A. Introduction

Despite the remarkable progress made during the past half century in the treatment of most degenerative diseases, including those of the cardiovascular system, the fact remains that for many patients the available treatment is largely palliative and/or unsuccessful long term. However, because many treatments are increasingly effective in dealing with the acute stages of life-threatening diseases, they often extend the life of the patient at the expense of leaving behind a chronic condition. These chronic sequels, particularly those resulting from an acute myocardial infarction (AMI), such as chronic heart failure (CHF) are frequently without effective treatment or leave organ transplantation as the only alternative available to restore function, with all the logistic, economic and biological limitations associated with this intervention (1).

With the continuous increase in average human lifespan and the progressive aging of the population in all developed countries, we are now facing an increasingly severe epidemic of chronic diseases whose treatment absorbs an ever-larger fraction of human resources and of the healthcare budget. Presently, there are >5 million patients in CHF post-AMI in the USA alone (2). More than 550,000 patients per year are added to this group, which has a similar prevalence in the EU countries. After the first episode of heart failure, this patient cohort has an annual average mortality rate of ~18% and in the USA alone absorbs ~$30 billion annually for their care (2). The root problem responsible for the poor outcome of the CHF post-AMI is a deficit of functional myocardial contractile cells (cardiomyocytes) and adequate coronary circulation to nurture them resulting in pathological cardiac remodelling, which, in turn, triggers the late development of cardiac failure in these patients (3). For this reason, for the past decade it has been a goal of cardiovascular research to find methods to replace the cardiomyocytes lost as a consequence of the MI in order to prevent or reverse the pathological cardiac remodelling which leads to CHF. Therefore, the need to identify new therapies has become a key research area in regenerative cardiovascular medicine and stem cell-based therapies are fast becoming an attractive and highly promising experimental treatment.

Until recently, a paucity of understanding about the cellular homeostasis of most adult solid tissues has been a major factor limiting the application of early breakthroughs in adult stem cell research and therapy, such as those applied to blood and bone marrow diseases, to a broader area of regenerative medicine. Until early in the last decade the prevalent view in the health sciences was that, although tissues like the bone marrow, intestinal epithelium and skin exhibit a robust self-renewal capacity based on the presence of adult (also called “tissue-specific”) stem cells, (4,5) they were an exception. The established paradigm was that the majority of solid tissues/organs either renewed very slowly (such as skeletal muscle, the endothelial lining of the vascular system), to the point of being physiologically irrelevant, or did not renew at all (like the myocardium and central nervous system). It was firmly believed that starting from shortly after birth many tissues did not harbor functional regenerating (stem) cells. A logical consequence of the above paradigm was the belief that for most organs the number and function of their parenchymal cells was in a downward spiral, which started in late infancy and continued until death. With the exception of the three main self-renewing tissues mentioned above, it necessarily followed that all therapeutic approaches to disease processes caused by a deficit in the number of functional parenchymal cells could be directed only toward improving and/or preserving the performance of the remaining functional cells in the tissue. Thus, it was broadly believed that to return the tissue or organ to the status quo ante, it would require the transplantation of either identical cells from another individual or transplantation of a cell type capable of differentiating into the cells whose shortage needed to be corrected. Heterologous or allogeneic organ and cell transplantation became the only avenue as our understanding of cells capable of differentiating into cardiac myocytes was limited. In fact, despite the multiple drawbacks of allogeneic cell/organ transplantation, its practice has become the clinical cutting edge for several medical specialties (6). However, the extreme shortage of donors, high costs, and the severe side effects of immunosuppression have limited this therapy to a small fraction of candidates in need of treatment. Thus, the positive reception and high expectations with the successful derivation of multipotent human embryonic stem cells (hESCs) (7-9). hESCs had the capacity to differentiate into most known cell types which was hoped to result in an unlimite supply of donor organs. Because of the ethical, immunological and teratogenic challenges posed by potential clinical use of hESCs, the euphoria started to dim. Then came the breakthrough which permitted the conversion of different adult somatic cells, such as fibroblasts, into multipotent cells, called induced pluripotent stem (iPS) cells, by the introduction of a very limited number of genes (now known to be responsible for the multipotent state of the stem cells) (10,11). With the development of iPS cells, it became possible to produce different types of parenchymal cells starting with an abundant and easy to obtain cell type from the same patient to be treated. Once converted into the parenchymal type needed, these cells could be potentially used for autologous cell therapy (12). Although the potential of the iPS cells as therapeutic agents remains high, it is already clear that many hurdles will need to be cleared before they can reach broad clinical application (13). Not least among these hurdles is the cost and lag time needed for producing such personalized therapy, which does not make it available for unpredicted acute diseases, such an AMI. The newer protocols to convert somatic cells directly into some types of parenchymal cells, even in situ, without apparently going through the multipotent stage by introducing tissue-specific transcriptional factors (14-16). It may be too soon to evaluate their clinical potential.

B. The Adult Heart is a Self-Renewing Organ

Out of the limelight and apart from the cultural and philosophical wars, over the past 15 years there has been a slow but steady re-evaluation of the prevalent paradigm about adult mammalian -including human- tissue cellular homeostasis. It has become slowly appreciated that the parenchymal cell population of most, if not all, adult tissues is in a continuous process of self-renewal with cells continuously dying and new ones being born. Once cell turnover was accepted as a widespread phenomenon in the adult organs, it was rapidly surmised that in order to preserve tissue mass, each organ that is constituted mainly of terminally differentiated cells (that is, cells that once differentiated lose their capability to re-enter the cell cycle and multiply) needed to have a population of tissue-specific regenerating cells. Not surprisingly, this realization was rapidly followed by the progressive identification of stem cells specific for each of these adult body tissues (17-22).

