AFM nano-mechanical study of the beating profile of hiPSC-derived cardiomyocytes beating bodies WT and DM1.

Myotonic Dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, characterized by a variety of multisystemic features and associated with cardiac anomalies. Among cardiac phenomena, conduction defects, ventricular arrhythmias, and dilated cardiomyopathy represent the main cause of sudden death in DM1 patients. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful in vitro model for molecular, biochemical, and physiological studies of disease in the target cells. Here, we used an Atomic Force Microscope (AFM) to measure the beating profiles of a large number of cells, organized in CM clusters (Beating Bodies, BBs), obtained from wild type (WT) and DM1 patients. We monitored the evolution over time of the frequency and intensity of the beating. We determined the variations between different BBs and over various areas of a single BB, caused by morphological and biomechanical variations. We exploited the AFM tip to apply a controlled force over the BBs, to carefully assess the biomechanical reaction of the different cell clusters over time, both in terms of beating frequency and intensity. Our measurements demonstrated differences between the WT and DM1 clusters highlighting, for the DM1 samples, an instability which was not observed in WT cells. We measured differences in the cellular response to the applied mechanical stimulus in terms of beating synchronicity over time and cell tenacity, which are in good agreement with the cellular behavior in vivo. Overall, the combination of hiPSC-CMs with AFM characterization can become a new tool to study the collective movements of cell clusters in different conditions and can be extended to the characterization of the BB response to chemical and pharmacological stimuli.

Epidermal growth factor-like domain 7 promotes migration and invasion of human trophoblast cells through activation of MAPK, PI3K and NOTCH signaling pathways.

Epidermal growth factor-like domain 7 (Egfl7) is a gene that encodes a partially secreted protein and whose expression is largely restricted to the endothelia. We recently reported that EGFL7 is also expressed by trophoblast cells in mouse and human placentas. Here, we investigated the molecular pathways that are regulated by EGFL7 in trophoblast cells. Stable EGFL7 overexpression in a Jeg3 human choriocarcinoma cell line resulted in significantly increased cell migration and invasiveness, while cell proliferation was unaffected. Analysis of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways showed that EGFL7 promotes Jeg3 cell motility by activating both pathways. We show that EGFL7 activates the epidermal growth factor receptor (EGFR) in Jeg3 cells, resulting in downstream activation of extracellular regulated kinases (ERKs). In addition, we provide evidence that EGFL7-triggered migration of Jeg3 cells involves activation of NOTCH signaling. EGFL7 and NOTCH1 are co-expressed in Jeg3 cells, and blocking of NOTCH activation abrogates enhanced migration of Jeg3 cells overexpressing EGFL7. We also demonstrate that signaling through EGFR and NOTCH converged to mediate EGFL7 effects. Reduction of endogenous EGFL7 expression in Jeg3 cells significantly decreased cell migration. We further confirmed that EGFL7 stimulates cell migration by using primary human first trimester trophoblast (PTB) cells overexpressing EGFL7. In conclusion, our data suggest that in trophoblast cells, EGFL7 regulates cell migration and invasion by activating multiple signaling pathways. Our results provide a possible explanation for the correlation between reduced expression of EGFL7 and inadequate trophoblast invasion observed in placentopathies.

Human embryonic stem cells recover in vivo acute lung inflammation bleomycin-induced.

Idiopathic pulmonary fibrosis (IPF) is characterized by alveolar epithelial cell injury, type II cell activation, apoptosis and bronchiolar epithelial cell proliferation, accumulation of extracellular matrix and fibroblasts. No current animal model recapitulates all of these cardinal manifestation of the human disease. However, bleomycin instillation in mice lung by intranasal way (ITN) represents the best experimental model of pulmonary fibrosis in which alveolar pneumocytes type II (ATII) are usually depleted. The aim of this study was to test the possibility to recover acute lung fibrosis after transplantation of human embryonic type II derived-pneumocytes in a murine model of bleomycin-induced damage. Our results indicate the striking "clinical" beneficial effect of differentiated HUES-3 cells into ATII in terms of lung function, weight loss and mortality in injured mice, suggesting this stem cell therapy as a promising, systemic and specific treatment of human pulmonary fibrosis.

Rescue of murine silica-induced lung injury and fibrosis by human embryonic stem cells.

Alveolar type II pneumocytes (ATII cells) are considered putative alveolar stem cells. Since no treatment is available to repair damaged epithelium and prevent lung fibrosis, novel approaches to induce regeneration of injured alveolar epithelium are desired. The objective of this study was to assess both the capacity of human embryonic stem cells (HUES-3) to differentiate in vitro into ATII cells and the ability of committed HUES-3 cells (HUES-3-ATII cells) to recover in vivo a pulmonary fibrosis model obtained by silica-induced damage. In vitro differentiated HUES-3-ATII cells displayed an alveolar phenotype characterised by multi-lamellar body and tight junction formation, by the expression of specific markers such as surfactant protein (SP)-B, SP-C and zonula occludens (ZO)-1 and the activity of cystic fibrosis transmembrane conductance regulator-mediated chloride ion transport. After transplantation of HUES-3-ATII cells into silica-damaged mice, histological and biomolecular analyses revealed a significant reduction of inflammation and fibrosis markers along with lung function improvement, weight recovery and increased survival. The persistence of human SP-C, human nuclear antigen and human DNA in the engrafted lungs indicates that differentiated cells remained engrafted up to 10 weeks. In conclusion, cell therapy using HUES-3 cells may be considered a promising approach to lung injury repair.

Cellular genetic therapy.

Cellular genetic therapy is the ultimate frontier for those pathologies that are consequent to a specific nonfunctional cellular type. A viable cure for there kinds of diseases is the replacement of sick cells with healthy ones, which can be obtained from the same patient or a different donor. In fact, structures can be corrected and strengthened with the introduction of undifferentiated cells within specific target tissues, where they will specialize into the desired cellular types. Furthermore, consequent to the recent results obtained with the transdifferentiation experiments, a process that allows the in vitro differentiation of embryonic and adult stem cells, it has also became clear that many advantages may be obtained from the use of stem cells to produce drugs, vaccines, and therapeutic molecules. Since stem cells can sustain lineage potentials, the capacity for differentiation, and better tolerance for the introduction of exogenous genes, they are also considered as feasible therapeutic vehicles for gene therapy. In fact, it is strongly believed that the combination of cellular genetic and gene therapy approaches will definitely allow the development of new therapeutic strategies as well as the production of totipotent cell lines to be used as experimental models for the cure of genetic disorders.