Comparisons with current literature

Two other similar studies on hES cells have been carried out recently: the International Stem Cell Initiative (ISCI) analysed 59 hES cell lines, including three of those analysed in the present ICH study, SHEF1, SHEF2 and CF1, although it incorrectly lists the latter as a female line (Adewumi et al 2007). Lorraine Young and colleagues at the Wolfson Centre for Stem cells, Tissue Engineering and Modelling (STEM) and Institute of Genetics at the University of Nottingham analysed 22 hES cell lines, including the 16 lines derived by Chad Cowan in 2004 (Cowan et al., 2004), plus an embryonal carcinoma line from Sheffield University.

In addition to assigning mono- or biallelic status to imprinted gene expression, both the STEM and ISCI studies assigned preferential status to expressed SNPs where the minor allele was less than 30% of that of the major allele. Notably, ISCI measured the contribution of minor alleles by calculating the area under cDNA sequence chromatograms. Such ‘partial imprinting’ was not distinguished in the ICH study. This was because in several cases the area under the cDNA sequence chromatogram varied depending on the direction of the sequence, due to the increased detection levels of certain dyes, and therefore was deemed unreliable. Instead, biallelic expression was assigned wherever the presence of the minor allele was observed, no matter how small. To account for this here, ISCI ‘partial imprinting’

data, and STEM ‘preferential’ samples are added to the ‘biallelic’ data in Table 8.2 to ease comparison of the two data sets with the present study. Any loss of repression, compared to that observed in the fetus in vivo, (Monk et al 2006 and own observations) was deemed important for the analysis of imprinting in cell lines as it may reflect a reduction in the stringency of imprinting. Functionally meaningful allele specific expression, whether it is complete or preferential, is a definitive measure of imprinting, however the functional relevance of such a loss of repression was beyond the scope of this study. Table 8.2 Comparison of hES imprinting to ISCI study (Adewumi et al 2007).

Mat

Number of samples is shown with percentages in parentheses. Included in ISCI data are the same samples at different time points. ICH data (the present study), and data from Nottingham University (STEM) include individual line data only. Maternally expressed genes, paternally expressed genes, not analysed. The overall patterns of allelic expression between the three studies is very similar, except for MEST, where the bias of biallelic to monoallelic expression is opposite between ISCI and the STEM and ICH studies.

The overall results of the ISCI, STEM and Sun et al studies, comparing the total number of monoallelic and biallelic genes across all the lines analysed are consistent with the findings here, as is shown in the last row of Table 8.2 (Adewumi et al., 2007; Kim et al., 2007b; Sun et al., 2006). Analysis of individual genes also gives similar results across all four studies, and the following genes - SNRPN, NDN, KCNQ1OT1, KCNQ1, CDKN1C and PEG3; are always monoallelic in hES cells (Adewumi et al 2007; Sun et al 2005, Kim et al 2007). The present study shows this to additionally be true of fMSC.

SLC22A18 is found to be biallelically expressed in all samples analysed in the present (ICH) study, and by Kim et al, (STEM study) although Adewumi and colleagues (ISCI study) find one monoallelic sample. PEG10 was found to be monoallelic in the ICH study and by Sun et al, but variable by the STEM study. For the other genes analysed, IGF2, MEST Isoform 1 and 2, GTL2 and H19, a mixed expression pattern was found in both ISCI and ICH studies, with a predominant monoallelic expression pattern for H19, consistent with that seen in the study by Sun et al. Results from allelic expression data from the STEM and ICH studies closely mirror one another. There are minor differences, i.e. ATP10C, GTL2 MESTIT1 and PEG10 display a mixed expression pattern according to the STEM study, but not in the ICH study. The allelic expression bias for these genes is the same for both data sets, and the small variations observed are likely due to the inter-individual differences between cell lines, whereby the analysis of further samples in each case would likely dilute any difference. As such, many more samples will have to be analysed to fully understand the reasons behind variation in allelic expression of these genes. As the ICH study included fewer informative samples for each, it is likely that analysis of more samples would reveal a pattern identical to the STEM data.

More differences were apparent between the ISCI and ICH/STEM studies.

IGF2 expression was predominately biallelic in hES cells in the STEM data, in fMSC in the ICH data, but mostly monoallelic in the ISCI hES cells. As previously

mentioned, a variation in the designation of expression bias between the three studies, where the ISCI study allowed inclusion of samples with a minor allele input of up to 14 % in their ‘monoallelic’ category, may account for the difference. The other main variation was in the expression of the MEST isoforms, which were also predominately biallelic in the ISCI study. The manuscript did not elaborate on which isoform was analysed, and non-isoform specific PCR would result in apparent biallelic expression of these transcripts.