Autologous and allogeneic cardiac stem cell therapy for cardiovascular diseases

Autologous and allogeneic cardiac stem cell therapy for cardiovascular diseases

Accepted Manuscript Title: Autologous and Allogeneic Cardiac Stem Cell Therapy for Cardiovascular Diseases. Authors: Ricardo Sanz-Ruiz MD, Francisco F...

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Accepted Manuscript Title: Autologous and Allogeneic Cardiac Stem Cell Therapy for Cardiovascular Diseases. Authors: Ricardo Sanz-Ruiz MD, Francisco Fern´andez-Avil´es PII: DOI: Reference:

S1043-6618(17)30397-3 http://dx.doi.org/doi:10.1016/j.phrs.2017.05.024 YPHRS 3607

To appear in:

Pharmacological Research

Received date: Revised date: Accepted date:

28-3-2017 14-4-2017 25-5-2017

Please cite this article as: Sanz-Ruiz Ricardo, Fern´andez-Avil´es Francisco.Autologous and Allogeneic Cardiac Stem Cell Therapy for Cardiovascular Diseases.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2017.05.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title: Autologous and Allogeneic Cardiac Stem Cell Therapy for Cardiovascular Diseases. Authors: Ricardo Sanz-Ruiz, MD; Francisco Fernández-Avilés, MD, PhD. Affiliations for authors: Department of Cardiology and Instituto de Investigación Sanitaria, Hospital General Universitario Gregorio Marañón. Universidad Complutense. CIBERCV. Madrid, Spain. Corresponding author: Francisco Fernández-Avilés, MD, PhD, FACC, FESC Cardiology Department, Hospital General Universitario Gregorio Marañón Dr. Esquerdo 46 28007 Madrid, Spain Telephone: +34 91 4265882. Fax: +34 91 5868276 E-mail: [email protected] Total word count: 4037 (excluding references and tables). Abstract word count: 278

ABSTRACT Stem cell therapy is one of the most promising therapeutic innovations to help restore cardiac structure and function after ischemic insults to the heart. However, phase I and II clinical trials with autologous “first-generation stem cells” have yielded inconsistent results in ischemic cardiomyopathy patients and have not produced the definitive evidence for their broad clinical application. Recently, new cell types such as cardiac stem cells (CSC) and new allogeneic sources have attracted the attention of researchers given their inherent biological, clinical and logistic advantages. Preclinical evidence and emerging clinical data show that exogenous CSC produce a range of protein-based factors that have a powerful cardioprotective effect in the ischemic myocardium, immunoregulatory properties that promote angiogenesis and reduce scar formation, and are able to activate endogenous CSC which multiply and differentiate into cardiomyocytes and microvasculature. Furthermore, allogeneic CSC can be produced in large quantities beforehand and can be administered “off-the-shelf” early during the acute phase of myocardial ischemia. The distinctive immunological behavior of allogeneic CSC and their interaction with the host immune system is supposed to produce immunomodulatory beneficial effects in the short-term, preventing long-term side-effects after their rejection. Preclinical studies have shown highly promising results with allogeneic CSC, and clinical trials are already ongoing. Finally, unraveling questions about the biology and physiology of CSC, the characterization of their secretome, the conduction of larger clinical trials with autologous CSC, the definitive evidence on the safety and efficacy of allogeneic CSC in humans and the possibility of repeated administrations or combinations with other cell types and soluble factors will pave the road for further developments with CSC, that will undoubtedly determine the future of cardiovascular regenerative medicine in human beings.

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Keywords: Cardiac Stem Cells; Cardiosphere-Derived Stem Cells; Allogeneic Stem Cells; ST-Segment Elevation Myocardial Infarction; Heart Failure.

INTRODUCTION Cardiovascular disease is the leading cause of mortality and morbidity in humans throughout developed countries, carrying an enormous psychological and socioeconomic burden to patients and healthcare systems. ST-segment elevation acute myocardial infarction (STEMI) and its chronic sequels of ischemic heart failure (HF) and sudden cardiac death account for more than 4 million deaths per year in Europe(1) and for one death every 40 seconds in the United States.(2) Despite major advances that have reduced early mortality of STEMI, 12% of patients die within 6 months and 25% of survivors progressively develop HF, a condition that entails a mortality rate of 50% in 5 years.(3) Current therapeutic strategies are only able to delay the progress of the disease but not to stop or reverse it. These data underscore an unmet clinical need for innovative and ideally curative biological therapies (i.e., stem cells, genes, growth factors and molecules) aimed at repairing the damaged cardiac tissue and at increasing the heart regenerative potential after STEMI. However, randomized clinical trials developed with “first-generation stem cells”, albeit undoubtedly safe, have yielded inconsistent results during the last years, the original enthusiasm being dampened by a cumulative body of evidence on the modest efficacy of these therapies in improving cardiac function and by the lack of a definitive proof-ofconcept on their clinical and prognostic benefits. These observations have led to the development of more potent and purified “second-generation stem cells”.(4) Among these, the application of resident cardiac stem cells aims at repairing the diseased myocardium by matching the target organ and by replacing the lost cardiomyocytes (CM), and has shown the most promising results so far in the field of human research with stem cell-based therapies and with different delivery technologies (Figure 1). This may not be surprising, since most stem cell therapy clinical trials have been designed with the final objective of generating new CM by the administration of non-cardiac cell types (i.e., bone marrow-derived and mesenchymal stem cells) (Table 1).(5-7) In this review, we summarize the origin and the phenotypic characteristics of cardiac stem cells, discuss on their ideal source (autologous or allogeneic) for clinical applications, outline preclinical and clinical experiences and indicate future directions of research.

