Stem cell homing and engraftment to the heart and new

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1 Review: Current Perspective Molecular Genetic Advances in Cardiovascular Medicine Focus on the Myocyte Piero Anversa, MD; Mark A. Sussman, PhD; Roberto Bolli, MD Stem cell homing and engraftment to the heart and new tissue formation capable of replacing the lost myocardium and improving the functional performance of the damaged heart represent the goals of regenerative medicine in cardiac pathophysiology. Theoretically, this outcome can be achieved by using any source of stem cells, embryonic or adult, or other cell types. In fact, several interventions have been used in an attempt to promote myocardial regeneration. These therapeutic strategies have used fetal cardiomyocytes; skeletal myoblasts; embryonicderived endothelial cells; bone marrow derived immature myocytes; fibroblasts; smooth muscle cells; endothelial progenitor cells; and hematopoietic, mesenchymal, and embryonic stem cells. 1 3 In all cases, promising results have been obtained, and the amelioration in cardiac performance was, at times, coupled with the structural and functional integration of the graft with the host myocardium. 4,5 Despite all these efforts, however, the most appropriate form of cellular therapy for myocardial injury remains to be identified, leaving unanswered the key question: What is/are the optimal cell/cells for cardiac repair? The difficulty encountered in identifying the ideal cell to rebuild the heart is certainly due to the many variables that have to be taken into account, including easy access to the source; the possibility of obtaining a significant number of cells; the time required for the cells to home, grow, and differentiate; and their long-term efficacy. The use of multipotent cells that are not resident in the heart but are derived from other organs such as the bone marrow has raised the problem of transdifferentiation and stem cell plasticity. In addition to the complexity of this issue, it is inevitable that when a strongly ingrained and sincerely believed theory is challenged, a variety of reactions are evoked, ranging from enthusiasm and hope to awe, suspicion, and hostility. Despite the evidence for adult stem cell plasticity, a group of skeptics has come forward, denying the existence of the phenomenon or classifying it as a biological curiosity. In particular, the inability to reproduce some data was immediately interpreted as a disproving of positive published results. Differences in protocol, methodological shortcomings, and suboptimal techniques have been overlooked, and strong objections were raised against the possibility of stem cell plasticity. Just a few voices spoke up in favor of transdifferentiation 6,7 : The fact that a phenomenon is quite rare in no way mitigates against its very existence: asteroid collisions with the Earth are rare, but try telling the dinosaurs they do not occur! 6 The Hematopoietic Stem Cell: The Pandora s Box or the Great Mystifier? In The Ray of Creation, the Absolute is the fundamental source of all creation. 8 From the Absolute, the process of cosmic creation branches and descends according to an ordered sequence of increasing complexity. Through the use of a metaphor, the Absolute corresponds to the hematopoietic stem cell (HSC), and the derivatives represent the numerous tissues that have been shown to transdifferentiate from this bone marrow entity, acquiring specific and complex architectural and functional characteristics. Brain, skeletal and heart muscle, liver, pancreas, kidney epithelia, lung and skin were found to be formed from bone marrow cells, calling into question the hematopoietic specificity of these blood-forming stem cells. 9,10 Whether Gurdjieff was a great spiritual teacher or a great mystifier has not been resolved yet. 11 Similarly, whether the HSC is deceiving us or can be a useful therapeutic tool remains to be determined. HSCs are a rare cell population of the bone marrow, comprising 1: to 1: of total blood cells. 12 To date, the HSC appears to be the most versatile stem cell in crossing lineage boundaries and the most prone to break the law of tissue fidelity. Thus, adult somatic HSCs may share the kind of developmental plasticity commonly seen in embryonic stem cells. The early studies on HSC transdifferentiation in new myocardium 4,13 generated an immediate wave of enthusiasm that was somehow encouraged by an overinterpretation of the data. Criticisms were then raised, and, among those, the use of a heterogeneous cell population was the most appropriate. This resulted in a second wave of skepticism that essentially rejected the notion of stem cell plasticity, introducing the possibility of cell fusion as the From the Cardiovascular Research Institute, Department of Medicine, New York Medical College, Valhalla, NY (P.A.); SDSU Heart Institute, San Diego State University, San Diego, Calif (M.A.S.); and the Division of Cardiology, Institute of Molecular Cardiology, University of Louisville, Louisville, Ky (R.B.). Correspondence to Piero Anversa, MD, Cardiovascular Research Institute, Department of Medicine, New York Medical College, Vosburgh Pavilion Room 302A, Valhalla, NY piero_anversa@nymc.edu (Circulation. 2004;109: ) 2004 American Heart Association, Inc. Circulation is available at DOI: /01.CIR

