stem cell therapy for the heart november . december 2004 293

www.lejacq.com ID:4276

Loren E. Wold, PhD;1 Wangde Dai, MD;1 Casilde Sesti, PhD;1 Sharon L. Hale, BS;1 Joan S. Dow, BS;1 Bradley J. Martin, PhD;2 Robert A. Kloner, MD, PhD1

From The Heart Institute, Good Samaritan Hospital, Cardiovascular Division, University of Southern California, Los Angeles, CA;1 and Osiris Therapeutics, Inc., Baltimore, MD2

Address for correspondence: Robert A. Kloner, MD, PhD, The Heart Institute, Good Samaritan Hospital, 1225 Wilshire Boulevard, Los Angeles, CA 90017E-mail: rkloner@goodsam.orgManuscript received October 19, 2004; accepted October 29, 2004

The field of stem cell research, par-ticularly as it relates to the car-

diovascular system, has exploded in recent years with numerous exciting experimental and clinical studies uti-lizing various types of stem cells. The purposes of this review are to summa-rize some of the major findings related to stem cell therapy in the heart and to delineate potential limitations of the various types of stem cells. The current state of the literature makes it clear that stem cell therapy is progressing rapidly to the clinical setting with early reports showing promising results.

Stem cells, a group of undifferenti-ated cells capable of long-term self-renewal, can be derived from a num-ber of host organs such as bone mar-row (mesenchymal, endothelial pre-cursor, and hematopoietic stem cells [HSCs]), adipose tissue, and skeletal muscle (myofibroblasts). These cells exhibit tissue-directed differentiation in such a way that stem cells isolated from the liver and reinjected into the

liver become hepatocytes, whereas the same cells, if injected into the heart, become cardiomyocytes.1 Stem cells have been used to regenerate neural tissue, skeletal muscle and bone,2 and recently the myocardium.3,4

Many challenges and questions remain to be answered concerning the use of stem cells in the regeneration of the myocardium. What is the maxi-mum therapeutic potential of stem cells? Are these cells able to home to sites of injury? What is the proper route of administration and/or combi-nation of cell types needed to ensure maximum regeneration of the myo-cardium? We will attempt to address these questions and present data from our group as well as others showing that the therapeutic use of stem cells is an issue that will not soon leave the biomedical research arena.

Types of Stem CellsBone Marrow-Derived Stem Cells. The bone marrow appears to contain

three types of stem cell populations: hematopoietic, endothelial progeni-tor, and mesenchymal (mesenchymal). The HSCs produce all of the types of formed elements of blood in the body. Endothelial progenitor cells differenti-ate into endothelial cells and have been isolated from circulating blood. Mesenchymal stem cells (MSCs) can be isolated from adult bone marrow and appear to have the potential for multiple lineages of differentiation.

HSCs are isolated from either the bone marrow or blood and are responsible for the constant renewal of blood cells. They exhibit constant self-renewal, differentiate into a vari-ety of specialized cells, mobilize out of the bone marrow into circulating blood, and can undergo apoptosis. HSCs have been used as a routine cancer therapy and treatment for dis-orders of the blood and the immune system. HSCs have also been shown to become other types of cells, includ-ing muscle, blood vessels, and bone;

R E V I E W P A P E R

Stem Cell Therapy for the Heart

Cellular cardiomyoplasty is an expanding field of research that involves numerous types of immature cells administered via several modes of delivery. The purpose of this review is to investigate the benefits of different types of cells used in stem cell research as well as the most efficient mode of delivery. The authors also present data showing that stem cells isolated from bone marrow are present at both 2 weeks and 3 months after engraftment in a myocardial infarction. These cells express muscle markers at both time points, which suggests that they have begun to differentiate into cardiomyocytes. Several ques-tions must be answered, however, before stem cells can be used routinely in the clinic. Once these questions have been

addressed, the use of stem cells in clinical practice can be realized. (CHF. 2004;10:293–301) ©2004 CHF, Inc.

Congestive Heart Failure (ISSN 1527-5299) is published bimonthly (Feb., April, June, Aug., Oct., Dec.) by CHF, Inc., Three Parklands Drive, Darien, CT 06820-3652. Copyright ©2004 by CHF, Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publishers. The opinions and ideas expressed in Congestive Heart Failure do not necessarily reflect those of the Editor and Publisher. For copies in excess of 25 or for commercial purposes, please contact Sarah Howell at showell@lejacq.com or 203.656.1711 x106.

stem cell therapy for the heart november . december 2004294

however, they are hard to distinguish from ordinary white blood cells due to their appearance and behavior in culture. A study by Murry et al.5 has raised a cautionary note about the use of these cells. HSCs were injected into normal and infarcted mice hearts and tracked using genetic techniques. This group showed that HSCs were not able to trans-differentiate into cardiomyocytes, which suggests that benefits observed in clinical trials may be due to factors other than formation of new muscle, a topic which is likely to remain controversial.

MSCs are a mixed cell popula-tion that generate bone, cartilage, fat, fibrous connective tissue, and the reticular network that supports blood cell formation. When cultured, MSCs can maintain a phenotype that is sta-ble and undifferentiated. There is no consensus, however, on the proper phenotype of a MSC and no adequate marker to allow the quantification of a purified population of cells.6 Testing in experimental ovine models has shown that undifferentiated human MSCs undergo site-specific differentiation into functional cardiac muscle,7 and can avoid destruction by the host immune system. Therefore, MSCs iso-lated from allogeneic bone marrow have potential clinical utility due to their lack of immunogenicity and ease of culture.6

Kudo et al.3 injected two types of bone marrow-derived stem cells into the injured myocardium, fresh and directly extracted Lin– cells and cul-tured MSCs, and evaluated the effect of cell engraftment on infarct size and the degree of fibrosis in the injured heart. Lin– cells, which by definition are enriched in HSCs,8 engrafted into the infarct, differentiated into car-diomyocytes and vascular cells, and reduced both infarct size and degree of fibrosis within the infarct. Orlic et al.9 showed that transplanted Lin–-c-kit+ bone marrow cells regenerated myo-cardium, endothelium, and smooth muscle cells, as well as improved cardi-ac performance in mice with coronary artery occlusion.

Myocytes with phenotypes similar to fetal ventricular cardiomyocytes have been produced by treating MSCs with 5-azacytidine, a demethylating agent.10 Tomita et al.11 transplanted fresh bone marrow cells, cultured MSCs, and cul-tured MSCs treated with 5-azacyti-dine into scars created by cryo-injury. In these studies, they observed that all three groups exhibited muscle-like phenotypes with expression of the car-diac-specific markers troponin-I and myosin heavy chain within the injured tissue, but only the 5-azacytidine group had improved developed pressures vs. control. Taken together, these results represent intriguing advances in the therapeutic use of bone marrow cells in the injured heart. The most effica-cious route of delivery and number of cells needed, however, is still unknown and requires further study.

MSCs have been shown to develop a fibroblast-like morphology within areas of myocardial infarction (MI) and a cardiomyocyte-like phenotype in the normal myocardial tissue adjacent to the infarct. The border that exists between the normal and injured myo-cardial tissue may represent an excel-lent environment for engraftment and differentiation into cardiomyocyte-like cells.9,12,13 Therefore, several investi-gators are determining the feasibil-ity of injecting stem cells directly into this border region and examining the effect on cell viability and differen-tiation capacity using magnetic reso-nance imaging technology and other techniques.12 MSCs transfected with Akt1 were able to restore myocardial volume at a rate four-fold greater than those cells transfected with the report-er gene lacZ. Thus Akt1-enhanced MSCs repaired infarcted myocardium, prevented remodeling, and almost completely normalized cardiac perfor-mance in a rat model of MI.14

Autologous bone marrow cells have been used to circumvent the immuno-logic rejection present when cells from one host are injected into a differ-ent recipient. Kuethe et al.15 injected autologous bone marrow cells via a balloon catheter into the infarct of

patients and found that left ventricu-lar ejection fraction (LVEF), regional wall motion in the infarct zone, con-tractility index assessed with dobuta-mine stress echocardiography, coro-nary blood flow reserve, and maximal oxygen uptake were all unchanged in cell-treated patients, suggesting that autologous bone marrow cells given via a balloon catheter directly into the infarct zone were not beneficial in regenerating the myocardium. Other groups, however, have shown that autologous bone marrow cells are ben-eficial in improving heart function in animals16 and humans.17–19

Bone marrow cells are a particularly attractive type of stem cell for research use because they can be obtained read-ily from animal and human sources and they have been shown to develop into several types of cells within the body. Their replication ex vivo, how-ever, is limited; there are no adequate markers to identify them, and the most efficacious mode of delivery is still in question. Therefore, it is important to answer some of these questions before bone marrow stem cells can be used in the clinical setting.

