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OPEN ACCESS ARTICLE
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TISSUE-SPECIFIC STEM CELLS |
aCardiovascular Research Institute, Department of Medicine, New York Medical College, Valhalla, New York, USA;
bInstitute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, USA
Key Words. Heart • Confocal and light microscopy • Mitosis • Chimerism • Bone marrow cell transdifferentiationEnhanced green fluorescent protein autofluorescence
Correspondence: Piero Anversa, M.D., Cardiovascular Research Institute, Vosburgh Pavilion, New York Medical College, Valhalla, New York 10595, USA. Telephone: 914-594-4168; Fax: 914-594-4406, e-mail: piero_anversa{at}nymc.edu
Received October 3, 2006;
accepted for publication November 7, 2006.
First published online in STEM CELLS EXPRESS November 22, 2006.
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ABSTRACT |
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INTRODUCTION |
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We will review the work that has promoted a shift in paradigm of the heart from a terminally differentiated postmitotic organ to a self-renewing organ. This dramatic change in understanding of cardiac behavior and function was originated by observations that were made in the 1940s and that have continued to accumulate over the years (for reviews, see [5, 6]). However, the disagreement that persists among the members of the scientific community today was present then and has persisted for nearly 60 years. The reason for the controversy is unclear, but it seems to reflect unshakable positions based on preconceived beliefs more than on careful analysis and proper consideration of published results [7]. In this article, we focus on methodological issues because, in the course of the controversy, recurrent statements have been made to undermine the technical protocols used in studies supporting the existence of cardiac regeneration. Consistently, the data in favor of myocardial regeneration are claimed to be the product of methodological artifacts [1–4, 8–11]. To properly evaluate the validity of these criticisms or lack thereof, methodologies need to be compared to provide readers with a better understanding of the substrate that governs the debate.
The old dogma has profoundly conditioned basic and clinical research in cardiology for the last three decades [7, 12]. Based on this paradigm, cardiomyocytes undergo cellular hypertrophy but cannot be replaced either by the entry into the cell cycle of a subpopulation of nonterminally differentiated myocytes or by the activation of a pool of primitive cells that become committed to the myocyte lineage. However, the efforts made to introduce a highly dynamic perspective of the heart have led to the identification and characterization of a resident pool of stem cells that can generate myocyte, and ECs and SMCs organized in coronary vessels [13]. This discovery has created a new, heated debate concerning the implementation of adult cardiac stem cells in the treatment of heart failure of ischemic and nonischemic origin.
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POSSIBILITY 1: THE HEART IS A TERMINALLY DIFFERENTIATED POSTMITOTIC ORGAN |
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Thus, the dogma was established that the postnatal heart is composed of a fixed number of myocytes and that, if myocytes die, they are permanently lost and the myocardium must maintain its vital role with a reduced number of cells. The remaining myocytes are not in G0 and cannot be triggered into the replicating phase [28]; they continue to perform their physiological function, undergo cellular hypertrophy, and ultimately die [29]. Based on this paradigm, the age of myocytes, organ, and organism was assumed to coincide, implying that myocytes in humans may have a lifespan that exceeds 100 years [30]. For several decades, no effort was made to reexamine this rather unusual view of the biology of the heart and cardiac homeostasis. Remarkably, there is not a single piece of evidence that demonstrates the inability of the heart to replace its dying myocytes. It seems rather extravagant that cardiomyocytes can contract 70 times per minute over 100 years and continue to be functional. During this period, they would have contracted 3.7 billion times and still be operative. If this were to be the case, adult myocytes would be essentially immortal cells.
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POSSIBILITY 2: THE HEART IS NOT A TERMINALLY DIFFERENTIATED POSTMITOTIC ORGAN |
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The qualitative results discussed so far were in sharp contrast with quantitative measurements of myocyte volume and number performed in human hearts obtained from patients who died because of decompensated cardiac hypertrophy and congestive heart failure [5]. In the late 1940s and early 1950s, Linzbach documented that, in the presence of a cardiac weight equal to or greater than 500 g, myocyte proliferation represented the predominant mechanism of increased muscle mass [38, 39]. An interesting aspect of this work is that these determinations were all based on two very simple but critical parameters: myocyte length, assessed by the distance between two nuclei of largely mononucleated myocytes, and myocyte diameter across the nucleus. These variables are easily recognizable in standard histologic sections and do not require high resolution and an impeccable preparation. Based on a cylindrical shape configuration, myocyte cell volume was computed. Moreover, the aggregate volume of the myocyte compartment of the myocardium was obtained, and the quotient of this quantity and myocyte cell volume yielded the total number of ventricular myocytes. Because of the simplicity of the approach, the original results were confirmed several years later in other laboratories in which more refined techniques were applied [40, 41]. In all cases, hearts weighing 500 g or more were characterized by a striking increase in myocyte number that was more prominent than cellular hypertrophy; this adaptation involved both the left and right ventricles [38–43].
