Unlocking female ES cells for research

Keniry AJ, Jansz N and Blewitt ME

Walter and Eliza Hall Institute.

Female mouse embryonic stem cells (mESCs) are significantly harder to grow and expand compared to their male counterparts. This discrepancy has resulted in the majority of mESC studies being performed in male cells, and therefore our appreciation of characteristics particular to female mESCs is severely lacking. This is worrying as in addition to being harder to maintain, female mESCs have also been found to be less karyotypically and epigenetically stable and display slower differentiation kinetics. Clearly female mESCs are very different to their male equivalents and demand significantly more research attention. To achieve this however, female mESCs must become more experimentally tractable through development of robust methods to both derive and maintain them. It is not fully understood what makes female mESCs less robust in culture, however there is some evidence that the second X chromosome is to blame as female XO mESCs behave similarly to male XY cells. Interestingly, female mESCs are the only female cell type that has not undergone the process of X chromosome inactivation, and thus are the only cells to express from both X chromosomes, suggesting that it may be the activity of the second X, as opposed to the second chromosome per se, that makes female mESCs less amenable to culture. In order to study female mESCs we have tagged each X chromosome with a different fluorescent reporter, such that we can monitor activity from each X rapidly and accurately by FACs. The system allows for rapid monitoring of cell fitness, karyotype and X inactivation status and has enabled us to establish robust methods for the derivation, culture and manipulation of female mESCs. We have also experimentally proven the utility of these cells for the study of pluripotency, differentiation, induced pluripotency and X inactivation. Moreover, we have used the cells to perform screens for epigenetic regulators of X inactivation. These screens have revealed genes from completely unexpected pathways and suggest unappreciated mechanisms are required for X chromosome inactivation and indeed gene silencing more broadly. We are now moving on to validating and characterising these discoveries; first in XCI and then in other epigenetic processes.