Not surprisingly, the cardiovascular research community, in agreement with the prevalent paradigm, for a long time has treated the adult mammalian heart as a post-mitotic organ without intrinsic regenerative capacity. The prevalent notion was that the >20-fold increase in cardiac mass from birth to adulthood and the further increases in response to different stimuli in the adult (such as hypertension, exercise), resulted exclusively from the enlargement of pre-existing myocytes (23-25). It was also accepted that this myocyte hypertrophy, in turn, was uniquely responsible for the initial physiological adaptation and subsequent deterioration of the overloaded heart. These beliefs were based on two generally accepted notions: a) all myocytes in the adult heart were formed during fetal life or shortly thereafter, were terminally differentiated and could not be recalled into the cell cycle (26,27) therefore, all cardiac myocytes had to be of the same or very close to the chronological age of the individual (28); b) the heart did not have any intrinsic parenchymal regenerative capacity because it lacked a stem/progenitor cell population able to generate new myocytes. Despite substantial published evidence that this prevalent view was incorrect, (29-34) it took the publication of Bergmann et al. in 2009, based on 14C dating in human hearts, showing that during a lifetime the human heart renews ~50% of its myocytes, to produce a significant switch in the prevalent opinion (35). However, because this “measured” self-renewal does not appear to be very robust, its physiological and clinical significance has remained in doubt.35 At the same time, it has been overlooked that the conclusion of Bergmann et al. on the rate of cardiac myocyte (CM) turnover depends on the validity of a complex mathematical formula, whose impact on the results dwarfs that of the measured data. Furthermore, their calculations identified the highest turnover rate during youth and early adulthood followed by a steady decrease with age. This conclusion is contrary to most or all the turnover values measured for other human tissues, including the heart (3). While Bergmann et al., (2009) calculated a yearly CM turnover of about 1%, others have calculated 4-10% (96,43). This spread of “measured” CM turnover rate raises significant scepticism about the validity of the methodologies used and leaves unanswered the physiological significance of CM replacement due to normal wear and tear in adulthood and after injury. Furthermore, the origin of the new myocytes and whether they originated from precursor cells or from the division of pre-existing myocytes was not addressed (35). Yet, a genetic fate mapping study in mice tracked new myocyte origin in the adult heart to a compartment of stem/precursor cells.

C. Endogenous Cardiac Stem/Progenitor Cells (eCSCs)

Over 99% of the myocytes of the normal adult mammalian myocardium are terminally differentiated cells that are permanently withdrawn from the cell cycle (26,27,36) Thus, the adult heart is composed predominantly of post-mitotic cells, but it, nevertheless, has a remarkable capacity for regeneration, both under normal conditions and in response to diverse pathological and physiological stimuli (33-35,37-39). It is becoming increasingly accepted now that new cardiomyocyte formation does not stop early in post-natal life, but continues throughout life. What still remains controversial is the amount and physiological significance of the cardiomyocyte turnover, as well as the origin of the newly formed myocytes (24,40). Three main sources of the new myocytes have been claimed: a) circulating progenitors, which through the bloodstream home to the myocardium and differentiate into myocytes; (32) b) mitotic division of the pre-exiting myocytes (41-43) and c) a small population of resident myocardial and/or epicardial multipotent stem cells able to differentiate into the main cell types of the heart: myocytes, smooth and endothelial vascular and connective tissue cells (38,44,45). It is clear now that the blood borne precursors, although well documented as a biological phenomenon,46 might be limited to very special situations (47) and their direct regenerative import very limited, if any (48). Division of fully mature pre-existing cardiomyocyte has not been convincingly documented and/or remains to be confirmed by different authors. What is more, all the evidence so far presented in support of mature mammalian myocyte division has been limited to show division of cells expressing cardiac specific sarcomeric proteins in their cytoplasm (37,41-43). This evidence is equally compatible with new myocyte formation from the pool of multipotent cardiac progenitor cells because it is a well-documented fact that newly born myocytes are not yet terminally differentiated and they are capable of a few rounds of mitosis before irreversibly withdrawing from the cell cycle (31).

The molecular mechanism of terminal differentiation of CMs has not yet been completely elucidated. Different experimental models indicate that cardiac muscle cell lineage determination genes directly interact with the retinoblastoma (Rb) pocket proteins to produce and maintain the terminally differentiated state (49,50). After more than 3 decades of research, there are hundreds of papers in reputed journals documenting that mature myocytes in and from the adult mammalian myocardium do not re-enter the cell cycle and in the very rare occasions they do, do not enter mitosis but undergo apoptosis, at least in part, due to a deficit in centriole formation which prevents generation of a mitotic spindle (51). This block of cell division (which does not necessarily block bi-nucleation and DNA endo-reduplication) has been shown both in vivo and in vitro in different mammalian species from mouse to human. This behavior of adult mature myocytes is in contraposition to the robust replication capacity of foetal and neonatal mammalian myocytes, which do not yet express RB. This replication window in the mouse extends up to 7-8 days postnatal (36,52) as well as those of adult invertebrates, fishes and reptiles.53 Furthermore, adult mammalian myocytes can also be co-axed to re-enter the cell cycle at a significant rate through genetic manipulations at the expense of the stability of their differentiated function (49). Notwithstanding the elegant studies documenting that adult cardiomyocytes divide in species other than mammals these findings cannot and should not be extrapolated to the adult mammalian heart (54).

Undoubtedly, the best documented source of regenerating myocardial cells in the adult mammalian heart, including the human, is a small population of cells distributed throughout the atria and ventricles of the young, adult and senescent mammalian myocardium, that have the phenotype, behavior and regenerative potential of bona fide endogenous cardiac stem cells (eCSCs) (44,45,55). The first report of these endogenous regenerating myocardial stem cells in the adult mammalian heart appeared in 2003 (56). These cells express stem cell receptor markers, namely the receptor for stem cell factor, the tyrosine kinase c-kit , and are negative for bone marrow-derived blood (CD45neg) and endothelial (CD34neg CD31neg) cell markers. Since then their existence has been confirmed by a number of independent groups (57). Although a variety of markers have been proposed to identify eCSCs in different species and throughout development (58-67), it still remains to be determined whether these different markers identify unique populations of eCSCs or, more likely, different developmental and/or physiological stages of the same cell type (68). Although the potential regenerative/repair role of the different putative cardiac stem cells identified by different membrane markers remains to be elucidated, it has been experimentally demonstrated that the progeny of a single c-kitposCD45negCD34neg eCSC (hereinafter eCSC) is able to differentiate into cardiac myocytes, smooth muscle and endothelial vascular cells in vitro and when transplanted into the border zone of an infarct regenerates functional contractile muscle and microvasculature of the tissue (56) Furthermore, the progeny of a single eCSC can completely restore extensive diffuse myocardial damage and restore the regenerative capacity to a heart from which its eCSCs have been ablated (38)

In a normal adult mammalian myocardium, at any given time, there is ~1 eCSC per every 1000-2000 myocytes (38). Most of these eCSCs are quiescent (>95%) while only a small fraction is actively cycling and differentiating to replace the myocytes and vascular cells lost by normal wear and tear. However, in response to stress (hypoxia, exercise, work overload, or other damage), a proportion of the resident eCSCs are rapidly activated, multiply and generate new muscle and vascular cells, (38,39,69) contributing to cardiac remodelling. This activation of the eCSCs is able to regenerate the myocardial cells lost as a consequence of major diffuse myocardial damage, which kills up to 10% of the myocardial mass (38). Their transplantation can regenerate the contractile cells lost as a consequence of a major AMI affecting up to 25% of the left ventricular mass (56).

Thus, the identification of eCSCs has contributed to a more widespread acceptance by the cardiovascular community that myocyte death and myocyte renewal are the two sides of the same proverbial coin of cardiac homeostasis in which the eCSCs play a central role. Taken together all these findings have eventually placed the heart squarely amongst other organs with regenerative potential such as the liver, skin, muscle, and CNS.

It is surprising that in the past decade the significant advances in understanding myocardial biology in general and cardiomyocyte biogenesis in particular have had a practically nil impact on the therapeutic attempts to replace/regenerate these myocyte losses. It is a well-documented fact that most reported, as well as planned, clinical trials for cell-based cardiac regeneration remains focused on the transplantation of different non-cardiac cell types, mainly bone marrow mononuclear cells and mesenchymal stem cells (70). This behavior remains based on the concept that the regenerative capacity of the adult heart is negligible, as is explicitly stated in most of these reports. In this intellectual environment it is not surprising that the cardiovascular regenerative medicine field has fully embraced the potential of ESCs and iPS cells (8,71,72). Unfortunately, the human use of these multipotent cells remains far in the future and faces many hurdles, which they might or might not be able to clear. Curiously, this is happening at the time when the regenerative potential of the eCSCs has been solidly documented at the experimental level (38,56) with auspicious indications in early clinical trials (73,74).

Unfortunately, the reports of multiple putative cardiac stem cells types identified by different membrane markers has produced confusion and until recently it had not been determined whether any of these different cell types is primary responsible for the cellular homeostasis of the myocardium throughout life and its repair in response to damage. To address this issue it was necessary to establish a cause-effect relationship between a given putative cardiac stem cell and myocyte generation in the adult; further, it had to be determined whether such cells are necessary and sufficient for myocardial cell homeostasis and repair.

Koch’s postulates, enunciated in 1884 to ascertain a cause-effect for infectious agents, have been lately used as general criteria to establish that a specific entity is the causative agent of a particular disease or biological phenomenon. Despite the fact that these postulates were formulated in a scientific environment vastly different from the present, they are still a powerful tool to test a cause-effect relationship between two biological phenomena. Koch’s postulates state that a causal agent must: (a) be present in every case of the disease/phenomenon; (b) be isolated from the disease host and grown in vitro; (c) the disease/phenomenon must be reproduced when the agent is delivered to a susceptible host; and (d) be recovered from the transmitted host.

In order to test whether the scepticism towards resident cardiac stem/progenitor cells is justified or, on the contrary, whether adult c-kitpos eCSCs fulfil Koch’s postulates as causal agents for cardiac homeostasis and regeneration, we employed experimental protocols of severe diffuse myocardial damage with preserved coronary circulation which, unlike an experimental infarct with permanent coronary ligation, spares the eCSCs (69). The proto-oncogene c-kit, or tyrosine-protein kinase Kit or CD177, is a cell marker used to identify certain types of blood progenitor cells in the bone marrow. This model was used in several transgenic mice and in wild-type mice and rats in combination with cell transplantation protocols. These tests have shown unambiguously that the eCSCs autonomously repair extensive cardiac diffuse damage, leading to complete cellular, anatomical and functional cardiac recovery (38). Furthermore: a) If the eCSCs are ablated, myocardial regeneration is stunted causing heart failure unless they are replaced by exogenous eCSCs; b) Selective suicide of these exogenous “replacement” eCSCs and their progeny abolishes regeneration and severely impairs ventricular performance; c) The transplanted eCSCs can be recovered from the recipient retaining their original characteristics and d) the recovered eCSCs maintain their original regenerative potential when re-transplanted into a new recipient. Therefore, these data confirmed that eCSCs fulfil all Koch’s postulates as the cell-type necessary and sufficient for myocardial repair and regeneration (38). These conclusions are supported by three main experimental facts: a) a model of myocardial injury with patent coronary circulation to test the spontaneous regenerative capacity of eCSCs; b) in situ labelling and genetic tracking the fate of c-kitpos eCSCs; and c) replacement of the endogenous eCSCs cohort by transplantation of exogenous and genetically tagged eCSCs (38).

Taken together the data obtained document that, contrary to common belief, the adult mammalian heart has a robust autonomous regenerative capacity which mainly resides in the Linnegc-kitposeCSCs. This capacity, however, is not sufficient to spontaneously repair in a significant degree the segmental tissue loses such as those produced by an AMI after occlusion of a main coronary artery. It should be remembered that this regeneration deficit is not specific to the myocardium because it is a general phenomenon which also affects the segmental loses of all other solid tissues (such as liver, testis, skin, etc.), no matter the abundance and regenerative potency of their tissue stem cells. In all solid tissues of the adult occlusion of a significant artery invariably results in tissue necrosis downstream and repair by scar formation without parenchymal regeneration to the status quo ante. The challenge, therefore, is to find clinically applicable protocols to manipulate the eCSCs and improve their regenerative potential sufficient to produce an autologous replacement of the cells lost by the insult, segmental or diffuse.

In light of the data reviewed above and further expanded below, where the role of the eCSCs has been tested by different methods and using several animal models, a recent publication (75) using a constitutive and a regulated Cre/lox system in a knockin into the c-kit locus of the mouse has casted doubts on the conclusions espoused here. Although these authors conclude that the adult myocardial c-kit positive cells give rise to myocytes and microvasculature in the adult, the data suggested that the contribution is too small to be of any clinical or physiological significance either for myocardial cell homeostasis or repair. More data is needed before final conclusions can be made in this area.

D. Allogeneic Stem Cell Therapy for Heart Failure and Disease

Bone marrow derived stem cells

Many of the so-called “regenerative myocardial therapies” currently undergoing clinical trials are using bone marrow-derived cells (BMDCs) of different types as the therapeutic agent and have produced mixed results (76,77). Although the cells used are commonly labelled as “stem cells”, most are a mixture of BM-derived mononuclear cells in which the frequency of multipotent cells is unknown but bound to be quite low. Controversy still surrounds and serious doubts have been raised about the cardiomyogenic potential of BMDCs (78,79). At present, the most advocated mechanism of choice for the action of the BMDCs on the myocardium is that of a ‘paracrine’ effect on the recipient’s myocardial cells (80-82). BMDCs release a complex mixture of cytokines and growth factors involved in cell survival, proliferation and migration, which enhance arteriogenesis (83,84) and myocyte survival (81,82). Recently, we have reported that the myocardial protection induced by BMDCs is triggered by secreted factors and mediated by the insulin-like growth factor receptor (IGF-1R) (82), an effect that can be blocked by inhibiting the activity of protein kinase C (PKC) and p38 mitogen activated protein kinase (MAPK) The protective effect of BMDCs is similar to the protection effect of myocardial conditioning produced by ischemia/reperfusion (IR). In essence, instead of the transplanted cells undergoing cardiomyogenic differentiation, through a yet incompletely defined paracrine mechanism, they protect cells at risk and improve myocardial contractility with amelioration of ventricular remodelling (decreasing fibrosis, myocyte hibernation and stunning), inhibition of the inflammatory response, increased cardiomyocyte survival and increased angiogenesis/neovascularisation. Interestingly, it has been suggested that these therapies also produce the activation of eCSCs to give rise to new vasculature and cardiomyocytes, leading to some endogenous/autologous regeneration (48,85,86).

Stimulation of the myocardial endogenous capacity for repair and regeneration

With the myocardium now recognized as a regenerating tissue which harbours eCSCs that can be isolated and amplified in vitro (45,57) and/or stimulated to replicate and differentiate in situ, (87,88) it has become reasonable to search for methods that can enhance and exploit this endogenous regenerative potential in order to replace the lost muscle after an AMI with autologous functional myocardium. Autologous is defined as “from the same individual”.

Although the results of two clinical trials with autologous cardiac stem/progenitor cells or their derivatives (73,74) have shown the safety of the procedure and very encouraging results in cardiac performance, it should be noted that they were very small Phase I/IIa trials designed to test short term safety and not effectiveness. Moreover, given the very small number of cells administered in both studies together with the known low cell survival/engraftment upon transplantation, it is clear that the effect on myocardial mass and contractile function detected on the treated patients cannot be due only to the direct contribution of the transplanted cells to the myocardial mass and function. Therefore, if the beneficial effect reported in the first patients proves to be real at longer time points and reproducible, it is likely due in large part to an indirect effect of the transplanted cells on the survival and functional recovery of stunned myocytes which otherwise might have been lost. While we wait for these results, the high cost and the long wait required to produce the number of cells needed for autologous cardiac stem/precursor cell therapy, will make imperative to compare the beneficial effects of this approach to that obtained with autologous and allogeneic BMDCs because of their easier availability, accessibility and lower cost of the procedure.

