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.