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Cardiovascular Research 2005 65(2):305-316; doi:10.1016/j.cardiores.2004.10.037
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Copyright © 2004, European Society of Cardiology

Can stem cells mend a broken heart?

Siamak Davania,*, Frédéric Deschaseauxb, David Chalmersb, Pierre Tiberghienb and Jean-Pierre Kantelipa

aLaboratoire de Pharmacologie, EA 479, IFR 133, Faculté de Médecine, CHU Jean Minjoz, 25000 Besançon, France
bEtablissement Français du Sang Bourgogne Franche-Comté, EA2284, INSERM U-645, IFR 133, Besançon, France

* Corresponding author. Tel.: +33 3 81 66 92 26; fax: +33 3 81 66 84 83. Email address: siamak.davani{at}ufc-chu.univ-fcomte.fr

Received 23 July 2004; revised 30 September 2004; accepted 25 October 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Embryonic stem cells...
 3. Adult stem cells
 4. Clinical trials
 5. Conclusion and future...
 Acknowledgments
 References
 
Myocardial infarction is the leading cause of heart failure in developed countries. The therapeutic measures of today are usually not sufficient to prevent left ventricular remodelling as they fall short of actual replacement of necrotic cardiac myocytes. However, current insights into stem cell plasticity have opened up new perspectives for regenerating the infarcted heart. Recently, a wide range of stem/progenitor cell types have been used for cardiac cell therapy (CCT), including embryonic or foetal stem cells, myoblasts, and bone marrow stem cells. To date, the choice of stem cells has yet to be optimised. This review details recent experimental data and discusses the clinical potential of the various stem cell sources for CCT.

KEYWORDS Myocardial infarction; Stem cells; Cardiac cell therapy


   1. Introduction
Top
 Abstract
 1. Introduction
2. Embryonic stem cells...
 3. Adult stem cells
 4. Clinical trials
 5. Conclusion and future...
 Acknowledgments
 References
 
Despite the advances made in the management of acute myocardial infarction (AMI), congestive heart failure secondary to ventricular remodelling following infarction continues to be a major medical problem world-wide [1]. By definition, at the time of end-stage heart failure, the effect of drug treatment is limited, and cardiac transplantation is the only viable alternative. However, this strategy is costly and severely limited by the availability of donor hearts. In addition, complications arising from the use of immunosuppressive agents may diminish the patient's quality of life. Recently, the technique of cardiac cell therapy (CCT) has been developed. This method consists of transplanting cells into the infarcted area of the myocardium to (i) increase or to preserve the number of cardiomyocytes, (ii) to improve vascular supply, and (iii) to augment the contractile function of the injured myocardium. CCT has therefore been proposed as a treatment option for end-stage heart failure [2–4]. Currently, different types of regenerating cells are being investigated for their capacity to proliferate and differentiate into functional cardiomyocytes [5–8]. In this review, we compare the various sources of regenerating stem/progenitor cells and their effect in experimental models of myocardial injury (summarised in Table 1). In addition, we discuss the progress of clinical studies using stem/progenitor cells for infarct repair (summarised in Table 2).


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  		Table 1 Selected studies investigating experimental cardiac regeneration by stem cells

 

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  		Table 2 Comparisons of cell therapy clinical trials for treatment of acute myocardial infarction (AMI) or chronic ischemic heart failure (CIHF)

 

   2. Embryonic stem cells (ES)
Top
 Abstract
 1. Introduction
 2. Embryonic stem cells...
3. Adult stem cells
 4. Clinical trials
 5. Conclusion and future...
 Acknowledgments
 References
 
The most obvious choice for CCT are ES as they are highly proliferative and totipotent, giving rise to all tissues of the adult body (for a review, see Ref. [9]). The attractiveness of using ES cells for CCT lies in the fact that, under appropriate conditions, they can be maintained as a cell line and then induced in specialised media to differentiate into a variety of cell types. The differentiation of ES cells has been extensively reviewed elsewhere [10–12]. Indeed, it is possible in vitro to produce Purkinje-like cells, pacemaker-like cells, and atrial/ventricular-like cells of the cardiac lineage. These cells express specific ion channels and structural proteins and maintain some of the hearts electrophysiological properties [11].

Several different approaches using ES or foetal cells for experimental CCT have now been assessed. For example, successful engraftment has been reported in a noninjured murine model [13]. Two months after transplantation of transgenic embryonic cardiomyocytes into an isogenic host myocardium, intercalated disks were formed between donor and host cardiomyocytes. These cells contained complete sarcomeres and abundant mitochondria and showed characteristic binucleation of adult cardiomyocytes. Although electrical coupling was not assessed, electrocardiogram (ECG) revealed no signs of arrhythmias.