In the mid- and late 1990s, new studies of the human heart examined the distribution of mononucleated and binucleated myocytes in 72 normal and 176 diseased hearts [43]. Aging, cardiac hypertrophy, and ischemic cardiomyopathy were characterized by the lack of changes in the relative proportion of mononucleated and multinucleated myocytes in the ventricular myocardium, confirming that the early and more recent measurements of myocyte proliferation were valid and did not represent and erroneous interpretation dictated by nuclear hyperplasia in the absence of myocyte division [6, 44]. Mononucleated cells constitute 
75% of myocytes in the human heart, differing significantly from mice [45], rats [46], dogs [47, 48], and pigs [49].
Mononucleated cells are smaller than binucleated cells, and this cellular property may influence the ability of myocyte to divide [50]. Human myocytes larger than 30,000 µm3 cannot reenter the cell cycle and proliferate [50]. In fact, cycling and mitotic myocytes are predominantly small and mononucleated, and this feature may account for the massive cardiac hypertrophy that can be achieved in humans (Fig. 1). It would be inefficient for large myocytes to divide once or at most twice to expand the cardiac mass. Heart weight in humans can increase nearly threefold, reaching values of 1,000 g or larger [5, 38, 41, 43]. Heart failure typically shows increases in myocyte number that vary from 20%–100% or more [5, 38, 41, 43, 51–53]. This phenomenon is not affected by age; in an analysis of 7,112 human hearts, from birth to 110 years of age, Linzbach and Akuamoa-Boateng have shown that extreme forms of organ hypertrophy are detectable up to the ninth decade of life, and heart weights of 500 and 600 g are present in patients at 100 years of age and older [54].
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However, the quantitative data collected in the human heart suggested that a reexamination of the mechanisms of myocyte growth needed to be considered and that additional studies needed to be performed to clarify the apparently contradictory results. For this purpose, experiments were conducted in animal models of cardiac failure and in human diseased hearts to provide relevant information in support of or against the notion of the regeneration potential of the adult myocardium. New methodologies were introduced to determine whether a subset of cardiomyocytes had the ability to reenter the cell cycle and divide and whether this process involved the activation of cell cycle-related genes, cyclins, and cyclin-dependent kinases and the expression of proteins modulating karyokinesis and cytokinesis [45, 50, 55, 66–80]. Rounds of cell division result in progressive telomere attrition, and critical telomeric shortening triggers irreversible growth arrest [81–83]. Thus, the telomere-telomerase axis, together with the telomere-binding proteins, is a fundamental component of the multiple mechanisms that regulate the regeneration of non-postmitotic organs; because of this function, this system was evaluated and found to be operative in the adult heart of animals and humans [50, 74–77, 79, 84]. Unfortunately, these multiple studies left the controversy unresolved.
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MYOCYTE DIVISION |
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In the last 10–12 years, however, mitotic images in human cardiomyocytes have been documented in acute and chronic ischemic cardiomyopathy [72, 89, 90], idiopathic dilated cardiomyopathy [72], chronic aortic stenosis with moderate ventricular dysfunction [50], myocardial aging [84], acromegaly (Fig. 2A), and diabetes (Fig. 2B). Using light microscopic examination of routine histologic sections, an extensive search was required for the recognition of occasional mitotic figures in myocytes. Because in the early successful observations, dividing myocytes were seen only in the fetal and diseased failing heart [89], the assumption was made that replicating myocytes are essentially undetectable in normal adult myocardium. This hypothesis, in fact, suggested a rather minor role of myocyte regeneration in cardiac homeostasis and pathology in humans. However, in spite of the caution exercised in the interpretation of these results, this work was considered to reflect the incorrect interpretation of proliferating interstitial fibroblasts as dividing myocytes [1, 2]. Surprisingly, this conclusion was reached based on electron microscopic images that illustrated nondividing fibroblasts in proximity to differentiated nonreplicating cardiomyocytes [1]. This represents the normal organization of the myocardium and makes it difficult to understand how this well-established morphological characteristic of cardiac structure can be used as an argument against myocyte division and a case in favor of its erroneous identification. So Artifact 1 was introduced in the literature as the only logical explanation for these unexpected results.
