Abstract
X chromosome inactivation (XCI) is a dosage compensation mechanism essential for embryonic development and cell physiology. Human embryonic stem cells (hESCs) derived from inner cell mass (ICM) of blastocyst stage embryos have been used as a model system to understand XCI initiation and maintenance. Previous studies of undifferentiated female hESCs at intermediate passages have shown three possible states of XCI; 1) cells in a pre-XCI state, 2) cells that already exhibit XCI, or 3) cells that never undergo XCI even upon differentiation. In this study, XCI status was assayed in ten female hESC lines between passage 5 and 15 to determine whether XCI variations occur in early passages of hESCs. Our results show that three different states of XCI already exist in the early passages of hESC. In addition, we observe one cell line with skewed XCI and preferential expression of X-linked genes from the paternal allele, while another cell line exhibits random XCI. Skewed XCI in undifferentiated hESCs may be due to clonal selection in culture instead of non-random XCI in ICM cells. We also found that XIST promoter methylation is correlated with silencing of XIST transcripts in early passages of hESCs, even in the pre-XCI state. In conclusion, XCI variations already take place in early passages of hESCs, which may be a consequence of in vitro culture selection during the derivation process. Nevertheless, we cannot rule out the possibility that XCI variations in hESCs may reflect heterogeneous XCI states in ICM cells that stochastically give rise to hESCs.
Highlights
Human embryonic stem cells are an invaluable tool for regenerative medicine and a model for early human embryogenesis [1]
Four cell lines (CSES3, CSES5, CSES6 and CSES14) did not express XIST both at the undifferentiated and differentiated state. These results clearly demonstrate that three different states of X chromosome inactivation (XCI) exist in Human embryonic stem cells (hESCs) even after a short culture period
We conclude that CSES2 and CSES7 cells are in the ‘‘naıve’’ state, showing XIST expression only upon differentiation; CSES1, 8, 10 and 11 cells are in the intermediate state, showing XIST expression at the undifferentiated state and further induction upon differentiation; and CSES 3, 5, 6, 14 cells are in the third ‘‘culture affected’’ state, showing no XIST expression regardless of undifferentiated or differentiated conditions
Summary
Human embryonic stem cells (hESCs) are an invaluable tool for regenerative medicine and a model for early human embryogenesis [1]. Due to the wide applications of hESCs, there have been increasing demands for more newly derived hESC lines. This interest allows comparison of different properties among various hESC lines and can potentially create a gold standard for the characterization of hESC lines. Efforts have been made to generate gene expression and epigenetic profiles for hESCs [5,6,7,8,9]. XIST gene expression varies among different hESC lines and even within the same cell line [5,11,12,13]
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