An important aspect of PSC research is their

An important aspect of PSC research is their theoretical ability to be differentiated into any cell type, including fgf receptor inhibitor expressing tissue-specific silencing of paternal Gsα, as described for Gsα. However, for such studies, it is critical to fully control the differentiation of these PSCs into specific cellular types of interest. Thus, our next step was to study methylation marks upon differentiation of PSCs. Our results indicate that methylation at the GNAS locus is affected both as a function of cell type (NSC versus MSC) and DMRs. Indeed, methylation changes at the NESP and A/B DMRs were observed only upon differentiation into NSC, not MSC. In addition, we observed an increase in AS methylation in all progenies (NSC and MSC), reaching hypermethylation levels in 4/8. Few reports, and none for the GNAS locus to our knowledge, have addressed the issue of DMR methylation upon “re”differentiation of PSCs into progenies in human cells. The pattern associating gain of methylation at NESP and loss of methylation at A/B and/or XL DMRs of GNAS is reminiscent to that of patients affected with sporadic PHP1B. Whereas it is tempting to speculate that changes upon reprogramming and epigenetic changes causing PHP1B are connected, the molecular mechanisms causing these changes are not identified. In contrast to our results in PSCs, loss of methylation at the AS DMR of GNAS is common in sporadic PHP1B (Maupetit-Méhouas et al., 2011) as observed for the A/B and XL DMRs, also methylated on the maternal allele). The mechanisms causing the epigenetic changes in PHP1B are under investigation and multiple. Some common mechanism might exist during reprogramming and PHP1B. Further studies analyzing the specific increase in AS methylation as well as the changes in other DMRs methylation observed in progenies from hESCs and hiPSCs may help understand the mechanisms whereby methylation at each DMR is controlled in physiology and the mechanisms leading to methylation defect in PHP1B. The notion that loss of methylation at the GNAS DMRs controls transcript expression is mostly intuitive, with little available direct evidence. Freson et al. (2008) showed decreased methylation at XL DMR and increased expression of the XL protein in platelets. Loss of methylation at exon A/B is associated with an increase in the levels of the noncoding exon A/B RNA and a loss of Gsα expression (Bastepe and Jüppner 2005; Fröhlich et al., 2010). Studies in mice have shown that paternal deletion of the exon 1A region results in reversal of Gsα allelic silencing with biallelic expression of Gsα (Liu et al., 2000; Williamson et al., 2004). Monoallelic expression of Gsα has been reported in a few studies and mainly mouse studies (for reviews, see Bastepe and Jüppner, 2005; Hayward et al., 2001; Levine, 2012; Linglart et al., 2013; Mantovani et al., 2012; Plagge and Kelsey, 2006; Weinstein et al., 2007). Human tissues expressing paternal Gsα allelic silencing are not easily accessible, and the correlation between transcript expression and DMR methylation is rarely reported. Predominant maternal origin of transcription of Gsα in human thyroid gland and gonads has been reported (Mantovani et al., 2002). Using the distinguishing parental single-nucleotide polymorphism rs7121, we correlated allelic expression and DMR methylation in hiPSCs and after differentiation for the three imprinted transcripts (A/B, XLsα, and NESP55) and also defined their parental expression as well as that of Gsα. Allelic expression of the maternally imprinted A/B transcripts varied as a function of the cell line. As indicated above, A/B DMR has a maternal-specific germline methylation. It is therefore expected that, in hiPSCs and progenies, the expression of the A/B transcripts originates predominantly from the paternal allele. This was observed in hiPSC clones and progenies derived from one fibroblast line, but not from the other. Importantly, however, we found that A/B transcript expression was correlated with the degree of methylation at the A/B DMR, indicating that allelic-silencing mechanism of A/B expression is methylation dependent.