplantation.

Myocardial Regneration by Stem Cell Mobilization Asahara et al46

Mouse

1 week

Beltrami et al31

Human

4 to 12 days

Jackson et al23

Mouse

4 weeks

Orlic et al37

Mouse

Stimulated bone marrow with G-CSF, SCF

4 weeks

Endothelial cell progenitor cells incorporated into infarct border zone. Proliferating cells identified in myocardium likely represent stem cells differentiating into cardiomyocytes. Mice had bone marrow transplant with side population cells before infarction. Mice treated with G-CSF, before SCF, and splenectomy before MI have increased wall thickness and improved function.

Abbreviations: LV, left ventricular; G-CSF, granulocyte-colony stimulating factor; SCF, stem cell factor; BMC, bone marrow cells; i.v., intravenous.

existence of ubiquitous reparative process based on the targeting of pluripotent stem cells or progenitor cells to any injured organ is an area of intense investigation. If such a generalizable reparative process does exist, it could mean that there exists a limited amount of time after infarction during which myocardial tissue can be regenerated by stem cell transplantation. Bone marrow– derived mesenchymal stem cells appear to offer a potential source of stem cells for myocardial repair. This population of stem cells has recently been shown to home to the site of myocardial infarction and engraft into the damaged tissue, a portion of which appear to differentiate into a cardiac myocyte-like cell. Engraftment of these cells appears to significantly improve left ventricular function in a pig model of myocardial infarction.28 Further, preliminary results suggest that mesenchymal stem cells offer the potential for allogenic transplantation because their presence

does not appear to initiate an immune response possibly owing to the lack of expression of costimulatory factor B7, which is required for T-cell stimulation by major histocompatibility complex (MHC) II.29,30

Regeneration of Damaged Myocardium by Stem Cell Mobilization There are a few studies that suggest that the concept that regeneration of damaged myocardium by stem cell mobilization is possible. Beltrami et al demonstrated the presence of proliferating cardiac myocytes in the hearts of humans who died 5 to 12 days after myocardial infarction.31 Previous studies have demonstrated that cardiac myocytes are unable to divide, and when stimulated to do so in vivo, they die of apoptosis.32 Given these observations, it is probable that the Ki-67 positive cells

28 identified as proliferating cells in the post-infarct human hearts were differentiating or proliferating progenitor cells originating from the bone marrow.31 Proof that bone marrow cells can target infarcted myocardium and differentiate into cardiac myocytes and vascular structures was recently demonstrated in the mouse model of myocardial infarction.23 Ten weeks before ligation of the LAD for 60 minutes, animals underwent bone marrow transplant with a highly enriched population of hematopoietic cells (side population cells) from the bone marrow of a syngeneic transgenic mouse whose cells expressed the reporter gene, lacZ. Therefore, any cells in the recipient animal that expressed lacZ had to be derived from the donor bone marrow. Two and 4 weeks after 60 minutes of LAD ischemia, lacZ-positive endothelial cells and cardiac myocytes were identified.23 Although the number of lacZ-positive cells was small (0.02% cardiac myocytes, 1% to 2% endothelial cells), these results suggest that if we could optimize release and targeting of stem cells from the bone marrow after myocardial infarction, we could significantly enhance repair and regeneration of myocardium. One potential mediator of bone marrow stimulation after myocardial infarction is vascular endothelial growth factor (VEGF). VEGF administration results in mobilization of CD34⫹ cells from the bone marrow of C57BL/6j mice with a peak at 4 days after initiating treatment.27 Further, consistent with a role for VEGF in inducing bone marrow release of progenitor cells, a recently published study demonstrated that in response to myocardial infarction in patients, there were increased numbers of CD34⫹ cells in the periphery with a peak at 7 days.22 This peak in circulating CD34⫹ cells was coincident with the peak in plasma levels of VEGF.22 Interestingly, treatment of mice with HMG CoA reductase inhibitors (statins) significantly increased the levels of circulating endothelial progenitor cells in the periphery.33,34 Whether this proves to be one of the non– lipid-lowering benefits of HMG CoA-reductase therapy after myocardial infarction35,36 remains to be determined. Another potential mechanism for increasing the frequency of bone marrow– derived stem cell engraftment into infarcted myocardium is the use of granulocyte-colony–stimulating factor (G-