THE CARDIAC STEM CELL NICHE. TYPES OF CARDIAC STEM CELLS Since the beginning of the XXI century, the cardiovascular research community has slowly come to accept that the adult heart is not a post-mitotic organ without intrinsic regenerative capacity but a self-renewing one with a population of tissue-specific regenerating cells. Indeed, resident cardiac stem cells have been identified in the adult mammalian heart, although no definitive consensus has been reached yet on their identity and actual regenerative effects.(8) Furthermore, and though it has been proved that new CM are formed throughout the life span of humans in response to different injuries,(9) the rate of this turnover is still highly debated and ranges from 1% to 40%

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per year.(8-11) Given that the contributions to the pool of newly formed CM of circulating progenitors (i.e., bone marrow-derived) and of the mitotic division of pre-existing CM (which may re-enter the cell cycle) are marginal,(12) it is now accepted that the most important documented source of regenerating CM is a small population of cells distributed throughout the atria and ventricles (i.e., the “cardiac stem cell niche”), that show stem cell characteristics: they are self-renewing, clonogenic and multipotent, and are able to differentiate into the three cardiogenic cell lineages of the normal adult heart. This compartment is formed by a mixture of different types of progenitor cells and is indispensable for myocardial cell homeostasis, repair and regeneration of the adult heart.(8) After the first report of these cells, at least eight cell populations have been identified according to different membrane markers and transcription factors: C-kit+ cells, Sca-1+ cells, side population cells, Isl1+ cells, cardiac mesangioblasts, cardiac resident colony-forming unit-fibroblast (cCFU-F), cardiosphere-derived cells and epicardial stem cells (Table 2). Due to the evident overlap of markers used for their characterization, it has been hypothesized that these populations may represent different phenotypic variations of only one multipotent resident cardiac stem cell, with the capacity to differentiate in vitro and in vivo into CM, smooth muscle cells and endothelial cells.(13) In the normal heart these cells are quiescent,(14) but are rapidly activated after myocardial damage (i.e., hypoxia, overload, ischemia, etc…)(15) generating new muscular and vascular cells. Unfortunately, this capacity - which declines with age - (16) is not enough to significantly repair large tissue loses. This is the rationale for the administration of exogenous cardiac stem cells after STEMI or once the adverse remodeling process has been initiated. Originally, “first-generation stem cells” were thought to exert their positive effect on cardiac repair by differentiation processes. However, and with the exception of pluripotent-derived cells (i.e., induced pluripotent and embryonic stem cells), these processes have been demonstrated in vivo at negligible rates. Conversely, injected cells secrete a variety of growth factors, cytokines and extracellular vesicles (“exosomes”) that re-awake resident cardiac stem cells, inhibit apoptotic signaling, decrease inflammation and modulate angiogenic pathways (the “paracrine hypothesis”) (Figure 2, Graphical Abstract). This is the rationale behind all trials with cardiac progenitor cells, to effectively deliver them into the ischemic area enabling the production and secretion of pro-survival, anti-inflammatory and regenerative growthfactors (i.e., IGF-1, HGF and the TGF-ß1 superfamily), which in turn will activate survival pathways in the cells at risk and also the endogenous cardiac stem cell niche. In the field of preclinical and clinical research, resident cardiac stem cells have been generically grouped under three denominations: cardiac stem cells (CSC), cardiospheres (CS) and cardiosphere-derived cells (CDC).(17) CSC (also “cardiac progenitor cells”) refer to all cardiac progenitors that are isolated from heart biopsies, are about 12-15 µm in diameter and are positive for stem cell markers (such as C-kit and Sca-1) and cardiac markers (such as Isl1, NKX2.5 and GATA4). Human CSC can migrate out of in vitro cultured human myocardial biopsies and form spheroids in suspension conditions.(18) Those spherical clusters are termed CS (50 to 200 µm in size, thus precluding their administration through the intracoronary route), and can be subsequently dissociated to obtain CDC (on average 20 µm in diameter). Interestingly, in these CS, C-kit+ cells are

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localized in the center of the spheroids and are positive for BrdU staining. Only these cells in the center are maintained in an undifferentiated state, whereas the cells at the surface layer are continuously undergoing differentiation. CDC have shown superior cardiomyogenic/vasculogenic differentiation and paracrine potential compared with bone marrow-derived, mesenchymal and adipose-derived cells in mice.(19)

