Conceptually, there are three major approaches that can be considered for inducing myocardial regeneration.
Stimulation of Endogenous Cardiomyocyte Proliferation
Whereas it has long been considered that the heart was a terminally differentiated organ, recent data have challenged this paradigm and elegant studies using 14C dating have provided evidence for some renewal of human cardiomyocytes spanning the lifetime but with a low turnover, as percentages of dividing cells decreased from 1% per year at the age of 25 to 0.45% at the age of 75 years.76 This has led some investigators to hypothesize that differentiated quiescent cardiomyocytes, which did not proliferate under resting conditions, could be induced to re-enter the cell cycle in response to extracellular mitogens. It has thus been demonstrated in mouse models of myocardial infarction that factors such as periostin77 or neuregulin78 triggered cardiomyocyte cell-cycle activity and that the resulting regeneration was associated with improved function, reduced fibrosis and infarct size, and increased angiogenesis. However, whether and how these basic science data can be translated into a safe and efficacious clinical therapy remains to be elucidated.
Transplantation of Putative Extracardiac Cell Niches Endowed with a Cardiomyogenic Differentiation Potential
In 2006, Witnisky and coworkers79 described in the mouse skeletal muscle a population of cells committed to a cardiomyogenic lineage. Unfortunately, using similar surface markers, we failed to show that such a cardiac-directed cell fraction existed in the human muscle.80 These findings are consistent with the increasing recognition that stem cells harbored in adult tissues are unlikely to cross lineages, except possibly under clinically irrelevant experimental conditions. It remains to be established whether this hurdle can be overcome by small molecules such as those of the sulfonyl hydrazone family, which have recently been reported as able to activate cardiac differentiation of human mobilized peripheral blood mononuclear cells,81 thereby effecting myocardial regeneration in a nude model of myocardial infarction.
Transplantation of Cardiac Stem/Progenitor Cells
Endogenous Sources: The Heart Itself
The 14C experiments mentioned in the preceding have actually been preceded in the early 2000s by studies from the group of Anversa who first claimed that despite being traditionally considered as a postmitotic organ, the heart actually harbored dividing cardiomyocytes evidenced by a positive expression for the cell proliferation marker Ki67, and that the proliferation rate of these cells was dramatically increased in patients with heart failure.82 The phenotypic characterization of these cells, found in multiple species, has yielded puzzling results in that different surface markers occasionally expressed in a mutually exclusive fashion have been identified (reviewed in Barile)83 and it is still uncertain whether this variability in marker distribution actually reflects distinct cell populations with specific functional properties or the ability of a unique pool of stem cells to express different markers at different stages of its maturation. C-kit + cells have been the most extensively investigated; because they lacked expression of hematopoietic markers, a bone marrow origin, such as that reported in sex-mismatched heart transplant recipients in whom 0.04% cardiomyocytes were found to be repopulated from extracardiac cells,84 has been excluded. Rather these c-kit + cells have been considered to represent a myocardial pool of progenitor cells committed to a cardiomyogenic lineage. Proof-of-concept was putatively provided by demonstration of donor-derived cardiomyocytes (along with smooth muscle cells and endothelial cells) when human cardiac progenitors were injected intramyocardially85 in a rat model of myocardial infarction and resulted in an improvement in contractile function, an attenuation of remodeling and histologic patterns of regeneration. This paradigm found a clinical application in July 2009 when a first patient was injected in Louisville, KY with his own cardiac stem cells derived from a right atrial biopsy that had been taken 4 months earlier during a CABG and subsequently expanded in Anversa's lab in Boston. Other investigators have adopted a slightly different approach in that instead of trying to isolate these putative cardiac stem cells, they grew fragments of myocardial tissue as cell clusters termed cardiospheres. These aggregates are composed of c-kit + progenitor cells primarily in their core and of a mix of cells expressing cardiac, endothelial, and mesenchymal markers at their periphery. Following preclinical studies showing that intramyocardial86 and intracoronary87 delivery of human and porcine cardiospheres could regenerate acutely and chronically infarcted myocardium, respectively, the group of Marban in Los Angeles moved to the clinics in June 2009 and performed a first intracoronary transfer of these cardiospheres 1 month after removal of a right ventricular specimen by an endomyocardial biopsy. A trial exploring the potential of this approach (CADUCEUS for CArdiosphere-Derived aUtologous Stem Cells to Reverse ventricular dysfunction) is currently under way.
Whereas the use of cardiac stem cells is appealing because of the cardiac lineage commitment of these cells and the avoidance of immunological issues owing to their autologous origin, several caveats must be mentioned. The major concern is that it is still uncertain whether these cardiac stem cells really exist in the adult diseased heart. In our experience, we have identified a few c-kit + cells in right atrial tissue taken during CABG or through endomyocardial biopsies,88 but all of them expressed hematopoietic markers and none expressed specific cardiac markers. Likewise, a high proportion of the c-kit + cells isolated from failing human hearts by Kubo and coworkers89 were positive for markers of the hematopoietic lineage, and although their number was fourfold higher that in nonfailing hearts, their reparative capacity was clearly inadequate because all failing hearts required transplantation. In keeping with these data, cells expressing both stem and differentiated cardiac markers have been identified in the human neonatal myocardium, but with a progressive decline in cell density over the first postnatal month,90 thereby raising serious doubts about the persistence of cardiac “stem” cells in adult patients with coronary artery disease, ie, those who would them need most. The fact that 4 months were required for growing these cells in the recently reported clinical experience also highlights the technical challenges associated with their isolation and scale-up and raises some doubts about a realistic widespread clinical use. Finally, a recent study has further challenged these findings by showing that spontaneously beating neonatal rat cardiospheres resulted from contamination by myocardial tissue fragments and that these cardiospheres actually represented aggregated fibroblasts rather than clonally expanded cardiac stem cells.91 Another more recently described variety of stem cells of cardiac origin is represented by those derived from the epicardium, but these cells also seem to improve function of infarcted hearts paracrinally as they fail to acquire a cardiac phenotype.92
Human embryonic stem cells (hESC) are derived from the inner cell mass of blastocyst-stage leftover embryos, which have been generated in the setting of assisted fertilization. Their most appealing feature is an intrinsic pluripotentiality that allows them to be committed in vitro toward the cardiac lineage in response to appropriate differentiation cues recapitulating those in play during the embryonic life and among which bone morphogenetic proteins (members of the TGF-β superfamily), Wnts, and fibroblast growth factors play a predominant role.93 It has thus been demonstrated that hESC could generate cells displaying the three major attributes of cardiomyocytes: (1) excitation-contraction coupling, demonstrated by the expression of ionic currents;94 intracellular calcium transients triggered by electrical activation and the resulting synchronous contractions; (2) responsiveness to the chronotropic effects of cardioactive drugs,95 and (3) the capacity to couple with neighboring cells through connexin 43-supported gap junctions.96 Following transplantation in rodent models of myocardial infarction, these cells have been shown to improve LV function, an effect usually associated with a beneficial effect on remodeling.97–99 Of note, mouse ESC have also been shown functionally effective in a nonischemic genetic cardiomyopathy model.100 One could legitimately argue that these cardioprotective effects have been reported with most cell types. However, an observation more specific for ESC is that, in contrast to their adult counterparts, they really differentiate into cardiomyocytes in transplanted areas, an event likely driven by cues originating from the infarcted tissue.101 However, injected ESC derivatives share with other cell types a limited amount of long-term engraftment,102 and this finding, along with the fact that increasing graft size has been reported not to positively correlate with a gain of function, has led to hypothesize that ESC-induced stimulation of vascularization was actually a major contributor to the cardiac functional improvement.103 Indeed, whereas ESC derivatives may well act paracrinally by activating endogenous cytoprotective pathways,104 one cannot fully exclude that they also directly contribute to cardiac contractility by generating a force transmitted to the surrounding tissues.105 Furthermore, it is important to recognize that the assessment of hESC transplantation is hampered by the lack of appropriate preclinical models as all of them are fraught with the confounding effect of xenotransplantation. For this reason, we have conducted experiments in nonhuman infarcted primates transplanted with primate ESC–derived cardiac progenitors, thereby mimicking an allogeneic situation closer to clinical practice. The data have confirmed the ability of these progenitors to achieve their differentiation into cardiomyocytes following in-scar engraftment. Furthermore, the finding made in a pig model of iatrogenic atrioventricular block, that intramyocardial transplantation of hESC-derived cardiac derivatives could restore electrical activity,106 provides compelling evidence for the ability of these cells to achieve an effective electromechanical integration within the recipient heart.
Asides from ethical considerations, the clinical use of hESC still raises several translational issues107 that fall into three main categories: (1) derivation and propagation of a line endowed with a cardiogenic differentiation potential, under good manufacturing practice (GMP) conditions; this first step of the process should allow to scale-up the number of cells without the use of xeno-components while maintaining their pluripotentiality and ensuring their genetic and epigenetic stability over time; (2) directed differentiation of the propagated pluripotent cells toward a cardiomyogenic lineage; (3) sorting of the cells, so as to yield a “pure” population of cardiac progenitors for clinical use. (Progenitors are likely preferable to a terminally differentiated cell type because of their ability to still proliferate in the host tissue to a certain extent, thereby effecting myocardial regeneration.) Several purification methods are available and rely on negative or positive selection, depending on whether they target the elimination of still undifferentiated cells or the enrichment of the lineage-committed progenitors from other unwanted cell types, respectively. Introduction of a reporter-suicide gene into stem cells that could serve as a safety net against cellular misbehavior is another option that warrants further investigation. In our experience, a positive selection approach based on identification of the surface marker SSEA-1 (or CD15) has turned out to be efficacious in that this antigen reliably labels cells that have entered a differentiation pathway,108 thereby allowing immunomagnetic sorting using an anti-CD15 antibody to selectively yield the clinically required cardiac progenitor cell population. The elimination of the contaminating cells is actually critical not only for efficacy, but also for safety reasons as a major concern with ESC is the development of a teratoma. Teratomas are complex tumors consisting of cell lines originating from the three initial germ layers. Although several factors (cell number, transplant site, degree of immunosuppression) may affect whether these tumors form after transplantation of ESC,109 a key role is played by the state of differentiation of the transplanted cells. Indeed, teratomas occur when cells are used in a still undifferentiated or poorly differentiated state and have thus retained an intrinsic capacity of uncontrolled proliferation, which can overcome the cardioinstructive signaling of the host heart. Conversely, animal models have failed to show evidence for teratomas following transplantation of hESC-derived cells committed to a given cell lineage,101,105 as demonstrated by their loss of pluripotent markers and the concomitant upregulation of the lineage-specific ones. These considerations highlight the importance of the purification step during preparation of a clinically usable ESC-derived cell therapy product.
Another major clinical issue raised by the use of this allogeneic cell source is immunogenicity. Despite initial hopes, it is now widely recognized that although undifferentiated ESC lack expression of MHC type II antigens and costimulatory molecules, they trigger a cellular and humoral immune response,110 which does not spare their differentiated progeny.111 Even though the lack of antigen-presenting cells within ESC-derived grafts may contribute to a reduced immunogenicity compared with adult tissues,111 it is still necessary to implement some form of immunosuppressive strategy to avoid cell loss by this immune process. Different approaches have thus been considered. Banking of hESC lines with a range of MHC profiles allowing an appropriate donor-recipient matching is likely not unrealistic, because about 150 randomly obtained cell lines could provide a worthwhile human leukocyte antigen (HLA) match for most potential recipients.112 This figure, however, was based on matching only selected MHC loci and thus does not dismiss that additional immune suppression would be required to overcome residual immunogenicity. Also, it may underestimate the ethnic diversity of most Western populations. Furthermore, such a banking approach raises complicated logistical and economic issues and, as such, first requires the convincing demonstration that the use of hESC-derived cells is therapeutically effective. Given the risks associated with a protracted use of immunosuppressive drugs, induction of tolerance in the recipient looks a particularly appealing approach should it be possible at the cost of minimal host conditioning.113 In practice, it might be achieved by the administration of hematopoietic stem cells derived from a given “donor” ESC line114 and the resulting induction of a microchimerism ensuring specific tolerance to cardiac (or other) progenitors derived from that same donor ESC line. The use of costimulation blockade for inducing peripheral tolerance mediated by regulatory T cells could be another strategy.115 In contrast, the creation of a “universal” cell line, for example by knockout of the β-2 microglobulin gene, which is essential for MHC-I presentation, remains a more uncertain perspective because MHC molecules also represent a surveillance mechanism whose deletion could create a favorable environment for viral infections and development of malignancies. Thus, from a practical standpoint, early clinical trials of hESC will likely have to rely on more conventional therapies based on immunosuppressive drugs. The challenge is then going to find an acceptable trade-off between the efficacy of these drugs and their well-known side-effects so as to optimize the risk-to-benefit ratio of the procedure. The additional use of autologous or allogeneic mesenchymal stem cells for further mitigating the immunorejection process is supported by a study in kidney transplant recipients,116 although we have experimentally failed to show such a benefit in a rat model of hESC transplantation.99
The recent presidential lift on the US federal funding ban on ESC research and the attendant increase in resources allocated to this area make likely that many of the remaining hurdles will be overcome in a not too distant future.
