Optimizing Cardiac Repair and Regeneration Through Activation of the Endogenous Cardiac Stem Cell Compartment
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- Ellison, G.M., Nadal-Ginard, B. & Torella, D. J. of Cardiovasc. Trans. Res. (2012) 5: 667. doi:10.1007/s12265-012-9384-5
Given the aging of the Western World and declining death rates due to acute coronary syndromes, the increasing trends in the magnitude and morbidity of heart failure (HF) are predicted to continue for the foreseeable future. It is imperative to develop effective therapies for the amelioration and prevention of HF. The search for the best cell type to be used in clinical protocols of cardiac regeneration is still on. That the adult mammalian heart harbors endogenous, multipotent cardiac stem/progenitor cells (eCSCs) and that cardiomyocytes are replaced throughout adulthood represent a paradigm shift in cardiovascular biology. The presence of eCSCs supports the view that the heart can repair itself if the eCSCs can be properly stimulated. Pending a better understanding of eCSC biology, it should be possible to replace autologous cell transplantation-based myocardial regeneration protocols with an “off-the-shelf,” readily available, and effective regenerative/reparative therapy based on activation of the eCSCs in situ.
KeywordsEndogenous cardiac stem/progenitor cellsGrowth factorsParacrineAllogeneicMyocardial regenerationMultipotent
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 the available treatment is largely unsuccessful. 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 either 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 .
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 post-AMI in CHF in USA alone . More than 550,000 patients per year are added to this group, which has a similar prevalence in the EU countries and, after the first episode of heart failure, has an average mortality rate of ∼18 % per year and in the USA alone absorbs ∼$30 billion annually for their care . 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 . For this reason, it has been a goal of cardiovascular research for the past decade to find methods to replace the cardiomyocytes lost as a consequence of the MI in order to prevent or reverse the pathological cardiac remodelling. 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 for heart disease and failure .
Until recently, a paucity of understanding about the cellular homeostasis of most adult tissues has been a major factor limiting the expansion of the early breakthroughs in adult stem cell research and therapy, such as those applied to the blood and bone marrow diseases, to other areas of medicine. Up to early in the last decade, the prevalent view 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 [5–7], they were an exception. The established paradigm was that the majority of the remaining tissues either renewed very slowly (such as the muscle and the endothelial lining of the vascular system), to the point of being physiologically irrelevant, or not at all. 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 that, for most organs, the number and function of their parenchymal cells was in a downward spiral starting 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 only directed toward improving and/or preserving the performance of the remaining functional cells in the tissue. Thus, 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 covered. Because it was believed that the cells needed for the second option did not exist for the majority of tissues, heterologous organ and cell transplantation became the only possible avenue. In fact, despite the multiple drawbacks of heterologous cell/organ transplantation, its practice has become the cutting edge for several medical specialties . 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 that received the successful derivation of multipotent human embryonic stem cells (hESCs) [9–11] with the capacity to differentiate in to most, if not all, known cell types promised an unlimited supply of donor parts. When the euphoria caused by this development started to dim, because of the ethical and immunological challenges posed by the use of hESCs, came the breakthrough of what permitted the conversion of different adult somatic cells, such as fibroblasts, into multipotent cells called induced pluripotent stem (iPS) cells by introduction of a very limited number of genes (now known to be responsible for the multipotent state of stem cells) [12, 13]. 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 could be potentially used for autologous cell therapy . Although the potential of the iPS cells as therapeutic agents remains high, it is already clear that many hurdles need to be cleared before they can reach clinical application .
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 been 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 constituted mainly of terminally differentiated cells needed to have a population of tissue-specific regenerating cells. Not surprisingly, this realization was rapidly followed by the progressive identification of stem cells in each of the adult body tissues [16–21].
For a long time, the cardiovascular research community has treated the adult mammalian heart as a postmitotic organ without intrinsic regenerative capacity. The prevalent notion was that the >20-fold increase in cardiac mass from birth to adulthood and in response to different stimuli in the adult heart results exclusively from the enlargement of pre-existing myocytes [22–24]. It was accepted that this myocyte hypertrophy, in turn, was uniquely responsible for the initial physiological adaptation and subsequent deterioration of the overloaded heart. This belief was 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 [25, 26]; therefore, all cardiac myocytes have to be of the same chronological age as the individual . (b) The heart has no intrinsic parenchymal regenerative capacity because it lacks a stem/progenitor cell population able to generate new myocytes. Despite published evidence that this prevalent view was incorrect [28–33], 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. However, because this “measured” self-renewal did not appear to be very robust, its physiological significance has remained in doubt. The conclusion of Bergmann et al.  on the rate of turnover depends on the validity of a complex mathematical formula, whose impact on the results dwarfs that of the measured data. Furthermore, their calculations identify the highest turnover rate during youth and early adulthood followed by a steady decrease with age. The latter conclusion, which is contrary to most or all the turnover values measured for all other human tissues, including the heart , has passed without a ripple. Whether the new myocytes formed originate from precursor cells or from the division of pre-existing myocytes was not addressed . However, a genetic fate mapping study in mice tracked new myocyte origin in the adult heart to a compartment of stem/precursor cells .
Tissue-Specific Endogenous Cardiac Stem/Progenitor Cells
Myocytes of the adult mammalian myocardium are terminally differentiated cells that are permanently withdrawn from the cell cycle [25, 26]. Thus, the adult heart is composed predominantly of postmitotic cells, but it, nevertheless, has a remarkable capacity for regeneration, both under normal conditions and in response to diverse pathological and physiological stimuli [30, 31, 34, 36]. It is becoming increasingly accepted now that new cardiomyocyte formation does not stop early in postnatal life but continues throughout life. What still remains controversial is the amount and physiological significance of the cardiomyocyte turnover and origin of the newly formed myocytes [24, 37]. Three main sources of origin of the new myocytes have been claimed: (a) circulating progenitors, which through the bloodstream home to the myocardium and differentiate into myocytes ; (b) mitotic division of the pre-existing myocytes [36, 38, 39]; 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 (i.e., myocytes, smooth and endothelial vascular and connective tissue cells) [40, 41]. It is clear now that the blood borne precursors, although well documented as a biological phenomenon , might be limited to very special situation , and their direct regenerative import is very limited, if any . Pre-existing cardiomyocyte division 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 that expressed proteins of the contractile apparatus in their cytoplasm [36, 38, 39]. 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 are capable of a few rounds of mitosis before irreversibly withdrawing from the cell cycle .
Undoubtedly, the best documented source of the 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, which have the phenotype, behavior, and regenerative potential of bona fide cardiac stem cells (eCSCs) [40, 41, 45]. The first report of these endogenous regenerating myocardial stem cells in the adult mammalian heart was in 2003 , and since, then their existence has been confirmed by a number of independent groups [47–55]. Although a variety of markers have been proposed to identify eCSCs in different species and throughout development [47–55], it still remains to be determined whether these markers identify different populations of eCSCs or, more likely, different developmental and/or physiological stages of the same cell type . Recently, another multipotent cell type, present in the epicardium and derived from the proepicardial organ has been described . The role of these cells in normal or pathological myocyte turnover remains to be elucidated.
The progeny of a single eCSC is able to differentiate into cardiac myocytes, smooth muscle, and endothelial vascular cells and, when transplanted into the border zone of an infarct, regenerates functional contractile muscle and the microvasculature of the tissue . In a normal adult myocardium, at any given time, most of the eCSCs are quiescent, and only a small fraction is active to replace the myocytes and vascular cells lost by wear and tear. In response to stress (hypoxia, exercise, work overload, or other damage), however, a proportion of the resident eCSCs are rapidly activated; they multiply and generate new muscle and vascular cells [58, 59], contributing to cardiac remodeling. The 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, and their transplantation can regenerate the contractile cells lost as a consequence of a major AMI affecting up to 25 % of the left ventricular mass [46, 58, 60].
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 proverbial coin of cardiac homeostasis in which the eCSCs play a central role . These findings have eventually placed the heart squarely among other organs with regenerative potential, such as the liver, skin, muscle, and central nervous system.
Stem Cell Therapy for Heart Failure and Disease
Bone-Marrow-Derived Stem Cells
“Regenerative myocardial therapies” currently undergoing clinical trials are mainly using bone-marrow-derived cells (BMDCs) of different types as the therapeutic agent and have produced mixed results [61–63]. Controversy still surrounds the cardiomyogenic potential of BMDCs [64, 65]. 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 [66–68]. BMDCs release a complex mixture of cytokines and growth factors involved in cell survival, proliferation, and migration, which enhance arteriogenesis [69, 70] and myocyte survival [67, 68]. In essence, instead of the transplanted cells undergoing cardiomyogenic differentiation, through a yet incompletely defined paracrine mechanism, they contribute to improving myocardial contractility and amelioration of ventricular remodeling (decreasing fibrosis, hibernation, and stunning), inhibition of the inflammatory response, increasing existing cardiomyocyte survival, and increasing angiogenesis/neovascularisation. Interestingly, it has also been suggested that these therapies also produce the activation of eCSCs to give rise to new vasculature and cardiomyocytes, leading to endogenous regeneration [44, 71, 72].
Recently, in a creative and innovative approach, Terzic and colleagues , convinced of the limited benefit of using naive BMDCs in patients with ischemic heart disease, have developed a successful protocol, based on the use of specific growth factors and cytokines, to precondition or “guide” human bone marrow mesenchymal stem cells (hMSC) into a cardiac phenotype. Guided, cardiopoietic hMSCs yielded sarcomere-containing myocytes capable of electromechanical response in vitro and improved ejection fraction up to 30 % over matched naive hMSC at 1 and 2 months after transplantation in nude mice with chronic MI . The benefit of cardiopoietic hMSC was particularly prominent in the subgroup with ejection fraction <45 % resulting in 1-year survival of 75 % in contrast to 48 % for naive hMSCs and 0 % for saline-treated counterparts . Histopathology revealed that global myocardial fibrosis and scar size were significantly reduced in cardiopoietic hMSC-treated hearts. Moreover, low human troponin staining was observed in the naive hMSC group, whereas the anterior wall of cardiopoietic hMSC-treated hearts revealed human-specific troponin I, α-actin, and ventricular myosin light chain (MLC-2v) positive cardiomyocytes, with connexin-43 gap junction formation . Therefore, hMSCs can be coaxed towards highly reparative cells with enhanced therapeutic efficacy. They are a desirable source for organ repair due to accessibility for harvest, propensity to propagate in culture, and favorable biological profile. A similar protocol has undergone a phase III clinical trial in patients with CHF with apparent beneficial effects but which results have not yet formally reached the public domain. However, this personalized-medicine approach with its complexity of cell husbandry and clinical procedures because of the long time needed to prepare the cells and its high cost will likely prove unsuitable for treating the large number of patients that need affordable and readily available products to treat the acute phase of the disease.
Therefore, both from a theoretical as well as practical point of view, the race is still on to find the “best” or even a “good” cardioregenerative protocol, which should be safe, effective, and affordable to reconstitute the myocardium and improve function following myocardial damage. With the myocardium now recognized as a regenerating tissue, harboring eCSCs that can be isolated and amplified in vitro  or stimulated to replicate and differentiate in situ , it has become reasonable to search for methods to exploit this endogenous regenerative potential to replace the lost muscle with autologous functional myocardium.
