Title Stable non-random X chromosome inactivation in human embryonic stem cells: culture artefact rather than a parent-of-origin specific event Study question In this study, we investigated the X chromosome inactivation (XCI) pattern in a large cohort of human embryonic stem cells (hESC) during long-term culture. Summary answer A stable XCI pattern was detected during long-term undifferentiated culture and after differentiation to somatic and trophoblast lineages: all fifteen lines displayed stage III XCI, with major loss of XCI marks, and those lines which were informative for the microsatellite markers we studied (11/15) showed a completely non-random XCI pattern. What is known already Female hESC cultures have previously been shown to display variable XCI patterns. Unlike mouse embryonic stem cells, most hESC already display XCI at the undifferentiated state. Moreover, a predominant occurrence of non-random XCI patterns has been reported; but the origin of this non-random pattern in hESC remained unresolved. Study design, size, duration The XCI pattern of fifteen female hESC lines was analyzed in the undifferentiated state, at different passages during long term culture, and after differentiation into somatic osteoprogenitor-like cells and towards trophoblast lineage. The observed XCI patterns were correlated to the DNA of the donors to identify the inactivated X chromosome. Participants/materials, setting, methods To analyze DNA methylation, we applied methylation-sensitive restriction, followed by PCR for two microsattelite markers, and bisulphite sequencing. Expression of XIST and TSIX was investigated using real time PCR, while minisequencing was used to determine mono- or biallelic gene expression. Histone modifications were analysed by immunostaining. Main results and the role of chance Methylation analysis revealed XCI in all 15 hESC lines. One line was non-informative for both markers; eleven lines displayed a completely skewed, non-random methylation pattern. From ten lines for which donor DNA was available, six lines displayed non-random methylation, and thus inactivation, of the male donor allele; four of the female donor allele. The remaining three lines did not display DNA methylation at the microsatellite markers, but further bisulphite sequencing analysis revealed partially methylated profiles, suggesting erosion of methylation rather than absence of XCI. Strikingly, in none of the lines the presence of repressing histone marks or XIST/TSIX expression could be observed. The same methylation pattern was observed at low and high passages and in undifferentiated as well as in differentiated samples in all lines. Limitations, reasons for caution We investigated the methylation status of the X chromosome using two microsatellite markers. Further refinement of the analysis at multiple loci spread over the whole chromosome could offer a more in-depth view of the XCI patterns in hESC. Wider implications of the findings We excluded a parent-of-origin-related mechanism for the non-random XCI appearance. Whether this pattern reflects the epigenetic status of the embryonic donor cells or an XCI-related culture advantage remains to be investigated. The presence of the culture-adapted XCI pattern, already at early passages, and its persistence in differentiated progeny cautions for the applicability of hESC. As this seems to result from suboptimal culture conditions, future efforts should focus on the optimization of hESC derivation and culture. Study funding/competing interest(s) This work was supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen) [grant number 1502512N] and the Methusalem grant of the Research Council of the Vrije Universiteit Brussel.