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Title: Regulation of pluripotent states in human embryonic stem cells
Author: Pinho, Sandra Isabel da Rocha Lourenco de
ISNI:       0000 0004 2688 8503
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2010
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A growing array of mouse and human pluripotent stem cell lines has been derived from the early embryo as well as from adult cells reprogrammed by ectopic expression of transcription factors – i.e. induced pluripotent stem (iPS) cells. These cell lines share the expression of key pluripotency markers and are able to self-renew and to generate differentiated progenies when induced. Their relationship to each other and whether they correspond to different pluripotent states with distinct developmental capacities and affiliations in vivo remains unclear, however. 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 across a panel of loci, as an indicator of chromatin accessibility. Of interest, this replication timing profiling was capable of discriminating pluripotent mouse ES (mES) cells from cells with a more restricted differentiation capacity. In this study, I have addressed whether distinct pluripotent states could be reliably discriminated at the chromatin level. In particular, I characterised the replication timing profile of a number of human ES (hES) cell lines alongside mES and mouse epiblast-derived stem (mEpiS) cell lines. I showed that mES cells have a steady and mostly early-replicating profile, regardless of their genetic background. In contrast, the profile of undifferentiated H1, H7 and H9 hES cell lines harboured an increased proportion of late-replicating loci during S-phase. 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 highlighted that hES and mEpiS cells share a common unstable or transitional state: primed on the verge of differentiation. This view was, however, further challenged by exploring how hES cell cultures could be modulated towards an ES-like versus epiblast-like state under different conditions. In particular, extensive and dynamic shifts of replication timing, from late to early, were consistently observed at many target loci in hES and hiPS cells upon increased Smad2/3 and p300 histone acetyltransferase activity. Importantly, these alterations were reversible and associated with differential gene expression profiles and functional properties of hES cells. Collectively, these data revealed the existence of distinct but interchangeable pluripotent hES cell states and proposed a key role for TGF-β/Activin signalling and the HAT p300 in modulating the balance between a naive versus primed state in hES cell cultures.
Supervisor: Azuara, Veronique Sponsor: FCT, Portugal ; IOG Trust Fund
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral