The Future of Cardiovascular Regenerative Medicine
2016; Lippincott Williams & Wilkins; Volume: 133; Issue: 25 Linguagem: Inglês
10.1161/circulationaha.115.019214
ISSN1524-4539
Autores Tópico(s)Tissue Engineering and Regenerative Medicine
ResumoHomeCirculationVol. 133, No. 25The Future of Cardiovascular Regenerative Medicine Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBThe Future of Cardiovascular Regenerative Medicine Richard T. Lee, MD and Kenneth Walsh, PhD Richard T. LeeRichard T. Lee From Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, and the Harvard Stem Cell Institute and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA (R.T.L.); and Whitaker Cardiovascular Institute, Boston University, Boston, MA (K.W.). and Kenneth WalshKenneth Walsh From Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, and the Harvard Stem Cell Institute and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA (R.T.L.); and Whitaker Cardiovascular Institute, Boston University, Boston, MA (K.W.). Originally published21 Jun 2016https://doi.org/10.1161/CIRCULATIONAHA.115.019214Circulation. 2016;133:2618–2625IntroductionFor centuries, biologists have studied salamanders that can regrow entire new limbs after amputation. Flatworms, when beheaded, grow a new head from the beheaded corpse (and the severed head grows a new body).1 All species, from bacteria to plants to animals, can regenerate to some degree, and it has been said that without regeneration, there could be no life.2 In fact, humans regenerate many of their tissues, including skin, blood, liver, and intestinal mucosa, routinely, efficiently, and often perfectly. Skeletal muscle regenerates remarkably well; every time we strain a muscle, muscle stem cells repair the injury, and this is a key component of the conditioning associated with exercise training. But the adult human heart appears to have little of the regenerative ability of our skeletal muscles. The challenge of this limitation has triggered considerable enthusiasm in using different types of stem cells to repair failing human hearts. We will not review this substantial effort in human cardiac cell therapy, because expert reviews have been published recently.3–5 Here, we attempt to predict where regenerative biology and its translational discipline, regenerative medicine, will take us in the future, with a focus on cardiovascular medicine.Regenerative biology has many definitions, but a simple one is the study of how organisms replace lost or damaged tissue with new tissue. We are now entering an era where regenerative biology is turning from science fiction into science, with realistic regenerative medical applications beginning to emerge. This change is based on a broader and more fundamental understanding of cell biology, and this new view has laid a foundation for the potential repair of organs that were previously viewed as being incapable of regeneration or even modest repair. The explosion of interest in regenerating diseased hearts and other organs is being fueled by advances in many fields, including nanotechnology, genetics, imaging, and other fields. But, at the center of this new regeneration movement are recent advances in understanding the biology of stem cells (Figure 1). Thus, before making predictions for the future of regenerative medicine, it is important to review some of the recent and stunning events in the stem cell field. At a fundamental level, these events have changed how we view diseased organs, because we now see in every cell, even the most diseased cells, the potential to become transformed into a different cell type.Download figureDownload PowerPointFigure 1. Stem cell biology and reprogramming. Although embryonic stem cell advances have enabled the generation of any cell type in the human body, the recognition that cells can be reprogrammed to other cells has opened the door to more diverse regenerative medicine approaches. Notably, fibroblasts can be directly converted to cardiomyocytes, without passing through a stem cell state.The Impact of Stem Cell BiologyStem cells, by definition, are cells that can self-renew, and form multiple types of differentiated cells, as well. Embryonic stem cells, are cells derived from the blastocyst before implantation into the uterine wall, and embryonic stem cells are capable of giving rise to all cell types in the body. This property is referred to as pluripotency. Mouse embryonic stem cells were first derived in 1981,6,7 and human embryonic stem cells were derived by Thomson and colleagues in 1988.8 Derivation of embryonic stem cells usually destroys the embryo, and, thus, research with embryonic stem cells has been controversial.9 Embryonic stem cells are derived from donated embryos; no human embryonic stem cell lines can be created without consent of the donor, and they are never made from aborted fetuses. Mouse and human embryonic stem cells can be easily directed into differentiating to cardiomyocytes through a series of sequential small-molecule, protein, or nucleic acid signals.10,11 Human cardiomyocytes derived from human embryonic stem cells have been shown to improve the function of infarcted rodent hearts,12 and have also been shown to improve the function of infarcted nonhuman primate hearts, although ventricular arrhythmias occurred.13Adult stem cells are cells that reside in adult tissues such as bone marrow, the hair follicle, and intestinal epithelium, and adult stem cells have the capacity of self-renewal and differentiation. Many putative adult stem cells have been reported to generate cardiomyocytes, with some of these candidate cells in clinical trials.14 Enthusiasm for adult cardiac stem cells has been justified to some extent, because this concept could lead to autologous cell transplantation for heart failure analogous to the widely used hematopoietic stem cell approach. However, in contrast to hematopoietic stem cells, cells proposed to be cardiac stem cells are less rigorously characterized and typically represent heterogeneous populations of cells that display a low level of differentiation into cardiac myocytes. Furthermore, the potential for adult cardiac stem cells may have been overstated, because recent data do not indicate that the adult mammalian heart has a highly regenerative stem cell population.15A landmark event in stem cell biology was the discovery of induced pluripotent stem cells (iPS cells) with properties of embryonic stem cells, as reported in 2006 by Shinya Yamanaka's team at Kyoto University. In this study, it was shown that an iPS cell can be generated from differentiated cells by using reprogramming factors.16 This monumental achievement meant that potentially any cell could be used to generate unlimited numbers of any differentiated cell type, without using embryonic stem cells (Figure 1). Yamanaka and John Gurdon, who had shown the potential of reprogramming in frogs in 1958,17 were awarded the Nobel Prize in 2012 for the discovery that mature cells can be reprogrammed to pluripotency. Since Yamanaka's transformative discovery, iPS technology has allowed the routine generation of human cardiomyocytes from iPS cells derived from human fibroblasts.18,19 Clinical trials for macular degeneration with retinal pigment epithelial cells derived from iPS cells are underway.20One of the consequences of iPS technology has been easing of the ethical tensions over the use of embryonic stem cells, because iPS cell generation requires only a skin fibroblast or another cell type from the donor. However, the impact of reprogramming of cells goes far beyond iPS cell generation for generating heart cells. We now recognize the potential to turn almost any cell into any other cell type, and this has opened the floodgates of new reprogramming strategies.21 Importantly, cells can now be directly reprogrammed to other types of cells without passing through a stem cell state, potentially limiting the generation of teratomas and cancers. For example, in the pancreas, exocrine cells that do not produce insulin can be directly reprogrammed to form β-cells for diabetes mellitus without removing the cells from the animal,22 and, in the brain, 1 type of neuron can be directly reprogrammed to another type of neuron.23 The direct conversion of adult skin fibroblasts to functional endothelial cells has also been documented.24 In the heart, direct reprogramming has allowed investigators to turn cardiac fibroblasts into cardiac myocytes.25,26 The concept that local fibroblasts, which generate the scar of the injured heart, can be directly converted into functional cardiac myocytes without returning to a pluripotent state, is a stunning example of the power of stem cell biology.In concert with advances in many fields, particularly human genetics, stem cell and reprogramming technologies will revolutionize cardiovascular biology over the next generation. It is obvious that the ability to generate billions of cardiac myocytes, and other cell types, as well, has the potential to replace damaged cardiovascular tissue by cell transplantation. Less obvious is the potential for stem cell science to transform virtually all areas of cardiovascular therapeutics, changing how diseases are studied and how therapies are developed. Even if injection of cells into damaged hearts is never a widely adopted therapy for patients, and even if we cannot grow new myocardium that is routinely sewn into hearts, the principles of regenerative biology will have a profound impact on how we view new therapies for cardiovascular disease. For example, it may be possible for us to stimulate endogenous regeneration mechanisms within the myocardium through proteins or gene delivery, leading to new cardiomyocyte formation without exogenous delivery of cells. In fact, this in situ regeneration approach could be much more economically attractive than cell therapy.We recognize that translating regenerative biology principles into