Peripheral Blood-Derived Stem Cells. Cells isolated from adult human peripheral blood have been shown to differentiate into nonhematopoi-etic tissues, such as epithelial cells of the gastrointestinal track and skin.20 One study found that CD34+ cells isolated from adult peripheral blood and injected via the tail vein in mice were able to home to the infarct zone and differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. In another set of studies, the same cell type was injected into the left ventricular (LV) cavity of mice without experimental MI; the cells did not migrate to the heart.20 These stud-ies show that adult human peripheral blood-derived stem cells can home to the site of injury and differentiate into different cell types, including cardio-myocytes. Interestingly, the adminis-tration of ascorbic acid has been shown to greatly improve the efficiency with

Congestive Heart Failure (ISSN 1527-5299) is published bimonthly (Feb., April, June, Aug., Oct., Dec.) by CHF, Inc., Three Parklands Drive, Darien, CT 06820-3652. Copyright ©2004 by CHF, Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publishers. The opinions and ideas expressed in Congestive Heart Failure do not necessarily reflect those of the Editor and Publisher. For copies in excess of 25 or for commercial purposes, please contact Sarah Howell at showell@lejacq.com or 203.656.1711 x106.

stem cell therapy for the heart november . december 2004 295

which peripheral blood-derived stem cells21 home to the site of injury.

Ogawa et al.22 investigated the effect of injection of peripheral blood-derived cells into a patient with dilated car-diomyopathy. The cells were isolated from the brother of the recipient and administered to the recipient patient. Three months after cell therapy, cardio-megaly was attenuated, LV volume was decreased, and LVEF was increased. After 10 months, however, the benefit of the cell therapy was lost. This study shows that peripheral blood-derived cells may be beneficial in the clini-cal setting; however, the timeframe of administration and effective cell num-bers must be further investigated.

Embryonic Stem Cells. Embryonic stem (ES) cells are the most primitive of all populations of stem cells. They develop as the inner cell mass of the human blastocyst at Day 5 after fer-tilization.6 At such an early stage, this type of stem cell has a vast amount of developmental potential and can give rise to cells of all three embryonic germ layers. When cultured in the appropri-ate media and culture conditions, ES cells can undergo an unlimited number of cell doublings and retain the capac-ity to differentiate into any cell type, including cardiomyocytes.23,24 Human ES cells, however, have a much lower capacity to convert into cardiomyo-cytes than those of mice.25,26

ES cells that were injected into the scar of an MI were able to reduce the size of the infarct as well as improve ven-tricular function and contractility in a rat model 6 weeks posttransplantation.26 ES cells are also thought to promote the release of vascular endothelial growth factor, which functions to protect the heart from damage.26 Another group has shown that ES cells can differentiate into cardiomyocytes and portray struc-tural and functional properties consis-tent with early cardiac tissue.27

ES cells are currently not approved for use in humans, due particularly to the requirement of immunosuppres-sion to avoid rejection of the trans-planted cells28 and to the development

of teratoma.29 The moral, ethical, and political debates about the use of ES cells in stem cell research have limited the ability to do research in this arena in the United States and have prompt-ed persons in the research community to seek other sources of stem cells.30

Umbilical Cord Blood Cells. Stem cells can be isolated from umbilical cord blood and have been shown to be rich in progenitor cells with character-istics of proliferation.31–33 These cells are easily obtainable, have the poten-tial for enhanced self-renewal and dif-ferentiation, and can be expanded in vitro34–36 and stored for use at a later time.37 There is less risk of immuno rejection with the use of umbilical cord blood-derived stem cells.38,39 CD34+ cells isolated from human umbilical cord blood have been shown to dif-ferentiate into mature endothelial and muscle cells and induce neovascular-ization in ischemic skeletal muscle32,35; however, their direct role in the heart is not known at this time.

Leor et al.40 showed that infused CD133+ cells derived from human umbilical cord blood could migrate, colonize, and survive in the myocar-dium of infarcted rats. Some of the cells infused into the area of damage within the heart were able to trans-differentiate and participate in neo-vascularization. They also prevented scar thinning and dysfunction of the left ventricle.40 Research with human umbilical cord blood-derived stem cells is still in its infancy; however, the results obtained so far have exhibited strong support for further delineating the potential role of these cells in the repair of a damaged myocardium.

