Search

Background: We previously demonstrated that the human amniotic fluid (hAF) from II trimester of gestation is a feasible source of stromal progenitors (human amniotic fluid stem cells, hAFSC), with significant paracrine potential for regenerative medicine. Extracellular vesicles (EVs) separated and concentrated from hAFSC secretome can deliver pro-survival, proliferative, anti-fibrotic and cardioprotective effects in preclinical models of skeletal and cardiac muscle injury. While hAFSC-EVs isolation can be significantly influenced by in vitro cell culture, here we profiled EVs directly concentrated from hAF as an alternative option and investigated their paracrine potential against oxidative stress. Methods: II trimester hAF samples were obtained as leftover material from prenatal diagnostic amniocentesis following written informed consent. EVs were separated by size exclusion chromatography and concentrated by ultracentrifugation. hAF-EVs were assessed by nanoparticle tracking analysis, transmission electron microscopy, Western Blot, and flow cytometry; their metabolic activity was evaluated by oximetric and luminometric analyses and their cargo profiled by proteomics and RNA sequencing. hAF-EV paracrine potential was tested in preclinical in vitro models of oxidative stress and dysfunction on murine C2C12 cells and on 3D human cardiac microtissue. Results: Our protocol resulted in a yield of 6.31 ± 0.98 × 109 EVs particles per hAF milliliter showing round cup-shaped morphology and 209.63 ± 6.10 nm average size, with relevant expression of CD81, CD63 and CD9 tetraspanin markers. hAF-EVs were enriched in CD133/1, CD326, CD24, CD29, and SSEA4 and able to produce ATP by oxygen consumption. While oxidative stress significantly reduced C2C12 survival, hAF-EV priming resulted in significant rescue of cell viability, with notable recovery of ATP synthesis and concomitant reduction of cell damage and lipid peroxidation activity. 3D human cardiac microtissues treated with hAF-EVs and experiencing H2O2 stress and TGFβ stimulation showed improved survival with a remarkable decrease in the onset of fibrosis. Conclusions: Our results suggest that leftover samples of II trimester human amniotic fluid can represent a feasible source of EVs to counteract oxidative damage on target cells, thus offering a novel candidate therapeutic option to counteract skeletal and cardiac muscle injury.

Abstract Extracellular vesicles (EVs), crucial mediators of cell‐to‐cell communication, hold significant diagnostic potential due to their ability to concentrate protein biomarkers in bodily fluids. However, challenges in isolating EVs from biological specimens hinder their widespread use. The preferred strategy involves direct analysis, integrating isolation and analysis solutions, with immunoaffinity methods currently dominating. Yet, the heterogeneous nature of EVs poses challenges, as proposed markers may not be as universally present as thought, raising concerns about biomarker screening reliability. This issue extends to EV‐mimics, where conventional methods may lack applicability. Addressing these challenges, the study reports on Membrane Sensing Peptides (MSP) as pan‐vesicular affinity ligands for both EVs and their non‐canonical analogs, streamlining capture and phenotyping through Single Molecule Array (SiMoA). MSP ligands enable direct analysis of circulating EVs, eliminating the need for prior isolation. Demonstrating clinical translation, MSP technology detects an EV‐associated epitope signature in serum and plasma, distinguishing myocardial infarction from stable angina. Additionally, MSP allow analysis of tetraspanin‐lacking Red Blood Cell‐derived EVs, overcoming limitations associated with antibody‐based methods. Overall, the work underlines the value of MSP as complementary tools to antibodies, advancing EV analysis for clinical diagnostics and beyond, and marking the first‐ever peptide‐based application in SiMoA technology.

Cardiovascular disease represents the foremost cause of mortality and morbidity worldwide, with a steadily increasing incidence due to the growth of the ageing population. Cardiac dysfunction leading to heart failure may arise from acute myocardial infarction (MI) as well as inflammatory- and cancer-related chronic cardiomyopathy. Despite pharmacological progress, effective cardiac repair represents an unmet clinical need, with heart transplantation being the only option for end-stage heart failure. The functional profiling of the biological activity of extracellular vesicles (EVs) has recently attracted increasing interest in the field of translational research for cardiac regenerative medicine. The cardioprotective and cardioactive potential of human progenitor stem/cell-derived EVs has been reported in several preclinical studies, and EVs have been suggested as promising paracrine therapy candidates for future clinical translation. Nevertheless, some compelling aspects must be properly addressed, including optimizing delivery strategies to meet patient needs and enhancing targeting specificity to the cardiac tissue. Therefore, in this review, we will discuss the most relevant aspects of the therapeutic potential of EVs released by human progenitors for cardiovascular disease, with a specific focus on the strategies that have been recently implemented to improve myocardial targeting and administration routes.

Abstract Background Exosomes (EXOs), tiny extracellular vesicles that facilitate cell–cell communication, are being explored as a heart failure treatment, although the features of the cell source restrict their efficacy. Fibroblasts the most prevalent non-myocyte heart cells, release poor cardioprotective EXOs. A noninvasive method for manufacturing fibroblast-derived exosomes (F-EXOs) that target cardiomyocytes and slow cardiac remodeling is expected. As a cardioprotective isothiocyanate, sulforaphane (SFN)-induced F-EXOs (SFN-F-EXOs) should recapitulate its anti-remodeling properties. Methods Exosomes from low-dose SFN (3 μM/7 days)-treated NIH/3T3 murine cells were examined for number, size, and protein composition. Fluorescence microscopy, RT-qPCR, and western blot assessed cell size, oxidative stress, AcH4 levels, hypertrophic gene expression, and caspase-3 activation in angiotensin II (AngII)-stressed HL-1 murine cardiomyocytes 12 h-treated with various EXOs. The uptake of fluorescently-labeled EXOs was also measured in cardiomyocytes. The cardiac function of infarcted male Wistar rats intramyocardially injected with different EXOs (1·1012) was examined by echocardiography. Left ventricular infarct size, hypertrophy, and capillary density were measured. Results Sustained treatment of NIH/3T3 with non-toxic SFN concentration significantly enhances the release of CD81 + EXOs rich in TSG101 (Tumor susceptibility gene 101) and Hsp70 (Heat Shock Protein 70), and containing maspin, an endogenous histone deacetylase 1 inhibitor. SFN-F-EXOs counteract angiotensin II (AngII)-induced hypertrophy and apoptosis in murine HL-1 cardiomyocytes enhancing SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a) levels more effectively than F-EXOs. In stressed cardiomyocytes, SFN-F-EXOs boost AcH4 levels by 30% (p

An in silico hybrid model to derive electrical biomarkers to be used as inputs for an unsupervised learning algorithm to classify differentiated hiPSC-derived cardiomyocytes from a mixed population of ventricular- and atrial-like cells.

Abstract The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)‐based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples. The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo‐electron microscopy (cryo‐EM); the assignment of specific nanomechanical readout to each object enables the simultaneous discrimination of co‐isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterised by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo‐EM. The described approach is label‐free, single‐step and relatively quick to perform. Importantly, it can be used to analyse samples which prove very challenging to assess with several established techniques due to ensemble‐averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma‐ and serum‐derived preparations.

Abstract Despite their clinical potential, Extracellular Vesicles (EVs) struggle to take the scene as a preeminent source of biomarkers in liquid biopsy. Limitations in the use of EVs origin from their inherent complexity and heterogeneity and from the sensitivity demand in detecting low to very low abundant disease‐specific sub‐populations. Such need can be met by digital detection, namely capable to reach the single‐molecule sensitivity. Here we set to compare, side by side, two digital detection platforms that have recently gained increasing importance in the field of EVs. The platforms, both commercially available, are based on the principles of the Single Particle Interferometric Reflectance Imaging Sensing (SP‐IRIS) and the Single Molecule Array technology (SiMoA) respectively. Sensitivity in immune‐phenotyping of a well characterized EV sample is reported, discussing possible applicative implications and rationales for alternative or complementary use of the two platforms in biomarker discovery or validation.

Cardiomyocyte renewal represents an unmet clinical need for cardiac regeneration. Stem cell paracrine therapy has attracted increasing attention to resurge rescue mechanisms within the heart. We previously characterized the paracrine effects that human amniotic fluid–derived stem cells (hAFSC) can exert to provide cardioprotection and enhance cardiac repair in preclinical models of myocardial ischemia and cardiotoxicity. Here, we analyze whether hAFSC secretome formulations, namely, hAFSC conditioned medium (hAFSC-CM) over extracellular vesicles (hAFSC-EVs) separated from it, can induce cardiomyocyte renewal. c-KIT+ hAFSC were obtained by leftover samples of II trimester prenatal amniocentesis (fetal hAFSC) and from clinical waste III trimester amniotic fluid during scheduled C-section procedures (perinatal hAFSC). hAFSC were primed under 1% O2 to enrich hAFSC-CM and EVs with cardioactive factors. Neonatal mouse ventricular cardiomyocytes (mNVCM) were isolated from cardiac tissue of R26pFUCCI2 mice with cell cycle fluorescent tagging by mutually exclusive nuclear signal. mNVCM were stimulated by fetal versus perinatal hAFSC-CM and hAFSC-EVs to identify the most promising formulation for in vivo assessment in a R26pFUCCI2 neonatal mouse model of myocardial infarction (MI) via intraperitoneal delivery. While the perinatal hAFSC secretome did not provide any significant cardiogenic effect, fetal hAFSC-EVs significantly sustained mNVCM transition from S to M phase by 2-fold, while triggering cytokinesis by 4.5-fold over vehicle-treated cells. Treated mNVCM showed disorganized expression of cardiac alpha-actinin, suggesting cytoskeletal re-arrangements prior to cell renewal, with a 40% significant downregulation of Cofilin-2 and a positive trend of polymerized F-Actin. Fetal hAFSC-EVs increased cardiomyocyte cell cycle progression by 1.8-fold in the 4-day-old neonatal left ventricle myocardium short term after MI; however, such effect was lost at the later stage. Fetal hAFSC-EVs were enriched with a short isoform of Agrin, a mediator of neonatal heart regeneration acting by YAP-related signaling; yet in vitro application of YAP inhibitor verteporfin partially affected EV paracrine stimulation on mNVCM. EVs secreted by developmentally juvenile fetal hAFSC can support cardiomyocyte renewal to some extension, via intercellular conveyance of candidates possibly involving Agrin in combination with other factors. These perinatal derivative promising cardiogenic effects need further investigation to define their specific mechanism of action and enhance their potential translation into therapeutic opportunity.

Following on reports that miR-424 expression promotes oncogenesis, Domenico Albino et al. find that extracellular vesicles (EVs) in the plasma of prostate cancer patients secrete miR-424. Using cell-based and animal models, they demonstrate that EV-mediated release of miR-424 can transfer oncogenic signals across cells to promote recurrence and metastatic progression.