real benefits for patients will take many years, and, in some cases, many decades. We know there will be disappointments and missteps along the way. Some of our predictions may seem as fanciful as science fiction, but we note that this is how implantable defibrillators and drug-eluting stents were once viewed. So, although we are cautious about what therapies are right around the corner, we are very optimistic that regenerative biology will change cardiovascular medicine.Prediction 1: Regenerative Medicine Will Initially Struggle to Become Practical RealityStem cell biology has set off an explosion of efforts to generate virtually every type of cell. Although stem cells provide enormous potential for all of medicine, we envision that many cardiovascular diseases will not be direct therapeutic targets for regenerative medicine over the next decade. For example, human cardiomyocyte pacemaker cells have been made from the keratinocytes from a hair plucked from a patient: a truly fascinating achievement.27 However, although electronic pacemaker technology is far from perfect, the reliability of a stem cell–derived sinoatrial node may never exceed or even approach our current pacemaker technology. The bar for replacing electronic pacemaker technology is extremely high, and, because pacemakers and leads will continue to improve, the bar will only move higher. Regenerative medicine therapies will need to be competitive in light of improvements in devices, surgical techniques, and standard pharmacological approaches (Figure 2).Download figureDownload PowerPointFigure 2. Needs unmet by nonregenerative therapies will define regenerative medicine opportunities. Regenerative treatments will emerge but will be most useful in areas where more traditional approaches leave voids of unmet needs; thus, progress in other fields will play a major role in determining where regenerative breakthroughs can occur. However, stem cell biology will influence all approaches, by allowing testing and validation with human tissue of diverse genetic backgrounds.Thus, the excitement of stem cell biology, and the dream of replacing diseased cells with healthy ones, will be met with an economic and practical reality. In many human diseases, including in the cardiovascular system, progress in more conventional therapies will make regenerative medicine uncompetitive. For example, consider the changes in the treatment of acute myocardial infarction over the past few decades. Once, only a minority of patients with myocardial infarction were candidates for percutaneous intervention because most people did not live close enough to hospitals with catheterization laboratories. With the demonstration of the benefits of primary percutaneous coronary intervention, by the year 2000, ≈80% of US residents lived within 60 minutes of a hospital capable of primary percutaneous coronary intervention.28 Because most patients with acute myocardial infarction currently do well if they make it to the catheterization laboratory and are successfully reperfused, the opportunity for regenerating myocardium immediately after myocardial infarction will be limited to patients with clear extensive damage or some subset of patients that can be predicted to have poor outcomes. In this manner, the success in treating acute coronary thrombosis has reduced the need for approaches to regenerate myocardium immediately after infarction. Of course, many patients still develop postinfarction heart failure, which continues to carry important short-term and long-term risks.29 However, if we can identify and intervene in these patients at the right time, heart muscle regeneration after infarction could prove to be a transformative therapy. If the risks and costs of the regenerative therapy were small, then even patients with very modest infarctions could be candidates for therapy.Progress in other areas of cardiovascular therapeutics will also continue, and this is why we predict that regenerative medicine will begin to focus on major areas where devices, medications, and other approaches will likely leave major unmet needs. These may include genetic cardiomyopathies, where diagnosis may be made long before the emergence of disease. Cell-based solutions for major problems will be expensive to develop, and economics is 1 reason that a limited number of diseases will emerge as targets for regenerative therapies through cells. Chronic ischemic cardiomyopathy will remain an enormous clinical problem, and, therefore, replacing ventricular scar tissue with new regenerated myocardium continues to be a major challenge to regenerative biology. In 2015, Menasche and colleagues30 reported the first case of a human with severe heart failure treated with embryonic stem cell therapy, using a progenitor cell-seeded patch sewn to the left ventricle at the time of bypass surgery. The patient did well postoperatively, with an improvement in ejection fraction and no apparent complications, and