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Title: Studying the direct effects of forces on embryonic stem cell behaviour
Author: Verstreken, Christophe
ISNI:       0000 0004 7651 9749
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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Cells experience different mechanical cues from their local environment, including shear flow, forces applied by neighbouring cells, and substrate stiffness. These external signals influence cell behaviour, also in embryonic stem (ES) cells, where they could potentially affect pluripotency or differentiation. The precise effects of external forces on ES cells are confounded by forces inducing secondary changes to attachment or cell-cell signalling, which themselves can also influence cell behaviour. In this study we developed a set-up to attach cells to elastic membranes using a novel functionalisation technique, and exposed them to single or cyclic stretch. We used this method to study the mechanosensitive response of ES cells. We found that stretching caused an immediate increase in the concentration of intracellular calcium, followed by a rapid decrease in some cells. On timescales of 1 - 2 h, stretching induced an increase in the expression of the immediate and early genes, but then cells became temporarily insensitive to subsequent mechanical signals. Stretching did not have a substantial impact on pluripotency and differentiation, as we showed using gene expression studies and a Rex1 reporter. To study how ES cells' susceptibility to mechanical signals depended on media condition, stretch duration and stretch type, we performed RNA sequencing and used gene ontology techniques to investigate the involvement of specific pathways. We found that forces have a broad impact on the overall transcriptome that is highly culture media-dependent. However, a core transcriptional response, including the biosynthesis of membrane components and stress pathways, was largely preserved across the different conditions. We supplemented our experimental findings with a conceptual model of force propagation in disordered environments, such as the nucleus of a cell. Using computational simulations, we studied how the large-scale behaviour of a disordered system depends on the microscopic structure. Contrary to common wisdom, we showed that disordered systems exhibit both positive and negative Poisson's ratios with equal probability. Overall, on short timescales, stretching affected ES cells' calcium concentration and transcription. On longer timescales, ES cells' response was small in magnitude but broad in scope, with limited effects on pluripotency. As such, our results suggest that mechanosensitivity in ES cells is mediated primarily by tissue-wide changes to morphology and attachment.
Supervisor: Chalut, Kevin Sponsor: EPSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: pluripotency ; forces ; stem cells ; biophysics ; granular physics