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Dissecting Pluripotency and Mammalian Embryonic Development via Droplet Microfluidics 10.17863/CAM.64458 Kohler, Timo 0000000319490655 Hollfelder, Florian 0000000213676312 Chalut, Kevin Pluripotency, the ability of a cell to differentiate towards any type of somatic cell is a transient feature of the developing embryo. In vitro, pluripotency can be captured in the form of embryonic stem cells (ESCs). In this study, I developed a microfluidic-based system to encapsulate ESCs into agarose microgels, three-dimensional scaffolds that are in terms of their mechanical and biochemical properties fundamentally different from conventional tissue culture in plastic dishes. Subsequently, I investigated how these microenvironmental changes influence pluripotency. Interestingly, microgel culture of ESCs was not just accompanied by drastic changes in morphology, but also a promotion in naïve pluripotency. RNA-sequencing of microgel cultured ESCs elucidated global transcriptional changes of which many affected members of the pluripotency network. I then identified plakoglobin, a homologue of b-catenin, as one of the strongest upregulated proteins upon microgel encapsulation. However, molecular functions of plakoglobin in embryonic stem cells remain largely elusive. To investigate plakoglobin’s potential role during naïve pluripotency, I created several ESC lines that constitutively expressed plakoglobin at varying levels. Cells expressing high amounts of plakoglobin, portrayed a distinct naïve phenotype with homogeneous transcription factor expression even under serum-based conditions. Single cell RNA-seq and the formation of blastocyst chimaeras were then used to confirm the re-establishment of the complete naïve network. In contrast, plakoglobin is absent or greatly reduced during primed pluripotency in epiblast-derived stem cells and conventional primate pluripotent stem cells. A finding that was further confirmed in the corresponding pre- and post-implantation embryo and naïve and primed marmoset and human pluripotent stem cells. Remarkably, forced expression of plakoglobin during primed pluripotency, unlike b-catenin, leads to stabilisation of the pluripotency network rather than differentiation. Finally, after having extensively elucidated plakoglobin’s role within the continuum of pluripotency I used the microgel system to co-encapsulate ESCs with extraembryonic endoderm (XEN) cells. Co-encapsulation of these cell types led to the formation of self-organising aggregates in which the XEN cells surrounded an inner core of ES cells. These aggregates, termed EX-structures, exhibited deposition of a basal lamina, acquired apical-basal polarity, and initiated lumen formation with subsequent lineage-specific differentiation. Taken together, I have developed a cross-species compatible, compartmentalised system, for the suspension-culture of microgel-encapsulated embryonic stem cells that is generated in microfluidic devices. This interdisciplinary approach led to the identification of plakoglobin as a hitherto unknown, evolutionary conserved, regulator of naïve pluripotency. Furthermore, I have shown that co-culture of ES and XEN in microgels can mimic spatiotemporal events reminiscent to the peri-implantation embryo. University of Cambridge 2019-11-11 Thesis Doctoral eng https://www.repository.cam.ac.uk/handle/1810/317345 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/53946552-2044-4264-bff5-d558a243300b/download 35f67a6ffa68861eef9cdb379eeb6d22 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b5326014-9455-455a-b21f-2643b923a266/download 353adac0d1ebdfd65ab16480263c3c87 https://www.rioxx.net/licenses/all-rights-reserved/ stem cells development microfluidics pluripotency