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Cardiovascular Research 2003 58(2):369-377; doi:10.1016/S0008-6363(02)00783-6
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Copyright © 2003, European Society of Cardiology

Stem cells and cardiac disorders: an appraisal

Michael J. Goldenthal and José Marín-García*

The Molecular Cardiology and Neuromuscular Institute, 75 Raritan Avenue, Highland Park, NJ 08904, USA

* Corresponding author. Tel.: +1-732-220-1719; fax: +1-732-220-2992. tmci{at}att.net

Received 28 August 2002; accepted 12 November 2002


   Abstract
Top
 Abstract
1 Basic studies
 2 Stem cell-transplantation...
 3 Applications of stem...
 4 Conclusions
 References
 
The use of stem cells has proved to be an important tool in investigating the events of early cardiac development, differentiation, and morphogenesis. In addition, stem cell transplantation in the treatment of certain cardiac disorders has shown early promise. We have attempted to present a balanced review of both basic studies and clinical–therapeutic potential of stem cells transplantation in the damaged heart.

KEYWORDS Stem cells; Myocytes; Mitochondria; Endothelial receptors; Ischemia


   1 Basic studies
Top
 Abstract
 1 Basic studies
2 Stem cell-transplantation...
 3 Applications of stem...
 4 Conclusions
 References
 
1.1 Cardiomyocyte differentiation of embryonic stem cells
Embryonic stem cells (ES) are valuable models to study the events of vascular morphogenesis, cardiac growth and differentiation and cardiac disorders. The delineation of early events in development and differentiation which are extremely difficult to assess in the mammalian heart are possible in ES cells. These studies have allowed progress in the identification and characterization of regulatory factors, elucidation of the pathways of transcriptional activation and the signal transduction events involved in cardiomyocyte differentiation.

Pluripotent ES cells can spontaneously differentiate, via embryo-like aggregates termed embryoid bodies, into early-stage cardiomyocytes in vitro by manipulation of the culture growth medium [1–4]. These cardiomyocytes can be of the pacemaker-atrium and ventricle-like type, being distinguishable by their specific patterns of action potentials [4,5]. In vitro cardiomyocyte differentiation has been established with both murine [1,3–5] and human [2] ES cells. To maintain their pluripotentiality, both mouse and human embryonic stem cells require cultivation on feeder layers (usually mouse fibroblast cells). Interestingly, the addition of a differentiation inhibiting cytokine i.e. leukemia inhibitory factor (LIF) can replace the feeder cells with mouse but not human ES cells [6]. The role that LIF plays in modulating ES cell and cardiomyocyte differentiation is dependent on the developmental stage. For instance, it can hamper early events in cardiomyocyte differentiation while promoting proliferation at more fully differentiated states [7]. It is also important to note that cardiomyocyte levels are increased in ES cultures supplemented with specific growth factors (e.g. EGF and retinoic acid) but that none of these factors direct ES cell differentiation to a single cell type [8]; cardiomyocytes can be subsequently purified from a heterogenous mixture of ES-derived cells.

Retinoic acid (RA) has been found to effect the efficiency of cardiomyocyte differentiation in a time and concentration dependent manner [4]. In vitro studies with mouse ES cells demonstrated that RA can specifically induce the formation of ventricular-specific cardiomyocyte [4], increasing cardiomyocyte number from ES cells and specifically enhanced ventricular cell differentiation albeit the number of pace-maker and atrial cells were reduced. In contrast, increasing RA concentration or RA treatment at the initial stages of embryoid body development (days 1–5) has been reported to inhibit cardiogenesis [4] and induces the formation of neuronal and glial cells from pluripotent ES cells and embryoid bodies in parallel with increased activity of PAX6, a transcription factor involved in central nervous system development [9]. RA is pivotally involved in early cardiac development with both positive and negative aspects and RA deprivation can induce cardiac malformations. Also, defective cardiac development has been found in association with increased levels of RA treatment (RA toxicity) with marked effect on expression of cardiac specific transcriptional factors [10,11]. RA primarily exerts its effect on development and cellular differentiation through the nuclear retinoic acid receptors (e.g. RXR) that modulate the transcription of an array of specific target genes by binding specific DNA sequences.