AUTOLOGOUS VERSUS ALLOGENEIC SOURCES OF CARDIAC STEM CELLS One of the purported reasons for the divergent results of stem cell therapy trials so far is the autologous origin of cell products. Autologous progenitor cells are free of ethical issues and non-immunogenic, but are primarily limited by their variable and unpredictable functionality, which is markedly affected by age, comorbidities (such as risk factors of atherosclerosis) and genetic signature inherent to the host. Moreover, the need for sampling tissue sources to obtain autologous cell products such as the bone marrow, adipose tissue or blood heavily limits their use in the acute setting of myocardial ischemia. This is critical and dramatically important in the case of CSC, which require invasive procedures for tissue harvesting (i.e., cardiac biopsies, surgical procedures) and long culture times for their expansion to meaningful numbers.(20) These issues have driven the interest of the scientific community towards banked, consistent and readily available allogeneic cell products, in which cells derived from healthy donors or unaffected organs can be strictly quality-controlled and manufactured in large quantities in a central facility to be immediately available as an “off-the-shelf” product for urgent applications including STEMI. The main advantage of this approach is that the functional variability of the cells is alleviated by preparing a master bank of validated fully-tested clinical-grade cells that allows a well-qualified product to be readily available for clinical applications even in the acute phases of the disease. Its disadvantage is the eventual immune response triggered by allogeneic cells.(21) Immunogenicity of allogeneic cells and tissues is based on allelic differences of major histocompatibility complex (MHC) antigens, also called the HLA (human leukocyte antigen) system. This system includes two types of cell surface glycoproteins: HLA class I molecules (present on all nucleated cells) and HLA class II molecules (expressed only on antigen-presenting cells and B lymphocytes). Thus, immunogenicity concerns were originally raised due to the possibility that allogeneic cells may induce the production of donor-specific antibodies (DSA, antibodies against mismatched donor HLA molecules), which could eventually interact with the allograft impacting its effect by immunological clearance.(22) However, a large body of in vitro and in vivo research indicates that allogeneic CSC are safe from an immunological standpoint, since they activate modulatory rather than deleterious cellular immune reactions.(23) First of all, CSC express medium levels of HLA class I and of the costimulatory/regulatory molecule PD-L1, but they do not express HLA class II or other co-stimulatory molecules like CD80/CD86. When delivered in the inflammatory and hypoxic environment of STEMI, CSC upregulate the expression of HLA class I and PD-L1 molecules, induce HLA class II molecules (producing the anti-inflammatory IL10) and activate effector regulatory T CD4+ and natural killer (NK) cells, also downregulating conventional CD4+ and CD8+ (cytotoxic) immune response.(24)

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Collectively, this distinctive allogeneic immune behavior of CSC, inducing a PD-L1dependent T cell immunomodulatory response and directing the NK cytokine secretion toward an anti-inflammatory profile,(25) is likely to contribute to repair inflamed myocardium.(22) Secondly, preclinical experiments have indicated that allogeneic CSC are sustained in the infarcted heart for around 3-5 weeks,(22,26) being afterwards eliminated, presumably by apoptosis. This fact has raised the concept of an “autologous” tissue obtained from an exogenous product. In other words, allogeneic injected exogenous CSC may exert their effect for short - but long enough - periods of time by initiating the paracrine loop of growth factor production in endogenous CSC, which in turn may act as real autologous regenerative effectors in situ, and then being cleared from the heart by the host immune system.(27) This elimination of allogeneic CSC also dramatically reduces the possibility of tumor formation. Indeed, long-term survival of transplanted cells has not been thoroughly documented.(8) Furthermore, and in order to assure the safety of this approach, new immunological technologies are being developed that could improve the performance of allogeneic cell products. For instance, a tailored in vitro flow cytometry-based assay allows us to determine the antigen specificity and the binding strength of circulating DSA to allogeneic transplanted CSC. Thus, we may be able to minimize the risk of allosensitization and to provide an immune-educated choice of “off-the-shelf” CSC that might ultimately optimize the benefit of the therapy by extending their persistence in the heart.(23) Also, a meticulous monitoring of the immune behavior of allogeneic CSC has been and must be included in phase I/II clinical trials. Finally, very few studies have directly compared autologous and allogeneic stem cell sources. In the preclinical scenario, a recent meta-analysis of large animal models has demonstrated that both types of cells exert similar beneficial effects in terms of LVEF when compared to control animals, although most of the preclinical studies included in this meta-analysis used non-cardiac stem cells.(28) In the clinical arena, just one clinical trial has compared the safety and the efficacy of the transendocardial injection of autologous and allogeneic bone marrow-derived mesenchymal stem cells (BM-MSC) in 37 nonischemic dilated cardiomyopathy patients, the POSEIDON-DCM trial.(29) The authors of this trial concluded that allogeneic BM-MSC resulted in better 6-min walk test results and in lower major adverse cardiac event (MACE) rates when compared to autologous BM-MSC, with no differences in LVEF or quality of life between these two groups. Overall, both treatments showed better outcomes than those of control patients. Interestingly, just one patient treated with allogeneic cells developed elevated levels (>80%) of DSA.

ANIMAL STUDIES: PRECLINICAL EVIDENCE Preclinical trials have been conducted in small and large animal models with CSC and CDC, both from autologous and allogeneic sources. Since 2003,(15) small animal trials have provided the proof-of-concept for cardiac regeneration with stem cells of cardiac origin, most of them with immunodeficient rodents and human or syngeneic cells (Table 3). All those studies showed promising positive results in terms of cardiac function and infarct size and led to the initiation of studies with allogeneic cells.(27,30) Interestingly,

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and in the absence of immunosuppression, allogeneic CDC caused only slight transient lymphohistiocitic infiltrations at 3 weeks that had completely subsided at 6 months.(27) In the case of CS, pathology showed no immune rejection signs and inflammatory cytokines at day 7 were lower than those of controls, suggesting an immunomodulatory effect of the CS, which also showed higher engraftment rates as compared to CDC.(30) Although testing regenerative therapies in rodent models of human diseases is necessary, biological quantitative and qualitative differences between the two species make large animal models mandatory for predicting the results of a clinical trial.(20) Thus, CSC and CDC have also been transplanted into dogs(31) and pigs with promising results (Table 3).(32-37) Initial experiments with autologous CDC showed that the intramyocardial route was more effective than intracoronary delivery.(32,33) However, CS must be delivered via intramyocardial injections (either tranespicardial or transendcoardial). In another study with autologous C-kit+ CSC, the intracoronary route yielded impressive positive results when injected 3 months after STEMI.(35) Allogeneic cells have also been investigated in swine models. In a study with allogeneic CDC and CS delivered with the NOGA navigating platform, a mild local immune response with no signs of systemic immunogenicity or toxicity were observed, with higher retention rates for CS with this methodology.(34) Allogeneic CDC have been shown safe and efficient when injected through the coronary arteries at doses of 12.5 million cells at week 3(36) and ranging from 5 to 10 million cells in the acute phase of STEMI, causing no microvascular obstruction and decreasing infarct size and adverse remodeling in pigs.(38) In the first study,(36) the authors observed a slight marginally significant (compared to placebo) focal lymphoplasmacytic infiltration in the absence of foci of myocyte damage or circulating donor-specific antibodies. Another group has investigated the safety and the efficacy of allogeneic C-kit+ CSC in the acute phase of STEMI (at day 0 and 7), showing no safety issues, a reduction of the infarct size and increased capillary density with CSC, and suggesting that intracoronary injection at 7 days after STEMI produced better results in terms of left ventricular volumes than those of injections at 2 hours.(37)

RANDOMIZED TRIALS: CLINICAL EVIDENCE The aforementioned positive results with autologous and allogeneic CSC and CDC rapidly led to the initiation of two important phase I clinical trials involving cardiac progenitor cells. The first one, the SCIPIO trial,(39) investigated the intracoronary transplantation of 1 million autologous C-kit+ CSC into 33 patients (20 treated and 13 controls) with chronic ischemic cardiomyopathy, 113 days after coronary artery bypass grafting (CABG). Using magnetic resonance imaging (MRI) significant improvements in left ventricular ejection fraction (LVEF) were observed 4 and 12 months after cell delivery (+8.2% and +12.3%, respectively). Also, important reductions in infarct size were noted at the same time points in treated patients (-7.8 g and -9.8 g, respectively).(40) The second trial, the CADUCEUS trial,(41) was also a phase I clinical trial that involved 17 patients, 8 controls and 9 treated with intracoronary delivery of autologous CDC at escalating doses of 12.5-25 million cells, 1.5-3 months after STEMI. At the 6month follow-up, cell therapy was shown to be safe, with no cases of cardiac tumors or

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MACE, including deaths. Furthermore, promising results were observed in MRI studies at 6 months, showing in treated patients a significant decrease in infarct size (-7.7% versus +0.3% in controls) and a significant increase in viable mass (+13 g versus +0.9 g in controls), albeit no changes in global LVEF and volumes were seen. These beneficial effects were confirmed at 1-year follow-up (scar size reduction of -11.1% versus -2.2% in controls, and viable mass increase of +22.6 g versus +1.8 g in controls), consistent with cardiac regeneration.(42) Based on the aforementioned preclinical results,(43) the ALCADIA trial addressed the safety and efficacy of the intramyocardial delivery of 5x105 autologous CSC/kg together with a controlled release formulation of basic fibroblast growth factor (bFGF), in the form of a biodegradable gelatin hydrogel sheet containing 200μg of bFGF that was implanted on the epicardium, covering the injection sites areas. Six ischemic HF patients who underwent CABG procedures were included, with no control group. Results of the ALCADIA trial were presented during the American Heart Association Scientific Sessions in November 2012 (LBCT-20032), but the final results have not been published yet. The treatment appeared to be safe and showed positive results in terms of LVEF (+9-12%) and infarct size (-3.3% of the total ventricular volume) at 6 months, and the maximal aerobic exercise capacity increased by 4.5 ml/kg/min. Given the small sample size and the absence of a control group, no conclusions can be drawn regarding the efficacy of this approach. Currently, several clinical trials with allogeneic CSC (or “cardiac-like progenitors”) are underway, some of them already having completed recruitment. The results of these trials, although with small sample sizes, are eagerly awaited, since they will increase our knowledge on cardiac stem cell-based therapies. CAREMI (NCT02439398) is a phase I/II placebo-controlled clinical trial that is evaluating the safety, feasibility and the efficacy of intracoronary delivery of allogeneic CSC in 55 patients with large STEMI, left ventricular dysfunction and at high-risk of developing HF. It comprises a dose-escalating phase (6 patients) and a randomized phase (49 patients), both having been completed so far. The ALLSTAR trial (NCT01458405) is a similar phase I/II placebo-controlled clinical trial that has already enrolled 134 STEMI patients with the final aim of assessing the safety and the efficacy of allogeneic CDC, also delivered through the coronary arteries between 4 weeks and 12 months after STEMI.(44) Preliminary phase I results of the ALLSTAR trial were presented during the American College of Cardiology Scientific Sessions in March 2014, and showed safety encouraging results with no myocarditis, ventricular arrhythmias or MACE at 1-month after CDC infusion. These two trials have been designed with similar safety (MACE) and efficacy (scar size) endpoints. DYNAMIC (NCT02293603) is phase I placebo-controlled clinical trial has enrolled 42 patients with idiopathic dilated cardiomyopathy to receive allogeneic CDC via the intracoronary route, and the HOPE trial (NCT) is a phase I clinical trial that has enrolled 24 patients with cardiomyopathy and left ventricular scar due to Duchenne muscular dystrophy, which have been treated with allogeneic CDC and compared to standard-ofcare (control group). Finally, the ESCORT trial (NCT02057900) was initiated in 2013. It is a unique proofof-concept phase I clinical trial in which human embryonic stem cell-derived