This study was extended into a dystrophic myocardial dog model, and transplanted foetal cardiomyocytes also formed intercalated disks without disruption of normal heart function [14].

Other experiments in the cryo-injured [15] or the coronary ligation model [16] showed that transplanted neonatal cardiomyocytes differentiate and express connexin-43, desmoplakin, and N-cadherin the major adhesion and gap junction proteins. This maturation resulted in intercalated disk formation and cell-to-cell electromechanical coupling with host cardiomyocytes. In addition, transplantation of foetal [17] or embryonic cardiomyocytes [18] improved myocardial function. Interestingly, it has also been demonstrated that transplanted adult cardiomyocytes survived in neither the injured nor the normal myocardium model [19].

The microenvironment plays a very precise role in the differentiation of multipotent adult stem cells [20]. All experiments should therefore be undertaken in the damaged heart model that replicates more exact conditions. In addition, transplanted cells sometimes have not been fully phenotyped to prove that they have undergone cardiomyogenic differentiation. For example, one investigation using embryonic cardiomyocytes showed that most cells conserve their initial phenotype, remain isolated, and do not differentiate into mature forms [18].

For the moment, there are little data concerning the functional (action potential and contractile properties) ability of transplanted cardiomyocytes. It is also essential to assess the outcome of such programs carefully. For example, two investigations have not detected arrhythmias in either mice [13] or dogs [14] using a surface ECG. However, this method is not ideal for detection of arrhythmias. Long-term monitoring (Holter monitors) would have been more useful in this setting. Finally, the early death of transplanted cells is a major problem of cardiac cell therapy [21] and has not been fully evaluated in these studies.

Despite the fact that all reported studies using embryonic or foetal stem cells have shown cell maturation, successful engraftment, and an improvement of myocardial function, there are several difficulties in using human embryonic stem cells. The first one is the ethical issue surrounding the use of human ES cells [22]. Secondly, ES cells are allogenic, and immunosuppressive therapy might be needed. This problem could be alleviated by establishing ES cell banks containing the full range of major histocompatibility antigens [23]. However, this may be futuristic since the establishment of human lines [24] has been more difficult than murine ones [25]. Thirdly, increased cell death due to ischemia has been observed when ES cell-derived cardiomyocytes were grafted into a normal myocardium [21]. Finally, human ES cells have also been shown to have the potential to form teratomas when injected into an immunocompromised mouse [24]. These drawbacks have lead researchers to seek alternative undifferentiated cells for CCT.


   3. Adult stem cells
Top
 Abstract
 1. Introduction
 2. Embryonic stem cells...
 3. Adult stem cells
4. Clinical trials
 5. Conclusion and future...
 Acknowledgments
 References
 
3.1. Skeletal myoblasts
Recently, there has been major interest in the use of adult stem cells because they can be used in an autologous setting, and many reports highlight their plasticity. However, there is still a great deal of controversy surrounding these claims. These include failures to reproduce results, contamination of donor samples by other rare stem cells, and the possibility that cell fusion accounts for cell switching and transdifferentiation [26].

Nevertheless, a logical substitute for CCT is to choose a source of progenitors closely related to cardiomyocytes. Skeletal myoblasts, also called satellite cells, are myocyte progenitor cells present in the basal lamina of adult muscle fibres that differentiate into mature myocytes by cell fusion. There are several advantages in using skeletal myoblasts in experimental studies: (i) they are highly resistant to ischemia [2], (ii) they can be harvested as autologous cells, avoiding the use of immunosuppressive drugs, (iii) they can be expanded and modified genetically in vitro [27], and (iv) they differentiate efficiently into adult skeletal muscle after transplantation [8]. Indeed, there are numerous studies using cell lines derived from satellite cells or autologous skeletal myoblasts. These demonstrated an improvement of myocardial function following engraftment in multiple animal models of myocardial injury [28].

The essential questions to be answered are if the transplanted skeletal myoblasts can transdifferentiate into a cell with a true cardiomyogenic phenotype and do they function electromechanically as cardiomyocytes after coupling with native cells.

Indeed, there are important differences between skeletal myoblasts and cardiomyocytes. The first is the lack of cell-to-cell junctions called intercalated disks, which couple electromechanically adjacent cardiomyocytes. Intercalated disks are composed of gap (for electrical coupling) and adhesion junctions (for mechanical coupling). These are essential for mechanical activity in the myocardium. The major proteins of gap and adhesion junctions are connexin-43 and N-cadherin. Skeletal myoblasts express both proteins in the early stages of development, and they are in fact essential for myoblast fusion [29,30]. The expression of these proteins decreases after formation of myotubes [31]. To date, however, they have not been detected in mature skeletal muscle grafts in the injured myocardium. Despite these limitations, some authors have suggested that myoblasts may play a role in the regenerative processes of injured muscle by migration into the damaged site. There, they could proliferate and fuse with damaged fibres inducing synthesis of myogenic-specific proteins [32].

Some authors have presented histological evidence for cardiomyocyte differentiation of the transplanted cells [33] and for the establishment of intercalated disks [2]. However, in one study, nonlabelled skeletal myoblasts were implanted, making it impossible to prove that the differentiated cells were not of recipient origin [33]. In a second study, a reporter gene was used to label cells. However, the differentiation of transplanted cells into cardiomyocytes was not fully proven [2]. In contrast, other investigations have indicated that transplanted skeletal myoblasts did not transdifferentiate into cardiomyocytes [34,35].

In the injured myocardium model, mature skeletal muscle grafts at 12 weeks of post-transplantation did not express N-cadherin or connexin-43 and failed to form junctions with host cardiomyocytes [36]. Skeletal myoblasts normally down-regulate the synthesis of these proteins as they differentiate into mature skeletal muscle [36]. On the other hand, skeletal myoblasts in vitro can express these proteins and form electromechanically junctions with cardiomyocytes [31].

In addition, formation of gap junctions between mature skeletal muscle and host cardiomyocytes may depend on the mode of delivery of skeletal myoblasts and the ischemic conditions of the myocardium. Indeed, connexin-43 has been detected between the skeletal muscle engrafted and host cardiomyocytes when the skeletal myoblasts were infused intracoronary into a noninjured myocardium [37,38].

A second difference between skeletal and cardiac muscle cells which may limit their use concerns L-type calcium channels. These underlie the essential process of excitation–concentration (EC) coupling in skeletal and cardiac muscle by calcium release from sarcoplasmic reticulum through ryanodine receptors (RyR). Cardiac EC coupling depends on extracellular calcium entry through dihydropyridine receptor (DHPR) calcium channels and on a functional interaction between DHPR and RyR. The mechanisms of EC are complex, involving the interaction of many membrane proteins. Furthermore, detailed information of their interaction with calcium has yet to be elucidated [39]. Indeed, electromechanical coupling has yet to be demonstrated between mature skeletal grafts and cardiac muscle cells [34,35].

Some investigations have highlighted the limitations in the use of skeletal myoblasts for cardiac cell therapy. This includes a delay due to the time needed for growth of myoblasts. Functional heart improvement depends heavily on the number of transplanted myoblasts [40]. In addition, the safety of using transplanted myoblasts for clinical trials has now been questioned [41], a factor largely ignored in initial experimental studies.

3.2. Bone marrow stem cells
The existence of stem cells has been historically best documented for the haematopoietic system [42]. However, the bone marrow contains several reconstructing stem cell types, with overlapping phenotypes, including haematopoietic stem cells (HSCs), endothelial stem/progenitor cells (EPCs), mesenchymal stem cells (MSCs), and multipotent adult progenitor cells (MAPCs) (for a review, see Ref. [43]).

There is some evidence for substantial "plasticity" or transdifferentiation of bone marrow stem cells. It has been shown, for example, that MAPCs can differentiate in vitro and in vivo into muscle, brain, and liver [44,45]. In addition, bone marrow transplantation studies in which donor cells were distinguishable from those of the recipient, e.g., using a sex-mismatch procedure, showed the presence of donor cells migrating to tissues, including endothelium [46], skeletal [47], and cardiac muscle [48]. In the damaged heart, different bone marrow cells might be able to form cardiomyocytes and/or vessels to improve impaired function. Bone marrow stem cells can be collected easily from adults, expanded in vitro if necessary, and readministered to the patient. However, improvements to CCT will depend on determining which fractions of stem cells or combinations of fractions are optimal.

A number of studies have now examined the potential of bone marrow-derived stem cell fractions to regenerate myocardium after experimental infarction.

3.2.1. Haematopoietic stem cells (HSCs)
The use of HSCs to repair an infarcted myocardium has been investigated in a murine model [49]. Lineage negative (Lin-) and stem cell factor receptor positive (c-kit+) primitive bone marrow stem cells were isolated from transgenic male donor mice carrying the gene for enhanced green fluorescent protein (EGFP+). Cells were transferred into female mice by injection into the noninfarcted myocardium bordering the injured heart tissue. The donor cells, monitored by the presence of EGFP and the Y chromosome, migrated into the damaged region and occupied up to 68 ± 11% of the infarcted region. Moreover, an average of 54 ± 8% of apparently new cardiomyocytes and endothelial and smooth muscle cells expressed EGFP. These cardiomyocytes were functionally competent and expressed cardiac transcription factors, including MEF2 (myocyte enhanced factor 2), GATA-4, and Csx/Nkx2.5, an early marker of myocyte development. Importantly, in contrast to skeletal myoblasts, these cells expressed connexin-43 that characterises the formation of gap junctions. Using Lin- c-kit+ bone marrow stem cells led to repair in 40% of the transplanted animals. One investigation has reported a case of myocardium repair by mobilizing progenitors with a cocktail of cytokines prior to infarction [50].

Another study has used a sub-population of murine HSCs that were isolated by their capacity to efflux compounds such as Hoechst 33352 or the mitochondrial fluorescent dye Rhodamine 123 (Rhod low). These low-level Hoescht-stained stem cells were CD34– but c-kit+ and Sca-1+ (stem cell antigen-1) [51]. This fraction of cells, termed side population (SP), is more enriched for HSCs than Lin- c-kit+ cells and had the capacity to both regenerate skeletal muscle [51] and repair an infarcted myocardium [52]. SP cells isolated from male mice marked with a β-galactosidase marker (LacZ) were transplanted into irradiated female mice. Ten weeks after bone marrow transplantation, chimeric mice were subjected to 1 h of ischemia followed by reperfusion. Two to four weeks later, donor-derived cardiomyocytes and endothelial cells were detected around the infarct. LacZ coupled with expression of {alpha}-actinin or Flt-1 marked the donor cardiomyocytes and endothelial cells, respectively. In summary, these studies apparently demonstrated the potential of HSCs to form cardiomyocytes and endothelial cells that home to and repair damaged myocardium.

Despite these encouraging results, the plasticity of HSCs has not been reproduced by other investigators. For example, in an elegantly designed study, the fate of HSCs from chimeric mice previously transplanted with a single GFP+ stem cell was evaluated in GFP– irradiated mice [53]. The authors reported that grafted HSCs contributed only to the formation of mature haematopoietic cells, and few GFP+ cells were detected in nonhaematopoietic tissues. Moreover, GFP+ cells did not express any antigens that mark neural, muscle, lung, or intestinal cells. In addition, the CD45+ haematopoietic marker was found to be conserved in all fluorescent cells. However, HSCs contributed to the production of a few isolated hepatocytes that expressed albumin. In addition, a rare GFP+ Purkinje cell was also detected in the brain. The authors concluded from their study that HSCs do not contribute to the regeneration of nonhaematopoietic tissues.

These findings have been substantiated recently by two other studies [54,55]. In the first, HSCs derived from the bone marrow that are Lin- c-kit+ were transplanted into either normal or an injured myocardium [54]. Cardiomyogenic differentiation of transplanted cells was followed by the expression of β-galactosidase driven from the cardiac-specific {alpha}-myosin heavy chain promoter. Parallel in vitro coculture experiments were conducted to investigate the cardiogenic transdifferentiation of Lin- c-kit+ cells. However, no activation of the {alpha}-MHC promoter was seen either in vivo or in vitro, leading to the conclusion that HSCs do not transdifferentiate into cardiogenic lineages.

In the second study, various c-kit-enriched fractions were used to follow the fate of HSCs in a murine myocardial infarction model (i.e., c-kit+, Lin- c-kit+, or Thy 1.1lo Lin- Sca-1+ c-kit+). Sorted cells from GFP+ mice were transplanted into wild-type animals. However, no apparent transdifferentiation occurred as evidenced by an absence of cardiomyogenic, smooth muscle cell, or endothelial cell specific markers in GFP+ cells.

Moreover, the majority of GFP+ cells retained both the CD45+ haematopoietic marker and the granulocyte marker (GR-1) [55]. In this study, injected HSCs had little effect on survival and myocardial function in infarcted mice compared to saline-treated controls. However, after 6 weeks, there were some improvements of fractional shortening and a little improvement in limiting ventricular dilatation.

These two groups also concluded that HSCs derived from bone marrow cells differentiate only into haematopoietic lineages, but this can still provide some long-term benefit to myocardial function.

Although these results are in contrast with those described elsewhere [49], they might be explained by the use of better methods of cell injection and detection [54,55].

Sometimes, benefit to the injured heart may be due to fusion of transplanted HSCs with host cardiomyocytes. Using a method based on Cre/lox recombination to detect cell fusion, it has been shown that bone-marrow-derived cells can fuse with host cardiomyocytes in the noninjured heart [56]. However, discrepancies between reports may be due to the different environments of the injured hearts or the cell type used.

In summary, these studies highlight the importance of using the appropriate tools to track the fate of transplanted cells before embarking prematurely on a clinical trial. More work is clearly needed on fully characterised HSC sub-populations.