Autologous Cardiac Stem Cell Therapy
Many questions about eCSC basic biology still remain unanswered, particularly their long-term effectiveness and regenerative potential. It is imperative that such issues be addressed quickly if the full potential of these cells is to be realized, manipulated, and applied clinically. In particular, it is imperative to document whether the teratogenic and neoplastic potential of the in vitro expanded eCSCs is low enough to make their use in humans safe. However, despite a lack of well-documented experimental information, clinical trials using autologous cardiac stem/progenitor cells are already underway [75, 76]. Sixteen patients with ischemic cardiomyopathy with postinfarction left ventricular (LV) dysfunction (ejection fraction ≤40 %) who had undergone coronary artery bypass grafting, had 500,000–1 million of autologous c-kit positive, lineage negative, and cardiac progenitor cells infused intracoronary, ∼4 months after surgery . The control group were not given any treatment. Left ventricular ejection fraction (LVEF) increased by 8 EF points at 4 months after infusion, whereas the LVEF did not change in the control patients, during the corresponding time interval. Moreover, LVEF increased by 12 EF points in eight of the treated patients at 1-year follow-up. Cardiac MRI (cMRI) of seven of the treated patients showed that infarct size decreased at 4 and 12 months .
In the prospective, randomized cardiosphere-derived autologous stem cells to reverse ventricular dysfunction trial, 17 patients (with left ventricular ejection fraction of 25–45 %) were infused into the infarct-related artery with up to 25 million, CD105-positive, autologous cardiosphere-derived cells (CDCs), 1.5–3 months after myocardial infarction . Eight patients received standard care and acted as the control group. Compared with controls at 6 months, MRI analysis of patients treated with CDCs showed significant reductions in scar size and mass, increased viable heart mass, regional contractility, and systolic wall thickening. However, changes in end-diastolic volume, end-systolic volume, and LVEF did not differ between groups at 6 months .
Although the results of these two pioneering clinical trials are encouraging because they were safe, it should be noted that they were phase I/IIa trials designed to test short-term safety and not effectiveness. Given the very small number of cells administered in both studies, and the known low cell survival/engraftment upon transplantation, it is clear that whatever effect on myocardial mass and contractile function detected on the treated patients cannot be the result of the direct contribution of the transplanted cells. Therefore, if the beneficial effect prove to be real and reproducible, it is likely to be due to an indirect effect of the transplanted cells on the survival and functional recovery of stunned myocytes, which otherwise might have been lost. Because of the high cost and the long wait for the availability of the cells for autologous cardiac stem/precursor cell therapy, it will become imperative to compare the beneficial effects of this approach to that obtained with BMDCs because of their easier availability, accessibility, and lower cost of the procedure. Furthermore, the widespread use and applicability of autologous cardiac stem cell therapy is highly debatable. Firstly, the procedure for cell acquisition, scale-up, and transplantation is complex, time consuming, and very expensive. The isolation and expansion of eCSCs to the number needed from catheter and surgical biopsies takes 1–3 months. 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 patients beyond those few required to establish proof-of-concept for the therapy and to a small group of individuals with abundant economical resources. Finally, eCSCs undergo senescence with severe pathological consequences [77–79]. Accordingly, for the cohort of patients (the aged population) most likely candidates for the regenerative therapy, >50 % of their eCSCs can be senescent and unable to participate in the regenerative process [77–79]. Thus, if eCSC “aging” is an age or cell cycle dependent process, which affects all or most of the eCSC population, most or all regenerative therapies based on eCSC isolation and expansion will likely result in further exhaustion of the self-renewal capability of these cells with an accelerated loss of their regenerative capacity.