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Isl1+/CD15+ progenitors - embedded in a fibrin patch - will be placed on the epicardium of 6 patients with severe ischemic left ventricular dysfunction at the time of CABG.

FUTURE DIRECTIONS Together with these novel stem cell types, other regenerative strategies are being investigated with the final aim of repairing the heart with cell products of cardiac lineage or with cardiomyogenic potential. These include the following: 1. Guided cardiopoiesis of autologous BM-MSC. Supported by preclinical studies, this next-generation approach for targeted cardiac repair entered the clinical arena with the C-CURE phase II clinical trial. In this trial, BM-MSC were treated with a cocktail of cardiogenic growth factors to guide them to the cardiomyocyte lineage. These cells were safely injected transendocardially into 48 ischemic HF patients showing an encouraging improvement of LVEF and a reduction of the left ventricular end-systolic volume (LVESV) in treated patients. Cell therapy also improved the 6-min walk distance and provided a superior composite clinical score encompassing cardiac parameters in tandem with New York Heart Association functional class, quality of life, physical performance, hospitalization, and event-free survival.(45) These findings led to the phase III CHART-1 clinical trial,(46) which has been recently published.(47) In this trial, 271 patients with severe ischemic HF were randomized to receive cardiopoietic BM-MSC transendocardial injections or sham procedure. The primary efficacy composite endpoint of all-cause mortality, worsening of HF, Minnesota Living with Heart Failure Questionnaire score, 6-min walk distance, LVESV and LVEF at 39 weeks was neutral. However, exploratory analyses suggested a benefit of cell treatment on the primary composite endpoint in patients with baseline LVEDV between 200 and 370 mL. Currently, the next phase of the CHART program is about to begin with the CHART-2 trial. 2. Combination therapies. Based on the rationale that MSC stimulate the cardioregenerative potential of C-kit+ CSC by regulating the cardiac stem cell niche, two preclinical studies in pigs have demonstrated that the combination of these cell types shows a synergistic effect, with better outcomes in terms of hemodynamic measurements and infarct size, increasing LVEF by twofold and cell engraftment by sevenfold when compared to the treatment with either cell type alone.(48,49) In this sense and in the clinical setting, the CONCERT-HF trial (NCT02501811) is an interesting phase II placebo-controlled clinical trial that is investigating the safety and the efficacy of the transendocardial injection of autologous C-kit+ CSC and BM-MSC, alone or in combination and compared to placebo, in 144 patients with ischemic HF. 3. Cell-free therapies. The identification of the growth factors and cytokines secreted by transplanted CSC that activate, expand and differentiate endogenous CSC should make possible the design of specific therapies based on those principal effector molecules. Should these components be identified and produced in large quantities, they could be available “off-the-shelf”, affordable in terms of production costs, easy to apply with percutaneous approaches and eventually advantageous for repeated administrations.(8)

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Initial experiences with cell-free products have been very recently developed by analyzing CPC-derived secretome and exosomes. Firstly, a detailed proteomic analysis of the secretome of CPC has shown that the injection of total conditioned medium (TCM) derived from human neonatal CPC into a rodent model of STEMI was more effective than TCM from human adult CPC or than CPC-derived exosomes in recovering cardiac function, stimulating neovascularization and reversing ventricular remodeling. This study also suggests that the heat-shock factor-1 is a powerful pathway that governs the composition of the CPC secretome.(50) Secondly, in a large animal model, CDC-secreted exosomes were injected by intracoronary or intramyocardial surgical delivery 30 minutes after reperfusion. Only intramyocardial injections decreased infarct size and preserved LVEF. In the second part of this study, exosomes delivered at week 4 post-infarction with the NOGA platform preserved LVEF and volumes while decreasing infarct size and increasing capillary density, changes that were absent in control animals.(51) Finally, proper growth factors may be used in combination with allogeneic CSC early after the ischemic insult to the myocardium, enhancing their beneficial effects, but this approach needs further research on the CSC secretome before a realistic application is possible.(8) 4. Repeated administration of CSC. Two preclinical studies in rodents have demonstrated that the repeated transplantation of CSC is more effective than a single administration. In the first one, three injections of allogeneic C-kit+ CSC 1 month after STEMI showed greater cumulative effects, which tripled LVEF improvements of one single injection.(52) Similar results were obtained with two sequential injections of allogeneic and syngeneic CDC 3 weeks after STEMI, in terms of LVEF and infarct size. Interestingly, repeated dosing of these cells did not induce immune rejection or humoral/cellular immune memory, suggesting that several dosing of allogeneic CDC is safe and effective even without immunosuppression.(53)

