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Induced Pluripotent Stem Cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell-like state. Fibroblast (FB) is the adult somatic cell most commonly used for reprogramming and is defined by spindle-shape morphology, plastic-adherence and expression of several markers. Nonetheless, such properties also define Mesenchymal Stem Cells (MSCs), that also share with dermal FB the ability to differentiate into osteoblasts, adipocytes and chondroblasts. Moving from the hypothesis that the most striking difference between FBs and MSCs is in the name, we isolated and cultured human dermal and visceral FBs from tissue waste fragments of patients undergoing surgical procedures, and evaluated their morphology and expression of markers typical of MSCs, at gene and protein level. Additionally, we analyzed synthesis and release of specific growth factors in culture medium and, finally, we tested FB ability to differentiate into osteoblasts, adipocytes and chondroblasts. Interestingly, all FBs in culture adhered to plastic culture dishes, expressed markers typical of MSCs, like CD90, CD105, CD146,SSEA-4, ECM2, ID-1, and were morphologically undistinguishable. As reported for MSCs, FBs released EGF, FGF-4, GDNF, HGF, IGF, TGF-b and VEGF, and were capable of differentiating into cells of mesodermal origin. Our findings raise the question of whether FBs and MSCs are not admittedly the same population.We might infer that previously described differences are due to different stages of differentiation, which also account for reports on high variability in both expression of mesenchymal markers by MSCs and efficiency of FB reprogramming to iPSCs.

Extracellular matrix (ECM) is an intricate mesh of collagenous and non-collagenous proteins, whose presence and amount vary according to type of tissue. ECM drew the attention of regenerative medicine scientists as natural scaffold suitable for stem cell delivery into damaged tissues. Although a multitude of protocols and combinations of chemical agents and physical methods have been tested and proved effective in the decellularization of human heart, none of the ones tried in our setting fulfilled the goal of obtaining a structurally preserved cardiac decellularized ECM (d-ECM). While testing already described procedures, we made several adjustments that led to the development of a novel, simpler and robust protocol to decellularize adult human heart. Specifically, we decellularized cardiac samples of the free wall of both ventricles of adult human hearts scaled down to 40x28x6mm, to fit into embedding cassettes used to avoid stirring stress and preserve structure. To shorten the procedure, a combination of SDS, Triton X-100 and antibiotics was used in simple and fast two-step protocol. After decellularization, d-ECM was fixed and processed for histological study or snap-frozen for molecular biology analysis or cytocompatibility test in vitro. Histochemistry and immunoistochemistry confirmed the absence of nuclei and the preservation of architecture and composition of d-ECM. Further, while DNA content in d-ECM was well below accepted standards, sGAG, elastin and growth factors were retained and d-ECM scaffolds supported cardiac primitive cell engraftment and survival. Hence, according to our evidence, our protocol is simple, fast, effective and is worth improving for clinical translation.

Introduction the ultrasound prenatal diagnosis of aphakia is a difficult diagnosis and often requires a genetic study of the karyotype. Case report we present a rare case of prenatal bilateral aphakia, confirmed after bird. The patient was observed by ultrasound during the 23rd week of pregnancy. Through transabdominal ultrasound the lens could not be visualized bilaterally. The remaining anathomy, explorable by ultrasound, was still regular. When aphakia is suspected, genetic counseling is essential. Conclusion a differential diagnosis between aphakia and anophtalmia is necessary. A TORCH complex evaluation can be useful. Amniocentesis is always required.

The essential requirement for a successful myocardial tissue engineering is that scaffolds and tissue constructs exhibit biomimetic activity toward cells. Our aim was to investigate the significance of cell-matrix interactions in cardiac tissue microenvironment in vitro. Cardiac fibroblasts from normal (CF-N) and pathological (CF-P) hearts with ischemic cardiopathy deposited extracellular matrix (Mx) and conditioned medium (Cm), which were then characterized by immunohistology, immunoblotting, ELISA-based protein array or mass spectrometry. Normal and pathological cardiac primitive cells CPC-N and -P) were cultured on CF-Mx-N and -P or in CF-Cm-N and -P and their proliferation, apoptosis, migration and maturation were evaluated by BrdU, TdT, scratch wound assays or RT-PCR, respectively. Laminin-1 and tenascin-X were detected in CF-Mx-P. The expression of IGFBP1, Il-6, EGF was higher in CF-Cm-P, while that of SCF, TGF-?3, BMP4 was higher in CF-Cm-N. Migration speed of CPC-P peaked at 21.3 ?m/h on CF-Mx-N. Proliferation of CPCs increased significantly on CF-Mx-P, while apoptosis diminished on both types of matrix. In CPC-N, maturation toward cardiomyocytes was activated by CF Mx-N and toward endothelial cells by CF-Mx-P; in CPC-P the trend toward smooth muscle cell-lineage was evident on both substrates. CF-Cm-N reduced apoptosis of CPC-N (2-fold), while CF-Cm-P reduced apoptosis of CPC-P (3.5-fold) and increased proliferation of CPC-N (1.6-fold, n=3, p

