MOLECULAR AND CELLULAR MECHANISMS REGULATING EPIGENETIC CELL CONVERSION

Phenotype definition is controlled by epigenetic regulations that allow cells to acquire their differentiated state. The process is reversible and cells can be driven back to a higher plasticity state with several different approaches, which include the interaction with the epigenetic set up through the use of epigenetic erasers, readers or writers. Among the many epigenetic modifiers presently available, in our previous experiments, we selected 5-azacytidine (5-aza-CR), which is a well-known DNA methyltransferase inhibitor, and has been previously shown to increase cell plasticity and facilitate phenotype changes in different cell types. Beside the epigenetic mechanisms driving cell conversion processes, growing evidences highlight the importance of mechanical forces that directly influence cell plasticity and differentiation. Aims of my PhD were: a) characterization of the molecular and cellular mechanisms regulating cell phenotype b) analysis of the possible relation between mechano-sensing and epigenetic control of cell plasticity and differentiation. The experiments carried out confirmed the global demethylating effect of 5-aza-CR, with a transient upregulation of pluripotency markers. At the same time increased transcription of TET2 and several histones was detected. This was accompanied by changes in enzymes controlling histone acetylation and cell morphological rearrangement. Interestingly, the study of DNA methylation profile and its regulatory genes, revealed that the use of a 3D micro-bioreactor promotes and stabilizes the maintenance of the acquired plasticity for a long period of culture. In particular, the use of an adequate soft substrate increased pancreatic conversion efficiency and induced the acquisition of a mono-hormonal phenotype, which is distinctive of terminally matured cell. Altogether these findings indicate that 5-aza-CR induced somatic cell transition to a higher plasticity state may be the result of multiple regulatory mechanisms that accompany the demethylating effect exerted by the modifier. The results described in my thesis also revealed that mechano-transduction-related responses modulate and maintain 5-aza- CR induced cell plasticity and significantly improve cell differentiation toward the pancreatic lineage