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1.4–1.5 µm in length. Normally, diastolic sarcomere length is 2.1–2.2 µm, and during systolic contraction, it becomes 1.8–1.9 µm [91]. Hearts with sarcomeres 1.4 µm in length are dead, since no contraction can be generated under this condition. Contraction bands produced by the inappropriate fixation of the myocardium were analyzed to reinterpret myocardial ultrastructure. Surprisingly, these artifacts were accepted and introduced in the literature as facts.
The frequency of mitosis in myocytes of normal human, mouse, or rat heart, as assessed by confocal microscopy, is low and averages 14/106 myocytes [72], 37/106 myocytes [45], and 85/106 myocytes (J.K., unpublished data), respectively. The values for dividing interstitial fibroblasts are similar to those of cardiomyocytes [89]. Based on these data, 350, 140, and 60 mm2 of tissue need to be examined to identify a dividing myocyte in the human, mouse, and rat heart, respectively. This is why the suggestion to implement electron microscopy for this analysis is not realistic. The area of myocardium that is sampled in each thin section by electron microscopy is 0.2 mm2, and the area of the microscopic field at a magnification of approximately x2,000 is 0.0054 mm2. Thus, the numbers of micrographs that would have to be collected to identify a single myocyte mitotic image are 66,000 pictures for humans, 25,000 for mice, and 11,000 for rats. If one wishes to use electron microscopy to challenge the existence of myocyte division and to demonstrate that proliferating fibroblasts were improperly interpreted as mitotic myocytes, that is what needs to be invested to obtain at least one example in support of or against this thesis [92].
Major advances in understanding myocyte replication in the adult human heart have been made by the use of confocal microscopy and immunolabeling in the analysis of the normal and diseased heart. With this approach, the identification of mitotic images in myocytes became easier and more accurate. These new results strengthened the notion that parenchymal cells are formed continuously in the normal myocardium and myocyte regeneration is markedly potentiated in the pathological failing heart [6, 50, 72, 84, 90]. This technology was also applied to the study of animal models of human diseases, and myocyte replication was unequivocally demonstrated in myocardial sections and isolated cell preparations [6, 45, 73, 93]. The latter observation conclusively refuted the claim that dividing myocytes represent proliferating fibroblasts inserted within or attached to terminally differentiated myocytes. The formation of the mitotic spindle by the arrangement of tubulin and the generation of the contractile ring by the accumulation of actin in the region of cytoplasmic separation were clearly documented, together with karyokinesis and cytokinesis (Fig. 2C–2E). However, these results did not change the position of certain groups, who continued to reject the regenerative ability of the adult mammalian myocardium [2, 4, 8, 9, 28]. These groups introduced the possibility of a new source of artifacts, Artifact 2, related to confocal microscopy; they stated that traditional light microscopy is greatly superior to confocal microscopy and, with this personal viewpoint, tried to invalidate the results obtained with the latter technique.
An important issue that requires discussion relates to the notion that the analysis of histochemical preparations by conventional light microscopy is superior to fluorescence labeling and confocal microscopy [94, 95]. The resolution of the micrographs provided by light microscopy is markedly inferior to that obtained by confocal microscopy, 0.62 versus 0.29 µm [37, 96]. In addition, only the very superficial layer of a tissue section can be examined by light microscopy (Fig. 3). This inherent problem with light microscopy was recognized immediately when confocal technology became available [97]. Clear examples were published in the Journal of Cell Biology in 1987 demonstrating the impossibility of identifying microtubules, mitotic spindle, cytoplasmic proteins, and chromosomal structures by light microscopy in cultured cells. Conversely, these morphological details were apparent when the same cells were examined by confocal microscopy [98]. The difference between epifluorescence light microscopy and confocal microscopy is even greater in tissue sections. When confocal microscopy was not available to us, we estimated the myocyte mitotic index in chronic heart failure to be 11 myocytes per million [89]. When comparable hearts were evaluated by confocal microscopy, the mitotic index reached a value of 152 cells per million [72]. Even more striking is the difference between the mitotic indices obtained by light and confocal microscopy in the border zone of acute infarcts in humans: light microscopy yielded 3.3 mitotic myocytes per million [89] and confocal microscopy 775 mitotic cells per million [90]. In all cases, the measurements of myocytes in mitosis were made in the same laboratory.