2 Anversa et al Stem Cells and Myocardial Regeneration 2833 Figure 1. Schematic representation of the process of differentiation of adult stem cells in cell lineages of the organ of origin, of the process of transdifferentiation of adult stem cells in cell lineages different from the organ of origin, and of the process of fusion of adult stem cells with terminally differentiated cells. The mermaid, half-woman and half-fish, is used as a symbol of cell fusion; Daphne, who transforms herself into a tree, is used as a symbol of cell transdifferentiation. actual mechanism of tissue repair. 14,15 The controversy became acrimonious, and the therapeutic potential of bone marrow derived cells (BMDCs) for the injured heart or other organs was questioned at multiple levels, including the selection of the injected cells, the surgical procedure, the remarkable findings that went beyond the most optimistic expectations, and the interpretation of the data. Unambiguous documentation that HSCs transdifferentiate and/or fuse is technically demanding (Figure 1), involving the assessment of the true identity of the metaplastic cell, the functional competence of the odd progeny, the identification of the fusion partners, and the demonstration that the converted cell is a hybrid. The latter may become an impossible undertaking if we accept that cellular or nuclear fusion initially leads to a higher DNA content that with time becomes euploid through the process of reduction division and expulsion of chromosomes. 16,17 Hybrid cells can give rise to daughter cells characterized by normal ploidy together with polyploid progeny. Whether the twist in fate occurs by transdifferentiation or fusion, reprogramming of chromatin configuration is required, mostly through activation of transcription factors driving the formation of specific progeny. In

3 2834 Circulation June 15, 2004 both cases, the mechanism is slow and limited in efficiency (Figure 1). Moreover, in the event of cell fusion, the bulky burden of the high nuclear DNA content implies genetic instability and reduced replicative potential. 18 This phenomenon has been demonstrated in neuronal regeneration induced by HSCs. 19 The high degree of plasticity of HSCs together with the overlap between the hematopoietic and cardiac developmental programs prompted us to test whether BMDCs could repair infarcted myocardium. In mice, both local and systemic delivery of BMDCs induced myocardial regeneration that replaced the necrotic area acutely after the ischemic event. 4,13 In the first case, an enriched population of lineage negative c-kit positive BMDCs was injected into the border zone and generated a new ventricular wall composed of small myocytes and an abundant network of patent coronary arterioles and capillaries. 4 The newly formed tissue contracted synchronously with the surviving myocardium and improved cardiac performance. Similar results were obtained by systemic administration of cytokines capable of mobilizing bone marrow cells. At 4 weeks, myocardial regeneration decreased mortality rates, infarct size, thinning of the wall, cavitary dilation, and diastolic wall stress. 13 Again, the reconstituted tissue had the structural and functional characteristics of the ventricular myocardium. These data proved the existence of myocardium-regenerating cells in the bone marrow and provided the foundation for clinical trials. The positive results obtained with the injection of BMDCs in humans confirmed and strengthened the experimental findings The observations in the mouse offer important insights into the controversial issue of cell fusion versus transdifferentiation. Numerous lines of evidence inherent in these results argue against cell fusion as the cause for the cardiac phenotype and pattern of gene expression of the injected or mobilized cells. 4,13 After coronary artery occlusion in rodents, all cells in the supplied region of the myocardium die in less than 5 hours, 3 leaving no partner cells for fusion. Moreover, adult myocytes have an average volume of m 3.If cell fusion occurs, the newly generated myocytes should have a cell volume of m 3. However, the volume of new myocytes reached a maximum of 2500 m 3 and a minimum of 200 m Also, in mice, the reconstitution of dead myocardium is characterized by the generation of 15 million new myocytes. This number is 5-fold higher than the total number of myocytes in the mouse left ventricle, , and 11-fold higher than the number of myocytes lost after infarction, Donor-derived cells divide rapidly and extensively, whereas, in general, tetraploid cells divide slowly and might not divide at all if one of the partners is a terminally differentiated myocyte. Although cell fusion could theoretically explain the colocalization of stem cell surface antigens with myocyte specific markers, it could not explain the functional improvement associated with stem cell injection or mobilization. Amelioration of cardiac function requires an increase in the number of myocytes, not the fusion of BMDCs with preexisting myocytes. Finally and most importantly, 92% of resident ventricular myocytes are binucleated and 6% are mononucleated. Conversely, 90% of new myocytes are mononucleated and 10% binucleated. Cell fusion would imply the generation of myocytes with 2 nuclei, 1 tetraploid and the other diploid, or myocytes with 3 diploid nuclei. This is not the case, excluding that cell fusion is implicated in cardiac repair after infarction. 3 5,13 However, cell fusion might be involved in the minuscule regeneration of cardiomyocytes associated with the administration of Sca-1 positive cells in a model of ischemia-reperfusion injury. 24 It is not surprising that studies performed with a single stem cell have yielded results different from those obtained with a population of cells. 7,25 Whether or not myocardial reconstitution involves cell fusion has clinical relevance because hybrid cells have limited potential for multiplication. 19 It would have been virtually impossible to form millions of myocytes in the infarcted heart by injecting 10 5 BMDCs 4 or by mobilizing with cytokines a restricted number of cells from the bone marrow. 13 The therapeutic goal of cellular cardiomyoplasty is the replacement of dead heart muscle with functionally competent myocardium. The critical issue with regard to the use of BMDCs is whether bone marrow derived myocytes are capable of reaching full differentiation and the adult phenotype or whether there are limitations in the process of transdifferentiation so that the new cells cannot go beyond early developmental stages. In long-term studies of myocardial infarction, mobilization of BMDCs has created myocardium, but the newly formed myocytes fail to acquire adult characteristics; myocyte cell volume does not exceed 1000 m Additionally, myocyte regeneration decreases with time while apoptosis of new cardiomyocytes increases, leading to a time-dependent reduction in reconstituted cardiac muscle mass. These preliminary data raise serious questions about the long-term efficacy of BMDCs. As recently stated, The therapeutic exploitation of resident populations of adult stem cells, either by stimulation of activity with biological agents or by transplantation, remains an option for investigation. 27 Together, this information supports the notion that injury to a target organ promotes alternative stem cell differentiation, emphasizing the plasticity of BMDCs. Although a better understanding of the biology of this repair mechanism is important, the complexity of the problem and the difficulty in predicting the use of stem cells on the basis of their ability to acquire a phenotype similar to the host organ has resulted in reservation on the applicability of non organ-specific stem cells for various diseases. Cardiac stem cells (Figure 2), on the other hand, are expected to be more efficient in rebuilding the damaged myocardium and more prone to acquire the adult cardiac phenotype than BMDCs. Elkab s Hidden Treasure: Cardiac Stem Cells The search for the ancestry in science follows paths similar to archeological discoveries. 28 The dispersed traces and imprints in the Egyptian sand resemble the scattered but consistent findings of the heart as a proliferative organ. The recognition that a subset of myocytes retains the ability to proliferate has led in backward motion to the identification of their founders, the cardiac stem cells (CSCs). 5,24,29 In fact, the search for the origin of dividing myocytes was unsatisfactory until CSCs were recognized. The possibility that an adult fully mature