Cardiac resynchronization therapy (CRT) has become a valuable addition to the treatment options for heart failure, in particular for patients with disturbances in electrical conduction that lead to regionally different contraction patterns (dyssynchrony). Dyssynchronous hearts show extensive molecular and cellular remodeling, which has primarily been investigated in experimental animals. Evidence showing that at least several miRNAs play a role in this remodeling is increasing. A comparison of results from measurements in plasma and myocardial tissue suggests that plasma levels of miRNAs may reflect the expression of these miRNAs in the heart. Because many miRNAs released in the plasma are included in extracellular vesicles (EVs), which protect them from degradation, measurement of myocardium-derived miRNAs in peripheral blood EVs may open new avenues to investigate and monitor (reverse) remodeling in dyssynchronous and resynchronized hearts of patients.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiomyopathy. The molecular mechanisms determining HCM phenotypes are incompletely understood. Myocardial biopsies were obtained from a group of patients with obstructive HCM (n = 23) selected for surgical myectomy and from 9 unused donor hearts (controls). A subset of tissue-abundant myectomy samples from HCM (n = 10) and controls (n = 6) was submitted to laser-capture microdissection to isolate cardiomyocytes. We investigated the relationship among clinical phenotype, cardiac myosin proteins (MyHC6, MyHC7, and MyHC7b) measured by optimized label-free mass spectrometry, the relative genes (MYH7, MYH7B and MYLC2), and the MyomiR network (myosin-encoded microRNA (miRs) and long-noncoding RNAs (Mhrt)) measured using RNA sequencing and RT-qPCR. MyHC6 was lower in HCM vs. controls, whilst MyHC7, MyHC7b, and MyLC2 were comparable. MYH7, MYH7B, and MYLC2 were higher in HCM whilst MYH6, miR-208a, miR-208b, miR-499 were comparable in HCM and controls. These results are compatible with defective transcription by active genes in HCM. Mhrt and two miR-499-target genes, SOX6 and PTBP3, were upregulated in HCM. The presence of HCM-associated mutations correlated with PTBP3 in myectomies and with SOX6 in cardiomyocytes. Additionally, iPSC-derived cardiomyocytes, transiently transfected with either miR-208a or miR-499, demonstrated a time-dependent relationship between MyomiRs and myosin genes. The transfection end-stage pattern was at least in part similar to findings in HCM myectomies. These data support uncoupling between myosin protein/genes and a modulatory role for the myosin/MyomiR network in the HCM myocardium, possibly contributing to phenotypic diversity and providing putative therapeutic targets.

Background: Coronavirus-2 (SARS-CoV-2) infection causes an acute respiratory syndrome accompanied by multi-organ damage that implicates a prothrombotic state leading to widespread microvascular clots. The causes of such coagulation abnormalities are unknown. The receptor tissue factor, also known as CD142, is often associated with cell-released extracellular vesicles (EV). In this study, we aimed to characterize surface antigens profile of circulating EV in COVID-19 patients and their potential implication as procoagulant agents. Methods: We analyzed serum-derived EV from 67 participants who underwent nasopharyngeal swabs molecular test for suspected SARS-CoV-2 infection (34 positives and 33 negatives) and from 16 healthy controls (HC), as referral. A sub-analysis was performed on subjects who developed pneumonia (n = 28). Serum-derived EV were characterized for their surface antigen profile and tested for their procoagulant activity. A validation experiment was performed pre-treating EV with anti-CD142 antibody or with recombinant FVIIa. Serum TNF-α levels were measured by ELISA. Findings: Profiling of EV antigens revealed a surface marker signature that defines circulating EV in COVID-19. A combination of seven surface molecules (CD49e, CD209, CD86, CD133/1, CD69, CD142, and CD20) clustered COVID (+) versus COVID (-) patients and HC. CD142 showed the highest discriminating performance at both multivariate models and ROC curve analysis. Noteworthy, we found that CD142 exposed onto surface of EV was biologically active. CD142 activity was higher in COVID (+) patients and correlated with TNF-α serum levels. Interpretation: In SARS-CoV-2 infection the systemic inflammatory response results in cell-release of substantial amounts of procoagulant EV that may act as clotting initiation agents, contributing to disease severity. Funding: Cardiocentro Ticino Institute, Ente ospedaliero Cantonale, Lugano-Switzerland.

