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Elucidating the Role of Plakoglobin in Embryonic Stem Cell Culture https://doi.org/10.17863/CAM.120337 Ellermann, Anna Lena Kathrin Hollfelder, Florian Pluripotency – the ability of cells to differentiate into all three germ layers – is of great interest, particularly in the fields of developmental biology and medicine. The in vivo continuum can be separated into three in vitro states that are also found in the embryo: naïve, formative, and primed pluripotency. Embryonic stem cells are a common tool in the field, typically cultured in conventional two-dimensional cell culture. Recently, naïve pluripotent stem cells were cultured in three-dimensional agarose microgels generated by droplet microfluidics. The three-dimensional matrix of the microgels supported the naïve pluripotency network and upregulated Plakoglobin. A cell-adhesion protein located in both adherens junctions and desmosomes. It is important for tight cell-cell adhesions and known for its function in tissues undergoing high mechanical stress. Plakoglobin also occurs in the cytosol, though its signalling functions remain mostly unknown. This thesis extends the work of the microgel cell culture, by exploring the system further and providing a deeper analysis of the results with a focus on the following four areas: (1) The microgel cell culture was further characterised and analysed, focussing on different microfluidic encapsulation techniques and stiffness of the agarose. Co-encapsulation and re-encapsulation processes were successfully improved, and different agarose stiffnesses tested for the encapsulation process. Atomic force microscopy analysis of the agarose suggested that the agarose matrix provides a very soft environment for the cells. (2) The upregulation of Plakoglobin was tested with different two-dimensional and threedimensional cell culture techniques, revealing that three-dimensional volumetric confinement is necessary for the upregulation of Plakoglobin. (3) Plakoglobin’s influence on supporting naïve pluripotency in mouse embryonic stem cells was analysed by culturing overexpression cells in media insufficient for maintaining naïve pluripotency, which normally leads to differentiation. However, Plakoglobin overexpression cells maintained self-renewal. The independence of Plakoglobin from its homologue b-catenin was tested with knock-out cell lines, which showed the support of naïve pluripotency similar to the parental overexpression cell line. (4) The role of Plakoglobin in the other two states of pluripotency – formative and primed – was tested. After the exit of naïve pluripotency, the cells strongly downregulated Plakoglobin despite high overexpression of the protein and high transcription levels in the cells. In conclusion, University of Cambridge 2024-11-30 Thesis Doctoral Doctor of Philosophy (PhD) Cambridge Trust Jane Bourque-Driscoll Fund from Jesus College https://www.repository.cam.ac.uk/handle/1810/387620 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3f2f0324-bd3b-463f-8a75-951e17b1ec7c/download 6d5df997b011922a3c94645d2d89f50d https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3e85dbf5-e9b5-42e7-9ebe-2ad3a3232db2/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved Plakoglobin Pluripotency embryonic stem cells Microfluidics cell-cell adhesion