4 Anversa et al Stem Cells and Myocardial Regeneration 2835 Figure 2. Clone of human c-kit POS cells. Green fluorescence corresponds to the distribution of the stem cell epitope, c-kit, on the plasma membrane of primitive cells. Nuclei are shown by red fluorescence of propidium iodide. myocyte has the ability to dedifferentiate and disassemble the well-organized longitudinally oriented myofibrillar structures and rows of mitochondria to reenter the cell cycle and divide is difficult to envision, biologically too complex, and mechanically disadvantageous to the heart. Similarly, the existence of a subpopulation of partially differentiated myocytes that maintains the capacity to proliferate from prenatal life is difficult to foresee as storage of repopulating cells in the myocardium. The presence of CSCs has challenged the generally accepted but never-proven paradigm that the heart is a postmitotic organ characterized by a predetermined number of parenchymal cells that is defined at birth 30,31 and is preserved throughout life until death of the organ and organism. Thus, the heart belongs to the group of constantly renewing tissues, in which the capacity to replace cells depends on the persistence of a stem cell compartment. 32 Under these conditions, regeneration conforms to a hierarchical archetype in which slowly dividing stem cells give rise to highly proliferating lineage-restricted progenitor cells that become committed precursors and, eventually, reach growth arrest and terminal differentiation. 5 Stem cells have a high capacity for cell division, and this property is preserved throughout the lifetime of an organism. Conversely, the less primitive cells, or transient amplifying cells, have a limited proliferative capacity but represent the largest group of dividing cells. This forms the basis of a new paradigm of the heart in which multipotent CSCs are implicated in the normal turnover of myocytes, endothelial cells, smooth muscle cells, and fibroblasts. The recognition of factors enhancing the activation of the CSC pool, their mobilization and translocation to areas of damage and growth would make the impossible dream of myocardial regeneration a feasible reality. 2,3 One of the relevant questions raised by the presence of CSCs in the myocardium concerns their origin, biological properties, and homeostasis. Particular emphasis has been given to their ability to regenerate adult myocardium, but how this process changes throughout the lifespan of the individual is currently unknown. What is clear is that the progeny of a single CSC isolated from the adult heart is able to form myocytes, smooth muscle cells, and endothelial cells in animals. 5 It is also clear from sex-mismatched human cardiac transplantations that CSCs stored in the atria can migrate from the recipient to the donor heart, home to the new myocardium, and differentiate into the 3 main cardiac cell lineages. 33,34 If this interpretation of human results is correct, why do not CSCs reach a damaged portion of the myocardium, such as an infarcted area, and rebuild the lost tissue completely or in part? The same question can be raised for highly proliferating organs including the skin, the intestine, the liver, and the spleen. Occlusion of dermal arteries in the skin, mesenteric artery in the intestine, hepatic artery in the liver, and splenic artery in the spleen all result in infarcts of the supplied tissue similar to what happens in the heart or in the brain, the two most recently discovered self-renewing organs. 29,39 The inevitable evolution in all these cases is the formation of a scar. However, the presence of multipotent adult stem cells in the heart and also in the brain would suggest that limited areas of damage in the heart or in the brain should be constantly repaired. Unfortunately, there is no demonstration of spontaneous regeneration of

5 2836 Circulation June 15, 2004 Figure 3. Cardiac stem cells and tissue damage. Ventricular myocardium of an old rat at 28 months of age contains an area of damage defined by yellow fluorescence of collagen type III and type I. Importantly, 4 c-kit POS stem cells are visible. Green fluorescence corresponds to the distribution of c-kit on the plasma membrane of primitive cells. c-kit POS cells are viable, as indicated by blue fluorescence of propidium iodide staining of their nuclei. the injured cardiac or cerebral tissue. Myocyte formation has been shown in the postinfarcted human heart acutely and chronically, 40,41 but the addition of these new cells is restricted to the spared portion of the injured ventricle. Similarly, spontaneous neuronal regeneration has been demonstrated in the human brain in the absence of damage. 