title: Female human pluripotent stem cells rapidly lose X chromosome inactivation marks and progress to a skewed methylation pattern during culture.
abstract: OBJECTIVE
  Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation?
  
  
  RESULTS
  We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture, often accompanied by erosion of XCI-specific methylation, and a frequent loss of random XCI in the cultures.
  
  
  BACKGROUND
  Variable XCI patterns have been reported in female hPSC, not only between different hPSC lines, but also between sub-passages of the same cell line, however the reasons for this variability remain unknown. Moreover, while non-random XCI-linked DNA methylation patterns have been previously reported, their origin and extent have not been investigated.
  
  
  METHODS
  We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines, during long-term culture and after differentiation, by gene expression analysis, histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence, XIST expression by real-time PCR, and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR, and more in depth by massive parallel bisulphite sequencing.
  
  
  RESULTS
  All hPSC lines showed XCI, but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation, it did not result in X chromosome reactivation. Moreover, lines without strong erosion of methylation frequently displayed non-random DNA methylation, which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture take-over of chromosome abnormalities and was independent of the parental origin of the X chromosome. Therefore, we suggest that a culture advantage conferred by alleles on the X chromosome or by XCI-related mechanisms may be at the basis of this phenomenon. Finally, differentiated populations inherited the aberrant XCI patterns from the undifferentiated cells they were derived from.
  
  
  CONCLUSIONS
  All hPSC lines in this study were cultured in highly similar conditions. Our results may therefore be specific for these conditions and alternative culture conditions might lead to different findings. Our findings are only a first step towards elucidating the molecular events leading to the phenomena we observed.
  
  
  CONCLUSIONS
  Our results highlight the significant extent of aberrant XCI in female hPSC. The fact that these aberrations are inherited by the differentiated progeny may have a significant impact on downstream research and clinical uses of hPSC. In order to achieve the full potential of hPSC, more insight into the XCI status and its stability in hPSC and its effect on the properties of the differentiated progeny is needed.
  
  
  METHODS
  Not applicable.
  
  
  BACKGROUND
  Our research is supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen, grant 1502512N), Generalitat de Catalunya (2014SGR-005214) and the Methusalem grant of the Research Council of the Vrije Universiteit Brussel, on name of K.S. L.V.H. is funded by EMBO (ALTF 701-2013). The authors declare no potential conflict of interest.
authors.affiliation: Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Belgium mieke.geens@vub.ac.be.
authors.forename: M
authors.initials: M
authors.lastname: Geens
authors.affiliation: Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Belgium Departament de Biologia Cellular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain Center for Regenerative Medicine in Barcelona, 08003 Barcelona, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Seriola
authors.affiliation: Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Belgium.
authors.forename: L
authors.initials: L
authors.lastname: Barbé
authors.affiliation: Departament de Biologia Cellular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Santalo
authors.affiliation: Center for Regenerative Medicine in Barcelona, 08003 Barcelona, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Veiga
authors.affiliation: Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Belgium.
authors.forename: K
authors.initials: K
authors.lastname: Dée
authors.affiliation: Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Belgium MRC Mitochondrial Biology Unit, Hills Road, Cambridge, UK.
authors.forename: L
authors.initials: L
authors.lastname: Van Haute
authors.affiliation: Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Belgium.
authors.forename: K
authors.initials: K
authors.lastname: Sermon
authors.affiliation: Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Belgium.
authors.forename: C
authors.initials: C
authors.lastname: Spits
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D040582:Inheritance Patterns; D008297:Male; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D061251:Primary Cell Culture; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D049951:X Chromosome Inactivation
chemical_list: D015415:Biomarkers; D006657:Histones; D062085:RNA, Long Noncoding; C077885:XIST non-coding RNA
keywords: X chromosome inactivation; XIST; differentiation; histone marks; human pluripotent stem cells; long-term culture; skewed methylation