A plausible interesting observation coming out

A plausible interesting observation coming out from this study is that DNA methylation imprint at H19 DMR may be more sensitive to loss of TET proteins in undifferentiated ES serine protease inhibitors than they are in differentiated cells, whereas DNA methylation imprint at Peg1 DMR may be more prone to hypermethylation in differentiated ES cells rather than in EBs. These effects may be due to the inherent differences in the stabilities of epigenetic modifications at these DMRs in undifferentiated ES cells, differentiated ES cells or EBs. DNA methylation imprint at H19 DMR may be more stably maintained in differentiated cells, whereas methylation at Peg1 DMR may be more stable in undifferentiated ES cells relative to the differentiated ES cells. In general, the increase of methylation at the imprinted regions may be less apparent in EBs than in ES cells. This could be the result of genome-wide DNA methylation during ES cell differentiation, similar to the gain of de novo genome-wide DNA methylation after implantation in mouse embryos (Law and Jacobsen, 2010; Li and Zhang, 2014; Smith et al., 2012). This increase in the levels of de novo DNA methylation during differentiation may stabilize the DNA methylation imprint at the imprinted regions (Gopalakrishnan et al., 2008; Latos et al., 2009), rendering them less susceptible to loss of TET proteins in the differentiated cells. These hypotheses could be tested in a future study. Hypermethylation was more consistently observed at the H19 and Peg1 DMRs in two TET TKO ES clones than in two TET DKO ES clones. This implies that three TET proteins may play a partially redundant role in DNA methylation imprint in ES cells although TET1 and TET2 may be responsible for most TET-mediated demethylation activities in ES cells. This observation is consistent with the expression patterns of these three TET proteins. Indeed, it is reported that notable expression of TET1 and TET2 is very much restricted to ES cells whereas TET3 is highly expressed in the adult tissues but only barely expressed in ES cells (Koh et al., 2011; Tsagaratou and Rao, 2013) Dawlaty et al., 2014. Hypermethylation at the H19 DMR in the TET mutant ES clones (TET DKO or TKO ES clones) in our study is consistent with what was reported in DKO mutant embryos previously published by another group (Dawlaty et al., 2013). Peg1 DMR was found to be variably hypermethylated in the TET mutant ES clones. Similarly, methylation at the Peg1 (also called Mest) DMR was also reported to be variably mildly increased in a subset of DKO mutant embryos (Dawlaty et al., 2013). These results suggest that loss of TET proteins has similar effects on DNA methylation imprint in ES cells and mouse embryos, and TET mutant ES clones can be used as a model system to investigate the functions of TET proteins on genomic imprinting. This is consistent with some other previously published studies using ES cells as a model system for studying genomic imprinting (Kohama et al., 2012; Latos et al., 2009; Mann, 2001; Quenneville et al., 2011; Stelzer et al., 2015; Stelzer et al., 2014; Strogantsev et al., 2015; Takikawa et al., 2013b; Zuo et al., 2012). We will continue to employ these TET mutant ES clones to dissect the dynamic maintenance mechanisms of DNA methylation imprint in our future research. The following are the supplementary data related to this article.
Acknowledgments The work in the author\'s laboratory is currently supported by the grant from NIH (R01GM093335). XL and GX conceived the study. LL, SM, CR, YZ, FB, SN and XL performed the experiments. XL wrote the manuscript.
Resource table: Resource Details Sixty-four IVF 3-day embryos (Fig. 1A) that were deemed PQ and thus unsuitable for either uterine transfer or cryopreservation were donated and cultured on hAEC. Morphological grading criteria used by embryologists were previously described Racowsky et al., 2003. We observed embryo outgrowth in 3 samples on the 4th day after plating. Just one embryo outgrowth survived (Fig. 1B) consecutive mechanic passages until colonies were obtained and the line Amicqui-1 was established. Our derivation efficiency was 1.5%, similar to that reported in the literature Lerou et al., 2008a. This line presented a normal karyotype 46, XY (Fig. 1C), and it was positive for unspecific alkaline phosphatase (Fig. 1D). Amicqui-1 cells expressed the core genes of pluripotency, OCT4, SOX2 and NANOG, detected by immunofluorescence (Fig. 1E) and by RT-PCR (Fig. 1F). Also, the line was positive for surface antigens TRA-1-60, SSEA-4 and the cellular adhesion molecule E-CADHERIN (Fig. 1E), and it expressed KLF4, STELLA, SALL4, TBX3, KLF2, FGF4 mRNAs for transcription factors involved in pluripotency (Fig. 1D). Later, we evaluated the differentiation capacity of Amicqui-1 cells through in vitro and in vivo assays. The tissues derived from embryoid bodies were positive for specific markers: mesoderm (T), ectoderm (PAX6 and TUJ-1) and endoderm (FOXA2 and SOX17) (Fig. 1G), while the teratomas contained differentiated cellular types from the three embryonic layers (Fig. 1H). Finally, we demonstrated that our ESC line maintained on a conventional feeder layer still expressed mRNAs for pluripotency factors (Fig. 1F).