Repositioning of Cefuroxime as novel selective inhibitor of the thyroid hormone activating enzyme type 2 deiodinase.

The iodothyronine deiodinases constitute a family of three selenoenzymes regulating the intracellular metabolism of Thyroid Hormones (THs, T4 and T3) and impacting on several physiological processes, including energy metabolism, development and cell differentiation. The type 1, 2 and 3 deiodinases (D1, D2, and D3), are sensitive, rate-limiting components within the TH axis, and rapidly control TH action in physiological conditions or disease. Notably, several human pathologies are characterized by deiodinases deregulation (e.g., inflammation, osteoporosis, metabolic syndrome, muscle wasting and cancer). Consequently, these enzymes are golden targets for the identification and development of pharmacological compounds endowed with modulatory activities. However, until now, the portfolio of inhibitors for deiodinases is limited and the few active compounds lack selectivity. Here, we describe the cephalosporin Cefuroxime as a novel D2 specific inhibitor. In both in vivo and in vitro settings, Cefuroxime acts as a selective inhibitor of D2 activity, without altering the enzymatic activity of D1 and D3. By inhibiting TH activation in target tissues, Cefuroxime alters the sensitivity of the hypothalamus-pituitary axis and interferes with the central regulation of THs levels, and is thus eligible as a potential new regulator of hyperthyroid pathologies, which affect thousands of patients worldwide.

Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation.

Breast cancer-associated fibroblasts (BCAFs), the most abundant non-cancer stromal cells of the breast tumor microenvironment (TME), dramatically sustain breast cancer (BC) progression by interacting with BC cells. BCAFs, as well as myofibroblasts, display an up regulation of activation and inflammation markers represented by α-smooth muscle actin (α-SMA) and cyclooxygenase 2 (COX-2). BCAF aggregates have been identified in the peripheral blood of metastatic BC patients. We generated an in vitro stromal model consisting of human primary BCAFs grown as monolayers or 3D cell aggregates, namely spheroids and reverted BCAFs, obtained from BCAF spheroids reverted to 2D cell adhesion growth after 216 h of 3D culture. We firstly evaluated the state of activation and inflammation and the mesenchymal status of the BCAF monolayers, BCAF spheroids and reverted BCAFs. Then, we analyzed the MCF-7 cell viability and migration following treatment with conditioned media from the different BCAF cultures. After 216 h of 3D culture, the BCAFs acquired an inactivated phenotype, associated with a significant reduction in α-SMA and COX-2 protein expression. The deactivation of the BCAF spheroids at 216 h was further confirmed by the cytostatic effect exerted by their conditioned medium on MCF-7 cells. Interestingly, the reverted BCAFs also retained a less activated phenotype as indicated by α-SMA protein expression reduction. Furthermore, the reverted BCAFs exhibited a reduced pro-tumor phenotype as indicated by the anti-migratory effect exerted by their conditioned medium on MCF-7 cells. The deactivation of BCAFs without drug treatment is possible and leads to a reduced capability of BCAFs to sustain BC progression in vitro. Consequently, this study could be a starting point to develop new therapeutic strategies targeting BCAFs and their interactions with cancer cells.

Influence of Tumor Microenvironment and Fibroblast Population Plasticity on Melanoma Growth, Therapy Resistance and Immunoescape.

Cutaneous melanoma (CM) tissue represents a network constituted by cancer cells and tumor microenvironment (TME). A key feature of CM is the high structural and cellular plasticity of TME, allowing its evolution with disease and adaptation to cancer cell and environmental alterations. In particular, during melanoma development and progression each component of TME by interacting with each other and with cancer cells is subjected to dramatic structural and cellular modifications. These alterations affect extracellular matrix (ECM) remodelling, phenotypic profile of stromal cells, cancer growth and therapeutic response. The stromal fibroblast populations of the TME include normal fibroblasts and melanoma-associated fibroblasts (MAFs) that are highly abundant and flexible cell types interacting with melanoma and stromal cells and differently influencing CM outcomes. The shift from the normal microenvironment to TME and from normal fibroblasts to MAFs deeply sustains CM growth. Hence, in this article we review the features of the normal microenvironment and TME and describe the phenotypic plasticity of normal dermal fibroblasts and MAFs, highlighting their roles in normal skin homeostasis and TME regulation. Moreover, we discuss the influence of MAFs and their secretory profiles on TME remodelling, melanoma progression, targeted therapy resistance and immunosurveillance, highlighting the cellular interactions, the signalling pathways and molecules involved in these processes.

The Microenvironment of Decellularized Extracellular Matrix from Heart Failure Myocardium Alters the Balance between Angiogenic and Fibrotic Signals from Stromal Primitive Cells.

Cardiac adverse remodeling is characterized by biological changes that affect the composition and architecture of the extracellular matrix (ECM). The consequently disrupted signaling can interfere with the balance between cardiogenic and pro-fibrotic phenotype of resident cardiac stromal primitive cells (CPCs). The latter are important players in cardiac homeostasis and can be exploited as therapeutic cells in regenerative medicine. Our aim was to compare the effects of human decellularized native ECM from normal (dECM-NH) or failing hearts (dECM-PH) on human CPCs. CPCs were cultured on dECM sections and characterized for gene expression, immunofluorescence, and paracrine profiles. When cultured on dECM-NH, CPCs significantly upregulated cardiac commitment markers (CX43, NKX2.5), cardioprotective cytokines (bFGF, HGF), and the angiogenesis mediator, NO. When seeded on dECM-PH, instead, CPCs upregulated pro-remodeling cytokines (IGF-2, PDGF-AA, TGF-ß) and the oxidative stress molecule H2O2. Interestingly, culture on dECM-PH was associated with impaired paracrine support to angiogenesis, and increased expression of the vascular endothelial growth factor (VEGF)-sequestering decoy isoform of the KDR/VEGFR2 receptor. Our results suggest that resident CPCs exposed to the pathological microenvironment of remodeling ECM partially lose their paracrine angiogenic properties and release more pro-fibrotic cytokines. These observations shed novel insights on the crosstalk between ECM and stromal CPCs, suggesting also a cautious use of non-healthy decellularized myocardium for cardiac tissue engineering approaches.

Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming.

Induced pluripotent stem cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell-like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency. As dermal fibroblast is the most used human cell for reprogramming, we hypothesize that the variability in reprogramming is, at least partially, because of the skin fibroblasts used. Human dermal fibroblasts harvested from five different anatomical sites (neck, breast, arm, abdomen and thigh) were cultured and their morphology, proliferation, apoptotic rate, ability to migrate, expression of mesenchymal or epithelial markers, differentiation potential and production of growth factors were evaluated in vitro. Additionally, gene expression analysis was performed by real-time PCR including genes typically expressed by mesenchymal cells. Finally, fibroblasts isolated from different anatomic sites were reprogrammed to iPSCs by integration-free method. Intriguingly, while the morphology of fibroblasts derived from different anatomic sites differed only slightly, other features, known to affect cell reprogramming, varied greatly and in accordance with anatomic site of origin. Accordingly, difference also emerged in fibroblasts readiness to respond to reprogramming and ability to form colonies. Therefore, as fibroblasts derived from different anatomic sites preserve positional memory, it is of great importance to accurately evaluate and select dermal fibroblast population prior to induce reprogramming.

Tendinopathy: Pathophysiology, Therapeutic Options, and Role of Nutraceutics. A Narrative Literature Review.

Tendinopathies are very common in general population and a huge number of tendon-related procedures take place annually worldwide, with significant socio-economic repercussions. Numerous treatment options are commonly used for tendon disorders. Besides pharmacological and physical therapy, nutrition could represent an additional tool for preventing and treating this complex pathology that deserve a multidisciplinary approach. In recent years, nutraceutical products are growing up in popularity since these seem to favor the prevention and the healing processes of tendon injuries. This narrative literature review aims to summarize current understanding and the areas of ongoing research about the management of tendinopathies with the help of oral supplementation.

Production of Cardiac Extracellular Matrix from Adult Human Fibroblasts for Culture Dish Coating.