3.2.2. Endothelial stem/progenitor cells (EPCs)
Bone marrow also contains EPCs, and these too are of interest for CCT. Recently, circulating EPCs were separated from peripheral blood-derived mononuclear cells using vascular endothelial growth factor receptor-2 (VEGFR2), CD34, and CD133 antigens [46,57–59]. In vitro, these cells differentiated into endothelial cells, and, in vivo, EPCs homed to sites of ischemia, where they become incorporated into neovessels and contribute to neovascularisation [60].

EPCs can be rapidly expanded in cultures containing VEGF-A [61]. In a nude rat model of myocardial ischemia, transplanted amplified human EPCs differentiated into endothelial cells, contributed to neovascularisation, and helped preserve left ventricular function [62]. Similarly, using the same model of myocardial ischemia in nude rats, it has been shown that bone marrow-derived angioblasts both decreased the apoptosis of myocytes while stimulating revascularisation [63].

Genetically modified EPCs may improve CCT. For example, EPCs altered to overexpress angiogenic factors (i.e.,VEGF) could induce neovascularisation in the ischemic myocardium by stimulating local angiogenesis (for a review, see Ref. [64]).

The use of different antibodies for endothelial markers and methods for tracking transplanted cells varies between studies. This makes the degree of neovascularisation due directly to EPCs difficult to estimate. Furthermore, the mechanisms by which EPCs improve neovascularisation remain unclear.

3.2.3. Mesenchymal stem cells (MSCs)
The bone marrow also contains another rare population of multipotent nonhaematopoietic CD34– CD45– stem cells called MSCs. These cells have the ability to differentiate into chondrocytes, osteoblasts, adipocytes, and fibroblasts that form the marrow stroma [44].

They can be used allogeneically and can be transduced by expression vectors that function after transplantation in vivo [65]. Here, rat MSCs were genetically modified to overexpress the Akt survival protein. This provided an improvement of myocardial function and inhibited cardiac remodelling.

In addition, MSCs can be induced in vitro and in vivo to differentiate into cardiac and skeletal muscle [6,66]. Interestingly, it has been possible in the mouse to obtain a cardiomyogenic cell line from MSCs by serial passage [6]. After treatment with 5-azacytidine, a DNA demethylating agent, which may enhance transdifferentiation, cells showed a fibroblast-like morphology and formed beating myotube-like structures connected by intercalated disks. Differentiated cells expressed atrial and brain natriuretic peptides, {alpha}- and β-MHC, plus {alpha}-cardiac and {alpha}-skeletal actin. Electrophysiological studies identified sinus node-like and ventricular cardiomyocytes-like action potentials. Moreover, the ultrastructural analysis of myotubes showed organized sarcomeres, central nuclei, numerous mitochondria, and atrial granules [6].

Cardiac engraftment of MSCs has been demonstrated in a rat model of myocardial infarction [67]. However, cultured MSCs were treated with 5-azacytidine and nuclei labelled with 5-bromo2'-deoxyuridine (BrdU) 24 h prior to transplantation. After 5 weeks of follow-up, BrdU-stained cells were detected in the scar and expressed troponin I. However, the improvement was not due to the connection between transplanted cells and the host myocardium since the transplantation of both treated and untreated MSCs induced angiogenesis in the transplanted area, explaining the cardiac function improvement in both groups.

In similar experiments, 5-azacytidine-treated MSCs induced in vivo cardiomyogenesis and vasculogenesis in the murine heart. However, these MSCs were CD34+, while untreated MSCs were CD34– [68]. The use of 5-azacytidine to transdifferentiate MSCs into cardiomyocytes raises some concerns about clinical safety. Reactivation of genes may not be entirely specific to the cardiac lineage. One could just as easily induce genes that drive cell proliferation with possible oncogenic consequences.

However, other studies in various animal models using transplanted MSCs demonstrated expression of muscle specific phenotypes without 5-azacytidine treatment. This emphasised the influence of the cardiac microenvironment on the differentiation of transplanted MSCs [7,69–71]. One study, for example, isolated MSCs from inbred Lewis rats expanded them in vitro before labelling with 4',6-diamidino-2-phenylindole (DAPI) prior to transplantation. After 4 weeks, marked cells expressed sarcomeric MHC, aligned with host cardiomyocytes, and formed intercalated discs [70].

Similar results have been obtained in a porcine myocardial infarction model. Here, histological and immunohistochemical analysis demonstrated the engraftment of MSCs in the scar tissue. These cells, 2 weeks after transplantation, expressed the muscle-specific proteins {alpha}-actinin, tropomyosin, troponin T, and phospholamban [71]. In this study, muscle-specific proteins were seen without the 5-azacytidine pretreatment. However, specific cardiomyogenic markers were not detected.

