Polarizing Macrophage Functional Phenotype to Foster Cardiac Regeneration.

There is an increasing interest in understanding the connection between the immune and cardiovascular systems, which are highly integrated and communicate through finely regulated cross-talking mechanisms. Recent evidence has demonstrated that the immune system does indeed have a key role in the response to cardiac injury and in cardiac regeneration. Among the immune cells, macrophages appear to have a prominent role in this context, with different subtypes described so far that each have a specific influence on cardiac remodeling and repair. Similarly, there are significant differences in how the innate and adaptive immune systems affect the response to cardiac damage. Understanding all these mechanisms may have relevant clinical implications. Several studies have already demonstrated that stem cell-based therapies support myocardial repair. However, the exact role that cardiac macrophages and their modulation may have in this setting is still unclear. The current need to decipher the dual role of immunity in boosting both heart injury and repair is due, at least for a significant part, to unresolved questions related to the complexity of cardiac macrophage phenotypes. The aim of this review is to provide an overview on the role of the immune system, and of macrophages in particular, in the response to cardiac injury and to outline, through the modulation of the immune response, potential novel therapeutic strategies for cardiac regeneration.

Adult Multipotent Cardiac Progenitor-Derived Spheroids: A Reproducible Model of In Vitro Cardiomyocyte Commitment and Specification.

BACKGROUND: Three-dimensional cell culture systems hold great promise for bridging the gap between in vitro cell-based model systems and small animal models to study tissue biology and disease. Among 3D cell culture systems, stem-cell-derived spheroids have attracted significant interest as a strategy to better mimic in vivo conditions. Cardiac stem cell/progenitor (CSC)-derived spheroids (CSs) provide a relevant platform for cardiac regeneration. METHODS: We compared three different cell culture scaffold-free systems, (i) ultra-low attachment plates, (ii) hanging drops (both requiring a 2D/3D switch), and (iii) agarose micro-molds (entirely 3D), for CSC-derived CS formation and their cardiomyocyte commitment in vitro. RESULTS: The switch from a 2D to a 3D culture microenvironment per se guides cell plasticity and myogenic differentiation within CS and is necessary for robust cardiomyocyte differentiation. On the contrary, 2D monolayer CSC cultures show a significant reduced cardiomyocyte differentiation potential compared to 3D CS culture. Forced aggregation into spheroids using hanging drop improves CS myogenic differentiation when compared to ultra-low attachment plates. Performing CS formation and myogenic differentiation exclusively in 3D culture using agarose micro-molds maximizes the cardiomyocyte yield. CONCLUSIONS: A 3D culture system instructs CS myogenic differentiation, thus representing a valid model that can be used to study adult cardiac regenerative biology.

A Mouse Model of Dilated Cardiomyopathy Produced by Isoproterenol Acute Exposure Followed by 5-Fluorouracil Administration.

Appropriate dilated cardiomyopathy (DCM) animal models are highly desirable considering the pathophysiological and clinical heterogeneity of DCM. Genetically modified mice are the most widely and intensively utilized research animals for DCM. However, to translate discoveries from basic science into new and personalized medical applications, research in non-genetically based DCM models remains a key issue. Here, we characterized a mouse model of non-ischemic DCM induced by a stepwise pharmacologic regime of Isoproterenol (ISO) high dose bolus followed by a low dose systemic injection of the chemotherapy agent, 5-Fluorouracil (5-FU). C57BL/6J mice were injected with ISO and, 3 days after, were randomly assigned to saline or 5-FU. Echocardiography and a strain analysis show that ISO + 5FU in mice induces progressive left ventricular (LV) dilation and reduced systolic function, along with diastolic dysfunction and a persistent global cardiac contractility depression through 56 days. While mice treated with ISO alone recover anatomically and functionally, ISO + 5-FU causes persistent cardiomyocyte death, ensuing in cardiomyocyte hypertrophy through 56 days. ISO + 5-FU-dependent damage was accompanied by significant myocardial disarray and fibrosis along with exaggerated oxidative stress, tissue inflammation and premature cell senescence accumulation. In conclusions, a combination of ISO + 5FU produces anatomical, histological and functional cardiac alterations typical of DCM, representing a widely available, affordable, and reproducible mouse model of this cardiomyopathy.