this landmark event raises the very real prospect that cardiac regeneration through embryonic stem cells could help patients with chronic cardiomyopathy. But even chronic ischemic cardiomyopathy will be a moving target. For example, after decades of research, the combined therapy angiotensin receptor antagonist–neprilysin inhibitor has led to a breakthrough success for heart failure with reduced ejection fraction in humans.31 Regenerative therapies for chronic heart failure will eventually have to face the same efficacy and economic reality tests that all new therapies encounter.Prediction 2: Stem Cell Biology, Irrespective of Therapy, Will Advance Our Understanding of DiseasesEven if stem cells do not become a routine therapy for cardiovascular patients, stem cell biology will change how we study diseases in the next decade. Most notably, stem cell biology allows one to model a disease in diverse human backgrounds, because stem cells can be generated from any patient, and cardiovascular cells can then be created from those patients. For example, human cardiomyocytes have been generated from patients with long-QT syndromes through reprogramming.32 This allows the study of a specific mutation in cells with the same genetic background as the patient, and potentially could allow one to identify drugs through in situ approaches that will have beneficial effects in that patient.Human disease modeling by stem cell technology may be particularly useful for common diseases that have complex genetic causes, rather than monogenic diseases (Figure 3). Studies of the human genome have taught us that, for many common diseases, such as type 2 diabetes mellitus or obesity, there is >1 gene that contributes to the genetic risk of disease. Instead, many diseases have familial patterns that are linked to many distinct regions in the genome, and each of these regions has only a moderate or sometimes even a very minor influence on the trait.33 For example, there are at least 46 different validated gene loci for the inheritance of coronary artery disease in humans, but these combined loci explain <10% of the familial inheritance of the disease.34Download figureDownload PowerPointFigure 3. Disease in a dish specific to individual patients. Even if most patients do not receive cellular therapies, regenerative biology will provide new testing platforms for therapy development. Generation of laboratory cells and tissues from specific patients provide the true genetic background of the disease. This approach is currently challenged by cost, time, and reproducibility, but advances over the next decade will make this a routine approach for many diseases.In view of this complexity, testing the interactions of many different genes is extremely challenging. Imagine all the combinations of genetically modified mice that would need to be generated to explore the interactions between ≥10 genes. Here, stem cell biology will have a major impact because it enables investigators to manipulate systematically many different genes, and it can be done within the identical genetic background of the patient. In this regard, creating stem cells from patients who have the disease will provide a more authentic system to study the human disease process than the laboratory mouse and other animal models. Furthermore, gene editing (discussed below) of those stem cells, will enable rigorous studies of complex disease mechanisms within the patient-specific tissues.At the current time, the cost and efficiency of taking hair or skin cells, reprogramming them to stem cells, and then generating heart cells to study specific drugs prohibits widespread use of this approach. Other major challenges to developing disease-in-a-dish models of cardiovascular diseases also exist, such as developing fully mature cardiomyocytes (most stem cell–derived heart cells are immature and do not develop into fully functional adultlike cardiac myocytes) and reducing the variability between different clones of iPS cells generated from the same cell.35 These barriers are sufficient to predict that modeling human diseases through iPS cells will be experimental for at least the next 5 years, and probably for the next decade. However, investigators are using 3-dimensional culture systems and other engineering techniques such as molecularly designed cellular scaffolds and mechanically stimulated cultures that are increasingly making stem cell–derived cardiac myocytes increasingly more similar to bona fide adult human myocardium.36 It is thus likely that cardiovascular therapies will be discovered and developed by testing on laboratory-developed human tissues from the patients who actually carry the disease. In addition, it is quite possible that therapies will someday be individualized for patients through testing on their own cells, in the same way that breast cancer cells are now studied to determine whether the patient will be hormone responsive. For example, Kass and colleagues37 