The myocardium is composed of cardiomyocytes and an even greater number of fibroblasts, the latter being responsible for extracellular matrix production. From the early stages of heart development throughout the lifetime, in both normal and pathological conditions, the composition of the extracellular matrix changes and influences myocardium structure and function. The purpose of the method described here is to obtain the substrate for the culture of cardiac cells in vitro (termed cardiac ECM), mimicking the myocardial extracellular matrix in vivo. To this end, fibroblasts isolated from the adult human heart were cultured to confluence on gelatin-coated dishes to produce the myocardium-specific extracellular matrix. The subsequent removal of cardiac fibroblasts, while preserving the deposited cardiac ECM, produced the substrate for studying the influence of the myocardium-specific extracellular matrix on other cells. Importantly, the composition of the fibroblast-derived coating of the culture dish changes according to the in vivo activity of the fibroblasts isolated from the heart, allowing subsequent studies of cell-matrix interactions in different normal and pathological conditions.

Potential Impact of Physical Activity on Measures of Well-Being and Quality of Life in People with Rare Diseases: A Nationwide Cross-Sectional Study in Italy.

BACKGROUND: Rare diseases constitute a heterogeneous group of approximately 7000-8000 conditions, distinguished by their low prevalence. Collectively, they present a significant global health challenge, affecting millions of people worldwide. It is estimated that rare diseases affect approximately 10% of the global population, which places a significant burden on individuals, families, and society. It is, therefore, important to consider strategies to improve the overall well-being and quality of life of individuals with rare diseases. One potential avenue for exploration is the incorporation of physical activity (PA). The scope of this study was to ascertain whether PA has a positive impact on measures of well-being and to determine its potential to enhance the quality of life of these individuals. METHODS: The data were collected via an online survey. The one-way ANOVA test for multiple groups and multivariate Poisson models were employed to identify the significant predictors of the outcomes of interest. RESULTS: The protective effects of PA become evident with a minimum of six hours of activity per week. Our data confirm that the weekly hours devoted to PA can serve as a significant protective factor for QoL. The study also provided some insights into the motivations behind patients' engagement in PA. These included improving QoL and physical well-being, as well as the desire to interact socially, with the goal of meeting friends or making new acquaintances. Finally, for adults and older adults, engaging in PA can also be a way to control weight. CONCLUSIONS: It is becoming increasingly clear that individuals with rare diseases stand to benefit greatly from PA, so it is only sensible to educate them on the advantages of an active lifestyle.

Cardiac primitive cells become committed to a cardiac fate in adult human heart with chronic ischemic disease but fail to acquire mature phenotype: genetic and phenotypic study.

Adult human heart hosts a population of cardiac primitive CD117-positive cells (CPCs), which are responsible for physiological tissue homeostasis and regeneration. While the bona fide stem cells express telomerase, their progenies are no longer able to preserve telomeric DNA; hence the balance between their proliferation and differentiation has to be tightly controlled in order to prevent cellular senescence and apoptosis of CPCs before their maturation can be accomplished. We have examined at cellular and molecular level the proliferation, apoptosis and commitment of CPCs isolated from normal (CPC-N) and age-matched pathological adult human hearts (CPC-P) with ischemic heart disease. In the CPC-P, genes related to early stages of developmental processes, nervous system development and neurogenesis, skeletal development, bone and cartilage development were downregulated, while those involved in mesenchymal cell differentiation and heart development were upregulated, together with the transcriptional activation of TGFß/BMP signaling pathway. In the pathological heart, asymmetric division was the prevalent type of cardiac stem cell division. The population of CPC-P consisted mainly of progenitors of cardiac cell lineages and less precursors; these cells proliferated more, but were also more susceptible to apoptosis with respect to CPC-N. These results indicate that CPCs fail to reach terminal differentiation and functional competence in pathological conditions. Adverse effects of underlying pathology, which disrupts cardiac tissue structure and composition, and cellular senescence, resulting from cardiac stem cell activation in telomere dysfunctional environment, can be responsible for such outcome.

Stem cell therapy in sports medicine: current applications, challenges and future perspectives.

Stem cells have demonstrated significant potential for tissue repair and regeneration, making them a promising therapeutic avenue in sports medicine. This review aims to provide a comprehensive overview of the current state of research on the application of stem cells in sports medicine. We will discuss the types of stem cells used, their mechanisms of action, and the clinical outcomes of stem cell therapy in different sports-related injuries. Furthermore, we will delve into the challenges and ethical considerations associated with stem cell therapy, as well as future directions and potential applications of stem cells in sports medicine.

Cardiac shock wave therapy: assessment of safety and new insights into mechanisms of tissue regeneration.

