Mechanisms of ES cell differentiation

Cells from the inner cell mass of the blastocyst and their in vitro counterpart embryonic stem (ES) cells have the ability to differentiate into all somatic cell types. Based on this, together with an unlimited potential to self-renew and accessibility for genetic manipulations, pluripotent cells hold an immense potential for applications in regenerative medicine. However, the mechanisms that drive proper differentiation are not sufficiently understood and precise and efficient directed differentiation is not currently possible. This is due to a lack of an understanding of the underlying molecular processes that mediate the exit from an unrestricted ground state into cellular differentiation and impose a definitive cellular identity. In my project I will study the molecular events that determine the exit from pluripotency and first lineage choices using two parallel strategies. In the first approach I will use an unbiased forward genetic screen to identify factors that are essential for lineage decisions into multiple cell types. I am planning to use a gene trap strategy exploiting a novel haploid ES cell resource which has greatly facilitated forward genetic screening in a mammalian setting. I will establish an experimental haploid ES cell system, carrying markers for unrestricted pluripotency and for early lineage determination in which developmental progression can be monitored in high resolution. The second strategy is to ask to what extent Pou5f1 (Oct4), the master transcription factor of the pluripotency network is involved in mediating lineage decisions. To study this, I will generate an ES cell line in which the endogenous Pou5f1 gene can be deleted and replaced by a protein-regulatable transgene. This strategy will enable manipulation of Oct4 expression with high temporal specificity during differentiation. Through these parallel approaches I hope to gain new insights into the molecular effectors of early differentiation decisions.