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Title: The effect of mechanical and shear forces on embryonic stem cells
Author: Hemsley, A. L.
ISNI:       0000 0004 5363 1500
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2014
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Embryonic stem (ES) cells hold great promise as a new paradigm in medicine. The first clinical trials using ES cells for regenerative therapies in humans have recently begun. Despite this, much of the basic research into the mechanical properties and the response of ES cells to the extra-cellular force applied during their routine culture and bioprocessing remains to be characterised. Atomic force microscopy (AFM) was used to apply specific force to OCT4-GFP and E14Tg2A mES cells. The formation of mechanically-induced blebs was observed in round, pluripotent cells. Blebs occur during the detachment of the plasma membrane from the main body of the cell. They are known to occur spontaneously and were found to form upon the application of ≥5nN force. Cytoskeletal studies investigating the distribution of actin, tubulin and pERM confirmed that undifferentiated mES cells have a less well-developed cytoskeleton, leaving them more susceptible to force-induced damage than differentiating or differentiated cells. Shear forces are routinely exerted on a cell population throughout passaging, expansion and differentiation. Therefore, it is essential to understand the effect of such forces as their influence may have dramatic consequences on ES cell viability, integrity and fate decisions, thus impacting their utility for regenerative therapies. For this reason, exposure to transient shear was also investigated. Shef-3 and Shef-6 hES cell lines were adapted to enzymatic passaging, facilitating the generation of sufficient cells which can tolerate single cell dissociation, to study the impact of cell culture induced shear forces. Single-cell suspensions ensured all hES cells were exposed to equal forces. Transient shear exposure had no noticeable impact on cell viability or on the induction of apoptosis. Significant differences in fold expansion rates (P≤0.005) and gene expression of the pluripotency markers OCT4 and Nanog were detected (P≤0.005). Assessment by qPCR revealed that shear exposure can influence cell fate decisions. Current thinking indicates that for regenerative therapies to be successful, ES cells will need to be differentiated into the required cell types prior to transplantation, thus confirming the significance of the findings presented herein.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available