194 EPIGENETIC REMODELING OF ADULT SOMATIC CELLS

Abstract

Mammalian differentiation is obtained through epigenetic regulations that shape the genome, which is identical in all cells, to distinct phenotypes and tissue specific identities. The differentiated state of mature cells in an adult organism is therefore acquired through epigenetic restrictions that lead to a gradual loss of differentiative potency. In agreement with this, recent experiments demonstrate that terminally differentiated cells can be induced to de-differentiate in vitro and increase their plasticity in response to epigenetic modifiers that are capable of reverting cells from their lineage commitment to a more plastic state. Here we describe experiments where we prepared porcine skin fibroblasts and granulosa primary cultures and exposed them to an inhibitor of DNA methylation, the 5-aza-cytidine (5-aza-CR), to increase cell plasticity. Taking advantage of the obtained increased permissivity window, we investigated the ability of 5-aza-CR treated cells to respond to specific differentiation conditions and be re-addressed to a different cell lineage either within the same germ layer or to a different germ layer. Cells were evaluated for their morphological changes and assessed using RT-PCR and immunocytochemical studies during the treatment. Following the exposure to 5-aza-CR the phenotype of both cell types changed. Treated cells displayed an oval or round shape, and appeared smaller with larger nuclei and granular and vacuolated cytoplasm. This was accompanied by an active expression of the main pluripotency-related genes OCT4, NANOG, SOX2, and REX1, originally undetectable in untreated fibroblasts and granulosa cells. 5-aza-CR treated granulosa cells cultured with recombinant human vascular endothelial growth factor to induce myogenic specification (different lineage within the same germ layer) suppressed the expression of granulosa specific marker (Cytokeratin) as well as of the pluripotency genes, and expressed MYOD, MYF5, and MYOG (earliest myogenic markers that are involved in the coordination of skeletal muscle development or myogenesis). In order to trans-differente 5-aza-CR treated fibroblasts to cells of a different germ layer, they were exposed to activin A to promote endoderm commitment. Cells down-regulated Vimentin (fibroblast marker) as well as pluripotent gene expression and transcribed Nestin (transiently involved in multi-lineage progenitor cell differentiation), SOX17, FOXA2 (induction of definitive endoderm), and HNF4A, HNF1 (primitive gut tube specific genes). Altogether these results suggest that it is possible to obtain a direct inter-lineage conversion by removing epigenetic restriction, using demethylating agents such as 5-aza-CR, and avoiding a stable pluripotent state. This novel approach may represent a promising tool for regenerative medicine because it does not involve the use of any transgenic modifications, retroviral transfection, or both.

Supported by Network Lombardo iPS (NetLiPS) Project ID 30190629.