Cell cycle remodelling during hESC differentiation: a lesson from single cells

Abstract

The cell cycle is a sequence of events that every cell goes through in order to duplicate its genomic content and segregate it into two daughter cells. Even though the molecular machinery involved in cell cycle control does not vary in different cells, cell cycle dynamics can be very different cells. Human embryonic stem cells (hESCs) have two unique capabilities: they self-renew and differentiate into the three germ layers upon receiving an external stimulus. While their self-renewal state is characterised by a very short cell cycle (~16h) mostly due to a short G1-phase and little checkpoint control, after differentiation they acquire the canonical cell cycle profile of somatic cells with long gap phases and active checkpoint control. Previous studies have highlighted the differences between the pluripotent and the somatic (differentiated) cell cycle but it is still unclear how cells remodel cell division networks to allow for structurally different cell cycles. In order to explore the mechanisms underlying cell cycle remodelling during hESCs differentiation, a combination of cell cycle biosensors and live cell imaging techniques was established to study how cell cycle remodelling happens in single cells during mesoderm differentiation of hESC. The main difference in cell cycle phases length between hESC and differentiated cells was found to be in G1-phase length. We show that the transition from the short G1-phase of hESCs to the remodelled G1-phase of differentiated cells happened in a switch-like manner. Finally, using perturbation approaches, we demonstrated that cell cycle regulators controlling Cdks activity have a role in G1-phase remodelling. We show that p15, p21 and cyclin A1, important Cdk regulators, are involved in G1-phase lengthening during differentiation. The work presented in this thesis set the foundations for future research looking at cell cycle dynamics in live, single cells during early developmental. The work also highlights hESCs as trackable, ideal system to study changes in cell cycle dynamics during embryonic to somatic transition.