Development of Cyclic Peptides Targeting the Epidermal Growth Factor Receptor in Mesenchymal Triple-Negative Breast Cancer Subtype.

Triple-negative breast cancer (TNBC) is an aggressive malignancy characterized by the lack of expression of estrogen and progesterone receptors and amplification of human epidermal growth factor receptor 2 (HER2). Being the Epidermal Growth Factor Receptor (EGFR) highly expressed in mesenchymal TNBC and correlated with aggressive growth behavior, it represents an ideal target for anticancer drugs. Here, we have applied the phage display for selecting two highly specific peptide ligands for targeting the EGFR overexpressed in MDA-MB-231 cells, a human TNBC cell line. Molecular docking predicted the peptide-binding affinities and sites in the extracellular domain of EGFR. The binding of the FITC-conjugated peptides to human and murine TNBC cells was validated by flow cytometry. Confocal microscopy confirmed the peptide binding specificity to EGFR-positive MDA-MB-231 tumor xenograft tissues and their co-localization with the membrane EGFR. Further, the peptide stimulation did not affect the cell cycle of TNBC cells, which is of interest for their utility for tumor targeting. Our data indicate that these novel peptides are highly specific ligands for the EGFR overexpressed in TNBC cells, and thus they could be used in conjugation with nanoparticles for tumor-targeted delivery of anticancer drugs.

Streptozotocin-Induced Type 1 and 2 Diabetes Mellitus Mouse Models Show Different Functional, Cellular and Molecular Patterns of Diabetic Cardiomyopathy.

The main cause of morbidity and mortality in diabetes mellitus (DM) is cardiovascular complications. Diabetic cardiomyopathy (DCM) remains incompletely understood. Animal models have been crucial in exploring DCM pathophysiology while identifying potential therapeutic targets. Streptozotocin (STZ) has been widely used to produce experimental models of both type 1 and type 2 DM (T1DM and T2DM). Here, we compared these two models for their effects on cardiac structure, function and transcriptome. Different doses of STZ and diet chows were used to generate T1DM and T2DM in C57BL/6J mice. Normal euglycemic and nonobese sex- and age-matched mice served as controls (CTRL). Immunohistochemistry, RT-PCR and RNA-seq were employed to compare hearts from the three animal groups. STZ-induced T1DM and T2DM affected left ventricular function and myocardial performance differently. T1DM displayed exaggerated apoptotic cardiomyocyte (CM) death and reactive hypertrophy and fibrosis, along with increased cardiac oxidative stress, CM DNA damage and senescence, when compared to T2DM in mice. T1DM and T2DM affected the whole cardiac transcriptome differently. In conclusion, the STZ-induced T1DM and T2DM mouse models showed significant differences in cardiac remodeling, function and the whole transcriptome. These differences could be of key relevance when choosing an animal model to study specific features of DCM.

Pharmacological clearance of senescent cells improves cardiac remodeling and function after myocardial infarction in female aged mice.

