Regenerative Medicine 1

Research title: 

Use of defined three-dimensional microenvironments to induce and maintain pluripotency and boost epigenetic conversion of adult cells

Tutor: Prof. Tiziana A.L. Brevini

 

Contact details

Prof Tiziana A.L. Brevini

Department of Health, Animal Science and Food Safety (VESPA)

University of Milan, Via Celoria 10, 20133 Milan, Italy

Tel. +39 02 50317970

e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

 

State of the art

Regenerative Medicine represents a promising approach to manage health problems, enabling the damaged tissues and organs to restore optimal function and integrity. However, in order to translate the present basic knowledge from the laboratory bench to the clinical application, we need solid in vitro models. In an organ or tissue, cells are surrounded by 3-D organization of ECM and neighbouring cells. Anchoring of cells to surrounding matrix is responsible for physical attachment and for transducing chemical and mechanical signals from the outer environment (1). Dimension in which cells are cultured is therefore crucial for fate determinant. The 3-D offers the possibility to explore the mechanisms underlying the process of tissue formation (migration, proliferation, adhesion, differentiation and apoptosis). It also favours cell-cell and cell-matrix interaction that may otherwise be precluded in monolayer culture. Furthermore, it also facilitates biological responses that might not be observable on 2-D substrates. One important contribution for the “closer-to-in vivo” behaviour of cells when grown as 3-D cultures is the matrices that are used for obtaining such cultures (2). More than 100 types of matrices are being used at present. The choice of such matrices is based on cell type and the nature of the study. The most commonly used are agarose, collagen, polyacrylamide, fibronectin, gelatin, laminin, and vitronectin. They all provide a biologically active environment for the cells to proliferate, differentiate and secrete cell specific extracellular matrix which can potentially be used for a variety of applications.

 

Janmey, P.A., et al., Annu Rev Biomed Eng. 2007..

Ravi, M., et al., Cytotechnology. 2014. 

 

brevini1

 

brevini2brevini3

Aims of the project

The project is designed to evaluate the use of substrate with different stiffness and 3D organization and investigate their impact on the acquisition of pluripotency and on the induction of a distinct adult differentiated cell phenotype.

The study will focus on five main aspects:

1)    the effect of matrix elasticity on the levels of DNA methylation and the expression of pluripotency related genes in cell exposed to epigenetic erasing;

2)    the interaction between a three-dimensional set-up and the epigenetic definition of cell fate;

3)    the ability of 3D cell rearrangement to induce and maintain a high plasticity state in erased cells. To this end we will be testing selected hydrophobic synthetic compound, to produce a facile and efficient micro-bioreactor;

4)    the impact of the selected stiffness substrates on the acquisition of a terminal epigenetic signature and a differentiated phenotype. In particular the project will assess the effect of 3D culture and substrate elasticity on differentiation efficiency towards a selected cell type and on the presence of markers related to mature terminal differentiation;

5)    in order to better understand the mechanisms involved in cell response to matrix elasticity, the project will analyze the activation of molecular pathways known to be related to mechano-transduction signalling and the possible crosstalk activated by the 3D rearrangement through exosome release.

 

Recent publications of the tutor in the field

1. Pennarossa G, Maffei S, Campagnol M, Tarantini L, Gandolfi F, Brevini TA. Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells. Proc Natl Acad Sci U S A. 2013 May 28;110(22):8948-53. doi: 10.1073/pnas.1220637110. Epub 2013 May 21.

2. Pennarossa G, Maffei S, Campagnol M, Rahman MM, Brevini TA, Gandolfi F. Reprogramming of pig dermal fibroblast into insulin secreting cells by a brief exposure to 5-aza-cytidine. Stem Cell Rev. 2014 Feb;10(1):31-43. doi: 10.1007/s12015-013-9477-9.

3. Brevini TA, Pennarossa G, Rahman MM, Paffoni A, Antonini S, Ragni G, deEguileor M, Tettamanti G, Gandolfi F. Morphological and molecular changes of human granulosa cells exposed to 5-azacytidine and addressed toward muscular differentiation. Stem Cell Rev. 2014 Oct;10(5):633-42. doi: 10.1007/s12015-014-9521-4.

4. Brevini TA, Pennarossa G, Acocella F, Brizzola S, Zenobi A, Gandolfi F. Epigenetic conversion of adult dog skin fibroblasts into insulin-secreting cells. Vet J. 2016 May;211:52-6. doi: 10.1016/j.tvjl.2016.02.014. Epub 2016 Mar 4.

5. Manzoni EF, Pennarossa G, deEguileor M, Tettamanti G, Gandolfi F, Brevini TA. 5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts. Sci Rep. 2016 Nov 14;6:37017. doi: 10.1038/srep37017.