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|Autori: ||Minonzio, Greta Maria|
|Tutor interno: ||CAMPOMENOSI, PAOLA|
|Co-tutore: ||SOLDATI, GIANNI|
|Titolo: ||Cardiomyogenic differentiation of human multipotent stromal cells: a new approach fore regenerative medicine in cardiovascular disease.|
|Abstract: ||Cardiovascular disease (CVD), which include hypertension, coronary heart disease (CHD), stroke, and congestive heart failure (CHF), are the leading and most common causes of mortality in both developing and industrialized countries, accounting for one third of all deaths globally.
Cardiomyocyte regeneration is limited in adult life and therefore the identification of a putative source of progenitors is of great interest to provide usable in vitro models and new perspectives for regenerative therapies. This appears to be a challenge due to the peculiar functional and anatomical heart properties. The goal of this project was to differentiate adult human Adipose tissue‐derived multipotent Stem Cells (ASCs) into cardiomyocytes or their close progenitors. Thus, the ultimate aim is to develop stem cell-based therapies for the cure of cardiovascular disease. More specifically, we investigated the application of two innovative approaches: the use of human growth factors in a well defined serum-free culture medium and the application of electrical and mechanical stimuli to mimic the physiological environment of the heart.
First of all, we developed a new protocol for the isolation of ASCs according with the standard GMP-procedure, which allows to extract a substantial number of cells for clinical application. The starting material was obtained from subcutaneous adipose tissue collected during an elective liposuction procedure. The isolated ASCs were characterized by flow cytometry and expanded until second passage. Several different cocktails of growth factors, cytokines and several culture conditions were tested for their ability to induce cardiomyogenesis. The goal was to identify the right combination of those factors and their time of application. One of the cardiogenic cocktails tested was indeed very effective in inducing the selected cardiac markers (e.g. NKX2-5 and MEF2C) and was chosen for the second part of this research work. The differentiation obtained was highly reproducible, as it was observed in 15 out of 15 samples from different patients. Interestingly, non induced ASCs expressed half of the selected cardiac markers (i.e. TBX5, GATA4 and BAF60C), suggesting the high Abstract V intrinsic potential and the inherent propensity of ASCs to differentiate into cardiac precursors. We further analysed the morphological changes that ASCs underwent during their differentiation by use of electron microscopy and immunofluorescence staining. By treating the cells with our selected induction cocktail, clear changes in their morphology were observed: the cells assumed a fusiform shape and became oriented.
The last milestone was to understand if the combination of the cardiac induction cocktail with a mechanical (and eventually electrical) stimulation on a scaffold would increase the efficiency of cardiogenic differentiation. For this, a customized device has been developed by the Swiss Stem Cell Foundation in collaboration with Scuola Universitaria Professionale della Svizzera Italiana (SUPSI) for the application of controlled mechanical and/or electrical stimuli to a scaffold over a desired lap of time. The scaffolds used were developed and characterized by Prof. Ciardelli’s Research Group (Materials in Bionanotechnology, Department of Mechanical and Aerospace Engineering, Politecnico di Torino). The preliminary results showed that the combination of growth factors, cytokines and mechanical stimulation for 10 days led to an at least 2 fold increase in mRNA expression of NKX2‐5, MEF2C, HAND2 and MHC when compared to the induction with cardiogenic cocktail alone on a simple 2D culture condition.
In conclusion, in this work we present a new protocol for the extraction of ASCs starting from adipose lipoaspirates, a new serum‐free based cardiogenic induction cocktail for ASCs and preliminary data about the differentiation in scaffold enhanced by mechanical stimulation opening a new perspective to cardiac regeneration.|
|Parole chiave: ||Adipose tissue, multipotent stromal cells, cardiovascular disease.|
|MIUR : ||BIO/11 BIOLOGIA MOLECOLARE|
|Corso di dottorato: ||Biologia Cellulare e Molecolare|
|Ciclo di dottorato: ||26|
|Università di conseguimento titolo: ||Università degli Studi dell'Insubria|
|Citazione: ||Minonzio, G.M.Cardiomyogenic differentiation of human multipotent stromal cells: a new approach fore regenerative medicine in cardiovascular disease. (Doctoral Thesis, Università degli Studi dell'Insubria, 2013).|
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