The main objectives of my research are the following:
1-muscle pathological ECM study with focus on rhabdomyosarcoma.
Rhabdomyosarcoma (RMS) is the most common and aggressive soft tissue sarcoma in childhood. Recently, the study of the tumor microenvironment and specifically of the extracellular matrix (ECM), highlighted the valuable role of the cross-talk between cells and their niche linking alteration of ECM composition to pathological outcomes. The interest in this new aspect starts widening the understanding of tumor progression and opens new avenues for developing innovative therapies. ECM obtained removing the cellular components from the native tissue by means of decellularization, represents the optimal 3D support for cell culture since the in vivo cancer microenvironment is recapitulated. This project already established a decellularization method to produce RMS ECM and start setting up recellularization of the obtained ECM, at first with human RMS cell line RH30 to set the recellularization conditions.
We will develop mainly two aspects:
- the 3D model will be further implemented with stromal cells (e.g. endothelial cells) to mimic the complexity of RMS TME and investigate, in a controlled fashion, the role of TME components during RMS malignant progression.
- ECM will be dissected to study both the proteins in the basal lamina and the glycoproteins present in the intercellular part with specific focus on glypicans.
Functional 3D model of rhabdomyosarcoma could help studying tumor development and could establish a platform for drug screening.
2- extracellular vesicles (EV) in regenerative medicine and muscle dysfunction models.
2.1-The need of new biomaterials to replenish the loss of muscle mass is currently a challenge. Indeed, after congenital malformations, trauma or tumor surgery the volume mass loss can be filled with synthetic materials already used in the clinical practice but the regain of function is still very difficult to reach. Nowadays the decellularization of tissues allows the obtainment of the highest biocompatible scaffold without the genetic material, such as the extracellular matrix (ECM). This biomaterial retains the biomechanical properties, proteins and biochemical factors that characterized the native tissue. Engineered tissues should actively integrate by inducing vascularization, cell recruitment and ECM production. On the other hand, adverse events such as foreign body response and fibrosis should also be prevented.
With the aim to improve the muscle condition/regeneration, the focus of the project is as follows:
- EVs from different cell sources (muscle precursor cells, amniotic fluid stem cells, cord blood mesenchymal stem cells, endothelial cells) will be considered and their biological activities evaluated. For instance, it is known that endothelial cell-derived EVs express VEGF favoring neo-angiogenesis.
- the presence of retained EVs in the decellularized matrix will be verified, and their biological activities in vitro will be evaluated.
- Investigation of:
- In vitro: the potential of including nanoparticles containing biological molecules produced by MSC-EVs in artificial scaffolds (engineered biomaterial);
- In vivo:
–mouse model of volume muscle loss. In our already set model of volume muscle loss, the integration of EV-modified ECM will be evaluated with specific attention toward fibrosis and angiogenesis process. Following the results, also the involved pathways will be studied.
In the future, in other muscle disease model (such as atrophy, cachexia, sarcopenia) the effect of engineered EV activity will be evaluated.
2.2-EV from healthy hMPC isolated from human biopsies will be compared with EV isolated from RMS cells (in normoxia and hypoxia). In vitro: the two EV groups in hMPC damaged with cardiotoxin will be tested and cell proliferation and differentiation will be analyzed. Genes and proteins devoted to muscle proliferation (Pax7, Ki67) and differentiation (MyoD, Myf5, MHC, Miogenin) will be analyzed by molecular biology, protein assay (IF, WB), cytofluorimetric methodology.
- Mattia Saggioro
- Stefania D’Agostino
- Federica Slaviero
- Fabio Magarotto
Selected PublicationsPozzobon M, Saggioro M, D'Agostino S, Bisogno G, Muraca M, Gamba P.. 2017. Alveolar Rhabdomyosarcoma Decellularization. Methods Mol Biol, doi: 10.1007/7651_2017_45
Bertin E, Piccoli M, Franzin C, Spiro G, Donà S, Dedja A, Schiavi F, Taschin E, Bonaldo P, Braghetta P, De Coppi P, Pozzobon M. 2016. First steps to define murine amniotic fluid stem cell microenvironment. Sci Rep, 15;6:37080
Bertin E, Piccoli M, Franzin C, Nagy A, Mileikovsky M, De Coppi P, Pozzobon M.. 2015. Reprogramming of mouse amniotic fluid cells using a PiggyBac transposon system. Stem Cell Research, 15(3): 510-3
Schiavo AA, Franzin C, Albiero M, Piccoli M, Spiro G, Bertin E, Urbani L, Visentin S, Cosmi E, Fadini GP, De Coppi P, Pozzobon M.. 2015. Endothelial properties of third-trimester amniotic fluid stem cells cultured in hypoxia. Stem. Cell Res Ther, 31;6:209
Piccoli M, Urbani L, Alvarez-Fallas ME, Franzin C, Dedja A, Bertin E, Zuccolotto G, Rosato A, Pavan P, Elvassore N, De Coppi P, Pozzobon M.. 2015. Improvement of diaphragmatic performance through orthotopic application of decellularized extracellular matrix patch. Biomaterials, 74:245-255
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