BackgroundCombined treatment with anthracyclines (e.g., doxorubicin; Dox) and trastuzumab (Trz), a humanized anti-human epidermal growth factor receptor 2 (HER2; ErbB2) antibody, in patients with HER2-positive cancer is limited by cardiotoxicity, as manifested by contractile dysfunction and arrhythmia. The respective roles of the two agents in the cardiotoxicity of the combined therapy are incompletely understood.ObjectiveTo assess cardiac performance, T-tubule organization, electrophysiological changes and intracellular Ca2+ handling in cardiac myocytes (CMs) using an in vivo rat model of Dox/Trz-related cardiotoxicity.Methods and ResultsAdult rats received 6 doses of either Dox or Trz, or the two agents sequentially. Dox-mediated left ventricular (LV) dysfunction was aggravated by Trz administration. Dox treatment, but not Trz, induced T-tubule disarray. Moreover, Dox, but not Trz monotherapy, induced prolonged action potential duration (APD), increased incidence of delayed afterdepolarizations (DADs) and beat-to-beat variability of repolarization (BVR), and slower Ca2+ transient decay. Although APD, DADs, BVR and Ca2+ transient decay recovered over time after the cessation of Dox treatment, subsequent Trz administration exacerbated these abnormalities. Trz, but not Dox, reduced Ca2+ transient amplitude and SR Ca2+ content, although only Dox treatment was associated with SERCA downregulation. Finally, Dox treatment increased Ca2+ spark frequency, resting Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and long-lasting Ca2+ release events (so-called Ca2+ “embers”), partially reproduced by Trz treatment.ConclusionThese results suggest that in vivo Dox but not Trz administration causes T-tubule disarray and pronounced changes in electrical activity of CMs. While adaptive changes may account for normal AP shape and reduced DADs late after Dox administration, subsequent Trz administration interferes with such adaptive changes. Intracellular Ca2+ handling was differently affected by Dox and Trz treatment, leading to SR instability in both cases. These findings illustrate the specific roles of Dox and Trz, and their interactions in cardiotoxicity and arrhythmogenicity.

Abstract Heart transplantation remains the treatment of reference for patients experiencing end‐stage heart failure; unfortunately, graft availability through conventional donation after brain death is insufficient to meet the demand. Use of extended‐criteria donors or donation after circulatory death has emerged to increase organ availability; however, clinical protocols require optimization to limit or prevent damage in hearts possessing greater susceptibility to injury than conventional grafts. The emergence of cardiac ex situ machine perfusion not only facilitates the use of extended‐criteria donor and donation after circulatory death hearts through the avoidance of potentially damaging ischemia during graft storage and transport, it also opens the door to multiple opportunities for more sensitive monitoring of graft quality. With this review, we aim to bring together the current knowledge of biomarkers that hold particular promise for cardiac graft evaluation to improve precision and reliability in the identification of hearts for transplantation, thereby facilitating the safe increase in graft availability. Information about the utility of potential biomarkers was categorized into 5 themes: (1) functional, (2) metabolic, (3) hormone/prohormone, (4) cellular damage/death, and (5) inflammatory markers. Several promising biomarkers are identified, and recommendations for potential improvements to current clinical protocols are provided.

The data and information presented here refer to the research article entitled: “Reactivating endogenous mechanisms of cardiac regeneration via paracrine boosting with the human amniotic fluid stem cell secretome” (Balbi et al., 2019, Apr 04). This dataset illustrates the in vitro paracrine effect exerted by the human amniotic fluid stem cell secretome on rodent neonatal cardiomyocytes, human endothelial progenitors and different subsets of cardiac progenitor cells. Cytokine/chemokine profiling of the human amniotic fluid stem cell secretome is provided as well. This data can provide useful insights in regenerative medicine as demonstrating the in vitro cardioprotective and proliferative secretory paracrine potential of human fetal stem cells. Keywords: Paracrine effect, Human amniotic fluid stem cells, Cardiac progenitor cells, Neonatal cardiomyocytes, Endothelial progenitors, Proliferation, Cardioprotection, Angiogenesis