have shown that the responses of stem cell–derived cardiomyocytes to antiarrhythmia drugs are predictive of management outcomes in a family cohort with a long-QT syndrome. Disease modeling based on iPS cells has largely been focused on cell autonomous (independent of other cells) features of myocardial disease such as QT length. The study of more complex multicellular genetic effects requires more tissuelike systems than individual cells.38As noted above, a powerful driving force in the development of stem cell–based laboratory models of human diseases will be economics. Many modern therapies are developed through careful mechanistic laboratory investigations in cells, followed by extensive testing in rodents and then in large animals. Although the cost increases with every step of this process, this course of investigation is used to reduce the risk that a therapy will fail in humans. Currently, the mouse remains the mainstay of laboratory investigation because of the ease of genetic engineering and widespread use of mouse experimental models. However, testing in mice is expensive relative to most cell culture experiments, and testing in large animals is much more expensive. Furthermore, mouse experimentation is severely limited by the inability to mimic the complexity of most human diseases, and most laboratory tests on mice are performed in a single genetically inbred background. Thus, exploring the effects of a drug or therapy on human tissue will likely become common when the cost, efficiency, and reproducibility of disease-in-a-dish models are all improved. Of course, many human diseases, including most cardiovascular disorders, like hypertension and heart failure, require study in a system with intact physiology. For this reason, animal models will continue to be essential, and stem cell biology is going to complement rather than replace animal research.Prediction 3: Stem Cell Therapy Will Provide an Avenue for the Application of Genome EditingFor serious genetic diseases, the ultimate goal is to precisely correct the defect through gene manipulation without harming the patient. The ability to manipulate the genome with ease has been an enormous challenge in molecular medicine, but recent stunning breakthroughs have opened genetic engineering to many possibilities. One of the most revolutionary discoveries has been the development of a gene-editing technology called CRISPR/Cas9.39,40 This technology is the adaptation of a bacterial defense system that allows the organism to cut the DNA of an invading virus. Through an understanding of the basic mechanisms that regulate this process, scientists have devised strategies that can be used to edit genes in potentially any organism and any type of cell in a rapid and accurate manner. The CRISPR/Cas9 system is sufficiently easy that, even though the technology was described in 2013, it has been simplified in a short time period and is now widely accessible. CRISPR/Cas9 gene editing can also be accomplished in the intact adult organism, and this proof of principle has been shown by reducing PCSK9 gene expression in the liver of the mouse, leading to dramatic reduction in cholesterol.41Gene editing combined with stem cell technology has the potential to revolutionize the care of monogenic diseases like sickle cell anemia in the near future. With generation of iPS cells from a patient, genetic defects can be corrected and then differentiated cells or tissues can then be transplanted into the patient (Figure 4). The first human application of genome editing, using the zinc finger technology that preceded CRISPR/Cas9, was used to inactivate CCR5, a coreceptor for the human immunodeficiency virus, on human T cells. The treatment was relatively safe, and the blood level of human immunodeficiency virus DNA decreased in most of the 12 patients enrolled in the study.42Download figureDownload PowerPointFigure 4. Gene editing to correct diseased cells. New technologies allow simple gene editing in stem cells, and this could enable unique approaches to many diseases. Stem cells from individual patients can be generated, with repair of one or even more genetic defects. The stem cells can then be directed to form cardiomyocytes or other types of cells, such as a hepatocyte with improved cholesterol metabolism. The cells with corrected function can be used to generate tissues for transplantation.Genome editing is so efficient and powerful that inventors of the technology, and other prominent scientists, as well, have called for a moratorium on use in humans until more consideration of the ethics of human gene editing can occur.43,44 There will have to be extensive discussion and research to evaluate the safety and ethical concerns of changing human DNA in a manner that goes beyond currently accepted gene therapy. No one wants to see gene editing for cosmetic reasons or to bestow traits that lead to superiority in sporting events to our children. However, no one