Stimulation of the Myocardial Endogenous Capacity for Repair and Regeneration
One potential therapeutic mechanism of action by which the different forms of transplantation cell therapy are now receiving a lot of attention is the activation, through a paracrine mechanism, of survival pathways in the cohort of cells at risk together with the endogenous regeneration compartment, represented by the eCSCs. A corollary of this hypothesis is that the identification of the molecules secreted by the transplanted cells should make possible the design of therapies, which eliminate the use of the cells and concentrate on the administration of the principal effector molecules these cells had identified. Interestingly, Smart et al.  found that “priming” with thymosinβ4 (Tβ4) followed by MI in mice resulted in significant activation of Wt1+ epicardial-derived progenitor cells, and these went onto differentiate into cardiomyocytes, in the infarct and border regions. Despite the intrinsic relevance of the 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 . 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 on the 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 as “off-the-shelf” and ready to use at all times without the lag time required for the cell therapy approaches; second, they should be affordable, in terms of the production costs of the medicinal product; third, such a 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); and fourth, because of the robustness of the regenerative response produced, it should be able to produce and/or recover ∼50–60 g of functional myocardial tissue, which is the minimum needed to change the course of the disease in a seriously ill patient.
Myocardial Regeneration Without Cell Transplantation—Using Growth Factors to Stimulate the Growth and Differentiation of the eCSCs
Testing regenerative therapies in mouse models of human diseases, although a necessary step in preclinical assays, 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 order of magnitude difference in mass between the two organisms, which make the challenges not only quantitatively but qualitatively different. Therefore, it is necessary that preclinical testing of therapies be carried out in a model, which is more similar in tissue biology, size, and physiology to the human than the rodent models commonly used.
Despite their effectiveness, the administration of IGF-1 and HGF has a significant drawback. Although it is very effective in the regeneration of the myocytes and microvessels lost, the rate of maturation of the newly formed myocytes 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 microislands of surviving pre-existing myocytes within the ischemic tissue or those in the border region, at 3 weeks after treatment, the length and diameter of the remaining new myocytes (∼85 % of those regenerated) are between one half and one fifth, respectively, of an adult myocyte, which means that their volume is significantly less than one tenth of their mature counterparts . 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 as to the regeneration of the lost ventricular mass, which lags behind very significantly. In consequence, the myocardial generation of force capacity, that is, the meaningful functional recovery, also lags significantly behind the regeneration of the cell numbers to the pre-AMI state. Despite the beneficial effect of the therapy in reducing the scar area, pathological remodeling, 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.
Myocardial Regeneration After Allogeneic Stem Cell Therapy
All the currently proposed autologous cell approaches are very attractive from the theoretical and biological standpoint. For those rare diseases with chronic and long-term evolution affecting hundreds or even thousands of potential patients to be treated, these personalized therapies, despite their high cost in medical and material resources, might even make sense from an economic standpoint. Unfortunately, this is not the case for diseases of high prevalence, such as the consequences of ischemic heart disease, with millions of patients/candidates for regenerative therapy. Not even the developed world has the resources needed to start a program of personalized regenerative medicine for the patients already in CHF who presently are left with heart transplantation as the only realistic option for recovery. Therefore, although the cell transplantation approaches outlined 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 ineffective to favorably 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 favorable 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 remodeling once it has developed but also soon after the acute insult in order to induce early regeneration of the cells lost in order to prevent or diminish the pathological remodeling.
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 method(s) of administration. As previously stated, to be widely available and compatible with current clinical standard of care for AMI, an intracoronary method for delivery in the cath laboratory at the time of the primary revascularization is the most feasible. In addition, direct myocardial injection at the time of revascularization surgery is highly realistic. Mesenchymal stem cells have a broad repertoire of secreted trophic and immunodulatory cytokines; however, they also secrete factors that negatively modulate cardiomyocyte apoptosis, inflammation, scar formation, and pathological remodeling . Moreover, it is questionable whether they are the most optimal cell to use in terms of survival and homing to and engraftment in the myocardium. Furthermore, cells can become entrapped in the microvasculature and impede cell entry into the myocardium.
