Novel cell lines isolated from mES cells exhibiting de novo methylation of the E-cadherin promoter.

Mouse embryonic stem (mES) cells and epiblast stem cells (EpiSCs) represent the na�ve and primed pluripotent states respectively. These cells self-renew via distinct signalling pathways and can transition between the two states in the presence of appropriate growth factors. Manipulation of signalling pathways has therefore allowed the isolation of novel pluripotent cell types such as FAB-SCs and IESCs. However, the effect of cell seeding density on pluripotency remains unexplored. In this study we have examined whether mES cells can epigenetically regulate E-cadherin to enter a primed-like state in response to low cell seeding density. We show that low density seeding in the absence of LIF induces decreased apoptosis and maintenance of pluripotency via Activin/Nodal, concomitant with loss of E-cadherin, STAT3 phosphorylation and chimera-forming ability. These cells, E-cadherin negative proliferating stem (ENPS) cells, can be reverted to a na�ve phenotype by addition of LIF or forced E-cadherin expression. However, prolonged culture of ENPS cells without LIF leads to methylation of the E-cadherin promoter (ENPS(M) cells), which cannot be reversed by LIF supplementation, and increased histone H3K27 and decreased H3K4 tri-methylation. Transcript analysis of ENPS(M) cells revealed a primed-like phenotype and their differentiation leads to enrichment of neuroectoderm cells. The generation of ENPS cells is similar to tumorigenesis as ENPS cells exhibit transcript alterations associated with neoplasia, hyperplasia, carcinoma and metastasis. We therefore describe a novel cell model to elucidate the role of E-cadherin in pluripotency and to investigate epigenetic regulation of this gene during mES cell differentiation and tumor metastasis. Stem Cells 2014.