Cardiac allograft rejection following heart transplantation is challenging to diagnose. Tissue biopsies are the gold standard in monitoring the different types of rejection. The last decade has seen an increased emphasis on identifying non-invasive methods to improve rejection diagnosis and overcome tissue biopsy invasiveness. Liquid biopsy, as an efficient non-invasive diagnostic and prognostic oncological monitoring tool, seems to be applicable in heart transplant follow-ups. Moreover, molecular techniques applied on blood can be translated to tissue samples to provide novel perspectives on tissue and reveal new diagnostic and prognostic biomarkers. This review aims to provide a comprehensive overview of the state-of-the-art of the new methodologies in cardiac allograft rejection monitoring and investigate the future perspectives on invasive and non-invasive rejection biomarkers identification. We reviewed literature from the most used scientific databases, such as PubMed, Google Scholar, and Scopus. We extracted 192 papers and, after a selection and exclusion process, we included in the review 81 papers. The described limitations notwithstanding, this review show how molecular biology techniques and omics science could be deployed complementarily to the histopathological rejection diagnosis on tissue biopsies, thus representing an integrated approach for heart transplant patients monitoring.

Extracellular vesicles (EVs) play a central role in intercellular communication, which is relevant for inflammatory and immune processes implicated in neurodegenerative disorders, such as Parkinson’s Disease (PD). We characterized and compared distinctive cerebrospinal fluid (CSF)-derived EVs in PD and atypical parkinsonisms (AP), aiming to integrate a diagnostic model based on immune profiling of plasma-derived EVs via artificial intelligence. Plasma- and CSF-derived EVs were isolated from patients with PD, multiple system atrophy (MSA), AP with tauopathies (AP-Tau), and healthy controls. Expression levels of 37 EV surface markers were measured by a flow cytometric bead-based platform and a diagnostic model based on expression of EV surface markers was built by supervised learning algorithms. The PD group showed higher amount of CSF-derived EVs than other groups. Among the 17 EV surface markers differentially expressed in plasma, eight were expressed also in CSF of a subgroup of PD, 10 in MSA, and 6 in AP-Tau. A two-level random forest model was built using EV markers co-expressed in plasma and CSF. The model discriminated PD from non-PD patients with high sensitivity (96.6%) and accuracy (92.6%). EV surface marker characterization bolsters the relevance of inflammation in PD and it underscores the role of EVs as pathways/biomarkers for protein aggregation-related neurodegenerative diseases.

Ischaemic cardiac disease is associated with a loss of cardiomyocytes and an intrinsic lack of myocardial renewal. Recent work has shown that the heart retains limited cardiomyocyte proliferation, which remains inefficient when facing pathological conditions. While broadly active in the neonatal mammalian heart, this mechanism becomes quiescent soon after birth, suggesting loss of regenerative potential with maturation into adulthood. A key question is whether this temporary regenerative window can be enhanced via appropriate stimulation and further extended. Recently the search for novel therapeutic approaches for heart disease has centred on stem cell biology. The “paracrine effect” has been proposed as a promising strategy to boost endogenous reparative and regenerative mechanisms from within the cardiac tissue by exploiting the modulatory potential of soluble stem cell-secreted factors. As such, growing interest has been specifically addressed towards stem/progenitor cell-secreted extracellular vesicles (EVs), which can be easily isolated in vitro from cell-conditioned medium. This review will provide a comprehensive overview of the current paradigm on cardiac repair and regeneration, with a specific focus on the role and mechanism(s) of paracrine action of EVs from cardiac stromal progenitors as compared to exogenous stem cells in order to discuss the optimal choice for future therapy. In addition, the challenges to overcoming translational EV biology from bench to bedside for future cardiac regenerative medicine will be discussed.

Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) has revolutionized the complex scientific field of disease modelling and personalized therapy. Cardiac differentiation of human iPSCs into cardiomyocytes (hiPSC-CMs) has been used in a wide range of healthy and disease models by deriving CMs from different somatic cells. Unfortunately, hiPSC-CMs have to be improved because existing protocols are not completely able to obtain mature CMs recapitulating physiological properties of human adult cardiac cells. Therefore, improvements and advances able to standardize differentiation conditions are needed. Lately, evidences of an epigenetic memory retained by the somatic cells used for deriving hiPSC-CMs has led to evaluation of different somatic sources in order to obtain more mature hiPSC-derived CMs.

Exosomes, nanosized membrane vesicles secreted by cardiac progenitor cells (Exo-CPC), inhibit cardiomyocyte apoptosis under stress conditions, promote angiogenesis in vitro, and prevent the early decline in cardiac function after myocardial infarction in vivo in preclinical rat models. The recognition of exosome-mediated effects has moved attempts at developing cell-free approaches for cardiac repair. Such approaches offer major advantages including the fact that exosomes can be stored as ready-to-use agents and delivered to patients with acute coronary syndromes. The aim of the present work was the development of a good manufacturing practice (GMP)-grade method for the large-scale preparation of Exo-CPC as a medicinal product, for a future clinical translation. A GMP-compliant manufacturing method was set up, based on large-scale cell culture in xeno-free conditions, collection of up to 8 l of exosome-containing conditioned medium and isolation of Exo-CPC through tangential flow filtration. Quality control tests were developed and carried out to evaluate safety, identity, and potency of both cardiac progenitor cells (CPC) as cell source and Exo-CPC as final product (GMP-Exo-CPC). CPC, cultured in xeno-free conditions, showed a lower doubling-time than observed in research-grade condition, while producing exosomes with similar features. Cells showed the typical phenotype of mesenchymal progenitor cells (CD73/CD90/CD105 positive, CD14/CD20/CD34/CD45/HLA-DR negative), and expressed mesodermal (TBX5/TBX18) and cardiac-specific (GATA4/MESP1) transcription factors. Purified GMP-Exo-CPC showed the typical nanoparticle tracking analysis profile and expressed main exosome markers (CD9/CD63/CD81/TSG101). The GMP manufacturing method guaranteed high exosome yield (>1013 particles) and consistent removal (≥97%) of contaminating proteins. The resulting GMP-Exo-CPC were tested for safety, purity, identity, and potency in vitro, showing functional anti-apoptotic and pro-angiogenic activity. The therapeutic efficacy was validated in vivo in rats, where GMP-Exo-CPC ameliorated heart function after myocardial infarction. Our standardized production method and testing strategy for large-scale manufacturing of GMP-Exo-CPC open new perspectives for reliable human therapeutic applications for acute myocardial infarction syndrome and can be easily applied to other cell sources for different therapeutic areas.

High aldehyde dehydrogenase (ALDHhi) activity has been reported in normal and cancer stem cells. We and others have shown previously that human ALDHhi cardiac atrial appendage cells are enriched with stem/progenitor cells. The role of ALDH in these cells is poorly understood but it may come down to the specific ALDH isoform(s) expressed. This study aimed to compare ALDHhi and ALDHlo atrial cells and to identify the isoform(s) that contribute to ALDH activity, and their functional role.Methods and Results: Cells were isolated from atrial appendage specimens from patients with ischemic and/or valvular heart disease undergoing heart surgery. ALDHhi activity assessed with the Aldefluor reagent coincided with primitive surface marker expression (CD34+). Depending on their ALDH activity, RT-PCR analysis of ALDHhi and ALDHlo cells demonstrated a differential pattern of pluripotency genes (Oct 4, Nanog) and genes for more established cardiac lineages (Nkx2.5, Tbx5, Mef2c, GATA4). ALDHhi cells, but not ALDHlo cells, formed clones and were culture-expanded. When cultured under cardiac differentiation conditions, ALDHhi cells gave rise to a higher number of cardiomyocytes compared with ALDHlo cells. Among 19 ALDH isoforms known in human, ALDH1A3 was most highly expressed in ALDHhi atrial cells. Knocking down ALDH1A3, but not ALDH1A1, ALDH1A2, ALDH2, ALDH4A1, or ALDH8A1 using siRNA decreased ALDH activity and cell proliferation in ALDHhi cells. Conversely, overexpressing ALDH1A3 with a retroviral vector increased proliferation in ALDHlo cells.Conclusions: ALDH1A3 is the key isoform responsible for ALDH activity in ALDHhi atrial appendage cells, which have a propensity to differentiate into cardiomyocytes. ALDH1A3 affects in vitro proliferation of these cells.

Cell therapy has been evaluated to enhance heart function after injury. Delivered cells mostly act via paracrine mechanisms, including secreted growth factors, cytokines, and vesicles, such as exosomes (Exo). Intramyocardial injection of cardiac-resident progenitor cells (CPC)-derived Exo reduced scarring and improved cardiac function after myocardial infarction in rats. Here, we explore a clinically relevant approach to enhance the homing process to cardiomyocytes (CM), which is crucial for therapeutic efficacy upon systemic delivery of Exo. By overexpressing exosomal CXCR4, we increased the efficacy of plasmatic injection of cardioprotective Exo-CPC by increasing their bioavailability to ischemic hearts. Intravenous injection of ExoCXCR4 significantly reduced infarct size and improved left ventricle ejection fraction at 4 weeks compared to ExoCTRL (p < 0.01). Hemodynamic measurements showed that ExoCXCR4 improved dp/dt min, as compared to ExoCTRL and PBS group. In vitro, ExoCXCR4 was more bioactive than ExoCTRL in preventing CM death. This in vitro effect was independent from SDF-1α, as shown by using AMD3100 as specific CXCR4 antagonist. We showed, for the first time, that systemic administration of Exo derived from CXCR4-overexpressing CPC improves heart function in a rat model of ischemia reperfusion injury These data represent a substantial step toward clinical application of Exo-based therapeutics in cardiovascular disease.

Cross-talk between different cell types plays central roles both in cardiac homeostasis and in adaptive responses of the heart to stress. Cardiomyocytes (CMs) send biological messages to the other cell types present in the heart including endothelial cells (ECs) and fibroblasts. In turn, CMs receive messages from these cells. Recent evidence has now established that exosomes, nanosized secreted extracellular vesicles, are crucial mediators of such messages. CMs, ECs, cardiac fibroblasts, and cardiac progenitor cells (CPCs) release exosomes carrying nonrandom subsets of proteins, lipids, and nucleic acids present in their cells of origin. Exosomes secreted from CMs are internalized by fibroblasts and regulate gene expression in these cells as well as in ECs. CPC-derived exosomes protect CMs against apoptosis while also stimulating angiogenesis. They are rich in cardioprotective and proangiogenic microRNAs such as miR-146, miR-210, and miR-132. When injected into infracted hearts in vivo, CPC-derived exosomes reduce infarct size and improve cardiac function. Thus, exosomes are emerging both as key mediators of intercellular communication in the heart and as therapeutic candidates for heart disease.

Cardiospheres (CSs) are self-assembling multicellular clusters from the cellular outgrowth from cardiac explants cultured in nonadhesive substrates. They contain a core of primitive, proliferating cells, and an outer layer of mesenchymal/stromal cells and differentiating cells that express cardiomyocyte proteins and connexin 43. Because CSs contain both primitive cells and committed progenitors for the three major cell types present in the heart, that is, cardiomyocytes, endothelial cells, and smooth muscle cells, and because they are derived from percutaneous endomyocardial biopsies, they represent an attractive cell source for cardiac regeneration. In preclinical studies, CS-derived cells (CDCs) delivered to infarcted hearts resulted in improved cardiac function. CDCs have been tested safely in an initial phase-1 clinical trial in patients after myocardial infarction. Whether or not CDCs are superior to purified populations, for example, c-kit+ cardiac stem cells, or to gene therapy approaches for cardiac regeneration remains to be evaluated.