Cardiovascular diseases (CVD) are predominantly an aging disease. Important sex-specific differences exist and the mechanism(s) by which this sex-by-age interaction influences CVD development and progression remains elusive. Accordingly, it is still unknown whether cell senescence, a main feature of cardiac male aging, is a significant feature also of the female aged mouse heart and whether senolytics, senescence-clearing compounds, promote myocardial repair and regeneration after myocardial infarction (MI) in aged female mice. To this aim, the combination of two senolytics, dasatinib and quercetin (D+Q) or just their vehicle was administered to 22-24 months old C57BL/6 female mice after MI. D+Q improved global left ventricle function and myocardial performance after MI whereby female cardiac aging is characterized by accumulation of cardiac senescent cells that are further increased by MI. Despite their terminal differentiation nature, also cardiomyocytes acquire a senescent phenotype with age in females. D+Q removed senescent cardiac non-myocyte and myocyte cells ameliorating cardiac remodeling and regeneration. Senolytics removed aged dysfunctional cardiac stem/progenitor cells (CSCs), relieving healthy CSCs with normal proliferative and cardiomyogenic differentiation potential. In conclusions, cardiac senescent cells accumulate in the aged female hearts. Removing senescent cells is a key therapeutic target for efficient repair of the aged female heart.

The negative regulation of gene expression by microRNAs as key driver of inducers and repressors of cardiomyocyte differentiation.

Cardiac muscle damage-induced loss of cardiomyocytes (CMs) and dysfunction of the remaining ones leads to heart failure, which nowadays is the number one killer worldwide. Therapies fostering effective cardiac regeneration are the holy grail of cardiovascular research to stop the heart failure epidemic. The main goal of most myocardial regeneration protocols is the generation of new functional CMs through the differentiation of endogenous or exogenous cardiomyogenic cells. Understanding the cellular and molecular basis of cardiomyocyte commitment, specification, differentiation and maturation is needed to devise innovative approaches to replace the CMs lost after injury in the adult heart. The transcriptional regulation of CM differentiation is a highly conserved process that require sequential activation and/or repression of different genetic programs. Therefore, CM differentiation and specification have been depicted as a step-wise specific chemical and mechanical stimuli inducing complete myogenic commitment and cell-cycle exit. Yet, the demonstration that some microRNAs are sufficient to direct ESC differentiation into CMs and that four specific miRNAs reprogram fibroblasts into CMs show that CM differentiation must also involve negative regulatory instructions. Here, we review the mechanisms of CM differentiation during development and from regenerative stem cells with a focus on the involvement of microRNAs in the process, putting in perspective their negative gene regulation as a main modifier of effective CM regeneration in the adult heart.

Long-term culture of patient-derived cardiac organoids recapitulated Duchenne muscular dystrophy cardiomyopathy and disease progression.

Duchenne Muscular Dystrophy (DMD) is an X-linked neuromuscular disease which to date is incurable. The major cause of death is dilated cardiomyopathy however, its pathogenesis is unclear as existing cellular and animal models do not fully recapitulate the human disease phenotypes. In this study, we generated cardiac organoids from patient-derived induced pluripotent stem cells (DMD-COs) and isogenic-corrected controls (DMD-Iso-COs) and studied if DMD-related cardiomyopathy and disease progression occur in the organoids upon long-term culture (up to 93 days). Histological analysis showed that DMD-COs lack initial proliferative capacity, displayed a progressive loss of sarcoglycan localization and high stress in endoplasmic reticulum. Additionally, cardiomyocyte deterioration, fibrosis and aberrant adipogenesis were observed in DMD-COs over time. RNA sequencing analysis confirmed a distinct transcriptomic profile in DMD-COs which was associated with functional enrichment in hypertrophy/dilated cardiomyopathy, arrhythmia, adipogenesis and fibrosis pathways. Moreover, five miRNAs were identified to be crucial in this dysregulated gene network. In conclusion, we generated patient-derived cardiac organoid model that displayed DMD-related cardiomyopathy and disease progression phenotypes in long-term culture. We envision the feasibility to develop a more complex, realistic and reliable in vitro 3D human cardiac-mimics to study DMD-related cardiomyopathies.

Myocardial regeneration protocols towards the routine clinical scenario: An unseemly path from bench to bedside.

