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Title: Investigating the regulation of distinct epigenetic states in human embryonic stem cells
Author: Autio, Matias
ISNI:       0000 0004 2736 8438
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Accumulating evidence suggest that pluripotency – the ability to generate all somatic cell types – is not a fixed state. Pluripotent cell populations encompass a heterogeneous group of cells with different phenotypic and functional properties in equilibrium. At least two phases of pluripotency, immature and primed for differentiation, have been identified during early mouse development, and these are typified by the two distinct stem cell populations – mouse embryonic (mES) and epiblast-­‐derived (mEpiS) stem cells – isolated from epiblast layers of pre-­‐ and post-­‐implantation embryo, respectively. Many lines of human ES (hES) cells have also been established in vitro from the inner cell mass of pre-­‐ implantation blastocysts yet the precise in vivo lineage affiliation of these cells remains largely unresolved. Profiling chromatin in a particular cell line has proven to be a valuable signature for cell identity and developmental stage. One approach has been to assay the timing of DNA replication during S-­‐phase of the cell cycle across a panel of loci, as an indicator of chromatin accessibility. This replication timing profiling was notably capable of discriminating pluripotent mES cells from cells with a more restricted differentiation capacity. This study sought to address whether distinct pluripotent states could be reliably discriminated at the chromatin level. In particular, the replication timing profiles of a number of hES cell lines were characterised and compared to those of mES and mEpiS cell lines derived from different genetic backgrounds. Profiles of undifferentiated H1, H7 and H9 hES cell lines typically harboured an increased proportion of late-­‐replicating loci during S-­‐ phase when compared to mES cells, which were confirmed to have a steady and mostly early-­‐replicating profile regardless of their genetic background. Moreover, hES cell replication profile greatly varied between cultures and cell lines; a level of replication timing variability also observed among mEpiS cells, as opposed to mES cells. These results highlight that hES and mEpiS cells most likely share a common unstable epigenetic state or transitional state primed on the verge of differentiation. The epigenetic state of hES cell lines was further interrogated by analysing cells grown under two different culture conditions, both however similarly relying on TGF-­‐β/Activin signalling. Results demonstrated that hES cells could adopt distinct yet reversible epigenetic signatures while retaining their pluripotency. In particular, extensive and dynamic shifts of replication timing, from late-­‐to-­‐early, were consistently observed at many target loci in hES cells as well as in human induced pluripotent cells (iPS) cells, upon increased SMAD2/3-­‐associated P300/CBP histone acetyltransferase (HAT) activity. This was accompanied by fluctuations in the expression of NANOG and REX1 (also known as ZFP42), and a change in hES cell’s functional properties, as judged by their responsiveness to differentiation-­‐inducing signals. Interestingly, inhibiting P300/CBP HAT activity by curcumin treatment in undifferentiated hES cells was sufficient to revert back to a late-­‐replicating profile associated with a histone hypoacetylated state. Stable knockdown of P300 in hES cell cultures, however, resulted in a gradual loss of the pluripotent identity through differentiation or apoptosis, preventing further analysis of effects on replication timing. Collectively, these data strongly support the view that different but interchangeable pluripotent states exist within hES cell cultures and suggest a role for P300/CBP HAT activity in determining distinct epigenetic states in hES cells. As mentioned above, REX1 was also significantly upregulated in H1 hES cells upon culture condition change. REX1 is a developmental stage-­‐specific marker that is expressed in the ICM of both mouse and human embryos, as well as in pluripotent mES, hES and iPS cells. However, its function in hES cells remains largely unclear. Human ES cells overexpressing REX1 were here generated to investigate the role of REX1 in regulating hES cell pluripotent identity. These cells expressed similar levels of key pluripotency markers than their normal counterparts and remained capable of self-­‐renewing and differentiating into the three germ layers in vitro. Interestingly, however, upon withdrawal of exogenous Activin A, REX1 overexpressing hES cells in contrast to control cells retained a comparatively high level of OCT4 and stained positive for alkaline phosphatase, a known marker of undifferentiated hES cells. Taken together, these results point to a possible role for REX1 in sustaining hES cell self-­‐renewal ability.
Supervisor: Azuara, Veronique Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral