Use this URL to cite or link to this record in EThOS:
Title: Mechanical and biochemical guidance of hMSC developmental trajectories monitored by cytoskeletal morphometrics
Author: Pereira Do Carmo Flores, Luís Rafael
ISNI:       0000 0004 8503 9640
Awarding Body: Queen Mary University of London
Current Institution: Queen Mary, University of London
Date of Award: 2019
Availability of Full Text:
Access from EThOS:
Access from Institution:
Morphology, cytoskeleton organisation and nuclear states are important biomarkers to characterise and modulate cell behaviours. Bilateral interactions between cells and their surroundings are critical aspects of biology, evidenced by the role of extracellular environment mechanical properties in directing human mesenchymal stem cell differentiation. Yet, mesenchymal stem cell differentiation studies have been mostly conducted on static linear-elastic hydrogels, neglecting important features of natural extracellular matrices. This thesis describes the development and characterisation of 2D collagen-coated agarose substrates with non-linear mechanical properties. Single-cell morphometric descriptors, extracted from cytoskeletal and nuclear images, were used to assess the combined impact of the substrate mechanical stimuli and biochemical differentiation factors on human mesenchymal stem cell morphological, cytoskeletal and nuclear states. A methodology based on Supervised Machine Learning algorithms was further employed to extend discrete population-based morphometric trends to single-cell developmental trajectories. Similar image quantification approaches were used to characterise the effects of Lifeact-GFP, a commonly used agent in fluorescent imaging of microfilaments, on cell F-actin organization, morphology and biophysical behaviour. The results in this work suggest that mesenchymal stem cells undergo dynamic non-monotonic morphometric changes under mechanical and biochemical stimuli. Contrary to established reports in linear-elastic hydrogels, cells responded similarly to collagen-coated agarose substrates covering a range of stiffnesses (1 to 30 kPa). This was accredited to the effects of the collagen coating on top of the hydrogels. Inclusion of osteogenic and adipogenic supplements conditioned the cells into assuming distinct morphologies through well-defined developmental trajectories, overriding the influence of the cell culture substrates. Single-cell developmental trajectories were akin to populational trends and helped reveal highly dynamic morphometric changes in greater detail. It was also shown that Lifeact induces changes in the cytoskeleton architecture and nucleus in a dose-response manner, ultimately leading to altered F-actin dynamics, reduced cell migration and increased cellular stiffness. Altogether, this work illustrates how morphometric analysis is uniquely poised to study morphological trends, such as characterising developmental trajectories or measuring the effects of mechanical and biochemical stimuli on cell behaviour.
Supervisor: Not available Sponsor: Marie Sklodowska-Curie
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Engineering and Materials Science ; mesenchymal stem cells ; regenerative medicine ; Tissue engineering ; Mechanobiology