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.

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.

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.

Surface functionalization of polyurethane scaffolds mimicking the myocardial microenvironment to support cardiac primitive cells.

Scaffolds populated with human cardiac progenitor cells (CPCs) represent a therapeutic opportunity for heart regeneration after myocardial infarction. In this work, square-grid scaffolds are prepared by melt-extrusion additive manufacturing from a polyurethane (PU), further subjected to plasma treatment for acrylic acid surface grafting/polymerization and finally grafted with laminin-1 (PU-LN1) or gelatin (PU-G) by carbodiimide chemistry. LN1 is a cardiac niche extracellular matrix component and plays a key role in heart formation during embryogenesis, while G is a low-cost cell-adhesion protein, here used as a control functionalizing molecule. X-ray photoelectron spectroscopy analysis shows nitrogen percentage increase after functionalization. O1s and C1s core-level spectra and static contact angle measurements show changes associated with successful functionalization. ELISA assay confirms LN1 surface grafting. PU-G and PU-LN1 scaffolds both improve CPC adhesion, but LN1 functionalization is superior in promoting proliferation, protection from apoptosis and expression of differentiation markers for cardiomyocytes, endothelial and smooth muscle cells. PU-LN1 and PU scaffolds are biodegraded into non-cytotoxic residues. Scaffolds subcutaneously implanted in mice evoke weak inflammation and integrate with the host tissue, evidencing a significant blood vessel density around the scaffolds. PU-LN1 scaffolds show their superiority in driving CPC behavior, evidencing their promising role in myocardial regenerative medicine.

Optimization of Human Myocardium Decellularization Method for the Construction of Implantable Patches.

Cardiac tissue engineering by means of synthetic or natural scaffolds combined with stem/progenitor cells is emerging as the response to the unsatisfactory outcome of approaches based solely on the injection of cells. Parenchymal and supporting cells are surrounded, in vivo, by a specialized and tissue-specific microenvironment, consisting mainly of extracellular matrix (ECM) and soluble factors incorporated in the ECM. Since the naturally occurring ECM is the ideal platform for ensuring cell engraftment, survival, proliferation, and differentiation, the acellular native ECM appears by far the most promising and appealing substrate among all biomaterials tested so far. To obtain intact scaffold of human native cardiac ECM while preserving its composition, we compared the decellularized ECM (d-ECM) produced through five different protocols of decellularization (named Pr1, Pr2, Pr3, Pr4, and Pr5) in terms of efficiency of decellularization, composition, and three-dimensional architecture of d-ECM scaffolds and of their suitability for cell repopulation. The decellularization procedures proved substantially different. Specifically, only three, of the five protocols tested, proved effective in producing thoroughly acellular d-ECM. In addition, the d-ECM delivered differed in architecture and composition and, more importantly, in its ability to support engraftment, survival, and differentiation of cardiac primitive cells in vitro.

Polyurethane-based scaffolds for myocardial tissue engineering.

Bi-layered scaffolds with a 0°/90° lay-down pattern were prepared by melt-extrusion additive manufacturing (AM) using a poly(ester urethane) (PU) synthesized from poly(ε-caprolactone) diol, 1,4-butandiisocyanate and l-lysine ethyl ester dihydrochloride chain extender. Rheological analysis and differential scanning calorimetry of the starting material showed that compression moulded PU films were in the molten state at a higher temperature than 155°C. The AM processing temperature was set at 155°C after verifying the absence of PU thermal degradation phenomena by isothermal thermogravimetry analysis and rheological characterization performed at 165°C. Scaffolds highly reproduced computer-aided design geometry and showed an elastomeric-like behaviour which is promising for applications in myocardial regeneration. PU scaffolds supported the adhesion and spreading of human cardiac progenitor cells (CPCs), whereas they did not stimulate CPC proliferation after 1-14 days culture time. In the future, scaffold surface functionalization with bioactive peptides/proteins will be performed to specifically guide CPC behaviour.

Cardiac fibroblast-derived extracellular matrix (biomatrix) as a model for the studies of cardiac primitive cell biological properties in normal and pathological adult human heart.