Recently, Nemer et al. reviewed the role that cardiac specific transcription factors plays in cardiomyocyte differentiation and cardiac development [12]. The expression of cardiac gene products occurs in a developmentally controlled program both in early myocardial development in vivo and in ES cell-derived cardiomyocytes. In both early embryogenesis and in early ES cells, expression of the cardiac specific transcription factors GATA-4, NKX 2.5 and members of the myocyte enhancer family (MEF-2), e.g., MEF2c precedes (and mediates in turn) the activation of expression of markers of early, intermediate and terminal cardiac cell differentiation including atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP), myosin light chain (MLC), {alpha}-myosin heavy chain, β-myosin heavy chain, and cardiac troponin C. Blocking specific GATA-4 transcription (with antisense transcripts) eliminates the formation of beating cardiac muscle cells and abolishes transcription of specific cardiac markers significantly reducing levels of MEF2c and NKX2.5. Although GATA-4 is one of the first cardiac transcription factors to be induced and plays a key role in cardiomyocyte differentiation, both loss-of-function and gain-of-function experiments have demonstrated that GATA-4 is not sufficient for the initiation of cardiac differentiation. ln the absence of GATA-4, embryonic stem cells can differentiate into mesoderm and can initiate the cardiogenic pathway but are unable to proceed beyond a precardiac (cardioblast) stage [12]. Similarly, while GATA-4 overexpression in embryonic stem cells potentiates cardiogenesis, its stimulatory effect on cardiac differentiation requires cell aggregation and the presence of other interacting factors [13]. Another important transcriptional activator, the cardiac homeodomain-containing NKX2.5 which is expressed in cardiac progenitor cells at early development, likely acts downstream of GATA-4 (since inactivation of NKX2.5 does not affect GATA-4 expression) and may play a role in late cardiac differentiation events (Fig. 1). It is important to note that both GATA-4 and NKX2.5 work in combination with other transcription factors; for instance, GATA-4 recruits MEF2 proteins to GATA binding sites to activate synergistically the promoters of several critical genes involved in both cardiac differentiation and in maintaining the cardiac phenotype during postnatal development including MLC, ANF, {alpha}-myosin heavy chain, desmin, troponin T and I and {alpha}-actin [14]. While MEF2c is likely downstream of both GATA-4 and NKX2.5, in mouse embryos containing null alleles of MEF2c, cardiomyocyte differentiation was disrupted, the ventricle failed to form and a subset of cardiac muscle genes (including ANF) were not expressed [15]. While these cardiac transcription factors have been the focus of much research, the mechanism by which these critical transcription factors themselves are regulated remains elusive. Moreover, several growth factors have been identified which play an important role in the initial induction of cardiac differentiation, e.g., bone morphogenetic proteins [16]. However, the precise pathway of the molecular cascades stemming from these growth factors leading to cardiac-specific gene expression (required for cardiac specification) remains to be elucidated. Molecules that are expressed in precardiac cells preceding the cardiac-specific transcription factors will be soon identified using such molecular techniques as differential display and microarray analysis allowing better definition of the molecular mechanism by which induction of cardiac differentiation is controlled.


Figure 1
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  		Fig. 1 Pathways leading to the differentiation of the cardiac cell. Shown are events at the plasma membrane (include growth and morphogenetic factor receptors and the signaling apparatus including the β-adrenergic system), the generation of reactive oxygen species (ROS) at the mitochondria and its impact on downstream events. Also depicted is the centrally located cardiomyocyte nucleus with its cascade of master regulatory transcription factors (e.g. GATA-4, NKX2.5 and MEF2c) and the activation of gene expression involved in the cardiomyocyte phenotype including connexin (major protein of the gap junction), ANF, BNP, sarcomeric and contractile prtoeins.

