Variations of X chromosome inactivation occur in early passages of female human embryonic stem cells

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.

Figure 1. XIST expression in CSES cell lines.

(A) XIST relative expression levels in CSES undifferentiated cells. (B) XIST relative expression levels in teratomas derived from CSES cells. (C) Pluripotency gene expression by Delta Ct in CSES cell lines in different passages as well as in H1 (male) cell line. (D) XIST expression by Delta Ct in all CSES cell lines analyzed compared to H1 (male) cell line. Significant values (p<0.01) are marked by asterisks.

Figure 2. Stability of XCI markers in CSES lines.

(A) CSES1 shows positive punctate staining for H3K27me3 (indicated by the arrow heads). CSES3 cells do not show punctate staining pattern for H3K27me3 in (B) late or (C) early passages. (D) Shown is XIST RNA coating of Xi by FISH in CSES1 cell line (indicated by the arrow heads). (E) CSES3 lack XIST RNA coating of Xi as indicated by FISH analysis. (F) CSES3 is positively stained for OCT4 (green) at p6. (G) CSES8 at p8 shows H3K27me3 punctate staining along with OCT4 staining; however, the relative expression of XIST is reduced in this cell line at p14. This observation is supported by a decrease in the cell population with H3K27me3 punctae in p17 (48% of the cells).

Figure 3. XIST promoter methylation analysis in CSES cell lines.

Schematic of XIST promoter CpG sites and bisulfite sequencing results of the corresponding CpG sites in the XIST promoter in each cell line. Open circle represents unmethylated CpG site and closed circle represents methylated CpG site.