Muscle-Derived CellsSkeletal muscle, unlike other types of muscle, is able to regenerate and repair itself after injury due to the presence of immature satellite cells or myoblasts (although, by definition, myoblasts are not a true stem cell since their lineage is committed). These cells lie quies-cent in normal skeletal muscle but, upon injury, reenter the cell cycle.

They are capable of fusing with other myoblasts or surrounding cells to pro-duce functional skeletal muscle. In vitro, skeletal myoblasts fuse to form contracting myotubes, which express characteristic gene patterns of skel-etal muscle including expression of the skeletal muscle transcription factor myogenin.41–43 Scorsin et al.44 showed that myoblasts were as effective as fetal cardiomyocytes in improving car-diac function, which they assessed via echocardiography in a rat model of coronary artery ligation. When transplanted, the myoblasts fused with myotubes, which was confirmed by staining for myosin heavy chain.45 The myoblasts, however, failed to estab-lish connections with the surrounding cardiomyocytes. It was also reported that a strikingly linear relationship was seen between the number of myo-blasts transplanted and the improve-ment in LVEF.46 Skeletal myoblasts have been shown to strongly resist ischemia, providing increased survival and functional engraftment in areas of poor oxygenation and blood flow, something often seen in patients with congestive heart failure and coronary artery disease.47

A recent study by Rubart et al.48 showed that skeletal myoblast cells expressing an enhanced green fluo-rescent protein, when transplanted into the hearts of nontransgenic mice, exhibited action potential-induced calcium transients in synchroniza-tion with adjacent cardiomyocytes. The duration of the calcium transient was in many cases indistinguishable from the resident cardiomyocytes. In a parallel study, myoblasts expressing enhanced green fluorescent protein were transplanted into hearts express-ing a cardiomyocyte-restricted β-gal reporter gene. It was observed that a small population of myocytes expressed both enhanced green fluorescent pro-tein and the β-gal reporter gene as well as connexin 43, suggesting that the transplanted myoblasts had coupled with the myocytes.

In a rat model of MI, skeletal myo-blast implantation resulted in the

Congestive Heart Failure (ISSN 1527-5299) is published bimonthly (Feb., April, June, Aug., Oct., Dec.) by CHF, Inc., Three Parklands Drive, Darien, CT 06820-3652. Copyright ©2004 by CHF, Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publishers. The opinions and ideas expressed in Congestive Heart Failure do not necessarily reflect those of the Editor and Publisher. For copies in excess of 25 or for commercial purposes, please contact Sarah Howell at showell@lejacq.com or 203.656.1711 x106.

stem cell therapy for the heart november . december 2004296

formation of viable grafts that decreased ventricular remodeling and increased cardiac function, particularly after exer-cise.49 Ghostine et al.45 injected autolo-gous skeletal myoblasts, which were harvested and expanded in culture, into infarcted myocardium of sheep. These myoblasts were able to colonize the infarcted area and improve LV systolic function, which correlated with the quantity of implanted myoblasts.50 Tambara et al.51 injected skeletal myo-blasts into the infarct of male Lewis rats and showed that freshly-isolated neo-natal skeletal myoblasts can survive in the host and fully replace the infarcted myocardium. They also showed that these cells were able to reverse the LV remodeling resulting from MI. Other groups have also shown that myocar-dial function is improved with skeletal myoblast transplantation.52–55 Reinecke et al.,56 however, showed that skeletal-muscle derived satellite cells were not able to transdifferentiate into cardio-myocytes after grafting into the infarct of normal syngeneic rats. It has also been shown that skeletal myoblasts can be delivered to the heart via either direct intramyocardial or intraarte-rial injection.52,57 Myoblasts represent an exciting possibility in cell therapy research: they can be readily obtained from a muscle biopsy, returned to the patient after in vitro expansion with-out the risk of immunorejection,29 and do not carry the ethical concerns of human ES cells. Several hurdles, how-ever, must be overcome. It must be definitively established whether skeletal myoblasts have the capacity to form connections with the surrounding car-diomyocytes and become functional, which has been shown by one group to be possible48 and improbable by another group.11 It is imperative that a connec-tion occur in order for the muscle cells to work in syncytium. In several studies using transplanted skeletal myoblasts, a dramatic increase in ventricular arrhythmias and premature deaths were observed in both animal models and in phase I clinical trials.58–61 Therefore, in some ongoing phase I clinical tri-als, it is mandatory that patients have

