Female mouse embryonic stem cells (mESCs) are significantly propagate compared to their male counterparts. This has resulted in the majority of mESC studies being performed in male cells, and therefore our knowledge of female mESCs is severely lacking. This is worrying as in addition to being harder to maintain, female mESCs are also less karyotypically and epigenetically stable and display slower differentiation kinetics. Clearly female mESCs are different to males and demand significantly more research attention. To achieve this however, female mESCs must become more experimentally tractable through development of robust methods.
It is not fully understood what makes female mESCs less robust in culture, however there is 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 hasn't undergone X chromosome inactivation, and are the only cells to express from both X chromosomes, suggesting that it is the activity of the second X, as opposed to the second chromosome, that makes female mESCs less amenable to culture.
To study female mESCs we have tagged each X chromosome with different fluorescent reporters, to monitor activity from each X rapidly and accurately by FACs. This allows rapid monitoring of cell fitness, karyotype and X inactivation status, enabling us to establish robust methods for the derivation, culture and manipulation of female mESCs. We have experimentally proven the utility of these cells for the study of pluripotency, differentiation, induced pluripotency and X inactivation. Moreover, we have performed screens for epigenetic regulators of X inactivation, revealing genes from completely unexpected pathways, suggesting unappreciated mechanisms are required for X chromosome inactivation and indeed gene silencing more broadly. We are now validating and characterising these discoveries; first in XCI and then in other epigenetic processses.