CONCLUSIONS Although controversies have arisen and many questions remain unsolved on the biology and physiology of cardiac progenitor cells, their seminal discovery has represented a paradigm shift in biology and a disruptive breakthrough in medicine, and has emerged as an outstanding opportunity for innovative regenerative therapies. Preclinical and clinical evidences have been so far highly promising for autologous cardiac regenerative cell products. Our increasing knowledge on the developmental origin and the biology of these cells, together with growing evidence on the immunological safety and efficacy of allogeneic cardiac progenitor cells and their secreted growth factors, will optimize the design and conduction of further clinical trials with larger patient numbers and longer follow-up times that will shed light on this fascinating new field of cardiovascular regenerative medicine.

Conflicts of interest: none to disclose

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ACKNOWLEDGEMENTS Figure 1 has been created with images that were kindly provided by Servier Medical Art (licensed under a Creative Commons Attribution 3.0 Unported License).

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Johnston PV, Sasano T, Mills K et al. Engraftment, differentiation, and functional benefits of autologous cardiosphere-derived cells in porcine ischemic cardiomyopathy. Circulation 2009;120:1075-83, 7 p following 1083. Lee ST, White AJ, Matsushita S et al. Intramyocardial injection of autologous cardiospheres or cardiosphere-derived cells preserves function and minimizes adverse ventricular remodeling in pigs with heart failure postmyocardial infarction. J Am Coll Cardiol 2011;57:455-65. Yee K, Malliaras K, Kanazawa H et al. Allogeneic cardiospheres delivered via percutaneous transendocardial injection increase viable myocardium, decrease scar size, and attenuate cardiac dilatation in porcine ischemic cardiomyopathy. PLoS One 2014;9:e113805. Bolli R, Tang XL, Sanganalmath SK et al. Intracoronary delivery of autologous cardiac stem cells improves cardiac function in a porcine model of chronic ischemic cardiomyopathy. Circulation 2013;128:122-31. Malliaras K, Smith RR, Kanazawa H et al. Validation of contrast-enhanced magnetic resonance imaging to monitor regenerative efficacy after cell therapy in a porcine model of convalescent myocardial infarction. Circulation 2013;128:2764-75. Crisostomo V, Baez-Diaz C, Maestre J et al. Delayed administration of allogeneic cardiac stem cell therapy for acute myocardial infarction could ameliorate adverse remodeling: experimental study in swine. J Transl Med 2015;13:156. Kanazawa H, Tseliou E, Malliaras K et al. Cellular postconditioning: allogeneic cardiosphere-derived cells reduce infarct size and attenuate microvascular obstruction when administered after reperfusion in pigs with acute myocardial infarction. Circ Heart Fail 2015;8:322-32. Bolli R, Chugh AR, D'Amario D et al. Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial. Lancet 2011;378:1847-57. Chugh AR, Beache GM, Loughran JH et al. Administration of cardiac stem cells in patients with ischemic cardiomyopathy: the SCIPIO trial: surgical aspects and interim analysis of myocardial function and viability by magnetic resonance. Circulation 2012;126:S54-64. Makkar RR, Smith RR, Cheng K et al. Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial. Lancet 2012;379:895-904. Malliaras K, Makkar RR, Smith RR et al. Intracoronary cardiosphere-derived cells after myocardial infarction: evidence of therapeutic regeneration in the final 1-year results of the CADUCEUS trial (CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction). J Am Coll Cardiol 2014;63:110-22. Takehara N, Tsutsumi Y, Tateishi K et al. Controlled delivery of basic fibroblast growth factor promotes human cardiosphere-derived cell engraftment to enhance cardiac repair for chronic myocardial infarction. J Am Coll Cardiol 2008;52:1858-65. Chakravarty T, Makkar RR, Ascheim DD et al. ALLogeneic Heart STem Cells to Achieve Myocardial Regeneration (ALLSTAR) Trial: Rationale and Design. Cell Transplant 2017;26:205-214.