an internal cardioverter-defibrillator implanted.61 Clinical trials using the transplantation of skeletal myoblasts have been initiated in Europe and the United States with preliminary obser-vations showing that ejection fraction is improved in patients with a previous MI.62 The potential for lethal arrhyth-mias, however, must be overcome to justify the use of myoblasts in the heart. Other problems exist in the use of skeletal myoblasts to replace existing myocardium. Skeletal muscle is not his-tologically similar to cardiac muscle and does not spontaneously or rhythmically beat like that of its cardiac counterpart. Skeletal muscle also may to tetanize or fatigue, unlike cardiac muscle. Several questions remain before skeletal myo-blasts can be actively used in the clini-cal setting. Once these questions have been addressed, however, the possibili-ties for myoblasts in stem cell therapy are promising.

The possibility of the heart regen-erating itself has been proposed by Beltrami et al.63 A subpopulation of resident cardiac stem cells, identified to be Lin–-c-kit+ has been found in areas of injury in the heart, suggesting that they are mobilized and become activated upon injury.64 One potential activator of these cells is the rapid induction of stem cell factor after myocardial ischemia.65 The origin and function of these cells is unknown, however, and must be investigated further to ascertain their potential therapeutic properties.

Modes of DeliveryThe issue of how to optimize delivery of the stem cell to the site of injury is an ongoing and active area of investiga-tion. If the cells are not able to reach the site of myocardial injury they will not be able to improve heart function. Several modes of delivery have been investigated, including direct intramus-cular injection into the heart muscle, IV administration through the jugular vein or other vein, and transendocardial and trans-epicardial injection into the endo-cardial or epicardial regions of the heart, as well as intracoronary injection.

Direct intramuscular injection into the damaged heart muscle has been used extensively in stem cell research. The main advantage of this means of delivery is the ability to deliver the cells directly to the damaged area. It requires surgical procedures that allow direct visualization of the heart, which can be time-consuming, particularly in the clinical setting. It has, however, been used with various cell types in both basic biomedical research66 and in the clinical setting67 with beneficial effects on the heart. Autologous bone marrow cells have also been inject-ed intramyocardially and produced improvement in myocardial perfusion in three out of five patients.68

IV administration of cells is the easiest mode of delivery; however, the main disadvantages are the length of time that it takes for the cells to migrate to the site of the injury and the ability of the cells to survive during this time frame. If these problems can be circum-vented, then the ability to inject cells directly into the vasculature would save time and money in the clinical setting and not require the patient to undergo open-heart surgery. The ability of the cells to home to the site of injury is a topic of intense research and will be investigated more thoroughly in the next section of this review.

The transendocardial and trans-epicardial mode of delivery of stem cells has been used in large animal experiments69 as well as in the clinical setting.70 The main advantages to these types of delivery are the direct visual-ization of injection to the affected area and an even distribution of the cells. The safety and feasibility of catheter-based transendocardial injection of stem cells was recently demonstrated in a large animal study.71 Currently, the clinical use of catheter-based tran-sendocardial injection is limited to one injection system, using electrome-chanical mapping to generate a three-dimensional LV reconstruction before the injection; however, this technique may induce arrhythmias including ventricular premature beats and ven-tricular tachycardia.72 The injection

Congestive Heart Failure (ISSN 1527-5299) is published bimonthly (Feb., April, June, Aug., Oct., Dec.) by CHF, Inc., Three Parklands Drive, Darien, CT 06820-3652. Copyright ©2004 by CHF, Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publishers. The opinions and ideas expressed in Congestive Heart Failure do not necessarily reflect those of the Editor and Publisher. For copies in excess of 25 or for commercial purposes, please contact Sarah Howell at showell@lejacq.com or 203.656.1711 x106.

stem cell therapy for the heart november . december 2004 297

of autologous bone marrow cells has been performed trans-endocardially as a part of several pilot and phase I studies; however, caution must be exercised since safety and feasibility data are still pending.72

A less invasive technique avail-able for cell delivery is the intra-coronary approach. In this technique, cells are delivered to the heart via an over-the-wire balloon catheter.73 This mode of delivery appears to be superior to IM and IV administration in clinical practice because the cells must flow through the infarct and peri-infarct regions during the first passage. It also allows for a more even distribution of cells throughout the infarct region,29 potentially explain-ing the lack of arrhythmia generation after transplantation. Strauer et al.73 administered bone marrow cells in the clinical setting via intracoronary injection and found that infarct size, coupled with an increase in stroke volume, was reduced after 3 months. Other groups have also shown that the intracoronary injection of stem cells is beneficial to heart function as it adequately delivers the cells to the desired area.74,75 Still unclear with this and all other modes of delivery are the appropriate number of cells needed as well as the time frame of administra-tion required to adequately deliver the most effective number of cells to the damaged myocardium.