Heart failure secondary to cardiomyocyte loss and/or dysfunction is the number one killer worldwide. The field of myocardial regeneration with its far-reaching primary goal of cardiac remuscularization and its hard-to-accomplish translation from bench to bedside, has been filled with ups and downs, steps forward and steps backward, controversies galore and, unfortunately, scientific scandals. Despite the present morass in which cardiac remuscularization is stuck in, the search for clinically effective regenerative approaches remains keenly active. Starting with a concise overview of the still highly debated regenerative capacity of the adult mammalian heart, we focus on the main interventions, that have reached or are close to clinical use, critically discussing key findings, successes, and failures. Finally, some promising and innovative approaches for myocardial repair/regeneration still at the pre-clinical stage are discussed to offer a holistic view on the future of myocardial repair/regeneration for the prevention/management of heart failure in the clinical scenario. Funding: This research was funded by Grants from the Ministry of University and Research PRIN2015 2015ZTT5KB_004; PRIN2017NKB2N4_005; PON-AIM - 1829805-2.

Unraveling and Targeting Myocardial Regeneration Deficit in Diabetes.

Cardiomyopathy is a common complication in diabetic patients. Ventricular dysfunction without coronary atherosclerosis and hypertension is driven by hyperglycemia, hyperinsulinemia and impaired insulin signaling. Cardiomyocyte death, hypertrophy, fibrosis, and cell signaling defects underlie cardiomyopathy. Notably, detrimental effects of the diabetic milieu are not limited to cardiomyocytes and vascular cells. The diabetic heart acquires a senescent phenotype and also suffers from altered cellular homeostasis and the insufficient replacement of dying cells. Chronic inflammation, oxidative stress, and metabolic dysregulation damage the population of endogenous cardiac stem cells, which contribute to myocardial cell turnover and repair after injury. Therefore, deficient myocardial repair and the progressive senescence and dysfunction of stem cells in the diabetic heart can represent potential therapeutic targets. While our knowledge of the effects of diabetes on stem cells is growing, several strategies to preserve, activate or restore cardiac stem cell compartments await to be tested in diabetic cardiomyopathy.

A novel phage display based platform for exosome diversity characterization.

We present an innovative approach allowing the identification, isolation, and molecular characterization of disease-related exosomes based on their different antigenic reactivities. The designed strategy could be immediately translated into any disease in which exosomes are involved. The identification of specific markers and their subsequent association with exosome subtypes, together with the possibility to engineer target-guided exosome-like particles, could represent the key for the effective adoption of exosomes in clinical practice.

Diabetes-Induced Cellular Senescence and Senescence-Associated Secretory Phenotype Impair Cardiac Regeneration and Function Independently of Age.

Diabetes mellitus (DM) affects the biology of multipotent cardiac stem/progenitor cells (CSCs) and adult myocardial regeneration. We assessed the hypothesis that senescence and senescence-associated secretory phenotype (SASP) are main mechanisms of cardiac degenerative defect in DM. Accordingly, we tested whether ablation of senescent CSCs would rescue the cardiac regenerative/reparative defect imposed by DM. We obtained cardiac tissue from nonaged (50- to 64-year-old) patients with type 2 diabetes mellitus (T2DM) and without DM (NDM) and postinfarct cardiomyopathy undergoing cardiac surgery. A higher reactive oxygen species production in T2DM was associated with an increased number of senescent/dysfunctional T2DM-human CSCs (hCSCs) with reduced proliferation, clonogenesis/spherogenesis, and myogenic differentiation versus NDM-hCSCs in vitro. T2DM-hCSCs showed a defined pathologic SASP. A combination of two senolytics, dasatinib (D) and quercetin (Q), cleared senescent T2DM-hCSCs in vitro, restoring their expansion and myogenic differentiation capacities. In a T2DM model in young mice, diabetic status per se (independently of ischemia and age) caused CSC senescence coupled with myocardial pathologic remodeling and cardiac dysfunction. D + Q treatment efficiently eliminated senescent cells, rescuing CSC function, which resulted in functional myocardial repair/regeneration, improving cardiac function in murine DM. In conclusion, DM hampers CSC biology, inhibiting CSCs' regenerative potential through the induction of cellular senescence and SASP independently from aging. Senolytics clear senescence, abrogating the SASP and restoring a fully proliferative/differentiation-competent hCSC pool in T2DM with normalization of cardiac function.