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Bartunek J, Behfar A, Dolatabadi D et al. Cardiopoietic stem cell therapy in heart failure: the C-CURE (Cardiopoietic stem Cell therapy in heart failURE) multicenter randomized trial with lineage-specified biologics. J Am Coll Cardiol 2013;61:2329-38. Bartunek J, Davison B, Sherman W et al. Congestive Heart Failure Cardiopoietic Regenerative Therapy (CHART-1) trial design. Eur J Heart Fail 2016;18:160-8. Bartunek J, Terzic A, Davison BA et al. Cardiopoietic cell therapy for advanced ischemic heart failure: results at 39 weeks of the prospective, randomized, double blind, sham-controlled CHART-1 clinical trial. Eur Heart J 2016. Karantalis V, Suncion-Loescher VY, Bagno L et al. Synergistic Effects of Combined Cell Therapy for Chronic Ischemic Cardiomyopathy. J Am Coll Cardiol 2015;66:1990-9. Williams AR, Hatzistergos KE, Addicott B et al. Enhanced effect of combining human cardiac stem cells and bone marrow mesenchymal stem cells to reduce infarct size and to restore cardiac function after myocardial infarction. Circulation 2013;127:213-23. Sharma S, Mishra R, Bigham GE et al. A Deep Proteome Analysis Identifies the Complete Secretome as the Functional Unit of Human Cardiac Progenitor Cells. Circ Res 2017;120:816-834. Gallet R, Dawkins J, Valle J et al. Exosomes secreted by cardiosphere-derived cells reduce scarring, attenuate adverse remodelling, and improve function in acute and chronic porcine myocardial infarction. Eur Heart J 2017;38:201-211. Tokita Y, Tang XL, Li Q et al. Repeated Administrations of Cardiac Progenitor Cells Are Markedly More Effective Than a Single Administration: A New Paradigm in Cell Therapy. Circ Res 2016;119:635-51. Reich H, Tseliou E, de Couto G et al. Repeated transplantation of allogeneic cardiosphere-derived cells boosts therapeutic benefits without immune sensitization in a rat model of myocardial infarction. J Heart Lung Transplant 2016;35:1348-1357.

FIGURE CAPTIONS Figure 1. Cardiac stem cell transplantation techniques with their main characteristics. In human research only the intracoronary and the transendocardial routes have been investigated. HF: heart filaure; CSC: cardiac stem cells; CS: cardiospheres; CDC: cardiosphere-derived cells; STEMI: ST-segment elevation acute myocardial infarction.

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Figure 2 (also Graphical Abstract). Putative mechanisms of action of autologous and allogeneic cardiac stem cells, once delivered to the damaged myocardium. Note that the contribution of direct differentiation processes to myocardial repair is quantitatively less important (thin arrows) than that of paracrine effectors (thick arrows). CSC: cardiac stem cells.

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Stem cell type

First generation Skeletal myoblasts Bone marrow mononuclear fraction Specific bone marrow-derived cells Circulating peripheral blood cells Adipose-derived stem cells Mesenchymal stem cells

Epicardial stem cells Second generation Cardiac stem cells Cardiosphere-derived cells Embryonic stem cells Induced pluripotent stem cells Third generation Phenotypically modified stem cells Allogeneic stem cells

Pluripotent cell-derived stem cells

Tissue of origin / cell subtype

Phase of clinical research

Overall results (safety / efficacy)

Skeletal muscle Bone marrow CD34+ CD133+ Endothelial progenitor and bone marrow-derived cells Adipose tissue (stromal vascular fraction) Bone marrow

II III III II II

Arrhythmogenic issues/positive results Safe/divergent results Safe/positive results Safe/positive results Safe/divergent results

II

Safe/positive results

III

Adipose tissue Cord blood Dental pulp Developing heart

II II II NA

Safe/positive results in phase II (phase III ongoing) Safe/positive results Safe/divergent results Safe/divergent results NA

Cardiac biopsies Cardiac biopsies Blastocysts Adult somatic tissues

II II NA NA

Safe/positive results Safe/positive results NA NA

Cardiopoietic mesenchymal stem cells Mesenchymal stem cells

III

Cardiac stem cells Embryonic-derived Isl1+/CD15+ progenitor cells

II I

Safe/positive results in certain subpopulations Safe/positive results in phase II (phase III ongoing) Safe/positive results No results available yet

III

Table 1. “Road-map” of investigated stem cell types for ischemic heart disease (STsegment elevation acute myocardial infarction, ischemic heart failure and refractory angina). NA: no current clinical trials ongoing.

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Cardiac stem cell subpopulation C-kit

Sca-1

Side populations

Isl1

Cardiac mesangioblasts

cCFU-F

CDC

Epicardial cells

Positive markers

Negative markers

In vitro differentiation potential

In vivo differentiation potential

Clinical trials (phase)

Sca-1 Abcg2 CD105 CD166 GATA4 NKX2.5low MEF2C C-kitlow PDGFR- GATA4 NKX2.5low MEF2C CD31

Lin Isl1 CD34 CD45

Cardiomyocytes Smooth muscle cells Endothelial cells Fibroblasts

Cardiomyocytes (rare) Smooth muscle cells Endothelial cells Fibroblasts (rare)

SCIPIO (I) CONCERT-HF (II) ALCADIA (I) CAREMI (I)

CD8 CD34 CD45 FLK1

Cardiomyocytes (rare) Smooth muscle cells Endothelial cells

None

CD34 CD45 Sca-1 Abcg2 C-kit GATA4 NKX2.5

GATA4 NKX2.5 CD31

Cardiomyocytes Smooth muscle cells Endothelial cells Hepatocytes Skeletal muscle Neural cells Adipocytes Cardiomyocytes Smooth muscle cells Endothelial cells Glial and neural cells

Unknown (rare in the adult heart)

None

CD31 Sca-1 C-kit

Cardiomyocytes Smooth muscle cells Endothelial cells

ESCORT (I)

