In vitro cultured progenitors and precursors of cardiac cell lineages from human normal and post-ischemic hearts.

The demonstration of the presence of dividing primitive cells in damaged hearts has sparked increased interest about myocardium regenerative processes. We examined the rate and the differentiation of in vitro cultured resident cardiac primitive cells obtained from pathological and normal human hearts in order to evaluate the activation of progenitors and precursors of cardiac cell lineages in post-ischemic human hearts. The precursors and progenitors of cardiomyocyte, smooth muscle and endothelial lineage were identified by immunocytochemistry and the expression of characteristic markers was studied by western blot and RT-PCR.The amount of proteins characteristic for cardiac cells (alpha-SA and MHC, VEGFR-2 and FVIII, SMA for the precursors of cardiomyocytes, endothelial and smooth muscle cells, respectively) inclines toward an increase in both alpha-SA and MHC. The increased levels of FVIII and VEGFR2 are statistically significant, suggesting an important re-activation of neoangiogenesis. At the same time, the augmented expression of mRNA for Nkx 2.5, the trascriptional factor for cardiomyocyte differentiation, confirms the persistence of differentiative processes in terminally injured hearts. Our study would appear to confirm the activation of human heart regeneration potential in pathological conditions and the ability of its primitive cells to maintain their proliferative capability in vitro. The cardiac cell isolation method we used could be useful in the future for studying modifications to the microenvironment that positively influence cardiac primitive cell differentiation or inhibit, or retard, the pathological remodeling and functional degradation of the heart

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

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 schemic 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, mand 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

Aorta and pulmonary trunk - comparison of wall structure in typical and atypical (Ross procedure) blood pressure conditions

The ascending aorta and pulmonary trunk develop from the common truncus arteriosus that later becomes symmetrically divided by the aorticopulmonary septum. Normally, the systolic pressure value and the diastolic pressure gradient in the aorta is much higher than that in the pulmonary artery. In the Ross procedure, patient’s diseased aortic valve is replaced with their own pulmonary valve and as a consequence the pulmonary trunk is placed in the aortic root position. Typically, a reinforcement of transposed pulmonary trunk is necessary to avoid vessel dilation. In order to investigate how the blood flow characteristics determine the vessel wall structure we examined by immunochemistry the wall of normal aorta (NA), normal pulmonary trunk (NPT), transposed pulmonary trunk (trPT) and transposed pulmonary trunk with reinforcement (trPT-R). Throughout the tunica media of NA, elastic fibers form numerous, conspicuous and orderly arranged wavy lamellae that parallel thin layers of smooth muscle cells between the internal and external elastic membrane. In the NPT, smooth muscle cells run amid collagen fibers and form layers that are thicker and irregular, with elastic fibers arranged accordingly. In the trPT, intima denudation and media disruptions were observed. In the tunica media, smooth muscle cells were abundant, but muscle fibers, with irregular profiles and no discernible alignment, were widely spaced with intervening collagen fibers. In the trPA-R, the endothelial lining was preserved and elastic fibers formed a thick and highly organized layer of concentric lamellae in the middle third of tunica media. The structure of normal aorta and pulmonary trunk, both elastic arteries, with common embryological origin, differs significantly in terms of smooth muscle and elastic sheets number and organization. The animal model of Ross procedure with pulmonary trunk in aorta position further underscores the role of blood pressure and mechanical stress in vessel wall modification

Decellularized Human Dermal Matrix as a Biological Scaffold for Cardiac Repair and Regeneration.

The complex and highly organized environment in which cells reside consists primarily of the extracellular matrix (ECM) that delivers biological signals and physical stimuli to resident cells. In the native myocardium, the ECM contributes to both heart compliance and cardiomyocyte maturation and function. Thus, myocardium regeneration cannot be accomplished if cardiac ECM is not restored. We hypothesize that decellularized human skin might make an easily accessible and viable alternate biological scaffold for cardiac tissue engineering (CTE). To test our hypothesis, we decellularized specimens of both human skin and human myocardium and analyzed and compared their composition by histological methods and quantitative assays. Decellularized dermal matrix was then cut into 600-mm-thick sections and either tested by uniaxial tensile stretching to characterize its mechanical behavior or used as three-dimensional scaffold to assess its capability to support regeneration by resident cardiac progenitor cells (hCPCs) in vitro. Histological and quantitative analyses of the dermal matrix provided evidence of both effective decellularization with preserved tissue architecture and retention of ECM proteins and growth factors typical of cardiac matrix. Further, the elastic modulus of the dermal matrix resulted comparable with that reported in literature for the human myocardium and, when tested in vitro, dermal matrix resulted a comfortable and protective substrate promoting and supporting hCPC engraftment, survival and cardiomyogenic potential. Our study provides compelling evidence that dermal matrix holds promise as a fully autologous and cost-effective biological scaffold for CTE

Biological properties of cardiac stem cells in normal and pathological conditions - matrix makes a difference

Cardiac cells and extracellular matrix (ECM) are reciprocally related and their characteristics are modified in response to developmental or pathophysiological cues. Adult human cardiac tissue regeneration mediated by cardiac stem cells (CSCs) is strictly regulated and, hypothetically, impaired by the ECM-CSC signalling in the pathological conditions. To test this hypothesis, we isolated cardiac fibroblasts (CFs) and CSCs from the atria of age-matched adult human normal (n=9) and pathological hearts (ischemic cardiomyopathy, n=11). The CFs were cultured in order to obtain ECM coating and conditioned medium, which were characterized by immunoblotting and ELISA, respectively. Next, we examined the effects of CF-derived ECM and CF-conditioned medium on normal and pathological CSC proliferation, apoptosis, and migration in vitro. The ECM produced by CFs from normal heart was composed mainly of fibronectin, laminin α2 and collagen I, while that produced by CFs from hearts with ischemic cardiomyopathy contained also laminin α1 and tenascin X. Compared to the normal CF-conditioned medium, that conditioned by pathological CFs contained twice as much IGF1 and HGF, and it stimulated proliferation and migration, while reducing apoptosis of CSCs. In the presence of pathological CF-derived ECM, there was a nearly 2-fold increase (p<0.05) in proliferation of normal and pathological CSCs, when compared to normal CF-derived ECM. Moreover, pathological CF-derived ECM reduced CSC apoptosis, specifically in cells from pathological heart. However, in the same conditions, the migration of pathological CSCs was significantly lower. These results indicate that the activity of CFs and its modification in chronic ischemic conditions determines biological properties of CSCs. Such an influence should be taken into consideration when attempting ischemic cardiac tissue stem cell-based regeneration

Positional memory of fibroblasts may affect efficiency of iPSC reprogramming

Induced Pluripotent Stem cells (iPSC) are pluripotent stem cells reprogrammed from adult somatic cells. Although iPSC hold great potential for applications in regenerative medicine, technical problems, mostly related to the low efficiency of reprogramming, are yet to be solved. Since the most used cells for iPSC reprogramming are skin fibroblasts (FB), and since FB preserve positional memory, we hypothesize that the anatomic origin of FB might influence iPSC reprogramming.We isolated FB from skin of five different sites (neck, arm, thigh, breast, abdomen) of 13 patients undergoing plastic surgery or from heart wall or ascending aorta wall of the explanted heart of 3 patients receiving heart transplantation. FB from different anatomic sites and control FB from neonatal foreskin, were cultured for one week to evaluate morphology, proliferation rate and proneness to apoptosis. Additionally, expression of vimentin, cadherin, smooth muscle actin and Factor VIII was investigated to exclude the presence of other cell types. Transcriptome analysis including genes involved in stemness maintenance, embryogenesis, cell growth, activation and development, was performed by real-time PCR. Despite the similar morphology of FB from different sites, and immunopositivity for vimentin, along with the absence of other cell type markers, FB isolated from abdomen and heart had 1.5-fold higher doubling time, while FB from heart, abdomen and breast were less susceptible to apoptosis. Intriguingly, Real-Time PCR revealed that in abdomen, breast, neck, arm and heart FB genes involved in cell growth, development, proliferation, and migration, as TM4SF1, GPC4, CSPG2, DDIT4, ID1 were up-regulated, while genes regulating embryogenesis and tissue morphogenesis, like VCAN, FN1, HOXA5, CD49a were up-regulated in FB isolated from abdomen, arm and heart. However, all FBs had transcripts of markers of Mesenchymal Stem Cells (MSC), as CD105 and CD90. Our results provide evidence that human adult FB from different sites have different genetic program. Therefore, FB may respond to reprogram technology in different manner, thus affecting reprogramming efficiency. While offering novel perspective of the reprogramming technology, our study also demonstrates that abdomen and breast FB share cardiac genetic signature of cardiac FB while expressing markers of MSC and they might represent the ideal cell for cardiac reprogramming

Bioresorbable reinforcement induces histological rearrangement of pulmonary autograft in an experimental model of Ross operation

The Ross procedure has emerged as a popular choice for aortic valve replacement in infants and children. However, pulmonary artery (PA) autograft dilation remains the major concern; hence, several modifications of the valve implantation techniques, such as reinforcing the autografts with a tubular synthetic mesh, have been reported. With the aim to prevent dilation and permit the normal growth of the neo-aortic root following pulmonary autograft implantation, we assessed the biological effect and long term performance of an external bioresorbable reinforcement for PA autograft in an experimental Ross model in growing animals. An experimental model of translocation of the pulmonary trunk as autograft in aortic position, funded on the Hook’s law and Laplace equilibrium, has been developed and performed under cardiopulmonary bypass in young lambs. The PA without reinforcement (n=5) was compared to PA reinforced with new scaffold polymer with an external armour of Polytetrafluoroethylene. The PA autograft diameter was measured using transoesophageal echography at day 0 and at 6 months and compared to the distal aortic diameter. Pathological analysis of the PA autograft reinforced was performed at 6 months and the results were compared to those of a control group with no reinforcement (n=5) Animal weight was 25+5 kg at day 0 and 58+10 kg at 6 months and the reference aortic diameter increased from 14+1mm at day 0 to 17+2mm at 6 months. With no reinforcement, an instantaneous PA graft distension (27,4+2mm) was noted followed by an aneurysmal formation at 6 months (38+3mm). Reinforcement with scaffold polymer on polidioxanone allowed maintaining the PA graft diameter close to the reference value (17+2mm at day 0). Immunohistochemistry revealed MMP-9 overexpression indicating the induction of a matrix remodeling process that are not detectable in the control group. Mallory staining revealed elastin deposition in the reinforced PA in comparison to the collagen present in the non-reinforced group, reliably suggesting a shift towards an elastic remodeling and arterialization. PicroSirius red staining reveled in the control group collagen fibers non- homogeneously distributed with a increased cellularity indicating inflammatory infiltrates. The reinforced PA displays more organized and dense collagen fibers in the “elastic zone” of the vessel and less pronounced cellular infiltrate. In conclusion, bioresorbable external polydioxanone-based reinforcement allowed a structural rearrangement of PA autograft consisting of media reorganization with an increase in the elastic wall component. Such histological outcome arguably prevented autograft dilation and conferred enhanced mechanical properties on the PA wal