In addition to the caveats raised above, the widespread clinical use and applicability of autologous cardiac stem/progenitor cell therapy is highly debatable. First, the procedure for cell acquisition, culture scale-up in vitro, and transplantation, is complex, time consuming and very expensive. The isolation and expansion of eCSCs starting from a catheter or surgical biopsy to the number needed for cell therapy takes 1 to 3 months when successful. Therefore, the cells are not available to be administered when they would be most effective; that is, when a patient with an AMI in progress arrives at the hospital. Furthermore, the cost of the procedure in human and material resources would make it unavailable to the majority of patients beyond those few required to establish the proof-of-concept for the therapy and to selected individuals with abundant economical resources. Finally, eCSCs undergo senescence with age and severe pathological consequences (89-91). Accordingly, for the cohort of patients (the aged population) most likely candidates for the regenerative therapy, >50% of their eCSCs will be senescent and unable to participate in the regenerative process.

Myocardial regeneration with allogeneic stem cell therapy

There is little doubt that all currently proposed autologous cell therapy approaches for different tissues and organs are very attractive from the theoretical and biological standpoint. For those rare diseases with chronic and/or long-term evolution affecting hundreds or even thousands of patient candidates for treatment, these personalized therapies, despite their high cost in medical and material resources, might make sense even from an economic standpoint. More controversial is whether autologous cell transplantation can become the therapy of choice for diseases of high prevalence, such as the consequences of ischaemic heart disease, with millions of patient candidates for regenerative therapy. Unfortunately, not even the richest countries of the developed world have the resources needed to start a program of personalized regenerative medicine available to many of the patients already in CHF who presently are left with heart transplantation as the only realistic option for recovery. Therefore, although the autologous cell transplantation approaches are very valuable as proof-of-concept and as research tools with the possibility of greatly improving a narrow subset of patients in need of therapy, we believe that all of the autologous cell strategies taken together, now and in the foreseeable future, are and will continue to be unavailable to favourably impact the societal health care problem posed by the consequences of CHF post-AMI.

Moreover, as outlined above, a consensus is gaining ground that most of the favourable effects of cell transplantation protocols used until now exert their beneficial effect by a paracrine mechanism of the transplanted cells over the surviving myocardial cells at risk and/or through the activation of the endogenous myocardial regenerative capacity represented by the eCSCs. If this is correct, then there seems to be little advantage in the use of autologous cells because a similar, and perhaps enhanced, effect can be obtained by the administration of the proper cell type isolated from allogeneic sources. These can be produced in large amounts beforehand, kept stored frozen before their use, and remain available at all times, which would allow their use not only for the treatment of the pathological remodelling once it has developed but soon after the acute insult in order to reduce the acute cell loss and also induce early regeneration of the cells lost by activation the eCSCs with the result of preventing or diminishing the pathological remodelling.

Unresolved clinical questions related to the use of allogeneic stem cells in the treatment of patients with AMI remain the identification of the optimal cell population and also the time and method(s) of administration. Allogeneic is defined as “from a different individual-genetically dissimilar”. As previously stated, to become widely available and to be compatible with current clinical standards of care for AMI, an intracoronary method for delivery in the catheterization laboratory at the time of the primary revascularization is the most advantageous. Also, direct intra-myocardial injection at the time of re-vascularization surgery is highly realistic. Mesenchymal stem cells (MSCs) secrete a broad repertoire of trophic and immunomodulatory cytokines. They also produce factors that negatively modulate cardiomyocyte apoptosis, inflammation, scar formation and pathological remodelling (92) However, it is questionable whether they are the optimal cells to use in terms of survival and homing to and engraftment into the myocardium. Furthermore, these cells can become entrapped in the microvasculature, block microcirculation and impede their entry into the extravascular space of the damaged myocardium (93).

To partially overcome the problems outlined above, Medicetty and colleagues (94) used a porcine model of AMI and delivered 20-200 million allogeneic, multi-potent, adult BMDCs (MultiStem) claimed to be non-immunogenic and to suppress activated T-cell proliferation and have anti-inflammatory and angiogenic properties as well), directly into the myocardium via the infarct related vessel using a transarterial microsyringe catheter-based delivery system, 2 days after AMI. Echocardiography showed significant improvements in regional and global LV function and remodelling at 30 and 90 days after myocardial injury (94). Rapidly after this pre-clinical study, Penn et al. conducted a multicenter phase I trial of adventitial delivery of MultiStem cells to patients 2 to 5 days after primary PCI. Administration of 20, 50, and 100 million cells to patients with EF <45% before the MultiStem injection, resulted in an absolute increase in EF of 4, 14, and 11%, respectively, measured at 4 months after the cell therapy (95).

Marban and colleagues have tested the safety and efficacy of using allogeneic, HLA mismatched Cardiosphere-Derived Cells (CDCs) in infarcted rats (96). These cardiospheres contain a mixture of cells some of which are eCSCs. Rats underwent permanent ligation of the LAD coronary artery and 2 million CDCs or vehicle were intramyocardially injected at 4 sites in the peri-infarct zone. 3 weeks post-MI, animals that received allogeneic CDCs exhibited smaller scar size, increased infarcted wall thickness and attenuation of LV remodelling. Allogeneic CDC transplantation resulted in a robust improvement of fractional area change (~12%), ejection fraction (~20%), and fractional shortening (~10%), and this was sustained for at least 6 months. Furthermore, allogeneic CDCs stimulated endogenous regenerative mechanisms (detected cardiomyocytes in the cell cycle, recruitment of c-kitpos eCSCs, angiogenesis) and increased myocardial levels of VEGF, IGF-1 and HGF (96).

Testing regenerative therapies in murine models of human diseases, although a useful step in pre-clinical assays, it is not an accurate predictor of their human effectiveness. This is so not only because of the potential biological differences between the two species but because of the three orders of magnitude difference in mass between the two organisms, which make the pathology and the challenges for repair not only quantitatively but qualitatively different. Therefore, it is necessary that pre-clinical testing of therapies be carried out in an animal model similar in tissue biology, size and physiology to the human than the rodent models commonly used. The pig, because of its size, rapid growth rate, well-known physiology and availability, has proven a very useful and frequently used pre-clinical large animal model for much pathology, particularly those involving tissue regeneration.

We have previously shown that eCSCs exert a paracrine survival effect on cardiomyocytes through increased insulin like growth factor 1 (IGF-1) secretion and induction of the insulin like growth factor 1 receptor (IGF-1R) signalling pathway (97). In fact, the eCSC, which express high levels of the transcription factor GATA binding protein 4 (GATA-4), have a more potent paracrine effect. Furthermore, unlike other cell types (77,98) eCSCs have a very high tropism for the myocardium, which is dependent on the receptor-ligand pair chemokine (C-X-C) motif receptor 4 (CXC4) and stromal cell-derived factor 1 (SDF1). Up to 90% of the eCSCs injected through the tail vein after acute myocardial diffuse damage home toward and nest into the damaged myocardium (38). This behavior of the injected eCSCs is in stark contrast to the 3-5% myocardial retention to BMDC and MSCs even when injected intracoronary (99).

Under proper culture conditions it is possible to clone and expand a single rodent, porcine or human eCSC to up to 1×1012 cells without detectable alteration of karyotype, loss of differentiating properties or the phenotype of the differentiated progeny (88). These cloned cells produce a repertoire of pro-survival, immunomodulatory, anti-inflammatory and cardiovascular regenerative growth factors [Our unpublished data]. For this reason, we decided to test whether these expanded cells in vitro, when administered into allogeneic animals, would be the source of a more complex and physiologic mixture of growth and differentiating factors which, through a paracrine effect would produce a robust activation of the eCSCs with more rapid maturation of their progeny. It was expected that once their short term effect had been produced and the auto/paracrine feedback loop of growth factor production has been activated in the eCSCs, the allogeneic cells would be eliminated (presumably by apoptosis) and that the regeneration triggered by activated eCSCs would be completely autologous. c-kitpos eCSCs do not express either MHC-I or the co-activator molecules and have strong immunomodulatory properties in vitro when tested in the mixed lymphocyte reaction [Our unpublished data]. We therefore expected the expanded cells to survive long enough in the allogeneic host to produce their paracrine effect before being eliminated by the host immune system.

Allogeneic, non-matched, cloned male EGFP-transduced porcine eCSCs (Fig. 1), were administered intracoronary to white Yorkshire female pigs, 30 minutes after MI and coronary reperfusion. Pig serum was injected to control pigs after MI (CTRL). The cells or sera were injected through a percutaneous catheter into the anterior descending coronary artery just below the site of balloon occlusion used to produce the AMI. We found a high degree of EGFPpos/c-kitpos heterologous HLA non-matched allogeneic porcine CSCs nesting in the damaged pig myocardium at 30 minutes through to 1 day after MI (Fig. 2).

Figure 1. Endogenous Cardiac Stem Cell Essential Phenotype. A) Light microscopy representative image of long term cultured pig eCSCs. B) Cytospin preparation and c-kit immunofluorescence of cloned eCSCs. C) Essential CD phenotype of a typical CSC preparation. *B and C are adapted from Ellison et al. (2011).

Figure 2. EGFP+/c-kit+ heterologous HLA non-matched porcine CSCs nest in the damaged pig myocardium at 30 minutes through to 1 day after MI. A-C, Representative images of EGFPpos (green) CSCs in the infarcted porcine heart at 30 mins (A), 24 hrs (B) and 3 weeks (C) after intracoronary injection. D, the number of EGFPpos CSCs in the infarcted porcine heart at 30 mins, 24 hrs and 3 weeks after intracoronary injection of 1 x 108 EGFPpos CSCs. E-F, Representative images of EGFPpos (green) CSCs in the spleen. No EGFPpos CSCs were found at 3 weeks in the heart (C and D) or other tissues (F). Nuclei are stained by DAPI in blue. The fluorescencence in the spleen at 24h might be influence by autofluorescence since similar images where detected in the spleen of some placebo animals. The heart images of the placebo are all negative for GFP and indistinguishable from panels C and F.

Periodic sampling of blood at the coronary sinus and systemic circulation during and an hour after the cell infusion failed to identify EGFP-CSCs in the coronary sinus or peripheral blood of the placebo animals, as expected. Most samples from the coronary sinus of the CSC-treated animals had <4 EGFP+ cells per 100 μl of blood and none of the systemic venous blood samples were positive for EGFP+ cells. Therefore, assuming a coronary flow through the sinus of 300 ml/min., less than one million EGFP-CSCs entered the systemic circulation. No EGFP-CSC cells were identified in the systemic circulation at 1 and 24 hours after the cells administration. An estimate of the cells homed to the myocardium, based on the histological data of the animals sacrificed 24 hours after EGFP-CSC administration indicates that >85% of the injected cells were in the myocardium. Although these estimates have a large margin of error, the qualitative results correlate well with the data obtained in rodents by injecting eCSCs through the tail vein of mice and rats with acute myocardial damage (38).

It should be noted that the density of EGFP-CSCs in the myocardium 30 min after injection (Fig. 2) is >500 fold higher than that of eCSCs in the normal myocardium (~1 eCSC/1000 myocytes or 4000 nuclei). Although the density of the injected cells has been reduced at 24 h. (Fig. 2), it is still >100 fold higher than the eCSCs in healthy myocardium.

At 3 weeks post-AMI, all the injected allogeneic cells had disappeared from the myocardium and peripheral tissues (i.e. spleen). Furthermore, qtPCR failed to detect the presence of DNA from the allogeneic cells (probing for Y chromosome and GFP sequences). However, there was significant activation of the endogenous GFPneg c-kitpos CSCs (eCSCs) following allogeneic EGFP-CSC treatment (Fig. 3), so that by 3 weeks after MI, there was increased new cardiomyocyte and capillary formation, which was not evident in the control hearts (Fig. 4). This cellular and histological improvement was correlated with a similar improvement of ventricular function, as shown in Table 1.

Figure 3. Activation of endogenous CSCs following intracoronary injection of c-kit+ heterologous HLA non-matched porcine CSCs, after MI in pigs. A, Representative image of CD45 (red; yellow arrows) negative, c-kit (green; white arrows) positive endogenous CSCs in the 3 wk infarcted region of the CSC-treated porcine myocardium. Nuclei are stained by DAPI in blue. B, The percent number of endogenous CD45 negative, c-kit positive CSCs significantly increased following EGFP+/c-kit+ heterologous HLA non-matched CSC treatment. *P<0.05 vs. CTRL. C & D, Representative images of endogenous progenitor cells differentiating into the myocyte (C; Nkx2.5, white; c-kit, green; α-sarcomeric actin, red) and capillary (D; Ets-1, white; c-kit, green; α-sarcomeric actin, red) lineages. Nuclei are stained by DAPI in blue.

Figure 4. Increased new cardiomyocyte and capillary formation after c-kit+ heterologous non-matched porcine CSC treatment. A & B, The percent number of newly formed BrdUpos (A; green, white arrows) myocytes (A; α-sarcomeric actin, red) significantly increased following CSC treatment. *P<0.01 vs. CTRL. C & D, Similarly, the percent number of Ki67pos (C, green, white arrow) myocytes (C; α-sarcomeric actin, red) significantly increased, after CSC treatment. *P<0.01 vs. CTRL. E & F. The fraction of newly formed BrdUpos (E; green, white arrows) capillaries (E; vWF, red) significantly increased following CSC treatment. *P&l;0.01 vs. CTRL. Nuclei are stained by DAPI in blue.

Moreover, through paracrine mechanisms, allogeneic c-kitpos HLA non-matched EGFP-CSC treatment preserved myocardial wall structure and attenuated remodelling by reducing myocyte hypertrophy, apoptosis and scar formation (fibrosis)(Fig. 5). In summary, intracoronary injection of allogeneic CSCs after MI in pigs, which is a clinically relevant MI model, activates the eCSCs through a paracrine mechanism resulting in improved myocardial cell survival, function, remodelling and regeneration.

Figure 5. Through paracrine mechanisms, c-kit+ heterologous HLA non-matched CSC ( treatment preserves myocardial wall structure and attenuates remodeling. A, CSC treatment led to significantly decreased myocyte hypertrophy in the border region. *P<0.05 vs. Control (CTRL). B, Representative H&E staining showing a band of hypertrophic myocytes in the border region of CTRL pig myocardium. C, CSC treatment significantly decreased percent number of apoptotic (caspase 3 positive) myocytes in the border region. *P<0.05 vs. CTRL. D & E, Representative images of sirius red staining to identify fibrotic tissue (red) and muscle (yellow) in the infarct region of CTRL (D) and CSC-treated (E) pig hearts. F, CSC-treated pig hearts had a decreased percentage area fraction of fibrosis in the infarct zone.*P<0.05 vs. Control (CTRL)

A potential risk of using large numbers of in vitro expanded CSCs is the appearance of transformed cells with uncontrolled growth. This risk is completely eliminated by the use of allogeneic cells, with a different HLA allele from the recipient, because they are all eliminated by the immune system without need for immunosuppression. Claims that some of the transplanted allogeneic cells have a long term survival in the host, have either not been reproduced or thoroughly documented (100-102). If the survival of these allogeneic cells, except for a small number of progeny-derived cells in some mothers,103 proved to be correct, many of the immunology concepts, which have ruled transplant biology until now will need to be revised. Furthermore, despite thorough pathological examination and contrary to many iPS- and ES-derived cell lines, the adult tissue-specific eCSCs have a very low or non-existent capacity to form tumours and/or teratomas in syngeneic or immunodefficient animals (58). (unpublished data)

Allogeneic CSC therapy is conceptually and practically different from any presently in clinical use. In allogeneic therapies the mechanism of action of the therapeutic cells is clearly paracrine and there is no direct participation of the allogeneic cells in the spared and regenerated tissue. Basically, this cell therapy is only a form of growth factor therapy, which is able to deliver a more complex mixture of growth factors than our present knowledge permits us to prepare. The factors produced by the allogeneic cells are designed first to salvage host cells (myocytes, microvasculature and connective tissue) at risk and to stimulate the endogenous stem cells of the target tissue. The transplanted cells themselves survive only transiently. Therefore, the allogeneic stimulus through salvage of cells at risk and activation of the host’s eCSCs produces a completely autologous myocardial regeneration. Once more information becomes available, the allogeneic cells could be used either alone or in combination with the available factor therapy to improve the activation of the eCSCs and the maturation of their progeny.

E. Myocardial Regeneration Without Cell Transplantation: Using Growth Factors to Stimulate the Growth and Differentiation of the eCSCs

The identification of molecules secreted by transplanted cells should make possible the design of therapies which could replace the use of transplanted cells in order to concentrate on the administration of the principal effector molecules these cells produce. Interestingly, Smart et al. found that ‘priming’ with thymosinβ4 (Tβ4) followed by MI in mice resulted in activation of Wt1+ epicardial-derived progenitor cells and these went onto differentiate into cardiomyocytes, in the infarct and border regions.66 Despite the intrinsic relevance of these novel findings, the induced differentiation of the epicardial-derived progenitor pool into cardiomyocytes by Tβ4 was limited (<1%), relative to the activated progenitor population as a whole.66 Therefore, it is pertinent to identify the most efficacious small molecules and factors, which are able to induce optimal eCSC activation and drive significant regeneration and maturation of the new myocardium.

Myocardial regenerative cell-free therapies effective through in situ activation, multiplication and differentiation of the resident eCSCs should have many advantages over those based on cell transplantation. First, therapeutic components should be available ‘off-the-shelf’ and ready to use at all times without the lag time required for the cell harvesting and/or expansion needed for cell therapy approaches. Second, they should be affordable, in terms of the production costs of the medicinal product. Thirdy, therapy should be easy to apply and compatible with current clinical standard of care for AMI, including the widespread use of percutaneous coronary interventions (PCI). Finally, because of the robustness of the regenerative response produced, it should be possible to salvage and/or produce de novo ~50-60g of functional myocardial tissue, which is the minimum needed to change the course of the disease in a seriously ill patient.

We have tested the regenerative effects of intracoronary administration of two growth factors known to be involved in the paracrine effect of the transplanted cells that express the corresponding receptors.88 Insulin-like growth factor I (IGF-1) and hepatocyte growth factor (HGF), in doses ranging from 0.5 to 2μg HGF and 2 to 8μg IGF-1, were intracoronary administered, just below the site of left anterior descendent occlusion, 30 minutes after AMI during coronary reperfusion in the pig. A single administration of this growth factor cocktail triggers a regenerative response from the c-kitpos eCSCs, which is potent and able to produce anatomically, histologically and physiologically significant regeneration of the damaged myocardium without the need for cell transplantation.88 IGF-1 and HGF induced eCSC migration, proliferation and functional cardiomyogenic and microvasculature differentiation. Furthermore, IGF-1/HGF, in a dose-dependent manner, improved cardiomyocyte survival, reduced fibrosis and cardiomyocyte reactive hypertrophy. Interestingly, the effects of a single administration of IGF-1/HGF on the replication and differentiation of the eCSCs and their progeny are still measurable 2 months after its application. This long term effect of a therapeutic agent known to have a half-live in the range of minutes uncovers the existence of a feedback loop triggered by the external stimuli that activates the production of growth and survival factors by the targeted cells, which explains the persistence and long duration of the regenerative myocardial response.88These histological changes were correlated with a reduced infarct size and an improved ventricular segmental contractility and ejection fraction assessed by cMRI at the end of the follow-up that were dose-dependent (88).

Despite their effectiveness, the administration of IGF-1 and HGF has a drawback. Although it is very effective in the regeneration of the number of myocytes and micro-vessels lost by the AMI, the rate of maturation and of the newly formed myocytes to reach the size of the spared ones is heterogeneous and quite slow. While the newly formed myocytes which are in contact with spared ones mature rapidly and can reach a diameter close to a normal pig cardiomyocyte, there is an inverse correlation between new myocyte size and their distance from the small islands of spared myocardium scattered within the ischemic zone. With the exception of those new myocytes in close proximity to spared micro-islands of surviving pre-existing myocytes within the ischemic tissue or those in the infarct border region, at three weeks after treatment the length and diameter of the new myocytes (~85% of those regenerated) is between 1/2 and 1/5, respectively, of an adult myocyte, which means that their volume is significantly less than 1/10th of their mature counterparts.88 Because of this slow maturation process, although the therapy is very effective in restoring the number of myocytes lost by the AMI this is not the case for the lost ventricular mass which lags behind very significantly. In consequence, the myocardial generation of force capacity, which is the measure of meaningful functional recovery, also lags behind the regeneration of the cell numbers to the pre-AMI state. These results suggest that there is a trophic effect elicited by the spared myocytes acting upon the newly formed ones that accelerates its maturation. This trophic effect/factor seems to have a very short effective radius of action, although the available data does not support the conclusion that it requires cell-cell contact. Thus, despite the beneficial effect of the therapy in reducing the scar area, pathological remodelling and partial recovery of ventricular function, there is little doubt that it would be desirable to obtain a more rapid recovery of the ventricular mass and the capacity to generate force.

F. Summary and Conclusions

The findings that the adult heart harbors a regenerative multipotent cell population composed by eCSCs and that mammalian, including human, cardiomyocytes are replaced throughout adulthood represents a paradigm shift in cardiovascular biology. The presence of this regenerative agent within the adult heart and the results obtained in different animal models support the view that the heart has the potential to repair itself if the eCSCs are properly stimulated. Indeed, it should be possible to replace autologous cell transplantation-based myocardial regeneration protocols, -which by definition cannot be applied in the acute phase of an MI where they would be most effective, with an “off-the-shelf”, readily available, unlimited and effective regenerative/reparative therapy based on allogeneic cells, a specific growth factor cocktail or a combination of both. For these therapies to be effective they should be robust enough that through a paracrine action are effective in producing the in situ activation of the resident eCSCs of the host. However, to turn this optimistic clinical scenario into clinical reality, it is mandatory to obtain a better understanding of eCSC biology in order to fully exploit their regeneration potential. This knowledge will ultimately lead to the development of realistic and clinically applicable and universally available myocardial regeneration strategies.

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