AN-2728 Supplier Recent reports demonstrate that culture of

Recent reports demonstrate that culture of mESCs in 2i conditions causes global hypomethylation but spares key regulatory elements, such as imprinted differentially methylated regions (Leitch et al., 2013; Yamaji et al., 2013). Further, gene-expression-array-based hierarchical clustering revealed that the pluripotent AN-2728 Supplier (mESCs and embryonic germ cells) cluster according to the media they are grown in and not to their embryonic origin (Leitch et al., 2013). In our case, growth in TX medium did not cause hypomethylation on a global level but seemed to specifically result in lower methylation of CGIs. Accordingly, only the methylation levels of CGIs, but not of contiguous sequences or promoters, allowed a distinct clustering between the two media. This might be due to inherent differences in the biology of TSCs and mESCs or might reflect the fact that the regular media used for TS culture already mirror the in vivo requirements to a very high extent. However, in TX media, the methylation levels of a set of CGIs more closely resembled the levels of in vivo E7.5 trophoblast cells, suggesting that TX conditions might reflect the in vivo situation even better than the regular media. Hypomethylation of CGIs is unlikely attributed to downregulation of the de novo methyltransferases Dnmt3a, Dnmt3b, and Dnmt3l, because they are not deregulated according to our microarray gene expression analysis. Additionally, a surprising similarity between the work by Leitch et al. (2013) and our analysis is the fact that among the nine upregulated transcripts in TX media were two factors (Dazl and Syce1) whose expression is also induced by culture of mESCs in 2i. Taken together, we have established a platform for the standardized routine culture of murine TSCs under defined conditions, which might open the possibility for derivation of TSCs from other mammalian species.
Experimental Procedures
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Acknowledgments