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

Abstract

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.

DNA methylation, insulin resistance, and blood pressure in offspring determined by maternal periconceptional B vitamin and methionine status

“…A small number of loci were altered in a large proportion of MD offspring, suggesting that some changes occur at highly susceptible regions of the genome. The nonrandom susceptibility of some loci to environmental perturbation is similar to our recent observations that, in blastocyst-derived human embryonic stem cells, notably Ͼ80% of the loci altered in undifferentiated cells persisted after differentiation (24). Because we wish to sustain the experimental animals in the current study to determine their later-life disease phenotypes, we have not yet been able to determine whether the methylation changes observed in fetal liver persist into adulthood.…”

Comparative analysis of DNA methylation profiles in peripheral blood leukocytes versus lymphoblastoid cell lines

“…[14][15][16][17][18][19] It has also been suggested that epigenetic modifications in cell lines may result from intrinsic factors associated with cell culturing, 4,20 including altered methylation maintenance in response to the availability of methyl donors in the culturing media, 21 and altered methylation over time with increasing passage number. 22 The observed differences in methylation between PBLs and LCLs may also be explained by cell-type specific methylation. LCL DNA samples used in this study were derived from purified B lymphocytes transformed by EBV, while the PBL samples did not undergo such enrichment and represent a range of leukocyte types.…”

A highly homozygous and parthenogenetic human embryonic stem cell line derived from a one-pronuclear oocyte following in vitro fertilization procedure

“…Imprinting refers to a phenomenon in which gene expression is regulated in a parentally specific, epigenetic manner. Although several previous studies have reported the epigenetic instability of in vitro cultured hESCs [29,30], imprinted genes seem less susceptible to in vitro culture conditions [27]. Thus, the expression pattern of imprinted genes in hESCs might faithfully reflect the original imprinting state of the embryo according to the results of a recent study, the error rate is normally 2-5% when using the same high-resolution SNP microarray to analyze hESCs [34].…”