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CARDIAC CHIMERISM AND MYOCYTE FORMATION |
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The surprising outcome of these findings was that the low extent of myocyte chimerism detected by some groups was used to question myocyte formation and to support the traditional view that the heart is a terminally differentiated postmitotic organ incapable of undergoing meaningful regeneration of any kind [2, 4, 8–10]. However, to accomplish this task, a rational explanation was necessary to account for the data pointing to a high number of male myocytes within female hearts transplanted in male recipients [99]. In fact, these observations indicated that a significant proportion of male myocytes was present in the female heart as a result of activation and commitment of the host male progenitor cells. Interestingly, the discrepancy in the degree of cardiac chimerism was immediately adopted to initiate another controversy and introduced in the literature Artifact 3. The promoters of this new source of technical error reached this conclusion by assuming that only the absence or minimal levels of chimerism were correct, whereas high levels of chimerism had to be necessarily wrong [94, 95, 104, 115]. The critics decided to take the prerogative of reinterpreting data published by other groups, pointing to a number of crass morphological errors. Surprisingly, these negative comments were published and, once again, these questionable criticisms became facts.
The most obvious technical differences among the studies published so far is the use of conventional light microscopy [94, 95, 100–102, 104] versus confocal microscopy [99, 109, 110]. The probes used for the detection of the Y chromosome in female hearts transplanted in male recipients were not always the same, and the procedures used for the recognition of the Y chromosome differed as well. These factors introduced critical variables in the acquisition of the data that, together with the method of analysis, have been a major cause for the discrepancy.
A serious technical and conceptual concern is that similar high values of Y-chromosome-positive myocyte nuclei have been obtained in control male human hearts, averaging 50%. However, the values reported in control male human hearts by conventional fluorescence microscopy and confocal microscopy in the absence of optical sectioning of the samples are unrealistic and therefore cast doubts on this series of studies. By these two methods of analysis, the fraction of Y-chromosome-positive myocyte nuclei is low and rarely exceeds 15% (Fig. 4A). This is due to the limited focal depth of the objective, 0.5 µm, [37] and the extent by which a nucleus has to be embedded within the section to be visible (i.e., the penetration factor) [44]. Moreover, with conventional fluorescence microscopy, the out-of-focus fluorescence blurs the image, complicating the identification of the Y-chromosome signal in nuclei (Fig. 4B). When histologic sections of male myocardium are examined by confocal microscopy, the superficial layer of the section, which corresponds to 0.6 µm, shows 12% Y-chromosome-positive myocyte nuclei (Fig. 4C). Importantly, the evaluation of the entire thickness of the section by the analysis of consecutive optical planes markedly increases the detection of Y-chromosome-labeled myocyte nuclei, reaching a cumulative value of 55% (Fig. 4D). Therefore, in several reports in which conventional fluorescence microscopy or confocal microscopy restricted to a single plane was used [94, 101, 102, 104], the finding of the Y chromosome in a large number of myocyte nuclei of control male hearts is at variance with reality. Because of this unacceptable premise, the extremely low number or absence of Y-chromosome-positive myocyte nuclei found in the transplanted female hearts can be seriously questioned.