CD34 CD44 CD31 Sca-1 C-kit Sca-1 PDGFR- C-kitlow CD44 CD90 CD29 CD105 Sca-1 C-kitlow Abcg2 CD31 CD34 CD90 CD105 Connexin 43 TCF21 TBX18 WT1

CD45

Cardiomyocytes Smooth muscle cells Endothelial cells

Unknown (rare in the adult heart, only described in neonatal specimens) Smooth muscle cells

CD31 CD45 FLK1

Cardiomyocytes Smooth muscle cells Endothelial cells Adipocytes Chondrocytes Osteocytes

Unknown: - stromal, perivascular and adipogenic cells in homeostasis - myofibroblasts after injury

None

CD45 CD133

Cardiomyocytes Smooth muscle cells Endothelial cells

Cardiospheres do not exist in the adult heart

CADUCEUS (I) ALLSTAR(I/II) DYNAMIC (I) HOPE (I)

-

Smooth muscle cells Endothelial cells

Smooth muscle cells Fibroblasts

None

None

Table 2. Endogenous cardiac stem cell subpopulations. cCFU-F: cardiac resident colony-forming unit-fibroblast; CDC: cardiosphere-derived cells.

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Species

Author (ref)

Cell type

Rat

Beltrami15 Dawn Rota Cheng

C-kit+, LinC-kit+ C-kit+ CDC

Davis Carr

CDC CDC

Malliaras27

SCID mouse

Time / number of cells Day 0, 1x105 Day 0, 1x106 Day 20, 4x104 Day 0, 1x106

Delivery

Findings

IM IC IM IM

Allogeneic CDC

Day 0, 1x106 Day 0, 2x106; day 2, 4x106 Day 0, 2x106

IM IM (day 0); IV (day 2) IM

Tseliou30

Allogeneic CS

Day 0, 4x104

IM

Oh Messina18

Sca-1+ CS

Day 0, 1x106 Day 0, 10 CS per

IV IM

Improvement of cardiac function Preservation of cardiac function Myocardial salvage and IS reduction Improved cell retention and functional benefit with magnetic targeting of CDC Improved LVEF and IS reduction Improvement of cell retention and survival, and of cardiac function IS reduction and increased wall thickness, no immunological concerns Improved LVEF and IS reduction, no immunological concerns Cell homing with fusion to host cells Preserved wall thickness and fractional

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Smith Li Chimenti Oskouei

Welt31 Takehara43

hCDC Sca-1+ hCDC and hCS C-kit+ and BM-MSC CDC, BM-MSC, AD-MSC and BM-MNC hCDC (from different donors) C-kit+ hCDC/bFGF hydrogel

Johnston32 Lee33

CDC CDC and CS

Williams49

Li19

Cheng Dog Swine

animal Day 0, 1x105 Day 0, 5x105 Day 0, 1x105 Day 0, 36000; day 0, 1x106 Day 0, 5x105; day 0, 5x105; day 0, 5x105; day 0, 1x106 Day 0, 1x105

IM IM IM IM IM

IM

Week 6, 1x106 Week 4, 2x107

IM IM IC IM

hCSC and/or hMSC Allogeneic CDC and CS

Week 4, 24.5x106 Week 4, 0.5x106 CDC/site, 20 sites Day 14, 1x106; day 14, 200x106 Day 28, 5 to 100200 CS

Bolli35

C-kit+

Month 3, 5x105

IC

Malliaras36

Allogeneic CDC

Week 3, 12.5x106

IC

Crisostomo37

Allogeneic C-kit+

IC

Kanazawa38

Allogeneic CDC

Day 0 and 7, 25x106 Day 0; 5, 7.5 and 10x106

Yee34

IM NOGA

IC

shortening Improvement of LVEF Poor cell retention and no benefits Three mechanisms of action demonstrated C-kit+ better results in terms of LVEF improvement and IS reduction CDC showed the greatest potential for myogenic differentiation and angiogenesis

hCDC from advanced HF patients showed the greatest benefit on cardiac function Improvement of LVEDV and LVEF bFGF improved hCDC retention and cardiac function (immunosuppressed pigs) Improved LVEF and IS reduction CS better results than CDC in terms of hemodynamics and regional function Improvement of cardiac function (x2) and engraftment (x7) with a combined approach CS better engraftment than CDC, similar IS reduction and viable mass improvement for both, and no effects on cardiac function Improvement of global function and signs of cardiac and vascular regeneration Improved LVEF and IS without immunological concerns Improvement of LVEDV with no changes in LVEF, more important when injected at 7 days Improvements of IS, microvascular obstruction and adverse remodeling

Table 3. Preclinical trials in small and large animal models with cardiac progenitor cells. All cells of autologous origin except otherwise indicated (allogeneic or xenogeneic [h: human]). SCID: severe combined immunodeficient; CDC: cardiospherederived cells; CS: cardiospheres; BM-MSC: bone marrow-derived mesenchymal stem cells; AD-MSC: adipose-derived mesenchymal stem cells; BM-MNC: bone marrowderived mononuclear stem cells; FGF: fibroblast growth factor; MSC: mesenchymal stem cells; IM: intramyocardial (surgical); IC: intracoronary; IV: intravenous; NOGA: transendocardial injections with the NOGA platform; IS: infarct size; LVEF: left ventricular ejection fraction; LVEDV: left ventricular end-diastolic volume.

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