 
Investigation of the developing mammalian cardiomyocyte's signaling cascade in response to β-adrenergic stimulation has compared ES cell response from early (embryoid bodies) and later developmental stages of the differentiating ES cells. The calcium current (measured by patch-clamp methodology) in early ES-derived cardiomyocytes were insensitive to isoproterenol, forskolin and 8-bromo-cAMP. Electrophysiological characteristics in late developmental stage of ES-cell derived cardiomyocytes were identical to postnatal cardiomyocytes suggesting that the signalling cascade components have become functionally coupled during cardiomyocyte development [17]. The hypo-responsivity of early cardiomyocytes to β-adreneregic agonists is especially noteworthy since a variety of cardiac disorders, including heart failure, are characterized by depressed function of β-adrenoreceptors and deficient production of cyclic AMP while altered G protein expression and coupling have been reported in the hypertrophic heart. In addition, it is well-known that both heart failure and cardiac hypertrophy are associated with a re-expression of the fetal gene program. However, recent evidence demonstrated that ES-derived cardiomyocytes manifest increased arrhythmogenic potential with marked heterogeneity in the initiation, duration and spontaneous activity of the action potential in contrast to adult myocytes [18], a caution to be considered in their potential use as donor cells in transplantation.

Interestingly, increased levels of oxidative stress can impact on cardiomyocyte development from embryoid bodies [19], e.g., Reactive oxygen species (ROS) is generated in increased quantities during embryoid body development, and incubation with the pro-oxidants H2O2 or menadione was found to enhance cardiomyogenesis. Similarly, reduced ROS levels resulting from addition of free radical scavengers significantly decreased the number of beating embryoid bodies. The role of ROS in ES cardiomyogenesis is consistent with recent data indicating that a rapid and significant increase in intracellular ROS is associated with growth factor or cytokine stimulation [20]. Phosphatidylinositol 3-kinase (PI-3-kinase) plays a pivotal role in the signaling and regulation of intracellular ROS levels and cardiomyogenesis [19]. The number of beating embyroid bodies was significantly reduced in parallel with decreasing ROS levels when ES cells are treated with LY294002, a specific inhibitor of PI-3-kinase. Raising the ROS levels in the presence of the PI-3-kinase inhibitor (by adding pro-oxidants) restored the levels of cardiomyocyte differentiation.

Also, it is important to note the relatively easy genetic manipulation of ES cells in constructing gene knock-out lines. This facilitates the examination of the functional role of specific targeted genes on cardiomyocyte differentiation and function without generating transgenic animals or as a critical intermediate in their generation.

1.2 Pluripotent embryonic carcinoma cell-lines can differentiate into cardiomyocytes
The pluripotent mouse embryonal carcinoma cell line P19 is a well-characterized model of cell differentiation and can give rise to the formation of all three germ layers thought to differentiate by the same mechanisms as normal embryonic ES. These cells ressemble the inner mass of the blastocyst and their differentiation mimics early events of embryogenesis. Under appropriate culture conditions, pluripotent P19 cell line can differentiate via embryo-like aggregates into spontaneously beating myocytes. In addition, clonal derivatives of P19 cells (PL19CL6) have been isolated which are more efficient at cardiomyocyte differentiation [21].

Several studies have utilized treatment with the solvent dimethyl sulfoxide (DMSO) to induce cardiac differentiation of P19 cells presumably through the activation of essential transcription factors (GATA-4 and Nkx-2.5). Exposure of P19 cells to RA leads to neural cell differentiation and selective endothelin receptor (ETB) expression [22]. In contrast, cardiomyocytes derived from this cell line have a pronounced induction of another class of endothelin receptor (ETA) which lead to increases in ANF levels. Recently, oxytocin was found to be a potent inducer of P19 cardiomyocyte differentiation [23] albeit how this is achieved remains to be defined. P19 derived cardiomyocytes exhibit action potentials similar to embryonic cardiomyocytes with full functional expression of adrenoceptors and calcium channels [24].

The opportunity to identify specific factors that mediate–promote cardiomyocyte differentiation has been pursued with P19 cells using differential display. A unique transcript and its gene have recently been characterized (Midori) whose gene product is developmentally regulated in the heart. Overexpression of Midori increased the efficiency of cardiomyocyte differentiation by P19 cells supporting its role in mediating that process [25].