Although low-energy extracorporeal cardiac shock wave (ECSW) therapy represents an attractive non-invasive treatment option for ischaemic heart disease, the precise mechanisms of its action and influence on the cardiac tissue remain obscure. The goal of this study was to evaluate the effects of SW application on cardiac function and structure. Four-month-old Fisher 344 rats were subjected to ECSW therapy. Echocardiographic measurements of cardiac function were performed at baseline and at 1 and 3 months after treatment. Signs of inflammation, apoptosis and fibrosis were evaluated by immunohistochemistry in the control and treated hearts. ECSW application did not provoke arrhythmia or increase the troponin-I level. At all time points, the left ventricular ejection fraction and fractional shortening remained stable. Histological analysis revealed neither differences in the extracellular matrix collagen content nor the presence of fibrosis; similarly, there were no signs of inflammation. Moreover, a population of cardiac cells that responded eagerly to ECSW application in the adult heart was identified; c-kit-positive, Ki67-positive, orthochromatic cells, corresponding to cardiac primitive cells, were 2.65-fold more numerous in the treated myocardium. In conclusion, non-invasive ECSW therapy is a safe and effective way of activating cardiac stem cells and myocardial regeneration. Because many factors influence cellular turnover in the ischaemic myocardium during the course of ischaemic heart disease, cardiac remodelling, and heart failure progression, studies to identify the optimal treatment time are warranted.

Non-modified RNA-Based Reprogramming of Human Dermal Fibroblasts into Induced Pluripotent Stem Cells.

The generation of pluripotent stem cells from adult somatic cells by cell reprogramming has put a whole new perspective on stem cell biology and stem cell-based regenerative medicine. Cell reprogramming acts through the introduction of key genes that regulate and maintain the pluripotent cell state. In this chapter, we describe the optimized protocol for the efficient isolation of fibroblasts from a skin punch biopsy and the subsequent easy and effective generation of integration-free induced pluripotent stem cell (iPSC) colonies forcing the expression of specific factors by non-modified RNAs.

Cardiac-derived extracellular matrix: A decellularization protocol for heart regeneration.

Extracellular matrix (ECM) is a fundamental component of the heart, guiding vital cellular processes during organ homeostasis. Most cardiovascular diseases lead to a remarkable remodeling of the ECM, accompanied by the formation of a fibrotic tissue that heavily compromises the heart function. Effective therapies for managing fibrosis and promoting physiological ECM repair are not yet available. The production of a decellularized extracellular matrix (d-ECM) serving as a three-dimensional and bioactive scaffold able to modulate cellular behavior and activities is considered crucial to achieve a successful regeneration. The protocol represents a step-by-step method to obtain a decellularized cardiac matrix through the combination of sodium dodecyl sulphate (SDS) and Triton X-100. Briefly, cardiac samples obtained from left ventricles of explanted, pathological human hearts were dissected and washed to remove residual body fluids. Samples were then snap-frozen and sliced by a cryostat into 350 µm thick sections. The sections obtained were decellularized using a solution containing 1% Triton X-100 and 1% SDS in combination, for 24 hours, until observing the color change from brownish-red to translucent-white. As a result, the protocol shows efficiency in preserving ECM architecture and protein composition during the whole process, suggesting that it is worthwhile, highly reproducible and produces a well- preserved decellularized extracellular matrix from cardiac samples. Notwithstanding, some limitations need to be addressed, such as the risk for microbial contamination and the unpredictable trend of the protocol when applied to decellularize samples other than myocardium, vessels, or skin. These issues require antibiotics mixture supplement during the procedure followed by UV sterilization, and appropriate adjustments for a tissue-specific utilization, respectively. The protocol is intended to produce a cardiac d-ECM for cell settlement, representing the ideal scaffold for tissue engineering purposes.

Cell Toxicity Study of Antiseptic Solutions Containing Povidone-Iodine and Hydrogen Peroxide.