In another experimental model, transfected human MSCs marked by LacZ were transplanted into the hearts of immunodeficient mice. The grafted cells expressed myosin heavy chains, desmin, {alpha}-actin, and phospholamban and had the typical sarcomeric organization of myotubes, indicating cardiomyogenic differentiation [7]. However, the survival rate of transplanted cells was very low (<0.5%), 4 days of post-transplantation.

In these reports, the ability of MSCs to express muscle-specific proteins and to improve myocardial function has been demonstrated. To our knowledge, however, in vivo studies showing the plasticity of MSCs are lacking. We have used a rat ligation model to implant MSCs. Thirty days after implantation, immunofluorescence analysis revealed that some engrafted cells expressed a smooth muscle phenotype ({alpha}-SM actin+) while others have acquired an endothelial phenotype (CD31+) [72]. Vessel density was also augmented in the MSCs-treated animals. Moreover, after this period, echocardiography demonstrated improved left ventricular performance only in the rats implanted with MSCs.

Taken together, these studies show that transplanted MSCs have different fates according to the microenvironment to which they locate. They expressed a smooth muscle phenotype in the scar which was absent from the vessels where an endothelial phenotype was displayed. In the noninfarcted myocardium, they exhibited a cardiomyocyte phenotype.

3.3. Cardiac stem cells (CSCs)
Until recently, our perception was that the adult mammalian heart was an organ without regenerative capacity. However, in the past few years, various reports demonstrated the existence of cycling ventricular myocytes both in the normal and pathologic adult heart [73]. Moreover, it has been shown that, in the regions adjacent to the infarcts from patients, 4% of myocyte nuclei expressed the Ki-67 cell proliferation marker. The reentry of myocytes into the cell cycle has been quantified as, respectively, 0.08% or 0.03% for the zones adjacent or distant to the infarcts [74]. In addition, it has also been reported that myocyte hyperplasia contributed to the cardiac hypertrophy perhaps due to the proliferation of CSCs [75]. These newly described stem cells are multipotent, giving rise to endothelial cells, smooth muscle cells, and functional cardiomyocytes. In addition, they supported myocardial regeneration after infarction in a rat model [76]. In mammals, the CSCs are Lin- c-kit+ (and Sca-1+ in rodents). CSCs are small cells (6 µm) distributed throughout the ventricular and atrial myocardium, with a higher density located in the atria and the ventricular apex. In contrast to HSCs, CSCs do not express either the pan-leukocyte antigen CD45 or the sialomucin-like protein CD34. In addition, these cells express GATA-4 and/or MEF2 when stimulated with oxytocin, a stronger inducer of cardiac differentiation than 5-azacytidine [77].

However, these cells remained CD34– as opposed to MSCs treated with 5-azacytidine, suggesting clearly that CSCs are distinct from MSCs [68].

The demonstration that the heart contains stem cells capable of regenerating large amounts of functional myocardium raises the interesting question as to why, in infarction, regenerative processes stop before the completion of the repair?

If this is due to insufficient numbers of CSCs to complete the repair, it offers the therapeutic potential of injecting untreated MSCs to augment the repair by CSCs.

Future research on CSCs will help to answer these questions and may provide the means for efficient heart regeneration.

3.4. Adverse events
To date, publications regarding adverse events in experimental studies have been relatively rare. However, the development of microinfarction has been reported when infusing MSCs directly into a dog's coronary artery [78]. During infusion, ST segment elevation and T wave changes were observed. Postmortem histology, 7 days post-transplant, showed evidence of myocardial infarction with fibroplasia, and macrophage infiltrates in the areas where MSCs were present. The authors concluded this may be due to infusing large MSCs directly into the coronary artery.

In addition, adverse calcification has been shown to be a problem in a rat model [79]. In this study, following myocardial infarction, inbred rats received either total bone marrow (BM) cells, BM-derived multipotent stem cells or saline. After 2 weeks, echocardiography revealed some myocardial calcifications in 28.5% of rats in the total BM cell group. Detailed histological analysis demonstrated that the calcifications in the periinfarct were surrounded by grafted cells. Several hypotheses have been proposed as an explanation for calcification: (i) MSCs contained in total BM cell fractions have the plasticity to differentiate into bone, (ii) the calcification was induced by an interaction between total BM cells and the infracted myocardium, and (iii) different bone marrow cell types interacted with each other in a novel environment.

It will be interesting to see if these findings will be confirmed in experiments using a larger population or a different animal model.

