To exploit pluripotent cells for regenerative medicine applications it will be necessary to understand the molecular mechanisms that govern pluripotency and lineage commitment within these cells. The mechanism by which LIF sustains ‘naïve’ pluripotency in mouse embryonic stem (mES) cell has recently been delineated; LIF signals to the core circuitry of pluripotency (Oct4, Sox2 and Nanog) via Jak/STAT3 and PI3K/Akt-mediated expression of Klf4 and Tbx3 respectively. E-cadherin has been shown to be required for LIF-dependent mES cell pluripotency since cell lines exhibiting low/no E-cadherin expression maintain pluripotency via Activin/Nodal. However, these cells maintain expression of the core circuitry of pluripotency, thus the role of E-cadherin in pluripotency remains elusive. To investigate this, we have characterised an E-cadherin negative proliferating stem (ENPS) cell line, generated by seeding wt mES cells at low density in the absence of LIF. These cells exhibit Activin/Nodal-dependent pluripotency marker expression but fail pluripotency tests such as EB differentiation and chimera generation and microarray analysis shows they lack naïve transcripts and express early lineage markers. We have also exploited two additional E-cadherin negative cell lines (Ecad-/- and EcadRNAi mES cells) to delineate the molecular mechanisms connecting E-cadherin to the core circuitry of pluripotency. These cells exhibit decreased expression of pluripotency markers Klf4 and Nanog, the latter a direct consequence of a lack of E-cadherin-mediated STAT3 activation. Interestingly, both ENPS and Ecad-/- mES cells can be ‘rescued’ to a naïve pluripotent state upon LIF stimulation. In ENPS cells, LIF supplementation induces restoration of E-cadherin expression, LIF-dependent pluripotency and EB and chimera generation abilities. In Ecad-/- mES cells, LIF supplemention restores LIF-dependent pluripotency via N-cadherin, thus demonstrating a novel role for N-cadherin in mES cell pluripotency. At high passage, ENPS cells (like some cancer cells) exhibit methylation of the E-cadherin promoter and PI3K-dependent increased survival compared to wt mES cells. Our findings provide a potential mechanism for the role of E-cadherin in induced pluripotent stem (iPS) cell generation, since STAT3 phosphorylation has recently been shown to be a limiting factor in this process. In addition, our data suggest E-cadherin can be manipulated to direct differentiation for regenerative medicine applications since ENPS cells exhibit a lineage bias towards neuroectoderm at the expense of endoderm specification.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:574339 |
| Date | January 2013 |
| Creators | Hawkins, Kate |
| Contributors | Merry, Catherine; Ward, Christopher |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-role-of-ecadherin-in-mouse-embryonic-stem-cell-pluripotency(45736e61-3fcf-45ac-ba69-3d3c89ce5105).html |
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