   2 Stem cell-transplantation studies: myocardial repair and treatment of cardiac disorders
Top
 Abstract
 1 Basic studies
 2 Stem cell-transplantation...
3 Applications of stem...
 4 Conclusions
 References
 
2.1 Cardiomyocyte transplants
It is generally agreed that the adult heart lacks the ability to regenerate. While there is recent evidence that a very limited proportion of cardiac cells maintain the ability to divide [26], the ability to easily grow cardiomyocyte cells in culture in quantities large enough to repair injured myocardium appears to be extremely limited. Recent study has suggested that the limitations of cardiomyocyte proliferation may ultimately be reversed by removing the cell-cycle block thereby allowing terminally differentiated cardiac cells to re-enter the cell cycle [27]. Early experiments with fetal cardiomyocyte transplantation showed success in the formation of stable grafts and nascent intercalated discs between grafted and host myocardial cells [28,29]. Since the cardiomyocytes used in the majority of successful transplantation studies have been of fetal origin, questions have been raised as to the extent of in vivo differentiation of the implanted cells into mature myocardium [30]. Investigation of the fate of neonatal rat cardiomyocytes implanted from male donors into normal female hearts as gauged by quantitative PCR analysis of the male-specific Sry gene showed that by 1 h after transplant, there was a considerable loss of donor cells at the graft site (57%) which decreased further to 24% over 24 h and to 15% over 12 weeks [31]. This study not only suggested the presence of substantial cell death after transplant, but also indicated that the techniques for cell injection need further optimization given the striking variability of graft efficiency. In addition, several investigators have addressed the concern that cardiomyocyte transplantation also may cause further problems at the site of myocardial grafting unless high levels of cardiomyocyte cell death (presumably due to ischemia) are avoided [32,33]. The demonstration of markedly increased cell death (using TUNEL analysis) at the graft site as well as the finding that no increase in graft size occurs with increasing number of injected cardiomyocytes [32] have prompted careful consideration of the clinical use of cardiomyocyte transplantation in treating the ischemic heart. In this regard, increased research focus is needed to develop successful strategies that can maximize grafted cardiomyocyte cell survival and enhance the differentiation process (both in vitro and in vivo).

2.2 Skeletal myoblast transplant
There is ample evidence that cells of transplanted skeletal muscle cells can differentiate and develop into striated cells within the damaged myocardium, and prevent progressive ventricular dilatation by improving heart function [34–37]. Skeletal muscle cells have been successfully delivered to myocardium by either intramural implantation or arterial delivery [35,36]. Skeletal muscle satellite cells can proliferate abundantly in culture, and can be easily grown from the patient themselves (self-derived or autologous) thereby reducing a potential immune response. Importantly, skeletal myoblasts are relatively ischemia-resistant (compared to cardiomyocytes) since they can withstand several hours of severe ischemia without becoming irreversibly injured as compared to cardiomyocytes (which injure rapidly within 20 min) [38]. Potential limitations in the use of skeletal muscle include the unknown myoblast cell survival, in situ myocardial integration (e.g. electrophysiologically), the stability of differentiated phenotype and long-term fate of the transferred myoblasts and the extent of myocardial functional benefits (e.g. contractility) with their transplant. Another limiting factor in the transplantation of skeletal myoblasts relates to the necessary concentration of cells delivered, (too much may be as problematic as too little) and the timing of growth (relative to the myocardial injury). In addition, in the repair of large myocardial infarct, putting new cells at a site of limited blood–nutrient–oxygen supply may (in some cases) not be sufficient for myocardial repair.

There is evidence that the cardiac environment is not only permissive for myogenic differentiation, but also can influence the developmental program of implanted myoblasts enabling them to better assist cardiac performance [35,39]. When neonatal skeletal myoblasts were transplanted into injured rat hearts, a gradual establishment of slow-twitch, fatigue-resistant muscle fiber phenotype in implanted cells occurred (with physiological characteristics similar to cardiac muscle including high OXPHOS capacity, fatigue resistance and use of β-MHC as a major contractile protein. Therefore, when grafted into injured hearts, skeletal myoblasts could establish new muscle tissue which contracted when electrically stimulated. Two decades ago, Menasche found that the latissimus dorsi skeletal muscle undergoes fiber type switching when conditioned for dynamic cardiomyoplasty. When conditioned by repeated electrical stimulation, there was a conversion from fast fiber–easy fatigability to slow fiber phenotype [37].

In addition, adult skeletal muscle contains a population of cells with several characteristics of bone-marrow hematopoietic stem cells [40]. However, Murry et al. and Reinecke et al. have not been able to observe the differentiation of skeletal muscle myoblasts to cardiomyocytes after cardiac grafting [39,41]. These authors conclude: (1) that skeletal satellite muscle cells injected into the heart differentiate into mature skeletal muscle and do not express cardiac-specific genes after cardiac grafting; (2) the demonstration of ‘trans-differentiation’ of skeletal muscle to cardiomyocyte was either ambiguous in defining cell lineage or not rigorous when using definitive characteristics to identify the cardiomyocyte phenotype.

2.3 Other ‘sources’ for stem cell transplant
2.3.1 Bone marrow cells
These multipotent stem cells are extremely responsive to their microenvironment for milieu-dependent differentiation [42–46]. Infarcted cardiac tissue regenerates with bone marrow cell (BMC) transplantation. Bone marrow stem cells have been found to differentiate to cardiomyocytes with 5-azacytidine treatment [42,43]. Signal transduction is markedly effected with BMC differentiation; qualitative and quantitative differences are found in the expression of adrenergic/muscarinic receptors in bone marrow-derived cardiomyocytes after exposure to 5-azacytidine [42].

Bone marrow hematopoietic stem cells can differentiate into cardiomyocytes, endothelium and smooth muscle when injected into ischemic ventricle [44,45]. Moreover, the growth response involving BMC interfered with ventricular scarring and stimulated some development of vascular structures within the infarcted area [45]. Albeit regarded as a major breakthrough, one major limitation of this study is that it was conducted with the mouse heart (which have limited size infarcts compared to human myocardium). Since marrow stromal cells can be easily obtained by bone marrow aspiration and also can proliferate in culture before being used as autologous implants, their use as a source for stem cell transplants appears to be both advantageous and promising [46].

A cardiomyogenic cell-line has been generated from marrow stromal cells grown in vitro in the presence of 5-azacytidine which express cardiomyogenic phenotypes (capable of forming myotubes that develop synchronous beating, producing ANF and BNP, staining with anti-actinin and anti-desmin antibodies, exhibiting a ventricular–like action potential and evidence of cardiomyocyte-like ultrastructure) [47]. These cells could be transplanted to myocardium in vivo replacing native cardiomyocytes lost by necrosis or apoptosis. BMCs from adult rats (after induction to differentiate into cardiomyocytes with in vitro growth in the presence of 5-azacytidine) were autologously transplanted into myocardial scar tissue. Transplantation of BMCs inhibited the ventricular scar from thinning, minimized ventricle dilatation and improved myocardial function [43]. The transplanted BMCs also induced angiogenesis which may contribute to the long-term survival of the transplanted cells in the scar. Recruitment of BMCs has also been examined in adult mdx mouse heart, a model of Duchenne's muscular dystrophy [48]. Dystrophic female mdx mice receiving BMC transplant from normal male mice showed incorporation of donor BMCs in heart and skeletal muscle as well as the presence in myocardium of single cardiomyocytes with donor nuclei derived from the BMCs. While this data is consistent with the growing body of evidence in support for the plasticity of adult stem cells, neither the relative number or the percentage of donor cells detected in the host myocardium nor the functional effects of the transplant were provided in this study.

Human mesenchymal stem cells derived from adult bone marrow can undergo in situ myogenic differentiation to a cardiomyocyte phenotype once transplanted into an adult heart [46,49]. In addition, human mesenchymal stem cells transplanted into fetal lamb (in utero) can engraft in multiple tissues, and can differentiate into cardiac and skeletal myocytes which can persist for over 1 year [50].

