Investigating the role of ERK signalling dynamics in the regulation of stem cell heterogeneity

Abstract

Stimulation of the ERK/MAPK pathway is required for the exit from pluripotency and onset of differentiation in mouse embryonic stem (ES) cells. Many markers of pluripotency show heterogeneous expression under standard ES cell culture conditions, reflecting varying tendencies for either pluripotency and being primed for differentiation. Inhibition of the ERK/MAPK pathway causes much of this heterogeneity to be lost, driving cells into a more pluripotent state. However, immunofluorescence revealed only a weak relationship with levels of pERK and pluripotency markers at the single cell level, implying the magnitude of ERK signalling may only have a weak input into cell state. The aim of this work was to gain insight into the relationship between ERK activity dynamics and ES cell differentiation. To enable this I implemented a FRET-based biosensor to monitor ERK signalling dynamics in single living cells. I found that ERK activity was highly heterogeneous, with considerable variability in ERK signalling between single cells within ES cell colonies. Surprisingly, the magnitude and dynamics of ERK signalling were not strongly coupled to the loss of pluripotency marker expression, implying the normal dynamic range of ERK signalling is not rate-limiting in single cells during differentiation. Using custom-built image analysis software, I demonstrated that ERK signalling dynamics were sensitive to the degree of cell crowding, and were similar in neighbouring cells. By imaging cell lineages, I showed that sister cells from a mitotic division were highly correlated in their ERK activity, an effect that was apparent whether cells remained adjacent or moved apart after division, suggesting cell autonomous inheritance of cell signalling state. Overall, these data imply a combination of cell lineage and niche contributes to the absolute level of ERK signalling in mouse ES cells.

Date of Award	2019
Original language	English
Awarding Institution	King's College London
Supervisor	Madeline Parsons (Supervisor)