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Title: “Potential biological meaning and origin of cardiac progenitor cells isolated as Cardiospheres"
Tutor: Giacomello, Alessandro
Keywords: Cardiac stem cells
Issue Date: 2011
Abstract: Several populations of progenitor cells have been identified in the adult heart, based on the expression of specific surface markers (c-kit, Sca1), or on functional properties, such as the ability to spontaneously migrate from tissue explants and grow in three dimensional structures, called cardiospheres (CSs). CSs represent a niche-like microtissue with a cardiogenic gradient of differentiation towards the periphery. It is not clear which is the origin of these endogenous progenitor populations and how they function in vivo, given the limited regenerative capacity of the heart. Several possibilities can be envisioned. These cells could be remnants of the embryonic development, or derive from the de-differentiation of cardiomyocytes. To test this hypothesis we used a double transgenic mouse expressing the recombinase MerCreMer under the cardiomyocyte-specific αMyosin-Heavy-Chain promoter. After a pulse with 4OH-tamoxifen (TAM) the activated Cre removes a stop signal between two LoxP sequences, allowing the expression of the reporter gene LacZ, in a spatial and temporal regulated manner. Ideally with this system all the differentiated cardiomyocytes should be irreversibly labeled after TAM treatment, even if they subsequently undergo de-differentiation. B-gal positive cells were very rare in culture, as shown by x-gal staining and real time PCR, suggesting that cardiomyocytes de-differentiation is not the main mechanism underlying CSs formation. However, given the limited sensibility of this method (the efficiency of recombination in the cardiomyocytes is 80% at maximum), we cannot conclusively exclude the occurrence of this phenomenon. Using another transgenic mouse strain, in which epicardial and epicardial-derived cells are labeled, we observed a large number of positive cells at all culture stages. Our hypothesis, supported by RT-PCR data, is that epicardial-derived cells undergo mesenchyma-to-epithelium (MET) transition during cardiospheres formation. This is consistent with a recent report presenting MET as a crucial mechanism in cell reprogramming.
Research interests:  Study of the mechanisms involved in myocardial ischemic diseases and of the potential strategies to promote cardiac repair, enhancing endogenous sources of regeneration and providing committed progenitor cells
Skills short description: Reliable, motivated, goal-oriented PhD scientist, committed to team work, with experience in stem cells research and expertise on techniques required for stem cells isolation, characterization and differentiation.
Personal skills keywords: - Intra-peritoneal injection and oral gavage on mice; organs and embryos harvesting. - Preparation of primary cultures (cardiac explants) and growth of cardiac progenitor cells and mESCs. - Langendorff preparation to disperse cells from the heart. - Cloning and transfection of cells in culture. - Cellular characterization by Immunohistochemistry, Immunofluorescence and FACS sorting. - Proteins extraction and Western Blotting. - RNA and DNA extraction and purification. - PCR (Polymerase Chain Reaction)/ RT-PCR (Sybr Green and Taqman). - Bacterial cultures and bacterial transformation Good knowledge of Bioinformatic tools. Use of ImageJ, OligoExplorer, BLAST. Basic knowledge of CorelDraw, CLUSTALW, Rasmol v.2.6, Swiss Pdb Viewer, Clone, Gene Runner.

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