Recent studies have demonstrated that progenitor stem cells of extra-cardiac origin can be incorporated into the human heart [51,52]. Some of these cells are found near or at the site of injury and might be involved in myocardial repair. These studies have utilized female hearts transplanted into male patients and found evidence of Y chromosome-containing cardiomyocytes; however the estimated levels of Y chromosome-containing cardiomyocytes differ substantially in quantity, by over three orders of magnitude (0.04% compared to 18%). Needless to say, methods to rigorously detect and to accurately quantitate cardiac cell populations of extra-cardiac origin are necessary. Defining the precise origin of the cardiomyocytes is also complicated by the recent finding that bone marrow cells can readily and spontaneously fuse with existing myocytes essentially adopting their phenotype (while retaining the BMC nuclear genome) [53].

2.3.2 Endothelial cells
Endothelial cells derived from embryonic stem cells from dorsal aorta and from differentiated cells of the human µmbilical vein can ‘trans-differentiate’ into beating cardiomyocytes when co-cultured with neonatal cardiomyocytes or when injected into postischemic adult mouse heart [54]. Adult tissues (e.g. neural stem cell and lung cells) showed no such capability. This study demonstrated activation of cardiac-specific genes (e.g. cardiac specific troponin), development of aligned sarcomeres, electrical coupling and in vivo differentiation when injected into post-ischemic ventricles. Cell-to-cell contact with neonatal cardiomyocytes is necessary for this process since conditioned medium does not support this differentiation. Another important finding from this study was that cellular signals that induce myocardial differentiation in endothelial cells are different than those that activate cardiogenesis in the early embryo. Fibroblast growth factor (FGF2) and bone morphogenetic protein (BMP4) which have been implicated in the activation of cardiogenesis in embryonic heart do not activate myocardial ‘trans-differentiation’ with endothelial cells. The nature of the activating signals for endothelial cell trans-differentiation to cardiomyocytes has not yet been elucidated. The finding that neural stem cells from brain showed little capability in ‘trans-differentiating’ into cardiomyocytes was somewhat unexpected given that previous reports established that neural stem cells from adult brain can give rise to liver, blood skeletal muscle and myocardium [55].

2.4 Methods and site of delivery of transplanted cells
While we have previously noted that transplanted skeletal myoblasts can be effectively introduced to the heart by either direct intramural injection or by arterial (usually coronary) delivery, the majority of studies surveyed in this review (employing BMC, mesenchymal, endothelial and cardiomyocytes) used direct injection into the ventricular wall. While a subset of these studies examined transplanted cells in normal heart tissues, a number also investigated transplantation into ischemic cardiac tissues, the majority employing injection of donor cells into the center of the ischemic region or scar. It remains to be seen whether this site impacts on the subsequent loss or retention and differentiation of the donor cells.


   3 Applications of stem cells in mitochondrial defects and toxicology
Top
 Abstract
 1 Basic studies
 2 Stem cell-transplantation...
 3 Applications of stem...
4 Conclusions
 References
 
Presently, information concerning mitochondrial structure and function in ES cells is limited, nor has the use of ES therapy been applied to treating cardiac diseases with extensive mitochondrial enzyme or DNA (mtDNA) abnormalities or for the testing of mitochondrial-based cardiotoxicity.

Cardiac disorders with a pronounced mitochondrial-based cytopathy and bioenergetic dysfunction have been reported. A subset of these have a genetic basis due to either defects in mtDNA or nuclear DNA [56]. Recently, it has been proposed to introduce mtDNA-repaired ES cells into a patient harboring a mtDNA mutation, thereby potentially transforming a diseased myocardium into a healthy one [57]. The mtDNA-repaired cells can be derived from the patient's own cells, whose endogenous defective mtDNA genome has been entirely eliminated by treatment with ethidium-bromide, and replaced by entirely wild-type mtDNA genes. In a similar vein, treatment of a patient's nuclear DNA defects would involve either specific genetic replacement of the nuclear gene defect (site-specific homologous recombination can be readily undertaken in ES cells) or if the precise site of the nuclear defect is not known, by replacement of the entire nucleus of the patient with a wild-type nucleus. In the near future ES cells therapy may be used in the treatment of mitochondrial-based cardiac diseases.