42 Therefore, a crucial issue of cardiac pathobiology must be discussed. Primitive and early committed cells accumulate acutely in the region bordering the infarct in humans and animals. After homing, these cells grow and differentiate in new myocytes and coronary vessels. However, a block exists at the sharp boundary that separates the viable myocardium of the border zone from the dead tissue of the infarct. CSCs, progenitors, and precursors do not cross this boundary because their translocation to the dead myocardium is impeded. Such an obstruction hampers the reconstitution of infarcted myocardium and the recovery of function. The entire phenomenon is obscure and of great clinical relevance for its impact on all organs. This apparent paradox may be explained on the basis that CSCs scattered throughout the infarct die in a manner identical to myocytes and vascular structures by apoptosis and necrosis, leaving no reserve for tissue reconstitution. The distant CSCs may sense the damage but are unable to migrate to this area, survive, grow, and differentiate. So far, we have found only two examples of CSCs homed to small sites of injury where they survived the hostile environment (Figure 3). A plausible attempt at actual myocardial regeneration of the infarcted heart may involve the activation of receptors on the surface membrane of primitive cells to facilitate their movement, viability, and successful competition with inflammatory cells and fibroblasts invading the infarcted region. If this tactic is effective, the healing process could be modified and cardiac repair would be promoted. A therapeutic strategy based on this principle has recently been applied successfully (Figure 4). It is intuitively apparent that this approach can easily be used in the acute phases of myocardial infarction, offering the unique advantage of promoting tissue regeneration immediately after the ischemic insult. If this protocol is effective, infarcts commonly incompatible with life may be rapidly reduced and patients rescued. Another approach to potentiate the regenerative capacity of stem cells uses ex vivo genetic modification to enhance survival and improve restoration of myocardial volume. 43 Manipulation of survival signaling pathways in future studies will enhance therapeutic application of stem cells by optimizing engraftment, regeneration, and persistence, with the goal of accelerating normalization of cardiac structure and function. Conclusions Is the search for the hidden treasure over? The recognition that the heart is a self-renewing organ advances dramatically our understanding of the biological mechanisms of myocardial growth and offers novel approaches for the treatment of cardiac diseases. The advances made in hematology in the past 2 decades have been a direct consequence of the identification of HSCs and their tremendous potential for bone marrow regeneration. Similarly, the discovery of CSCs can radically change the practice of cardiology and the treatment of patients with ischemic and nonischemic cardiomyopathy. CSCs can be isolated from small cardiac biopsy specimens and can be expanded in vitro for subsequent clinical use. Percutaneous injection of autologous CSCs may enable the reconstitution of dead or scarred myocardium and halt the inevitable unfavorable evolution of the infarcted heart. Moreover, the replacement of poorly functional, markedly hypertrophied myocytes of the severely decompensated heart with new, younger, more powerful muscle cells and coronary resistance and nonresistance vessels may positively interfere with the onset of terminal failure and death. The

6 Anversa et al Stem Cells and Myocardial Regeneration 2837 Figure 4. Band of regenerating myocardium in the infarcted area of a mouse heart treated with cardiac stem cells mobilizing growth factors. Red fluorescence corresponds to cardiac myosin heavy chain antibody staining of the cytoplasm of newly formed myocytes; regenerating band is indicated by arrowheads. Thin layer of surviving myocytes is visible (asterisks). Nuclei are shown by green fluorescence of propidium iodide. Nuclear colocalization of propidium iodide and BrdU labeling of new growing myocytes is shown by bright fluorescence in the inset. isolation and cloning of human CSCs underscore the likelihood of this novel form of cellular therapy. Whether the CSC pool is worn out in end-stage failure and/or the growth reserve of the remaining CSCs is exhausted remains an important question. Under these extreme conditions, nonresident stem cells may also be necessary for treatment and the bone marrow is a logical source in view of the ability of progenitor hematopoietic cells to generate myocardium and intramural coronary arteries, arterioles, and capillary structures. Acknowledgments This study was supported by National Institutes of Health grants HL-38132, AG-15756, HL-66923, AG-17042, and AG References 1. Rosenthal N. Prometheus s vulture and the stem-cell promise. N Engl J Med. 2003;349: Anversa P, Nadal-Ginard B. Myocyte renewal and ventricular remodeling. Nature. 2002;415: Nadal-Ginard B, Kajstura J, Leri A, et al. Myocyte death, growth and regeneration in cardiac hypertrophy and failure. Circ Res. 2003;92: Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature. 2001;410: Beltrami AP, Barlucchi L, Torella D, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. 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