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CSF). In a recent study, the upregulation of bone marrow expansion using G-CSF, stem cell factor and splenectomy before inducing myocardial infarction led to increased engraftment of bone marrow– derived stem cells into the infarct zone.37 In the treated animals, the infarct zone was thicker, and the hearts demonstrated improved hemodynamics during systole and diastole. Surprisingly, this study demonstrated a significant decrease in mortality at 15 days in those mice that received G-CSF compared with control subjects (approximately 25% v 80%). The applicability of this finding to clinical populations is unclear given the extensive literature that has shown a significant benefit from neutropenia in ischemia/reperfusion models of myocardial infarction.38,39 Further, the 80% mortality in control mice is significantly greater than that observed by 3 independent groups using the mouse LAD ligation model to yield infarcts of similar size.40-42 Clearly during acute myocardial infarction the concept of stem cell mobilization, as achieved by administering G-CSF, is eminently pragmatic for clinical applicability and warrants further investigation.

Future Directions Autologous cell transplantation offers significant hope for the treatment of damaged myocardium. The pioneering studies discussed above have set the stage for further development of the use of skeletal myoblast and bone marrow– derived stem cells in this patient population; however, many significant challenges remain and are outlined in Table 3.

Biology of Tissue Repair Studies need to focus on the basic biology involved in stem cell regeneration of myocardium after ischemia. Potential near-term focus of these studies should include determining the signaling pathways involved in (1) stimulation of stem cell expansion leading to the release of CD34⫹ cells or perhaps even more pluripotent stem cells; (2) homing of stem cells to injured tissue; and (3) differentiation of progenitor cells to cardiac myocytes and endothelial cells. Enhancing our understanding and control of these steps will lead to optimization of stem cell mobilization, the most noninvasive of all the strategies outlined above.

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Table 3. Near and Long-Term Goals Leading to Optimization of Autologous Cell Transplantation in Humans Initial Focus Basic biology Homing mechanism of stem cells to infarct zone Stem cell differentiation into cardiac myocytes Clonal stem cell expansion Animal studies Engraftment late after MI and LV remodeling Effect of gene overexpression on cell engraftment and LV function Optimize bone marrow release of progenitor cells Clinical trials Initially as an adjunct to CABG Dosing of delivered cells v efficacy Develop imaging modalities to quantify cell engraftment

Indication/Goal Develop noninvasive means of cell delivery; may characterize a general organ repair process. Maximize regeneration of myocardial tissue. Optimize bone marrow response to MI. Maximize potential to treat patients with congestive heart failure. Autologous cell transplantation could be a vector for gene therapy to further optimize LV function. Necessary for the development of stem cell mobilization strategy. These trials should focus on demonstrating long-term safety of autologous cell transplantation. Initial efficacy trials should attempt to correlate cell dosing with efficacy. A mechanism to quantify cell transplantation in vivo is critical for interpreting efficacy of future strategies to optimize engraftment developed in animals.

Abbreviations: MI, myocardial infarction; LV, left ventricular; CABG, coronary artery bypass graffing.

We also propose that the unraveling of these mechanisms will potentially lead to the discovery of a generalized process of organ regeneration in response to ischemia.

Timing of Autologous Cell Transplantation Perhaps one of the most pressing issues that can be addressed by animal studies is related to the timing of autologous cell transplantation after myocardial infarction. The goal of these studies needs to be whether autologous cells can engraft into dilated and scarred myocardium, and, if engraftment is successful. The second is whether left ventricular function is augmented. To date, there are no data to suggest that autologous cell transplantation with either skeletal myoblasts or stem cells will improve the left ventricular function of the typical patient who had a last myocardial infarction 1 to 2 years ago and who now presents with signs and symptoms of congestive heart failure without evidence of ongoing ischemia. If animal studies demonstrate an inability of autologous skeletal myoblast or stem cells to engraft into dilated and scarred myocardial tissue, studies will be needed to determine whether it is possible to induce cell engraftment. For example, perhaps reestablishing in the damaged myocardium the signaling pathways for stem cell homing that are

upregulated soon after myocardial infarction could lead to improved cell engraftment.

Gene- and Cell-Based Therapies Additional studies in animals should focus on complementary strategies to autologous cell transplantation that may lead to optimization of left ventricular function after autologous cell transplantation. Because skeletal myoblasts need to be expanded in vitro before transplantation, this offers an ideal opportunity to introduce expression vectors that encode for angiogenic factors such as VEGF and angiopoietin or for factors that could reverse endothelial dysfunction including inducible or endothelial nitric oxide synthase. The patchy nature of skeletal myoblast engraftment suggests that the expression of such proteins could be spread throughout the infarct zone.