According to recent findings, pathological processes may deplete adult heart of stem cells. The scope of the present study was to assess proliferation, apoptosis and commitment of cardiac stem cells (CSCs) resident in normal (n=11) and pathological hearts with ischemic cardiomyopathy (n=20). Immunofluorescence and immunoblotting of CSCs isolated from normal and pathological hearts revealed higher expression of cardiac-specific markers in the latter. Proliferation of CSCs isolated from pathological hearts was 3.2±0.7-fold higher, while apoptosis induced by oxidative stress was 1.8±0.2-fold higher than in the normal cells. Asymmetric division, with unequal Notch distribution between daughter cells, occurred frequently in CSCs from ischemic hearts, while symmetric division was typical of normal cells. Gene expression was examined by stem cells specific PCR-based microarray. We confined our analysis to genes with at least 1.7-fold differential expression. Genes downregulated in pathological hearts belonged to the early stages of developmental process (snail1 homolog, jagged-1, ephrin b1), cell cycle control (mdm2, p15 and p16), neurogenesis, skeletal development, bone remodeling and cartilage development functional classes. Upregulated genes, among which OTF2, endothelin receptor, CD105 and MRG1, were involved in developmental maturation, mesenchymal cell differentiation, heart development and circulatory system processes. Major finding emerging from the analysis was the activation of the epithelial-mesenchymal transition and the upregulation of its inducer, TGFβ. The expression of genes related to TGFβ BMP-mediated signal transduction pathway was examined by PCR Array. The analysis confirmed the upregulation of genes as TGFβ-1 and -3, Nodal, ID1 and c-Myc, suggesting the activation of mesenchyme formation and CSCs differentiation towards cardiac cell lineages. In conclusion, CSCs in the normal and in the pathological heart differ in several respects and the differences reflect the activation of cardiac stem cells pool in the chronic pathological conditions. Given the apparent failure of intrinsic heart regeneration, further studies are warranted to optimize the strategies for CSCs application in regenerative medicine.

The precise spatial and temporal expression of extracellular matrix proteins and their receptors, integrins, is critical for proper heart structure and cardiac cell function. We have analyzed and compared the distribution of CD117 positive cells in the human adult normal (n=6) and pathological hearts with chronic ischemic cardiomyopathy (n=10), in relation to the expression and localization of laminin and integrin isoforms. Examination of tissue sections by immunofluorescence revealed that the left atrium contained the highest number of CD117 (+) cells. Moreover, their number in the diseased hearts was significantly higher than in the normal hearts (about 10-fold higher in the mid section of the left ventricle and 6-fold higher in the apex). Within the same heart, CD117 (+) cells were up to 57-fold more numerous in the subepicardium than in the myocardium. The presence of laminin α1 chain, typical for developing myocardium, was observed in the subepicardium and interstitial spaces of the normal hearts and in the subepicardium and endomysium in the pathological hearts, where the branches of laminin-1 were also spread between the subepicardial space and the main myocardium, co-localizing with CD117 (+) α6 integrin (+) cells. While most of the cells in the subepicardium expressed also α6 integrin subunit, within the myocardium α6 integrin was detected on about 50% of CD117 (+) cells in the diseased hearts. In the normal hearts, the percentage of α6 integrin (+) cells was always much lower than in the pathological conditions and identical between the subepicardium and myocardium. The expression of laminin α1β1 chains detected by immunoprecipitation and western blot in the extracts of atria was always higher in the normal hearts, while within the mid-section of the left ventricle it was higher in the pathological hearts. In vitro, laminin-1 coating of the culture dish stimulated migration and proliferation of the cardiac CD117 (+) cells and their migration was inhibited in the presence of the antibodies blocking α6 integrin function. We conclude that chronic post-ischemic remodeling of the heart involves the mechanisms aiming at the regeneration of the myocardium and affecting all: cardiac CD117 (+) cells, extracellular matrix proteins and their receptors. Our observations support the hypothesis that the regeneration process follows the mechanisms operative in the early heart development, during which the epicardiumderived cells give rise to the cells of cardiac lineages and laminin-1 guides primitive cells in their migration from the subepicardial space into the myocardium.