Lyotropic Liquid Crystals: A Biocompatible and Safe Material for Local Cardiac Application.

The regeneration of cardiac tissue is a multidisciplinary research field aiming to improve the health condition of the post-heart attack patient. Indeed, myocardial tissue has a poor ability to self-regenerate after severe damage. The scientific efforts focused on the research of a biomaterial able to adapt to heart tissue, thus guaranteeing the in situ release of active substances or growth promoters. Many types of hydrogels were proposed for this purpose, showing several limitations. The aim of this study was to suggest a new usage for glyceryl monooleate-based lyotropic liquid crystals (LLCs) as a biocompatible and inert material for a myocardial application. The main advantages of LLCs are mainly related to their easy in situ injection as lamellar phase and their instant in situ transition in the cubic phase. In vivo studies proved the biocompatibility and the inertia of LLCs after their application on the myocardial tissue of mice. In detail, the cardiac activity was monitored through 28 days, and no significant alterations were recorded in the heart anatomy and functionality. Moreover, gross anatomy showed the ability of LLCs to be bio-degraded in a suitable time frame. Overall, these results permitted us to suppose a potential use of LLCs as materials for cardiac drug delivery.

From Spheroids to Organoids: The Next Generation of Model Systems of Human Cardiac Regeneration in a Dish.

Organoids are tiny, self-organized, three-dimensional tissue cultures that are derived from the differentiation of stem cells. The growing interest in the use of organoids arises from their ability to mimic the biology and physiology of specific tissue structures in vitro. Organoids indeed represent promising systems for the in vitro modeling of tissue morphogenesis and organogenesis, regenerative medicine and tissue engineering, drug therapy testing, toxicology screening, and disease modeling. Although 2D cell cultures have been used for more than 50 years, even for their simplicity and low-cost maintenance, recent years have witnessed a steep rise in the availability of organoid model systems. Exploiting the ability of cells to re-aggregate and reconstruct the original architecture of an organ makes it possible to overcome many limitations of 2D cell culture systems. In vitro replication of the cellular micro-environment of a specific tissue leads to reproducing the molecular, biochemical, and biomechanical mechanisms that directly influence cell behavior and fate within that specific tissue. Lineage-specific self-organizing organoids have now been generated for many organs. Currently, growing cardiac organoid (cardioids) from pluripotent stem cells and cardiac stem/progenitor cells remains an open challenge due to the complexity of the spreading, differentiation, and migration of cardiac muscle and vascular layers. Here, we summarize the evolution of biological model systems from the generation of 2D spheroids to 3D organoids by focusing on the generation of cardioids based on the currently available laboratory technologies and outline their high potential for cardiovascular research.

In vitro CSC-derived cardiomyocytes exhibit the typical microRNA-mRNA blueprint of endogenous cardiomyocytes.

miRNAs modulate cardiomyocyte specification by targeting mRNAs of cell cycle regulators and acting in cardiac muscle lineage gene regulatory loops. It is unknown if or to-what-extent these miRNA/mRNA networks are operative during cardiomyocyte differentiation of adult cardiac stem/progenitor cells (CSCs). Clonally-derived mouse CSCs differentiated into contracting cardiomyocytes in vitro (iCMs). Comparison of "CSCs vs. iCMs" mRNome and microRNome showed a balanced up-regulation of CM-related mRNAs together with a down-regulation of cell cycle and DNA replication mRNAs. The down-regulation of cell cycle genes and the up-regulation of the mature myofilament genes in iCMs reached intermediate levels between those of fetal and neonatal cardiomyocytes. Cardiomyo-miRs were up-regulated in iCMs. The specific networks of miRNA/mRNAs operative in iCMs closely resembled those of adult CMs (aCMs). miR-1 and miR-499 enhanced myogenic commitment toward terminal differentiation of iCMs. In conclusions, CSC specification/differentiation into contracting iCMs follows known cardiomyo-MiR-dependent developmental cardiomyocyte differentiation trajectories and iCMs transcriptome/miRNome resembles that of CMs.