wants children to experience the debilitating effects of genetic diseases, such as Duchene muscular dystrophy. In this regard, Olson and colleagues45 have used CRISPR/Cas9 genome editing to reverse Duchene muscular dystrophy in mice, with improvements in both skeletal muscle and myocardium. Thus, it appears inevitable that genome editing will become a clinical approach for many very serious human diseases, but defining guidelines for gene editing is very prudent as the technology rapidly advances. Gene editing of differentiated somatic cells such as cardiomyocytes and fibroblasts will be more acceptable than gene editing of the germline, which has potential for harmful effects for not only that embryo, but also for future offspring of that embryo.Genetic causes of cardiomyopathy are major contributors to heart failure, and thus it is attractive to consider gene editing for correction of lethal genetic cardiomyopathies. However, the myocardium is a much more challenging organ for this approach than, the hematopoietic system, for example, where stem cells are in routine clinical use. Extensive controversy arose over the past decade on whether the heart, like skeletal muscle, is continuously regenerated by endogenous stem cells that became cardiomyocytes. The recent advances in gene editing make the issue of the origins of our heart cells into much more than an academic question. If the myocardium were continuously being refreshed by stem cells, then the concept of gene editing those stem cells and repairing the lethal mutation would be viable, because those repaired stem cells would then replace the diseased cardiomyocytes. Unfortunately, it appears that mammalian myocardium does not have a stem cell pool driving constant replacement of cardiomyocytes at a clinically significant rate.15 It now appears that the stem cell contribution to myocardial homeostasis in the adult may be very modest, and that most cardiomyocytes arise from preexisting cardiomyocytes. Thus, replacing most of the diseased cardiomyocytes with stem cell–derived cardiomyocytes with genetic correction will not be an easy task. However, the concept should certainly not be abandoned, because it appears that, slowly, about half of the cardiomyocytes are replaced throughout a human lifetime.46 Thus, if a cardiomyopathy can be diagnosed at a very early age, one could envision approaches where transplant gene–corrected cardiomyocytes could slowly replace a substantial portion of the myocardium and thereby circumvent the disease that would develop later in life. There remain important questions about cell therapy, including how to maximize true long-term integration of transplanted cells into the complex environment of the myocardium.Prediction 4: Regenerative Medicine Will Promote the Convergence of Diverse FieldsAn interesting social phenomenon is the separation of scientific and clinical disciplines into silos that provide stable, albeit insulated, environments for collaboration and advancement within a given field. However, new technology often forces integration of those fields, in the same way that transcatheter aortic valve replacement is forcing convergence of cardiac surgery and interventional cardiology. Similarly, the regenerative medicine movement will force collaborations between many fields (Figure 5). Developmental biologists can define factors necessary to guide a critical biological step in regeneration, but they will need bioengineers and material scientists to help them implement a practical method of delivery. Geneticists will discover genes that may cause a disease, but they will need stem cell biologists to help them determine how the gene contributes to the disease mechanism.Download figureDownload PowerPointFigure 5. Regenerative medicine will drive integration. Achieving regeneration in patients will require integration of many skills. Most of these are obvious, but one particularly important integration step will be merging immunology with regenerative approaches.This convergence of fields toward regeneration will stimulate innovations. Engineers will improve catheter technology to provide cell and regenerative proteins not only to any specific region of the heart, but also to other organs. Molecular imaging will advance such that localizing and monitoring individual cells in humans may become feasible. And cells themselves may be engineered to serve as biological monitors of their own health and the status of the environment.One crucial integration event in the next decade will be the merging of immunology with regenerative medicine approaches. Clearly, the innate response is an essential component of a regenerative response to injury, and recent studies have provided a molecular framework by which immune modulators promote cellular reprogramming.47,48 However, inflammation can be viewed as a double-edged sword, and all cell-based regenerative therapies must cons
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