To partially overcome this, Medicetty and colleagues  used a porcine model of AMI and delivered 20–200 million allogeneic, multipotent, adult BMDCs (MultiStem; that are nonimmunogeneic and can suppress activated T-cell proliferation and have anti-inflammatory and angiogenic properties as well), directly to 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 remodeling at 30 and 90 days after myocardial injury . Rapidly following on from this preclinical study, Penn et al.  conducted a multicenter phase I trial of the effects of adventitial delivery of MultiStem in patients 2–5 days after primary PCI. In patients with EF determined to be <45 % before the MultiStem injection, at 4 months after AMI, a 1, 4, 14, and 11 % absolute increase in EF was observed following injection of 20, 50, and 100 million cells, respectively .
Recently, Malliaras and colleagues  have tested the safety and efficacy of using allogeneic, mismatched CDCs in infarcted rats. Rats underwent permanent ligation of the LAD coronary artery, and 2 million CDCs or vehicle were intramyocardially injected at four sites in the peri-infarct zone. Three weeks post-MI, animals that received allogeneic CDCs exhibited smaller scar size, increased infarcted wall thickness, and attenuation of LV remodeling. 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 (cardiomyocyte cycling, recruitment of c-kitpos eCSCs, angiogenesis) and increased myocardial VEGF, IGF-1, and HGF .
We have previously shown that eCSCs that express high levels of the transcription factor GATA-4 exert a paracrine survival effect on cardiomyocytes through increased IGF-1 secretion and induction of the IGF-1R signalling pathway . Furthermore, unlike other cell types [62, 85], eCSCs have a very high tropism for the myocardium (our unpublished findings). Under proper culture conditions, it is possible to clone and expand a single rodent, porcine, or human eCSC to up to 1 × 1010 cells without detectable alteration of karyotype, loss of differentiating properties, or the phenotype of the differentiated progeny . These cloned cells produce a repertoire of prosurvival and cardiovascular regenerative growth factors (our unpublished data). For this reason, we decided to test whether these in vitro expanded cells, 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 locus or coactivator 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.
A possible risk of using large numbers of in vitro expanded CSCs is the appearance of transformed cells with the potential to form abnormal growths. This risk is completely eliminated by the use of allogeneic cells, with a different HLA allele from the recipient, because they all get eliminated by the immune system without immunosuppression. Claims that some of the transplanted allogeneic cells have a long-term survival in the host have not been reproduced or thoroughly documented [83, 87, 88]. If their survival proves 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 ECS-derived cell lines, the adult tissue-specific eCSCs have a very low or nonexistent capacity to form tumors and/or teratomas in syngenic or immunodefficient animals (our unpublished data and ).
Allogeneic CSC therapy is conceptually and practically different from any presently in clinical use. The proposed cell therapy is only a different form of growth factor therapy 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 to stimulate the endogenous stem cells of the target tissue, but the transplanted cells themselves survive only transiently and do not directly participate in the production of progeny that contributes to the regenerated tissue. Once more information is 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.
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 represent a paradigm shift in cardiovascular biology. The presence of this regenerative agent within the adult heart supports the view that the heart has the potential to repair itself if the eCSCs can be properly stimulated. Indeed, it is predicted that, in the near future, it should be possible to replace cell transplantation-based myocardial regeneration protocols with an “off-the-shelf,” readily available, unlimited, and effective regenerative/reparative therapy based on specific growth factor administration or on the paracrine secretion by allogeneic CSC transplantation able to produce the activation in situ of the resident eCSCs. However, before reaching this optimistic clinical scenario, it is mandatory to obtain a better understanding of eCSC biology in order to fully exploit their regeneration potential. The latter will ultimately lead to developing realistic and clinically applicable myocardial regeneration strategies. Cardiac regenerative medicine is set to revolutionize the treatment of cardiac diseases, and such research will have significant and long-term impact on socioeconomics and patient well-being. Indeed, therapies that are based on findings from high quality research will undoubtedly cut deaths from cardiovascular disease, reduce recovery times, increase life expectancy, and quality of care and save money.