Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Recent work has demonstrated that ES cells are intrinsically self-sufficient when shielded away from differentiation-inductive signals. The combinatorial challenge of two important signal pathway inhibitors (2i) under defined cell culture conditions allows maintenance of the ground state of ES cell pluripotency. These inhibitors target the GSK and MAPK signalling pathways. ES cells differentiate upon withdrawal of these two inhibitors.
Taking advantage of this defined system, we carried out a genome-wide RNAi library screen to identify signal-dependent regulators. We focussed on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase pathway in this process. Several rounds of screening strategies and subsequent gene ontology analysis revealed the involvement of a wide variety of pathways which prominently featured members of signalling pathways, transcriptional regulators and chromatin-associated factors.
More detailed analysis identified MAP kinase phosphatases as a focal point of regulation that determines ERK activation kinetics and subsequent early cell fate decisions.