Understanding the status of the X chromosomes in human ESCs and preimplantation embryos

In placental mammals, dosage compensation occurs by silencing one X-chromosome in female cells, a process known as X-chromosome inactivation. Unlike female mouse embryonic stem cells (ESCs), which possess two active X chromosomes and undergo XCI upon induction of differentiation, female human ESCs exhibit various epigenetic states of the X chromosome, indicating a surprising epigenetic instability of these cells under normal culturing conditions. Since this epigenetic variation could have implications for the use of female human ESCs in regenerative medicine, disease studies, and basic research, in this proposal, we are aiming to determine how the epigenetic variability of the X chromosome arises during derivation and maintenance of human ESCs, the causes and consequences of deregulation of XCI in human ESCs, and to devise methods of stabilizing Xist expression in human ESCs. During the first funding period, we have extensively characterized the epigenetic state of the X chromosome in many established and newly derived human ESC lines as well as in human blastocysts. Together, our findings reveal new insights into the relationship between different X chromosome states in undifferentiated female human ESCs, clarify how they arise during ESC derivation, and define the implications of these X chromosome status for differentiated cells. The findings from our study have implications for the utilization and quality assessment of human ESCs.

The application of human embryonic stem cells (hESCs) requires reliable cell sources that do not change over time and initiate proper transcriptional and chromatin changes upon induction of differentiation. Therefore, it is important to understand how and when aberrancies such as the epigenetic instability of the X chromosome arise, and to define their consequences for differentiation processes and the differentiated progeny. To this end, our goal is to understand how the inactive X chromosome is regulated in human pre-implantation embryos, during derivation of hESCs from blastocysts, and during their maintenance. We have applied a large number of single cell, genome-wide, and population-wide approaches to understand this problem at a systematic and comprehensive level. Our findings define the relationship between different X-inactivation states in female hESCs and demonstrate the consequences of different X-inactivation states for hESC differentiation. Moreover, we have started to assess strategies that would prevent the instability of the inactive X chromosome and allow normal dosage compensation upon differentiation of hESCs.

The application of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) requires reliable cell sources that do not change over time and initiate proper transcriptional and chromatin changes upon induction of differentiation. However, female hESCs and hiPSCs exhibit an epigenetic instability of the X chromosome. Therefore, it is important to systematically define the epigenetic abnormalities that hESCs and hiPSCs carry, to understand how and when the epigenetic instability of the X chromosome arises during the derivation of these cells, to define the consequences if the different X chromosome states for differentiation, and to find ways to overcome the epigenetic instability. To this end, we have applied a large number of single cell, genome-wide, and population-wide approaches to understand this problem at a systematic and comprehensive level. Our findings define the relationship between different X-inactivation states in female hESCs and hiPSCs and demonstrate the consequences of different X-inactivation states for differentiation. Moreover, we have developed a strategy that erases the instability of the inactive X chromosome and enables faithful X chromosome dosage compensation in differentiating hESCs and hiPSCs, which is critical for the use of these cells in regenerative medicine, disease studies, and basic research.