Induced Pluripotent Cells
In 2006, Yamanaka made a real breakthrough in the stem cell field when he reported that mouse embryonic and adult fibroblasts could acquire properties similar to those of ESC after retrovirus-mediated transfection of genes encoding the four transcription factors Oct3/4, Sox2, Klf4, and c-Myc.117 Since then, several groups have embarked in this research on induced pluripotent stem (iPS) cells and have successfully reprogrammed human adult somatic cells into an embryonic-like state, including from patients with a variety of neurodegenerative and genetic diseases.117 Reprogrammed cells can then be redirected toward a given cell lineage, including cardiomyocytes, and this cardiomyogenesis process actually involves a temporal sequence of gene expression (specific for cardiac mesoderm, cardiac transcription factors, and cardiac-specific structural proteins, successively), which parallels that observed during differentiation of hESC.118
The use of iPS could potentially allow to address two limitations of ESC, ie, immune rejection and ethical concerns raised by the use of human embryos, with still the caveat that one does not know whether the reprogramming-redifferentiation process could not cause expression of otherwise hidden-self and lead to autoimmunity events. However, their clinical application also faces important obstacles. Some of them are shared with ESC, such as teratoma formation following unwanted transplantation of still undifferentiated cells; other hurdles are rather unique to iPS cells and specifically include the risk of incomplete/aberrant reprogramming that may impair their ability to be redifferentiated toward the required cell type. It also remains to determine which is the best somatic cell source to use (eg, skin fibroblasts, keratinocytes, blood cells) and how to increase the still low efficiency of the reprogramming process while improving its safety. This is indeed a key question as a major risk of the initially used retroviruses and lentiviruses is transgene reactivation leading to tumorigenesis, hence the more recent interest in plasmids, proteins,119 and even chemicals that represent a mandatory step on the pathway to production of iPS for clinical use. However, even these virus-free approaches may induce genetic alterations and therefore would call for a sequencing of the whole genome of iPS cell clones to detect these alterations before any clinical application.
These considerations suggest that although iPS cell technology may be soon ready for prime time toxicology screening and patient-specific disease modeling, its application to “regenerative” medicine still requires many challenges to be surmounted. Nevertheless, encouraging data have already been reported. Thus, the potential therapeutic value of iPS cells has yet been successfully tested in three noncardiac disease models (sickle cell anemia, Parkinson's disease, and hemophilia A). These data have more recently been extended to the heart by Nelson and coworkers,120 who have pioneered the transplantation of fibroblast-derived iPS cells in a murine model of myocardial infarction and shown that it resulted in an improvement in LV function compared with the parental fibroblasts, associated with a differentiation of the iPS cell progeny into cardiomyocytes. However, because this study did not include “true” ESC-derived cardiomyocytes as another control, additional experiments remain necessary to thoroughly assess the comparative efficacy of these two types of pluripotent stem cells. This is particularly relevant in view of recent data that indicate that regardless of their origin or the method by which they have been generated, iPS retain a gene expression pattern distinct from that of hESC and that extends to the expression of noncoding (mi)RNAs.121 Therefore, it is critical to assess the functional significance, if any, of these differences. Finally, although iPS have the major theoretical advantage over hESC of being patient-specific, one should not underscore the limitations of autologous cell therapy products when large patient populations are targeted; these limitations include intrinsic interindividual variability in cell functionality, which complicates the production of consistent cell products, logistical constraints inherent in cell shipments, and the huge cost of customized batch controls.