Restriction landmark genome scanning identifies culture-induced DNA methylation instability in the human embryonic stem cell epigenome

Widespread provision of human embryonic stem cells (hESCs) for therapeutic use, drug screening and disease modelling will require cell lines sustainable over long periods in culture. Since the short-term, in vitro culture of mammalian embryos can result in DNA methylation changes, the epigenetic stability of hESCs warrants investigation. Existing hESC lines have been derived and cultured under diverse conditions, providing the potential for programming differential changes into the epigenome that may result in inter-line variability over and above that inherited from the embryo. By examining the DNA methylation profiles of > 2000 genomic loci by Restriction Landmark Genome Scanning, we identified substantial inter-line epigenetic distance between six independently derived hESC lines. Lines were found to inherit further epigenetic changes over time in culture, with most changes arising in the earliest stages post-derivation. The loci affected varied between lines. The majority of culture-induced changes (82.3-87.5%) were stably inherited both within the undifferentiated cells and post-differentiation. Adapting a line to a serum-free culture system resulted in additional epigenetic instability. Overall 80.5% of the unstable loci uncovered in hESCs have been associated previously with an adult tumour phenotype. Our study shows that current methods of hESC propagation can rapidly programme stable and unpredictable epigenetic changes in the stem cell genome. This highlights the need for (i) novel screening strategies to determine the experimental utility and biosafety of hESCs and (ii) optimization and standardization of procedures for the derivation and culture of hESC lines that minimize culture-induced instability.