Taken together, experimental studies performed so far for myocardial regeneration by cell therapy generally show that improvement of cardiac function is not exclusive to one cell type because a variety of progenitors accomplished this. Progress in this field carries the proviso that detecting the fate of transplanted cells is essential. This must be done with a sensitive reporter transgene rather than immunostaining that sometimes generates false positives. Determining the precise fate of these cells goes hand in hand with the safety aspects of a clinical trial [80].


   4. Clinical trials
Top
 Abstract
 1. Introduction
 2. Embryonic stem cells...
 3. Adult stem cells
 4. Clinical trials
5. Conclusion and future...
 Acknowledgments
 References
 
The experimental studies described above have encouraged numerous nonrandomised phase I clinical trials that tested the feasibility and safety of autologous cell transplantation for treatment of chronic ischemic heart failure (CIHF) or acute myocardial infarction (AMI).

4.1. Chronic ischemic heart failure (CIHF)
One of the earliest reports implanted autologous skeletal myoblasts into the postinfarction scar during a coronary artery bypass. Contraction and viability in the grafted scar were assessed by echocardiography and positron emission tomography. The clinical status of the patient after 5 months of follow-up was improved [81]. After this encouraging result, a nonrandomised phase I trial was extended to 10 patients [41]. Inclusion criteria were the following: left ventricular ejection fraction <35%, presence of nonviable and akinetic scar postinfarction and an indication for concomitant coronary artery bypass grafting (CABG), and ischemic but viable myocardium. A mean of 871 x 106 of autologous myoblasts was injected into the scar during bypass grafting. At an average follow-up of 10.9 months, contraction and viability were improved in the cell-implanted scars. However, four patients had episodes of ventricular tachycardia early after surgery, which is manageable by defibrillator implantation. This may be due to differences between the transplanted myoblasts and host cardiomyocytes discussed in the Adverse events section below.

One group has used purified autologous CD133+ stem cells in a study in six patients who had experienced an acute transmural myocardial infarction between 10 days and 3 months prior to the procedure [82]. These patients were good candidates for CABG. After CABG, CD133+ cells were injected into the infarct border. The 9–16 months of follow-up after surgery showed no ventricular arrhythmias, and all patients reported an improvement in their daily activities. In this study, there was little effect on local contractility. The improvement of the myocardial perfusion was probably due to angiogenic stimulation by CD133+ cells. However, formal demonstration of this effect was not performed. Other studies have employed different means of cell delivery. Transendocardial catheter-based cell administration has been used to deliver freshly isolated bone marrow mononuclear cells (BM-MNCs) CD34+ CD45+. Fourteen patients with severe ischemic heart failure were entered into a nonrandomised trial. Half of the patients received cells [83]. After 2 months of follow-up, there was a significant reduction in total reversible defect and an improvement in global left ventricular function. The same treatment has been tried in eight patients with stable pharmacological refractory angina [84]. The 3 months of follow-up reported a decrease in the number of anginal episodes per week and a reduction in the number of nitroglycerin tablets consumed. There was no evidence for arrhythmias, the mean left ventricular ejection fraction was unchanged, and there was an improvement in target wall motion and thickening.

Finally; one study evaluated the feasibility of administering autologous total bone marrow cells by percutaneous transendocardial delivery in no-option patients with advanced coronary disease [85]. During a 3-month follow-up, the authors reported a change in the Canadian Cardiovascular Society angina score and an improvement of stress-induced ischemia. However, this was a nonrandomised study, and the risk benefit ratio was also low.

4.2. Acute myocardial infarction (AMI)
Transplants using bone marrow-derived progenitor/stem cells have also been conducted. Ten patients, for example, were entered into a nonrandomised trial using autologous BM-MNCs [86]. The control group included patients who refused cell therapy and were treated by standard therapy. BM-MNCs, containing CD133+/CD45+ stem cells, were isolated by Ficoll density separation, cultured overnight, and administrated by high-pressure infusion into the infarct-incriminated artery. After 3 months, the size of infarcted region decreased only in BM-MNCs-treated patients. Stroke volume also increased significantly, but the ejection fraction was unchanged.

In another nonrandomised trial, an evaluation was made of the effects of intracoronary administration of bone marrow-derived cells (CD34+ CD45+) or circulating blood-derived cells (CD105+ CD31+ KDR+ vWF+) in 20 patients with reperfused acute myocardial infarction [87]. After 4 months, there was a significant improvement of regional wall motion and left ventricular ejection fraction in both groups compared to the controls. Myocardial viability increased in the infarct area in both experimental groups. End-systolic volumes remained unchanged in the cell-treated groups, and no untoward adverse events were reported by patients.

All phase I studies detailed above are limited by their nonrandomised design. However, a larger randomised controlled clinical trial has now been reported [88]. In this study, 60 patients underwent percutaneous coronary intervention with stent implantation for acute myocardial infarction. They were randomised into two groups, either receiving or not autologous BM-MNCs. All patients received standard pharmacological postinfarction treatment. After 6 months, the authors reported a significant increase in the ejection fraction in the patients who received BM-MNCs. However, the left ventricular diastolic volume was not different between groups, indicating a lack of improvement of ventricular remodelling during follow-up. No adverse events have been reported so far in this study.

4.3. Route of cell delivery
The success of CCT may depend on injecting a high cell concentration into the target zone. Therefore, local administration of transplanted cells is preferable to the intravenous systemic cell delivery.

4.3.1. Intramyocardial implantation
During open heart surgery, the ischemic area is accessible, and cells can be delivered by multiple injections. However, the apparition of arrhythmias and regurgitation of the cell solution limited the therapeutic effects [81].

4.3.2. Intracoronary cell delivery
This technique requires a high concentration of cells to be injected directly into the target area and can be performed during percutaneous coronary intervention [86–88].

4.3.3. Transendocardial catheter-based cell administration
The safety and the feasibility of this technique has been recently assessed in patients using intramyocardial cell/gene therapy [83–85,89]. However, cells can be damaged due to the injection of cell solution under high pressure. In addition, endocardial injection, like intramyocardial delivery, may induce arrhythmias.

4.3.4. Intravenous systemic delivery
Technically, this has been the easiest route of delivery. However, the pulmonary first-pass effect means only a low percentage of cells reached the injured myocardium. Therefore, this route of administration also required large numbers of cells.

4.4. Adverse events
In the phase I trial, using skeletal myoblasts for CCT ventricular arrhythmias developed in patients between 11 to 22 days post-transplantation, constituting a serious adverse event [41]. This may be explained by the lack of gap junctions between transplanted myoblasts and resident cardiomyocytes or the difference between the action potential of the two cell types.

In contrast, arrhythmias were not per se a problem with clinical trials using BM-MNCs. However, administration of any cells by the transendocardial route could induce arrhythmias.

The major concern of using BM-MNCs may be the development of angiogenic neoplasias since bone-marrow-derived EPCs can contribute to tumor neovascularisation [90]. However, no angiogenic neoplasia has yet been reported in clinical trials.

One trial which utilised peripheral blood stem cell mobilised by granulocyte-colony stimulating factor (G-CSF) for CCT has been stopped [91]. In this study, an intracoronary infusion of stem cells after coronary stenting was performed. An improvement of myocardial function and angiogenesis was observed, but this was accompanied by an unacceptable rate of stent restenosis. The authors hypothesised that the restenosis might be due to differentiation of progenitor cells into smooth muscle cells within the stented segment.


   5. Conclusion and future perspectives
Top
 Abstract
 1. Introduction
 2. Embryonic stem cells...
 3. Adult stem cells
 4. Clinical trials
 5. Conclusion and future...
Acknowledgments
 References
 
The aim of CCT is to regenerate or replace cardiomyocytes and endothelial cells in the damaged heart. There are several important issues common to all cell sources, which need to be addressed. This includes the longevity of intracardiac grafts, the capacity of engrafted cells to differentiate and maintain a mature cardiac phenotype while at the same time integrating with the host myocardium and contributing to contractile function. The response of engrafted cells to physiological and pathological stimuli also needs to be ascertained. This procedure induces angiogenesis, so it theoretically carries a risk of inducing tumors at the injection site or elsewhere and will require vigilance. However, ultimately, we will need to gain a more fundamental understanding of stem cell (particularly, CSCs) proliferation and differentiation to control it both in vitro and in vivo. Optimal therapy may then involve transplantation of more efficient stem cells or mixtures of different stem cell fractions that complement each other in restoring cardiac function.

In conclusion, skeletal myoblasts, bone marrow MSCs, HSCs, and CSCs, although limited in their differentiation capacity, provide a safer and ethically acceptable alternative to ES cells for CCT. Improvements in CCT will also depend on efficient delivery of stem cells to the infarcted area. Concerning the clinical trials, some may be premature because accurate experimental data are still lacking for stem cells derived in particular from bone marrow. Larger randomised animal trials should be performed using selected stem cell populations, which so far have been shown to be safe.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Embryonic stem cells...
 3. Adult stem cells
 4. Clinical trials
 5. Conclusion and future...
 Acknowledgments
References
 
We are grateful to the Société Française de Cardiologie, La Fondation Pharmacia and Les Laboratoires Pfizer for the support of our research on cell therapy.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Embryonic stem cells...
 3. Adult stem cells
 4. Clinical trials
 5. Conclusion and future...
 Acknowledgments
 References
 

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