Commercially pure titanium implants presenting a relatively smooth, machined surface or a roughened endosseous surface show a large percentage of clinical success. Surface properties of dental implants seem to affect bone cells response. Implant topography appears to modulate cell growth and differentiation of osteoblasts affecting the bone healing around the titanium implant. The aim of the present study was to examine the effects of 1cm diameter and 1mm thick titanium disks on cellular morphology, adhesion and bone phenotypic expression of human osteoblast-like cells, SaOS-2. SaOS-2 cells were cultured on commercially 1 cm pure titanium disks with three different surface roughness: smooth (S), sandblasted (SB) and titanium plasma sprayed (TPS). Differences in the cellular morphology were found when they were grown on the three different surfaces. An uniform monolayer of cells recovered the S surface, while clusters of multilayered irregularly shaped cells were distributed on the rough SB and TPS surfaces. The adhesion of SaOS-2 cells, as measured after 3h of culture, was not affected by surface roughness. ECM components such as Collagen I (CoI), Fibronectin (FN), Vitronectin (VN) and Tenascin (TN) were secreted and organized only on the SB and TPS surfaces while they remained into the cytoplasm on the S surfaces. Osteopontin and BSP-II were largely detected on the SB and TPS surfaces, while only minimal production was observed on the S ones. These data show that titanium surface roughness affects bone differentiation of osteoblast like-cells, SaOS-2, indicating that surface properties may be able to modulate the osteoblast phenotype. These observations also suggest that the bone healing response around dental implants can be affected by surface topography.

We investigate the boundary stabilization of a particular subset of 3×3 linear hyperbolic systems with varying coefficients on a bounded domain. The system is underactuated since only one of the three hyperbolic PDEs is actuated at the boundary. The setup considered in the paper occurs in control of multiphase flows on oil production systems. We use a backstepping approach to design a full-state feedback law yielding exponential stability of the origin.

OBJECTIVE: . To investigate the in vitro effect of granulocyte macrophage-colony stimulating factor (GM-CSF) on the deposition of extracellular matrix (ECM) in fibroblasts obtained from the skin of patients with systemic sclerosis (Ssc), compared to healthy controls. METHODS: Dermal fibroblasts obtained from 14 patients with SSc (7 with the diffuse form and 7 with CREST syndrome) and from 7 controls were studied. Both SSc and normal skin fibroblast cultures were stimulated for 4 and 8 days with 100 ng/ml GM-CFS. GM-CSF receptor (GM-CSFR) expression was determined by Western blot of cell lysates. Immunofluorescence was used to determine GM-CSFR expression and to investigate the deposition of ECM (type I collagen, fibronectin, and tenascin). Quantitative analysis of ECM was performed by ELISA. Expression of type I collagen and metalloproteinase 1 (MMP-1) mRNA was determined by real-time quantitative PCR. RESULTS: Deposition of ECM by normal fibroblasts appeared not to be influenced by stimulation with GM-CSF; in contrast, after stimulation with GM-CSF SSc fibroblasts showed increased deposition of fibronectin and tenascin, while type I collagen production was decreased; these results were found with both immunofluorescence and ELISA. Quantitative PCR revealed that GM-CSF inhibited the expression of mRNA type I collagen in SSc fibroblasts but not in normal fibroblasts, whereas levels of the main collagenolytic enzyme, MMP-1, were not affected. CONCLUSION: These results suggest that in SSc fibroblasts GM-CSF exerts a blocking effect on the deposition of type I collagen, through an inhibitory action on mRNA, while the production of other components of ECM such as fibronectin and tenascin is increased by stimulation with this cytokine.