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250 µm3, and the signal for the Y chromosome has a volume of 0.5 µm3, occupying 0.2% of the entire nucleus. When the Y chromosome is present in a mid-section of a nucleus, the area of the Y-chromosome signal is 1 µm2, and the area of the nucleus profile is 75 µm2 [116]. Thus, the Y chromosome occupies 1.3% of the nuclear area (Fig. 4E). It follows that the probability of hitting the Y chromosome in myocyte nuclear profiles of a tissue section can be calculated. Importantly, the chance of a positive signal varies as a function of section thickness and is influenced by the orientation of the nuclei in myocytes. These variables can be computed according to the following equations [44, 116]. |
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. Supplemental online Table 1 lists the predicted minimal, maximal, and average values of Y-chromosome-positive myocyte nuclei in control male human hearts according to section thickness, nuclear orientation, and modality of analysis. Surprisingly, there is a dramatic inconsistency between the expected values of Y-chromosome labeling of myocyte nuclei in normal conditions and in published results. Because of the questionable validity of control values in these studies, the number of male cells identified in female-transplanted hearts suffers from lack of reliability and necessitates reevaluation. This issue is certainly more relevant than the claim concerning the difficulty of distinguishing myocyte nuclei from interstitial cell nuclei [1, 2, 94, 115]. As recently emphasized, an experienced pathologist can easily differentiate an interstitial cell from a cardiomyocyte or discriminate an interstitial cell nucleus from a myocyte nucleus, particularly by confocal microscopy [117]. This is confirmed by the clear recognition of the Y-chromosome signal and nuclear profiles by confocal microscopy [99, 109, 110] and the rather ill-defined Y-chromosome labeling and nuclear boundary by light microscopy [94, 95, 104]. In some cases, by light microscopy, the Y chromosome cannot be distinguished from the nucleolus [94] or properly identified because of little contrast, weak intensity of the signal, and diffuse pattern of staining [104].
Several other factors have to be considered when human myocardium is examined. The age of the patients, time from transplantation, level of rejection, immunosuppressive regimen, and, most importantly, the condition of the tissue to be studied may all contribute to the variability in the levels of cardiac chimerism reported in the last several years. Poor fixation and/or delayed fixation affect the integrity of the DNA and, thereby, the probability of detecting any nucleotide sequence [118]. DNA degradation precedes the alterations in protein antigenicity [119] so that Y-chromosome labeling of myocyte nuclei may constitute a significant underestimation of the actual degree of myocyte chimerism present in the organ [109, 117]. Likewise, prolonged preservation of the myocardium in formalin fixative produces excessive cross-linking of proteins [120], and this condition diminishes the accessibility of the Y-chromosome probe to the nuclear DNA [118, 121]. It is surprising that these crucial factors, together with elementary morphometric principles, have been ignored in the studies pointing to the absence or minimal levels of cardiac chimerism following sex-mismatched heart allograft or bone marrow transplantation.
An elegant protocol has been introduced in one study of cardiac chimerism following sex-mismatched bone marrow transplantation to correct, at least in part, this problem. The fluorescence in situ hybridization (FISH) assay was performed with two distinct probes, which detected, respectively, the Y chromosome and the X chromosome in myocyte nuclei [103, 109]. Nuclei negative for the X chromosome had severely altered DNA and had to be excluded from the analysis. When this approach was used, the extent of cardiomyocyte chimerism was found to be 30-fold higher than in other comparable studies [108]. Because questions have been raised concerning some of the values obtained in our study of cardiac chimerism following sex-mismatched heart transplantation [99], we have re-examined cases with the highest levels of Y-chromosome-positive myocyte nuclei using this dual-color FISH assay and evaluated the percentage of XX-positive and XY-positive myocyte nuclei (Fig. 5A, 5B). The original data were confirmed and strengthened by this more sophisticated approach. In addition, the measurements of the number of X and Y chromosomes in nuclei allowed us to exclude cell fusion as the mechanism of male myocyte formation in the female heart. Importantly, optical sectioning by confocal microscopy prevented any possible misinterpretation of interstitial cell nuclei as myocyte nuclei (Fig. 5C–5L).
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The recognition that cardiac chimerism occurs rapidly and in a noticeable manner has had a dramatic impact on our understanding of the biology of the heart and mechanisms of myocardial repair. This work has formed the foundation for the concept of myocardial regeneration and has created the opportunity for testing the role that HSCs have in the restoration of the injured heart [123]. The notion that HSCs retain a significant degree of developmental plasticity and can acquire cell lineages distinct from the organ of origin has promoted the entire field of cell therapy of the failing heart [124]. Opponents of this emerging new paradigm in cardiology, however, continued to claim that these concepts are the product of artifacts [1, 2, 10, 94, 95, 108, 125]. A recurrent unqualified statement is that they have introduced in their work "rigorous" and "stringent" criteria, which implies that others do not adhere to the same high scientific standards. The old paradigm has to survive, and the notion that myocytes cannot be generated postnatally, in adulthood or senescence, must be defended. An example of this approach can be found in a recent report in which levels of EC chimerism of 25% and vascular SMC chimerism of 3.5% have been found in sex-mismatched heart transplants in the absence (0.0016%) of myocyte chimerism [95].
