The Regulation of X Chromosome Inactivation in Human Development and Pluripotent Stem Cells
The following thesis addresses how the epigenetic state of the female X chromosome reflects the developmental stage captured by human embryonic stem cells (hESCs). This topic has been controversial, as many epigenetic states of the X chromosome have been proposed to exist in female human pluripotent stem cells; however, none of the described states captures the state of the X chromosome in the human embryo. To investigate the basis for this disconnect between human pluripotent stem cells and human development, we used single cell resolution techniques in conjunction with mapping of DNA methylation and global transcription to examine the epigenome and transcriptome of pre-implantation embryos, cells during derivation, and in established hESCs. We demonstrate that X chromosome inactivation (XCI) initiates early during derivation of female hESCs, and unlike mouse development, progresses from a naï¿½ve state where XIST is expressed on two active chromosomes to a state where XIST is expressed on one inactive X chromosome. Therefore, the majority of established female hESCs display a pattern resembling the mouse post-implantation embryo, with one X chromosome silenced, which is associated with the spreading and coating of the non-coding RNA (ncRNA) XIST. However, this pattern erodes during progressive culture of female hESCs, with Xist RNA loss and subsequent partial activation of genes on the Xi, to an extent that is unpredictable. We also find that de novo XCI can be stochastically disrupted during hESC derivation, leading to a state where XIST is permanently silenced thus that XCI cannot occur in vitro. Accordingly some established female hESC lines carry two active X chromosomes in the self- renewing state, but, unlike the prevailing model suggests, do not undergo X chromosome inactivation (XCI) nor undergo dosage compensation upon induction of differentiation. Finally, we find that no existing method is capable of reproducing the epigenetic environment of the pre-implantation embryo in established human pluripotent stem cell lines, and conclude that a female human pluripotent stem cell that is competent to undergo XCI has not been attained. My work places the seemingly confusing findings regarding human pluripotency and the X chromosome into context and also highlights the need for a revision of culture conditions to stabilize the naï¿½ve, pre-implantation state of pluripotency in human cells.