Physical Exercise and Cardiac Repair: The Potential Role of Nitric Oxide in Boosting Stem Cell Regenerative Biology.

Over the years strong evidence has been accumulated showing that aerobic physical exercise exerts beneficial effects on the prevention and reduction of cardiovascular risk. Exercise in healthy subjects fosters physiological remodeling of the adult heart. Concurrently, physical training can significantly slow-down or even reverse the maladaptive pathologic cardiac remodeling in cardiac diseases, improving heart function. The underlying cellular and molecular mechanisms of the beneficial effects of physical exercise on the heart are still a subject of intensive study. Aerobic activity increases cardiovascular nitric oxide (NO) released mainly through nitric oxidase synthase 3 activity, promoting endothelium-dependent vasodilation, reducing vascular resistance, and lowering blood pressure. On the reverse, an imbalance between increasing free radical production and decreased NO generation characterizes pathologic remodeling, which has been termed the "nitroso-redox imbalance". Besides these classical evidence on the role of NO in cardiac physiology and pathology, accumulating data show that NO regulate different aspects of stem cell biology, including survival, proliferation, migration, differentiation, and secretion of pro-regenerative factors. Concurrently, it has been shown that physical exercise generates physiological remodeling while antagonizes pathologic remodeling also by fostering cardiac regeneration, including new cardiomyocyte formation. This review is therefore focused on the possible link between physical exercise, NO, and stem cell biology in the cardiac regenerative/reparative response to physiological or pathological load. Cellular and molecular mechanisms that generate an exercise-induced cardioprotective phenotype are discussed in regards with myocardial repair and regeneration. Aerobic training can benefit cells implicated in cardiovascular homeostasis and response to damage by NO-mediated pathways that protect stem cells in the hostile environment, enhance their activation and differentiation and, in turn, translate to more efficient myocardial tissue regeneration. Moreover, stem cell preconditioning by and/or local potentiation of NO signaling can be envisioned as promising approaches to improve the post-transplantation stem cell survival and the efficacy of cardiac stem cell therapy.

WIND (Workflow for pIRNAs aNd beyonD): a strategy for in-depth analysis of small RNA-seq data.

Current bioinformatics workflows for PIWI-interacting RNA (piRNA) analysis focus primarily on germline-derived piRNAs and piRNA-clusters. Frequently, they suffer from outdated piRNA databases, questionable quantification methods, and lack of reproducibility. Often, pipelines specific to miRNA analysis are used for the piRNA research in silico. Furthermore, the absence of a well-established database for piRNA annotation, as for miRNA, leads to uniformity issues between studies and generates confusion for data analysts and biologists. For these reasons, we have developed WIND ( Workflow for p IRNAs a Nd beyon D), a bioinformatics workflow that addresses the crucial issue of piRNA annotation, thereby allowing a reliable analysis of small RNA sequencing data for the identification of piRNAs and other small non-coding RNAs (sncRNAs) that in the past have been incorrectly classified as piRNAs. WIND allows the creation of a comprehensive annotation track of sncRNAs combining information available in RNAcentral, with piRNA sequences from piRNABank, the first database dedicated to piRNA annotation. WIND was built with Docker containers for reproducibility and integrates widely used bioinformatics tools for sequence alignment and quantification. In addition, it includes Bioconductor packages for exploratory data and differential expression analysis. Moreover, WIND implements a "dual" approach for the evaluation of sncRNAs expression level quantifying the aligned reads to the annotated genome and carrying out an alignment-free transcript quantification using reads mapped to the transcriptome. Therefore, a broader range of piRNAs can be annotated, improving their quantification and easing the subsequent downstream analysis. WIND performance has been tested with several small RNA-seq datasets, demonstrating how our approach can be a useful and comprehensive resource to analyse piRNAs and other classes of sncRNAs.