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HEMATOPOIETIC STEM CELLS AND MYOCYTE FORMATION |
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Criticisms have focused on the need to use genetic markers to demonstrate whether HSCs can form heart muscle [2–4, 9, 10]. Genetic markers have been used in the questioned positive studies [129, 135], as well as in the negative reports that have been claimed to be valid [138–140]. Both positive and negative publications used immunostaining, but in the former case, autofluorescence was assumed to be a factor, whereas in the latter case, the technique used was considered immaculate. Unfortunately, the basis for this conclusion remains an enigma. Although EGFP labeling was applied in several of the papers that opposed HSC plasticity [139, 140, 145], one of these laboratories introduced the notion that green autofluorescence is commonly present in skeletal muscle samples and therefore the detection of EGFP-positive cells in the heart or skeletal muscle following implantation of EGFP-positive HSCs is by necessity an artifact [146]. This concept was adopted immediately by the International Society of Stem Cell Research [147] and emphasized in original studies and reviews [2, 10].
The rationale for Artifact 5 is incomprehensible. The green autofluorescence observed in histologic sections of skeletal muscle was largely due to the use of an inappropriate fixation protocol for immunolabeling studies. A mixture of paraformaldehyde and glutaraldehyde (Fig. 7A) was used rather than phosphate-buffered formalin (Fig. 7B), which carries minimal background autofluorescence. But most importantly, immunolabeling of glutaraldehyde-fixed tissue never works (Fig. 7C, 7D) because of the high level of cross-linking of proteins generated by glutaraldehyde [148]. Green autofluorescence can be clearly distinguished from EGFP-expressing cells by the use of specific green fluorescence protein antibodies as opposed to direct identification of the EGFP protein by epifluorescence light microscopy (Fig. 7E–7G). Therefore, immunolabeling can be easily optimized so that the emission signal is orders of magnitude greater than background fluorescence, or autofluorescence can be totally excluded by direct labeling of antibodies with quantum dots (Fig. 7H–7K).
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CARDIAC STEM CELLS AND MYOCYTE FORMATION |
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The discovery that cardiac stem cells (CSCs) are present in the adult myocardium led to the proposal of a new artifact. The formulation of Artifact 6 required vivid imagination and unusual logic; since myocytes cannot be formed [1, 2], the preparations of CSCs must be a product of an artifact associated with the in vitro isolation [10]. The critics then go on to suggest that this artifact may, however, have some positive outcomes. But to question the presence and role of CSCs in the adult heart, Artifact 6 could not be restricted to the in vitro work and had to be extended to the demonstration that CSCs regenerate infarcted myocardium in vivo [13]. This task was accomplished by stating that cells that expressed cardiac myosin heavy chain, connexin 43, and N-cadherin and responded to electrical stimulation by shortening and relengthening were in reality fibroblasts [10].
In summary, the controversy concerning stem cell therapy for the damaged heart has its foundations in the debate that originated nearly 80 years ago when it was concluded that myocardial regeneration ceases at birth and cannot occur in adulthood or senescence [31]. The biochemical characterization of cardiac hypertrophy performed in the late 1960s and 1970s has contributed to reaffirming the notion that myocardial growth postnatally can be accomplished only by enlargement of the pre-existing myocytes [36], strengthening the notion of the heart as an organ formed by a predetermined number of myocytes incapable of reentering the cell cycle and dividing. Molecular cardiology was built on this inherited inviolable paradigm [57], which has been strongly supported by traditional cardiovascular pathologists reluctant to introduce modern technology in the analysis of the structure of the normal and diseased heart. Surprisingly, myocyte death and myocyte formation, which are the two critical variables that control cardiac cell number, are rarely evaluated concurrently to obtain a dynamic view of the heart in its various phases of life [45, 75, 76, 155]. By this simple approach, it would become clear whether the heart can function with the same cells throughout the lifespan of the organ and organism or whether an alternative possibility has to be considered.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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Abstract
Introduction