Clinical Trials Initial clinical trials to study the potential role of autologous skeletal myoblast transplantation in the treatment of ischemic cardiomyopathy are ongoing. The focus of these trials is to perform autologous skeletal myoblast transplantation as an adjunct to coronary artery bypass grafting (CABG). By focusing on patients who are to un-

30 dergo elective CABG, the negative effects of the time it takes for skeletal myoblast expansion are somewhat eliminated. This clinical trial design is optimal for studying the long-term safety profile of autologous skeletal myoblast transplantation. If this treatment strategy in humans proves to be arrhythmogenic, it should be apparent in these studies. Furthermore, this population offers the field the ability to discover whether any technical concerns regarding skeletal myoblast expansion or cell injection exist. The danger of this approach is that it will be difficult to demonstrate clear clinical efficacy related to the skeletal myoblast transplantation because coronary revascularization with or without surgical left ventricular remodeling being performed simultaneously. It is critical that proponents of autologous cell transplantation help the community of cardiologists and cardiac surgeons (as well as the lay press and industry) understand that the goal of these early trials is not to demonstrate efficacy. There has been significant interest and enthusiasm from many interested parties in the potential to regenerate damaged myocardium; it would be unfortunate if the momentum gained in the field over the last 2 to 3 years were lost owing to unrealistic expectations. Unfortunately, the initial reports of bone marrow transplantation have been more frequent in the lay press than the peer-reviewed literature. At the recent American Heart Association Annual Meeting, 3 reports of autologous stem cell therapy were presented. Hamano et al reported their experiences with the injection of a mononuclear enriched preparation of autologous bone marrow as an adjunct to CABG in 5 patients. They found a significant benefit to myocardial perfusion in 3 of the 5 patients studied.2 Importantly, the benefits seen in this study cannot be separated from the benefit of CABG. To date, no data have been presented on the use of G-CSF or stem cell factor in patients after acute myocardial infarction. As discussed above, although this strategy may offer significant benefit, the unknown consequences of the granulocytosis, including potentially increased tissue destruction and leukostasis, suggest that studies focused on obtaining further preclinical data are warranted. It would be reasonable to expect that once clinical safety is determined, the first clinical trial designed to demonstrate efficacy of cell transplantation will focus on the correlation between cell

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dosing and left ventricular function. This type of trial design early in efficacy trials will be necessary to begin to optimize future protocols. We anticipate that before initiation of efficacy trials in humans, animal studies will have determined whether there exists an optimal time for autologous cell transplantation after myocardial infarction. The development of a system for quantifying cell engraftment in vivo is yet another challenge that needs to be addressed. Cell engraftment has to be determined in an in vivo system, and, to date, quantifying engraftment requires ending the experiment. Further, no clinical or experimental system for the quantification of cell engraftment in a human population exists. As we begin to plan to test the efficacy of autologous cell transplantation in clinical populations, having the ability to correlate the level of cell engraftment with clinical outcome will be necessary.

Conclusion It was once believed that tissue damaged during a myocardial infarction was permanently lost. The concept that myocardial tissue is regenerated at low levels as a physiological response to injury sets the foundation for stem cell mobilization or transplantation to amplify this process. It is a real tribute to the visionaries that began this work a short 10 years ago. Since myocardial infarction remains the leading cause of death and disability, further intensive basic and clinical investigation of autologous cell transplantation is clearly warranted.

References 1. Menasche P, Hagege AA, Scorsin M, et al: Myoblast transplantation for heart failure. Lancet 357 (9252): 279-280, 2001 2. Hamano K, Nishida M, Hirata K, et al: Preliminary results of clinical trials of therapeutic angiogenesis achieved by the implantation of self bone marrow cells for ischemic heart disease. Circulation 104:1169, 2001 3. Koh GY, Klug MG, Soonpaa MH, et al: Differentiation and long-term survival of C2C12 myoblast grafts in heart. J Clin Invest 92:1548-1554, 1993 4. Li RK, Jia ZQ, Weisel RD, et al: Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol 31:513-522, 1999 5. Kocher AA, Schuster MD, Szabolcs MJ, et al: Neovascularization of ischemic myocardium by human

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