Regulation of DNA methylation and X chromosome inactivation in human embryonic stem cell

Human embryonic stem cells (hESCs) can self-renew indefinitely while maintaining pluripotency in vitro. However, genetic and epigenetic abnormalities exist after long term culturing of hESCs. Therefore, it is of paramount importance to define and monitor epigenetic changes in hESCs and differentiated progenies. My thesis work examines dynamic changes in DNA methylation and X-inactivation of hESCs and their derivatives.;In this thesis, I first studied the changes of DNA methylation in promoter CpG islands during the conversion and expansion of hESCs into neural precursor cells (NPCs). A wave of de novo methylation in CpG islands takes place during the differentiation. Furthermore, the methylation level in a subset of the promoter CpG islands is higher in hESC-NPCs than primary NPC cells with the reduced expression of associated genes. These genes are involved in cell metabolism, cellular structure, and signal transduction. Collectively, my data show that abnormal CpG island methylation occurs in a subset of genes during the differentiation/expansion of hESC-NPCs; thus the methylation status of cultured hESC derivatives should be monitored and corrected for regenerative medicine.;Secondly, I studied the regulation of XCI in female hESC lines. Three hESC lines cultured in our lab (HSF6, H7, and H9) have completed XCI regardless of the presence or absence of XCI markers. Moreover, the expression of XCI markers is unstable and prone to disappear under sub-optimal culture conditions. Coincident with the loss of XCI markers, DNA methylation in a subset of X-linked CpG islands was decreased with the reactivation of a subset of silenced X-linked alleles. Furthermore, the loss of XCI markers accompanies changes in DNA methylation and expression of autosomal genes.;Thirdly, I compared genome-wide CpG island methylation between hESCs and adult leukocytes, and found a subset of CpG islands differentially methylated between hESCs and leukocytes. The differential methylation at promoter CpG islands is associated with differential gene expression in different cell types, suggesting that DNA methylation contributes to tissue-specific gene expression.;In conclusion, my thesis work demonstrates that epigenetic mechanisms, including DNA methylation and XCI, can significantly affect proper gene expression in both hESCs and their derivatives. Moreover, by monitoring DNA methylation and XCI status we can ensure the safety of the patients who use hESCs for regenerative medicine.