Stem cells could also be readily employed in the pharmacological testing and evaluation of cardiotoxic compounds [58–60]. As an alternative to in vivo studies, a test utilizing the differentiation of ES cells into cardiomyocytes (to test chemical toxicity in vitro) has been developed. Interestingly, RA was strongly embryotoxic inhibiting cardiac cell differentiation at very low concentrations (several orders of magnitude) compared to the levels needed to exert cytotoxic effects on the viability of the ES cells [59].


   4 Conclusions
Top
 Abstract
 1 Basic studies
 2 Stem cell-transplantation...
 3 Applications of stem...
 4 Conclusions
References
 
While both embryonic and adult stem cells have shown exciting possibilities in their projected use in cardiac transplantation and repair, at present there are limitations in their use (Table 1). In addition to the difficulties presently raised by significant ethical, legal and distribution issues with the use of embryonic cells, there are technical challenges that will have to be surmounted. Both embryonic and adult stem cells generate heterogenous populations of cells of which cardiomyocytes represent a small fraction [61]. It has been estimated that millions of cardiac cells would be required to repopulate and repair a single damaged human heart. The ability to direct specific cardiomyocyte differentiation (with either adult or embryonic cells) will require further knowledge of the signaling and transcriptional events involved in cardiomyocyte differentiation. The promising developmental plasticity of adult stem cells (and of multipotent adult progenitor cells (MAPC) that co-purify with mesenchymal stem cells [62]) may allow circumvention of the ethical and availability issues currently associated with embryonic cell studies as well as providing an autologous source of highly proliferative cells bypassing the issues of immune rejection (which could arise with embryonic stem or cultured cells of heterologous origin).


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  		Table 1 Myocardial cell transplants: advantages and limits associated with cell-type

 
In addition, rigorous criteria are needed in further studies of the efficacy of cell transplantation, effects on stability and function of the transplanted cells and in the unequivocal identification of transplanted cells. To evaluate the most effective sources of transplanted cells, the following are necessary:
1. A careful analysis of isoforms of contractile protein genes, such as of myosin and alpha-actin comparing their phenotype with that of fetal ventricular cardiomyocytes.
2. Expression of cardiac transcription factors (e.g. Nkx2.5, eHAND, dHAND, GATA-4) and examination of both the timing and interaction of cardiac-specific transcription factors in the cascade pathway (Fig. 1).
3. Immunocytological and ultrastructural analyses to examine whether the selected cardiomyocyte cultures are highly differentiated and evaluating the proportion of cells which are cardiomyocyte and fibroblast as well as gauging their origin (double-labelling experiments very effective).
4. Analysis of specific ion currents and action potentials characteristic of specific cardiomyocyte populations. Regenerated cardiomyocytes need to be capable of responding to appropriate adrenergic and muscarinic stimulation.

Finally, it is important to underscore that the expression of specific transcription factors and structural markers of cardiac differentiation does not represent proof of functionality. Establishing the best cell type for transplantation may require refinement of our imaging technology, as well as further studies to document the efficiency in the production of cardiomyocytes and their stability and functioning in the myocardium. It would seem prudent to remember that the primary objective of the cell transplantation process is to incorporate cells into myocardium that can perform cardiac work, respond appropriately to adjacent cardiomyocytes and be appropriately responsive to physiological stimuli. Also, studies addressing what kinds of myocardial injury are most likely to be effectively treated by stem cell transplantation and which are least likely are necessary. Approaches to cell transplantation therapy to treat ischemic myocardium must take into account the rapid death of ischemic tissue and in cases of myocardial infarction, the size of infarct. Whether results obtained in animal models of ischemia, cryoinjury or cardiomyopathy will be reproducible within the setting of human coronary artery occlusion and extensive myocardial infarction remains to be seen.

Time for primary review 27 days.


   References
Top
 Abstract
 1 Basic studies
 2 Stem cell-transplantation...
 3 Applications of stem...
 4 Conclusions
 References
 

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