Impaired Differentiation: Understanding a Single Cell State Transition Merrit Romeike 1,2, Celine Sin 1,2, Michele Hurth 1,2, Martin Leeb 1,2, Joerg Menche 1,2, Christa Buecker 1,2 1 Max Perutz Labs Vienna, 2 University of Vienna Development is characterized by distinct, coordinated cell state transitions. During each transition, an existing gene expression program is dismantled, and a new cellular identity has to be established. We use the exit from naïve pluripotency as a highly accessible and controllable model system for a single cell state transition: mouse embryonic stem cells cultivated under defined conditions are homogenous naïve pluripotent and maintained by a well-established core gene regulatory network. Upon change of culture conditions, this network is rapidly dismantled, and cells irreversibly commit to differentiation into formative pluripotency, a less characterized cell state. Despite extensive screening for factors required for exiting naïve pluripotency, so far not a single factor has been identified which completely abrogates differentiation ability. Differentiation impaired mutants rather exhibit a phenotype which is mostly described as prolonged expression of pluripotency markers or clustering of bulk transcription profiles with naïve wildtype. Single cell methods open up a window into understanding these aberrant cell states. We collected fine-tuned differentiation time courses of control and selected differentiation impaired mutants, targeting the major signalling pathways involved in this transition. We built a shared trajectory across these genotypes and could recapitulate a delay in cell state of mutants in comparison to time matched control cells. With this, we now address fundamental questions: are observed differences based on differences in cell state, or are they directly affected by the altered gene regulatory networks? How are these networks responding to the challenge of genetic manipulations? How is the remarkable robustness of this cell state transition ensured? Taken together, our work will elucidate the mechanism of a single cell state transition and its intrinsic robustness.