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Title: Genomic imprinting in human stem cells and human peripheral blood leukocytes
Author: Frost, Jennfier May
ISNI:       0000 0004 2675 9912
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2009
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Genomic imprinting in mammals is the monoallelic expression of genes in a parent-of origin dependent manner. Imprinting can be transient and tissue specific, indicative of its role in specific developmental regimes. Placental specific imprinting has been found to be non-conserved between humans and mice. A new model of human trophoblast, trophoblast stem cells (hTS), differentiated from human embryonic stem (hES) cells, allows analysis of placental specific imprinting during the earliest stages of placentation. The use of cell lines to characterise imprinting in vivo is limited by epigenetic alterations during cell line derivation and culture. hES cells may harbour exceptional dichotomy in epigenotype compared to their in vivo counterpart, the preimplantation embryo, due to their derivation from superovulated embryos during genome-wide epigenetic remodelling. There are very limited options for analysis of imprinting in vivo in human tissue, and the most practical and available resource is human peripheral blood. Imprinted gene expression and in normal healthy blood is currently uncharacterised. Analysis of the Kcnq1/KCNQ1 cluster, which contains six ubiquitous and eight murine placental specific imprinted transcripts, in hTS cells showed that imprinting was not conserved for the placental specific transcripts. In addition, one of the ubiquitously imprinted transcripts was not imprinted in hTS cells or undifferentiated hES cells. Subsequent genome-wide imprinting analysis in undifferentiated hES cells and human fetal mesenchymal stem cells (fMSC), not derived from superovulated conceptions or during genome remodelling, found abnormal biallelic expression of several imprinted genes, to an extent consistent between both types of stem cell. In fMSC, differentially methylated imprinting control regions (ICRs) were unexpectedly normal. In hES cells, however, both hypo- and hypermethylation was detected at several ICRs. Imprinted gene expression following differentiation and expression of pluripotentiality conferring transcription factors were measured to further assess the potential of fMSC. Imprinting did not change following differentiation, however, pluripotency transcription factor expression was almost negligible compared to that in hES cells. Imprinting in peripheral blood was characterised by virtually undetectable expression of most transcripts, biallelic expression of those which could be detected and only a minority of genes remaining imprinted. These findings provide an overview of imprinted gene expression in human stem cells, complimenting previous work on hES cells. Whilst imprinted gene expression is universally disrupted by cell culture, the results suggest that methylation at ICRs may be sensitive to derivation associated specifically with hES cells, as it was normal in the fMSC lines. This lack of correlation between methylation at ICRs and imprinted expression was also mirrored in the hES cells as aberrant methylation patterns were stochastic, and did not correlate with the abnormal imprinted expression. This indicates that the loss of imprinting in cultured cells is caused by an epigenetic mechanism other than aberrant methylation. In peripheral blood, the often biallelic nature of imprinted gene expression limits the use of this tissue as a control, and also of this feature as an indicator of disease. Six of the 36 transcripts analysed remained monoallelic in blood giving them potential as biomarkers, so their imprinting status in disease should be characterised further.
Supervisor: Regan, Lesley Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral