We previously demonstrated that porcine fibroblasts exposed to 5-aza-cytidine (5-aza-CR), an inhibitor of DNA methylation, increase their plasticity and can be re-addressed to pancreatic lineage. Here we investigate the mechanisms involved in the acquisition of a higher plasticity state by 5-aza-CR treated fibroblasts. Furthermore we characterized the cellular and molecular events driven by the differentiation protocol following thereafter. Cells were analyzed at different time points: untreated fibroblasts, after 5-aza-CR exposure and then on day 1-2-3-4-5-6-7 of pancreatic induction. DNA global methylation modifications were evaluated with an antibody against 5-Methylcytidine used for DNA dot blot analysis and immunolocalization studies. Cells were also immuno-characterized with primary antibodies against Vimentin, Oct4, Nanog, Sox17 and Hnf4, and gene expression level changes were evaluated in parallel. Our results show that 5-aza-CR induced a decrease in 5- Methylcytidine positivity that gradually returned to the levels observed in untreated fibroblasts within 3 days. Consistently, a down-regulation of vimentin was observed, together with an increase of Oct4 and Nanog, which remained clearly expressed until day 4 and were down-regulated thereafter. At the same time Sox17 and Hnf4, involved in the induction of definitive endoderm and primitive gut tube, respectively, displayed a reverse trend with a signal becoming gradually more intense. Our observations demonstrate that 5-aza-CR effect on DNA methylation is transient and initial levels are restored within 3 days. The combined and sequential action of the molecule with an induction protocol enables efficient inter-lineage conversion and controlled cell differentiation.
Mechanisms involved in inter-lineage conversion and differentiation of porcine fibroblasts / T.A.L. Brevini, G. Pennarossa, S. Maffei, F. Gandolfi. ((Intervento presentato al 9. convegno Congresso nazionale AMV tenutosi a Roma nel 2013.
|Titolo:||Mechanisms involved in inter-lineage conversion and differentiation of porcine fibroblasts|
PENNAROSSA, GEORGIA (Secondo)
GANDOLFI, FULVIO (Ultimo)
|Data di pubblicazione:||2013|
|Settore Scientifico Disciplinare:||Settore VET/01 - Anatomia degli Animali Domestici|
|Enti collegati al convegno:||Associazione italiana dei morfologi veterinari|
|Citazione:||Mechanisms involved in inter-lineage conversion and differentiation of porcine fibroblasts / T.A.L. Brevini, G. Pennarossa, S. Maffei, F. Gandolfi. ((Intervento presentato al 9. convegno Congresso nazionale AMV tenutosi a Roma nel 2013.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|