Morphological and molecular changes of pig skin fibroblasts exposed to 5-AZA cytidine and addressed to subsequent pancreatic differentiation

Recent experiments demonstrate that terminally differentiated cells can be induced to de-differentiate in vitro and increase their plasticity in response to synthetic molecules capable of reverting cells from their lineage commitment to a more pluripotent state. In response to specific conditions de-differentiated cells can then be re-addressed to a different cell type. However only scattered information are available on the morphological changes and the ultrastructural remodelling required when cells transit along the differentiation pathways, adopting the modifications needed in order to adequately adapt to a different and specific state. In the present study we prepared skin fibroblast primary cultures and exposed them to 5-aza-cytidine (5-aza), an inhibitor of DNA methylation, to increase cell plasticity. We then investigated the ability of 5-aza treated cells (5-azatC) to be re-addressed to pancreatic beta cell-like cells (r-betaC), in response to specific differentiation conditions. Ultra-structural modifications were evaluated in parallel with molecular analysis for the expression of differentiation stage-specific markers. Elucidation of the morphological changes that take place in cells undergoing de-differentiation events is essential for a better understanding of the biology of these process paving the way to the successful use of stem cells in regenerative medicine and tissue replacement therapies.