title: Progressive accumulation of epigenetic heterogeneity during human ES cell culture.
abstract: Human embryonic stem cells (hESCs) can be maintained in culture over a large number of passages while maintaining apparently normal colony morphology. However, recent reports describe variability in epigenetic states in comparisons among different human ES cell lines. These epigenetic differences include changes in CpG methylation, expression of imprinted genes, and the status of X chromosome inactivation (XCI). We report here that the status of XCI in the female hESC line H9 (WA09) is hypervariable. We find that XIST expression can differ between individual culture isolates of H9. In addition, we find that XIST expression status can vary even between different colonies present within the same H9 culture, effectively rendering the culture mosaic. H9 cultures that lack XIST expression, but have cytological evidence of completed XCI, can also exhibit altered response to BMP4, a growth factor known to induce differentiation of hESCs to a trophectodermal lineage. In the same cultures we find biallelic expression of X-linked genes suggesting that these lines consist of mixtures of cells that retain inactivation of one of two X chromosomes following random choice. Prolonged culture of the XIST-negative isolates to high passage numbers did not result in changes in global epiproteomic signatures, demonstrating rather stable levels of post-translational nucleosome modifications within the culture-adapted hESC lines. The results show that epigenetic variants arise within human ES cell cultures after cell line derivation. In addition, the results indicate that apparently normal cultures of hESCs may contain mixtures of cells with differing epigenetic states. Assays of epigenetic integrity are warranted as quality control measures for the culture of hESCs.
authors: Center for Regenerative Biology, University of Connecticut, Storrs, CT 06269-4243 , USA.
authors: Borko
authors: B
authors: Tanasijevic
authors: Bo
authors: B
authors: Dai
authors: Toshihiko
authors: T
authors: Ezashi
authors: Kimberly
authors: K
authors: Livingston
authors: R Michael
authors: RM
authors: Roberts
authors: Theodore P
authors: TP
authors: Rasmussen
mesh_terms: D000818:Animals; D001483:Base Sequence; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D020732:Cytogenetic Analysis; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D044127:Epigenesis, Genetic / Q000187:drug effects; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D050172:Genes, X-Linked; D006657:Histones / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism
chemical_list: D055415:Bone Morphogenetic Protein 4; D006657:Histones; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; C077885:XIST non-coding RNA