Several groups have shown that pro-ducing engineered functional cardiac muscle cells is feasible as well as effi-cacious.76–80 Embryonic chick cardiac myocytes cultured in collagen gels dis-played characteristic physiologic and pharmacologic responses to stimuli.79 Rat ventricular cardiomyocytes cultured on polystyrene microcarrier beads in bioreactors formed a three-dimensional, spontaneously-contracting aggregates of cardiac cells.76 Another group has shown that culturing neonatal rat cardiomyo-cytes on polyglycolic acid scaffolds in bioreactors resulted in contracting three-dimensional tissue81 that functioned and resembled normal cardiac muscle.77 These data show that the ability to

engineer cells in the laboratory setting is very much a reality and will continue to be important as people involved in stem cell research continue to delineate the proper type and mode of delivery of the cells to the injured myocardium.

Cell Homing: A Likely EventIt has been shown that stem cells injected via the bloodstream are able to localize in the area of an MI82,83 without the exogenous introduction of recruit-ing agents; scientists have termed this phenomenon “cell homing.” It is highly likely that the damaged tissue releases some form of a signal (possibly an inflammatory cytokine) to attract the injected cells to an area of injury. Upon receiving this signal, the stem cells are able to work their way throughout the bloodstream, arrive at the site of injury, and begin the repair process.

As part of a pilot study evaluating different routes of cell delivery, we evaluated the potential of rat mesen-chymal stem cells to home to the site of myocardial ischemic damage. Rat allogeneic bone MSCs were expanded in culture and labeled with DAPI (4ʹ,6-diamidino-2-phenylindole-2-hydro-chloride) and DiI (1,1ʹ-dioctadecyl-

3,3,3ʹ,3ʹ-tetramethylindocarbocyanine perchlorate) fluorescent markers. The cells were then injected via IV into rats 2 days after experimental MI. Two weeks later, the hearts were harvested and frozen sections were assessed by fluorescent microscopy. Donor cells were identified in the hearts of four of five rats (Figure 1), which shows that the cells could target the site of injury and engraft when delivered systemi-cally. These results also suggest that the injected cells are able to survive and potentially differentiate into car-diac cells, even when injected into the vasculature distal to the MI.

Recent Experience With Fetal, Neonatal, and Stem CellsStudies in our laboratory have focused on the ability of neonatal, fetal, and bone marrow-derived mesenchymal stem cells to graft into the infarct region and contribute to cardiac func-tion. Muller-Ehmsen et al.84 showed that neonatal rat cardiomyocytes were able to engraft into the infarct of syn-geneic Fischer 344 rats and survive for at least 6 months after ligation of the coronary artery. In the treated

Figure 1. Donor cells administered via IV through the tail vein shown here in 2-day-old myocardial infarctions of rats and observed under fluorescent microscopy (original magnification 400×). Cells are labeled with DiI (red marker) and DAPI (blue marker). DiI and DAPI are expanded in the text.

Congestive Heart Failure (ISSN 1527-5299) is published bimonthly (Feb., April, June, Aug., Oct., Dec.) by CHF, Inc., Three Parklands Drive, Darien, CT 06820-3652. Copyright ©2004 by CHF, Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publishers. The opinions and ideas expressed in Congestive Heart Failure do not necessarily reflect those of the Editor and Publisher. For copies in excess of 25 or for commercial purposes, please contact Sarah Howell at showell@lejacq.com or 203.656.1711 x106.

stem cell therapy for the heart november . december 2004298

rats, the wall of the left ventricle was significantly thicker, and the rat hearts exhibited an improved ejection frac-tion (assessed by contrast angiography) and reduced paradoxical systolic bulg-ing of the infarct. Reffelmann et al.85 showed that after 4 weeks, transplanta-tion of neonatal cardiomyocytes into the infarct of Fischer rats resulted in an improvement of regional myocar-dial blood flow as well as decreased LV systolic and diastolic volumes, coupled with thickening of the infarct and lower infarct expansion index.

Our laboratory has also investigated the role of fetal cells on ventricular

remodeling and function in the heart of rats. Yao et al.86 injected fetal cardiac cells into the 1-week-old infarcts of Fischer rats and found that over the course of 10 months the transplanted cells were able to increase infarct wall thickness, reduce LV dilatation, and improve LVEF. Yao et al.87 also injected fetal cardiac cells into the pericardium of infarcted adult synge-neic rats and showed that the cells sur-vived and began differentiating as well as expressing α-sarcomeric actin and connexin 43. These data suggest that fetal cardiomyocytes are able to survive in the damaged organ and differentiate into viable cardiac cells.

In a pilot study, we injected bone marrow-derived MSCs into the scar formed from 1-week-old infarcts in female Fischer rats and assessed hemo-dynamic and histological changes in the infarcted hearts 2 weeks after the injection of the cells. At the comple-tion of the study, contrast angiography was performed, and some hearts were snap frozen and assessed for histo-logical differences in the expression of markers of the cardiomyocyte phe-notype by Osiris Therapeutics, Inc. There were no differences in heart rate, blood pressure, or ±dP/dt between the groups; contrast angiography revealed no differences in ejection fraction or postmortem diastolic or systolic LV volumes. The cells were evident by hematoxylin and eosin staining (Figure 2A) and were positive for α-sarco-meric actin (Figure 2B). Trichrome staining revealed a large infarction (Figure 3A) with cells present (DAPI and DiI convergent staining, Figure 3B). The muscle marker α-Actinin (Figure 3C) was not expressed. These results suggest that MSCs injected into the infarct are able to survive and begin differentiating in the region of the infarct 2 weeks after engraftment where they express some, but not all, muscle markers. A recent pilot study in our laboratory has investi-gated whether these MSCs are able to survive for 3 months in the infarct of rats. We show in Figures 4A–C and 5A–C that the cells are still present in the infarct after 3 months and have begun to differentiate, as evidenced by positive staining for α-Actinin (Figure 4C) and MF20 (Figure 5C), a marker for myosin heavy chain. These data coupled with the 2-week data show that MSCs can survive for up to 3 months after an infarct and have the potential to regenerate the damaged myocardium.

Clinical Studies Involving Stem CellsNumerous phase I clinical studies have been conducted and are being planned to investigate the use of stem cells in the clinical setting. Several reviews have

Figure 2A. Hematoxylin and eosin staining (original magnification 400×). 2B. Staining for α-sarcomeric actin (original magnification 400×) in the infarct region of female syngeneic Fischer rats 2 weeks after IM injection of cells directly into the infarct.

Congestive Heart Failure (ISSN 1527-5299) is published bimonthly (Feb., April, June, Aug., Oct., Dec.) by CHF, Inc., Three Parklands Drive, Darien, CT 06820-3652. Copyright ©2004 by CHF, Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publishers. The opinions and ideas expressed in Congestive Heart Failure do not necessarily reflect those of the Editor and Publisher. For copies in excess of 25 or for commercial purposes, please contact Sarah Howell at showell@lejacq.com or 203.656.1711 x106.

stem cell therapy for the heart november . december 2004 299

listed ongoing or recently completed tri-als.29,88 Perin et al.17,89 recently reported that they have injected autologous bone marrow mononuclear cells using elec-tromechanical mapping into areas of ischemic myocardium in patients with endstage ischemic cardiomyopathy89 and heart failure17 and have seen a ther-apeutic effect with improved myocardial perfusion and exercise capacity, at 6 and 12 months,89 as well as increased global LV function.17 Numerous new studies are actively recruiting patients or will

begin shortly. Dr. Douglas Losordo of Caritas St. Elizabeth’s Medical Center of Boston, MA will conduct a phase I clinical trial to assess the efficiency of

autologous bone marrow cells (CD34+) administered via a catheter to the dam-aged regions of the heart. Patients

Figure 3A. Trichrome staining of a rat showing a large infarct region (original magnification 100×). 3B. Convergence staining of DiI (red) and DAPI (blue) in the infarct region (original magnifi-cation 400×). 3C. Negative staining of the infarct region for the muscle mark-er α-Actinin (original magnification 400×). DiI and DAPI are expanded in the text.

Figure 4A. DAPI (red; original magni-fication 400×). 4B. α-Actinin (green; original magnification 400×). 4C. Convergent staining of DAPI and α-Actinin (yellow; original magnification 400×) in the heart of rats that received Mesenchymal stem cells (MSCs) 1 week after experimental myocardial infarc-tion and assessed at 3 months. DAPI is expanded in the text.

Figure 5A. DAPI (red; original mag-nification 400×). 5B. MF20 (green; original magnification 400×). 5C. Convergent staining of DAPI and MF20 (yellow; original magnification 400×) in the heart of rats that received Mesenchymal stem cells 1 week after experimental myocardial infarction and assessed at 3 months. DAPI is expanded in the text.

Congestive Heart Failure (ISSN 1527-5299) is published bimonthly (Feb., April, June, Aug., Oct., Dec.) by CHF, Inc., Three Parklands Drive, Darien, CT 06820-3652. Copyright ©2004 by CHF, Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publishers. The opinions and ideas expressed in Congestive Heart Failure do not necessarily reflect those of the Editor and Publisher. For copies in excess of 25 or for commercial purposes, please contact Sarah Howell at showell@lejacq.com or 203.656.1711 x106.

stem cell therapy for the heart november . december 2004300

must have class III or IV angina and have total occlusion of an epicardial coronary artery with a high risk for percutaneous coronary angioplasty. Bioheart, Inc. of Weston, FL is currently recruiting patients for a study entitled the Myogenesis Heart Efficiency and Regeneration Trial (MYOHEART), which will assess the ability of trans-planted autologous skeletal myoblasts to repair postinfarct deterioration of cardi-ac function in patients with congestive heart failure. Patients admitted into the study must have an implantable cardio-verter-defibrillator in place and show New York Heart Association symptom class II or III and have a LVEF between 20%–40%. Bioheart, Inc. has also pro-posed a study to investigate the safety of autologous skeletal myoblast implanta-tion via epicardial injection during coro-nary artery bypass graft surgery and its

effect on regional myocardial function. These patients must also already have an implantable cardioverter-defibrilla-tor in place as well as a planned coro-nary artery bypass graft procedure for revascularization with a LVEF between 20%–40%. The National Heart, Lung and Blood Institute is also sponsoring a study investigating the effect of exercise on stem cell mobilization and heart function in patients undergoing cardiac rehabilitation. In this study, the level of endogenous endothelial progenitor cells will be monitored in patients currently involved in cardiac rehabilitation with concomitant repetitive exercise pro-grams (www.clinicaltrials.gov).90 Each of these studies is designed to determine the most efficient type of stem cell and mode of delivery as well as time of therapy to maximize the benefits of cel-lular cardiomyoplasty.

Conclusions and Future DirectionsThe field of stem cell research has exploded in recent years; several types of cells are currently being assessed for their beneficial effects on myocardial damage resulting from MI. We have presented data concerning several types of cells and their role in the ameliora-tion of heart dysfunction; however, the most efficacious type of stem cell and the most beneficial mode of delivery remain unclear. These questions must be answered to optimize the eventual use of stem cells for the treatment of a wide array of cardiovascular diseases.

Acknowledgments: The authors would like to acknowledge the generous sup-port from the National Institutes of Health (1R01-HL073709–01 and 1RO1-HL61488) as well as the expert technical assistance of Osiris Therapeutics, Inc.

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Congestive Heart Failure (ISSN 1527-5299) is published bimonthly (Feb., April, June, Aug., Oct., Dec.) by CHF, Inc., Three Parklands Drive, Darien, CT 06820-3652. Copyright ©2004 by CHF, Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publishers. The opinions and ideas expressed in Congestive Heart Failure do not necessarily reflect those of the Editor and Publisher. For copies in excess of 25 or for commercial purposes, please contact Sarah Howell at showell@lejacq.com or 203.656.1711 x106.

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Congestive Heart Failure (ISSN 1527-5299) is published bimonthly (Feb., April, June, Aug., Oct., Dec.) by CHF, Inc., Three Parklands Drive, Darien, CT 06820-3652. Copyright ©2004 by CHF, Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publishers. The opinions and ideas expressed in Congestive Heart Failure do not necessarily reflect those of the Editor and Publisher. For copies in excess of 25 or for commercial purposes, please contact Sarah Howell at showell@lejacq.com or 203.656.1711 x106.