The increasing incidence of periprosthetic joint infections (PJIs) has led to a growing interest in developing strategies to prevent and treat this severe complication. The surgical site's application of antiseptic solutions to eliminate contaminating bacteria and eradicate the bacterial biofilm has been increasing over time. Even though it has been proven that combining antimicrobials could enhance their activities and help overcome acquired microbial resistance related to the topical use of antibiotics, the toxicity of integrated solutions is not well described. This study aimed to evaluate the cytotoxicity of solutions containing povidone-iodine (PI) and hydrogen peroxide (H2O2), alone or in combination, after 1.3 and 5 min of exposure. Chondrocytes, tenocytes, and fibroblast-like synoviocytes were used for cytotoxicity analysis. Trypan blue stain (0.4% in PBS) was applied to evaluate the dead cells. All solutions tested showed a progressive increase in toxicity as exposure time increased except for PI at 0.3%, which exhibited the lowest toxicity. The combined solutions reported a reduced cellular killing at 3 and 5 min than H2O2 at equal concentrations, similar results to PI solutions.

Human Cardiac Progenitor Cell-Derived Extracellular Vesicles Exhibit Promising Potential for Supporting Cardiac Repair in Vitro.

Although human Cardiac Progenitor Cells (hCPCs) are not retained by host myocardium they still improve cardiac function when injected into ischemic heart. Emerging evidence supports the hypothesis that hCPC beneficial effects are induced by paracrine action on resident cells. Extracellular vesicles (EVs) are an intriguing mechanism of cell communication based on the transport and transfer of peptides, lipids, and nucleic acids that have the potential to modulate signaling pathways, cell growth, migration, and proliferation of recipient cells. We hypothesize that EVs are involved in the paracrine effects elicited by hCPCs and held accountable for the response of the infarcted myocardium to hCPC-based cell therapy. To test this theory, we collected EVs released by hCPCs isolated from healthy myocardium and evaluated the effects they elicited when administered to resident hCPC and cardiac fibroblasts (CFs) isolated from patients with post-ischemic end-stage heart failure. Evidence emerging from our study indicated that hCPC-derived EVs impacted upon proliferation and survival of hCPCs residing in the ischemic heart and regulated the synthesis and deposition of extracellular-matrix by CFs. These findings suggest that beneficial effects exerted by hCPC injection are, at least to some extent, ascribable to the delivery of signals conveyed by EVs.

Biomimetic design of bioartificial scaffolds for the in vitro modelling of human cardiac fibrosis.

In vitro models of pathological cardiac tissue have attracted interest as predictive platforms for preclinical validation of therapies. However, models reproducing specific pathological features, such as cardiac fibrosis size (i.e., thickness and width) and stage of development are missing. This research was aimed at engineering 2D and 3D models of early-stage post-infarct fibrotic tissue (i.e., characterized by non-aligned tissue organization) on bioartificial scaffolds with biomimetic composition, design, and surface stiffness. 2D scaffolds with random nanofibrous structure and 3D scaffolds with 150 µm square-meshed architecture were fabricated from polycaprolactone, surface-grafted with gelatin by mussel-inspired approach and coated with cardiac extracellular matrix (ECM) by 3 weeks culture of human cardiac fibroblasts. Scaffold physicochemical properties were thoroughly investigated. AFM analysis of scaffolds in wet state, before cell culture, confirmed their close surface stiffness to human cardiac fibrotic tissue. Following 3 weeks culture, biomimetic biophysical and biochemical scaffold properties triggered the activation of myofibroblast phenotype. Upon decellularization, immunostaining, SEM and two-photon excitation fluorescence microscopy showed homogeneous decoration of both 2D and 3D scaffolds with cardiac ECM. The versatility of the approach was demonstrated by culturing ventricular or atrial cardiac fibroblasts on scaffolds, thus suggesting the possibility to use the same scaffold platforms to model both ventricular and atrial cardiac fibrosis. In the future, herein developed in vitro models of cardiac fibrotic tissue, reproducing specific pathological features, will be exploited for a fine preclinical tuning of therapies.

Patent Foramen Ovale-A Not So Innocuous Septal Atrial Defect in Adults.

Patent foramen ovale (PFO) is a common congenital atrial septal defect with an incidence of 15-35% in the adult population. The development of the interatrial septum is a process that begins in the fourth gestational week and is completed only after birth. During intrauterine life, the foramen ovale allows the passage of highly oxygenated blood from the right to the left atrium and into the systemic arteries, thus bypassing the pulmonary circulation. In 75% of the general population, the foramen ovale closes after birth, and only an oval depression, called fossa ovalis, remains on the right side of the interatrial septum. Patent foramen ovale can be associated with various clinically important conditions, including migraine and stroke, or decompression illness in divers. The aim of this review is to summarize the PFO developmental and anatomical features and to discuss the clinical risks associated with this atrial septal defect in adults.

Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine.

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of surrounding cells due to its content of diverse bioactive molecules. Due to these characteristics, the ECM has been recently considered a promising candidate for the creation of biological scaffolds to boost tissue regeneration. Emerging studies have demonstrated that decellularized human tissues could resemble the native ECM in their structural and biochemical profiles, preserving the three-dimensional (3D) architecture and the content of fundamental biological molecules. Hence, decellularized ECM can be employed to promote tissue remodeling, repair, and functional reconstruction of many organs. Selecting the appropriate decellularization procedure is crucial to obtain acellular tissues that retain the characteristics of the ideal microenvironment for cells. The protocol described here provides a detailed step-by-step description of the decellularization method to obtain a reproducible and effective cell-free biological ECM. Skin fragments from patients undergoing plastic surgery were scaled down and decellularized using a combination of sodium dodecylsulfate (SDS), Triton X-100, and antibiotics. To promote the regular and homogeneous transport of the solution through the samples, they were enclosed in embedding cassettes to ensure protection from mechanical insults. After the decellularization procedure, the snow-white color of skin fragments indicated complete and successful decellularization. Additionally, decellularized samples showed an intact and well-preserved architecture. The results suggest that the proposed decellularization method was effective, fast, and reproducible and protected samples from architectural damages.

Correlation between Official and Common Field-Based Fitness Tests in Elite Soccer Referees.

Official tests are used to assess the fitness status of soccer referees, and their results correlate with match performance. However, FIFA-approved tests expose the referees to high physical demands and are difficult to implement during the sportive year. The aim of our study was to evaluate the correlation between the 6 × 40-m sprint and Yo-Yo Intermittent Recovery Level 1 (IR1) official tests and other field-based tests that require no or little equipment, are not time-consuming, and impose low physical demands. All tests were performed by male referees from the Regional Section of the Italian Referee Association (n = 30). We observed a strong correlation between 6 × 40-m sprint and Illinois agility tests (r = 0.63, p = 0.001) and a moderate correlation between Yo-Yo IR1 and hand-grip strength in the dominant (r = 0.45, p = 0.014) and non-dominant hand (r = 0.41, p = 0.031). Interestingly, only a moderate correlation (r = -0.42, p = 0.025) was observed between the FIFA official tests, 6 × 40-m sprint and Yo-Yo IR1. These results suggest that Illinois agility and hand-grip tests could represent simple and low-physical-impact tools for repeated assessment and monitoring of referee fitness throughout the sportive season.

Epithelial-mesenchymal transition of epicardial mesothelium is a source of cardiac CD117-positive stem cells in adult human heart.

Epithelial-mesenchymal transition is implicated in the remodelling of tissues during development and in the adult life. In the heart, it gives origin to progenitors of fibroblasts, coronary endothelium, smooth muscle cells, and cardiomyocytes. Moreover, epicardially-derived cells determine myocardial wall thickness and Purkinje fibre network. Recently, the presence of numerous cardiac stem cells in the subepicardium of the adult human heart has been described and the hypothesis that epicardially-derived cells can contribute to the population of cardiac stem cells in the adult heart has been advanced. In an effort to test this hypothesis and establish a possible link between epicardium, epicardially-derived cells and cardiac stem cells in the adult human heart we have examined epicardial mesothelial cells in the normal and pathological adult human heart with ischemic cardiomyopathy in vivo and we have induced and documented their epithelial-mesenchymal transition in vitro. Noticeably, epicardial cells were missing from the surface of pathological hearts and the cells with the expression of epithelial and mesenchymal markers populated thick subepicardial space. When the fragments of epicardium from the normal hearts were cultured on the specific substrate formed by extracellular matrix derived from cardiac fibroblasts, we obtained the outgrowth of the epithelial sheet with the mRNA and protein expression characteristic of epicardium. TGFß induced cellular and molecular changes typical of epithelial-mesenchymal transition. Moreover, the epicardially-derived cells expressed CD117 antigen. Thus, this study provides evidence that cardiac stem cells can originate from epithelial-mesenchymal transition of the epicardial cells in the adult human heart.