Novel Cell Lines Isolated From Mouse Embryonic Stem Cells Exhibiting De Novo Methylation of the E-Cadherin Promoter

Abstract

Mouse embryonic stem cells (mESCs) and epiblast stem cells represent the na€ ıve and primed pluripotent states, respectively. These cells self-renew via distinct signaling pathways and can transition between the two states in the presence of appropriate growth factors. Manipulation of signaling pathways has therefore allowed the isolation of novel pluripotent cell types such as Fibroblast growth factor, Activin and BIO-derived stem cells and IESCs. However, the effect of cell seeding density on pluripotency remains unexplored. In this study, we have examined whether mESCs 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 leukaemia inhibitory factor (LIF) induces decreased apoptosis and maintenance of pluripotency via Activin/Nodal, concomitant with loss of E-cadherin, Signal transducer and activator of transcription phosphorylation, and chimera-forming ability. These cells, E-cadherin negative proliferating stem cells (ENPSCs) can be reverted to a na€ ıve phenotype by addition of LIF or forced E-cadherin expression. However, prolonged culture of ENPSCs without LIF leads to methylation of the E-cadherin promoter (ENPSC M ), which cannot be reversed by LIF supplementation, and increased histone H3K27 and decreased H3K4 trimethylation. Transcript analysis of ENPSC M revealed a primed-like phenotype and their differentiation leads to enrichment of neuroectoderm cells. The generation of ENPSCs is similar to tumorigenesis as ENPSCs exhibit transcript alterations associated with neoplasia, hyperplasia, carcinoma, and metastasis. We therefore describe a novel cell model to elucidate the role of Ecadherin in pluripotency and to investigate epigenetic regulation of this gene during mESC differentiation and tumor metastasis.

The Role of E-Cadherin in Maintaining the Barrier Function of Corneal Epithelium after Treatment with Cultured Autologous Oral Mucosa Epithelial Cell Sheet Grafts for Limbal Stem Deficiency

“…These bindings increase cytoskeleton interaction with tight and gap junction proteins, thus increasing cell adhesion. Abundant literature shows that E-cadherin is involved in cell polarity [ 21 – 23 ], cell differentiation [ 24 , 25 ], and cell proliferation [ 26 ]. In the present study, we found that E-cadherin was significantly expressed in CAOMECS, at higher levels than normal healthy epithelial cells.…”

Cell signalling pathways underlying induced pluripotent stem cell reprogramming

“…Interestingly, human dermal fibroblasts (HDFs) have been shown to give rise to naïve human iPS cells when reprogrammed in the presence of LIF, FGF2 and TGFβ1 plus inhibitors of c-Jun NH2-terminal kinase, p38, MAPK and glycogen synthase kinase 3 (3i) [85] , thus demonstrating that the cell signalling context is critical to the determination of naïve and primed pluripotency rather than the two states representing a species difference. The derivation of various novel stem cell lines, including intermediate epiblast stem cells which exhibit dual responsiveness to LIF and ACTIVIN/NODAL signalling [86] , has challenged the concept of 2 distinct pluripotent states, instead suggesting that a spectrum of pluripotency exists, an idea we develop in Hawkins et al [87] . Thorough investigation into this spectrum of pluripotency, and therefore the transition from pluripotent cells to differentiated cells, should accelerate the delineation of mechanisms occurring throughout the reverse process, from a somatic cell to an iPS cell.…”

Novel peptides for deciphering structural and signalling functions of E-cadherin in mouse embryonic stem cells

“…We have previously shown that pluripotency is maintained in Ecad −/− mESCs and ENPS cells (mESCs exhibiting de novo methylation of the E-cadherin promoter) through the TGFβ signalling pathway 14 24 , specifically via Activin/Nodal, and that self-renewal is regulated via the FGFR1 pathway. However, mESCs treated with Epep or DECMA-1 did not induce Activin/Nodal-dependent pluripotency or FGFR1-dependent regulation of self-renewal despite ablation of STAT3 phosphorylation with both E-cadherin inhibitor treatments.…”

“… 19 have observed that disruption of E-cadherin mediated cell-cell contact does not influence many intracellular protein interactions with E-cadherin and concluded that most of these interactions are independent of cell-cell adhesion 19 . Therefore, it is possible that Epep and DECMA-1 inhibition of E-cadherin in mESCs does not disrupt cytoplasmic E-cadherin-protein interactions sufficiently for the switch to Activin/Nodal-dependent pluripotency and FGFR1-dependent self-renewal observed in Ecad−/− mESCs and ENPS cells 14 24 . Alternatively, this may reflect the activation of unknown signalling pathways capable of maintaining pluripotency and self-renewal in these cells.…”

“…Unless otherwise stated, mESC lines were grown on gelatinised tissue culture grade 6-well plates at 37 °C/5% CO 2 , as previously described 24 . mESCs cells were treated with 10 μM peptide in mESC culture medium which was replenished every 24 h. Inhibition of specific pathways in mESCs was achieved by incubation with 10 μM LY294002 (PI3K inhibitor), PD9805 (MEK inhibitor) and SB431542 (TGFβ1 R inhibitor).…”