of the observed variations were specific to hESCs or hiPSCs, and none of the 20 CpG sites in the promoter region of NNAT interrogated by the 27K and 450K DNA Methylation array passed our imprinted site filters. Using linear regression, we were able to correlate the imprinting status of DIRAS3, L3MBTL and PEG3 with specific in vitro manipulations, while the DNA methylation status of the other imprinted genes in our analysis were independent of the identifiable variables. The strongest association seen was between DIRAS3 hypermethylation and culture in the original hESC medium, which contained LIF and FBS, in contrast to the currently used hPSC media, which contain knockout serum replacement and purified FGF2. Given the limited numbers of samples and cell lines representing each variable in the regression models, it will be necessary in future studies to systematically test specific variables in a well replicated manner in order to identify causal relationships between specific derivation/reprogramming and culture conditions and epigenetic aberrations.