Cardiac tissue regeneration is guided by stem cells and their microenvironment. It has been recently described that both cardiac stem/primitive cells and extracellular matrix (ECM) change in pathological conditions. This study describes the method for the production of ECM typical of adult human heart in the normal and pathological conditions (ischemic heart disease) and highlights the potential use of cardiac fibroblast-derived ECM for in vitro studies of the interactions between ECM components and cardiac primitive cells responsible for tissue regeneration. Fibroblasts isolated from adult human normal and pathological heart with ischemic cardiomyopathy were cultured to obtain extracellular matrix (biomatrix), composed of typical extracellular matrix proteins, such as collagen and fibronectin, and matricellular proteins, laminin, and tenascin. After decellularization, this substrate was used to assess biological properties of cardiac primitive cells: proliferation and migration were stimulated by biomatrix from normal heart, while both types of biomatrix protected cardiac primitive cells from apoptosis. Our model can be used for studies of cell-matrix interactions and help to determine the biochemical cues that regulate cardiac primitive cell biological properties and guide cardiac tissue regeneration.

Flatfoot in children: anatomy of decision making.

Concern about a child's foot posture is a common reason for frequent consultations for an array of health care professionals; sports medicine specialists are often the first to recognize and advise on foot pathology. In the decision making process, it is essential to distinguish between the different types of flatfoot deformity: paediatric or adult, congenital or acquired, flexible or rigid. Although flatfoot in children is a common finding, evidence for the techniques of the reliable and reproducible assessment of the foot posture is scant. This general review presents the factors involved in the forming and supporting of the foot arches, discusses the protocols useful in the evaluation of the foot posture, and indicates how to differentiate between flatfoot cases needing treatment and cases that need only reassurance.

Localization and origin of cardiac CD117-positive cells: identification of a population of epicardially-derived cells in adult human heart.

During heart morphogenesis, epicardial cells undergo epithelial-mesenchymal transition giving origin to a population of epicardially derived cells that play a crucial role in the development of most cardiac cell lineages. Considering the hypothesis that epithelial-mesenchymal transition of epicardial mesothelium can generate cardiac primitive cells in the adult heart, we have examined in vivo and in vitro the epicardium and subepicardium of normal human adult hearts and of pathological hearts from patients with chronic ischemic heart failure for the presence of CD117-positive cells with epithelial and mesenchymal markers expression. The number of CD117-positive cells increased significantly in the subepicardium of pathological hearts and sloped down towards myocardium, remaining still elevated with respect to normal hearts. While cells with typical epithelial proteins expression formed an intact layer on the surface of the normal hearts, CD117-positive cells were localized mainly in the subepicardium and expressed mesenchymal markers in the pathological hearts. Epithelial-mesenchymal transition, induced in vitro by several growth factors known to accumulate in the ischemic myocardium, gave origin to epicardially-derived cells with CD117 expression. These data support the hypothesis of epicardial origin of cardiac primitive cells in the adult human heart.

Epicardial cells are missing from the surface of hearts with ischemic cardiomyopathy: a useful clue about the self-renewal potential of the adult human heart?

The search for ideal cell candidate for heart regeneration, as well as for putative cardiac stem cell responsible for cardiac tissue homeostasis, is occupying both basic scientists and clinicians. Growing number of studies and publications indicate epicardium-derived cells as cardiac stem cells. While it is beyond doubt that these cells contribute to normal development of the heart during organogenesis, it remains an open question whether mesothelial epicardial cells can preserve their embryonic potential and if they can undergo epithelial-mesenchymal transition, giving origin to cardiac cell lineages, also in the adult human heart. Recent observations in vitro confirm this hypothesis, but direct evidence from the adult human heart is difficult to obtain. We report the absence of epicardial cells from the surface of adult human hearts with ischemic cardiomyopathy and the accumulation of cells with epithelial and mesenchymal markers in the subepicardium. We argue that these findings may correspond to the activation of the epithelial-mesenchymal transition in the chronic pathological conditions requiring cardiac cell regeneration, followed by epicardial cell pool exhaustion. Hence, observation of the epicardium of patients with cardiovascular disease, although not offering immediate diagnostic advantage, could provide some urging answers concerning the self-renewal potential of the adult heart.