Statins Stimulate New Myocyte Formation After Myocardial Infarction by Activating Growth and Differentiation of the Endogenous Cardiac Stem Cells.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert pleiotropic effects on cardiac cell biology which are not yet fully understood. Here we tested whether statin treatment affects resident endogenous cardiac stem/progenitor cell (CSC) activation in vitro and in vivo after myocardial infarction (MI). Statins (Rosuvastatin, Simvastatin and Pravastatin) significantly increased CSC expansion in vitro as measured by both BrdU incorporation and cell growth curve. Additionally, statins increased CSC clonal expansion and cardiosphere formation. The effects of statins on CSC growth and differentiation depended on Akt phosphorylation. Twenty-eight days after myocardial infarction by permanent coronary ligation in rats, the number of endogenous CSCs in the infarct border zone was significantly increased by Rosuvastatin-treatment as compared to untreated controls. Additionally, commitment of the activated CSCs into the myogenic lineage (c-kitpos/Gata4pos CSCs) was increased by Rosuvastatin administration. Accordingly, Rosuvastatin fostered new cardiomyocyte formation after MI. Finally, Rosuvastatin treatment reversed the cardiomyogenic defects of CSCs in c-kit haploinsufficient mice, increasing new cardiomyocyte formation by endogenous CSCs in these mice after myocardial infarction. In summary, statins, by sustaining Akt activation, foster CSC growth and differentiation in vitro and in vivo. The activation and differentiation of the endogenous CSC pool and consequent new myocyte formation by statins improve myocardial remodeling after coronary occlusion in rodents. Similar effects might contribute to the beneficial effects of statins on human cardiovascular diseases.

Unravelling the Biology of Adult Cardiac Stem Cell-Derived Exosomes to Foster Endogenous Cardiac Regeneration and Repair.

Cardiac remuscularization has been the stated goal of the field of regenerative cardiology since its inception. Along with the refreshment of lost and dysfunctional cardiac muscle cells, the field of cell therapy has expanded in scope encompassing also the potential of the injected cells as cardioprotective and cardio-reparative agents for cardiovascular diseases. The latter has been the result of the findings that cell therapies so far tested in clinical trials exert their beneficial effects through paracrine mechanisms acting on the endogenous myocardial reparative/regenerative potential. The endogenous regenerative potential of the adult heart is still highly debated. While it has been widely accepted that adult cardiomyocytes (CMs) are renewed throughout life either in response to wear and tear and after injury, the rate and origin of this phenomenon are yet to be clarified. The adult heart harbors resident cardiac/stem progenitor cells (CSCs/CPCs), whose discovery and characterization were initially sufficient to explain CM renewal in response to physiological and pathological stresses, when also considering that adult CMs are terminally differentiated cells. The role of CSCs in CM formation in the adult heart has been however questioned by some recent genetic fate map studies, which have been proved to have serious limitations. Nevertheless, uncontested evidence shows that clonal CSCs are effective transplantable regenerative agents either for their direct myogenic differentiation and for their paracrine effects in the allogeneic setting. In particular, the paracrine potential of CSCs has been the focus of the recent investigation, whereby CSC-derived exosomes appear to harbor relevant regenerative and reparative signals underlying the beneficial effects of CSC transplantation. This review focuses on recent advances in our knowledge about the biological role of exosomes in heart tissue homeostasis and repair with the idea to use them as tools for new therapeutic biotechnologies for "cell-less" effective cardiac regeneration approaches.