Pluripotency is the ability of a cell to differentiate into derivatives of all three somatic lineages and germ cells. In vivo, pluripotent cells exist transiently in the epiblast of the developing embryo and in rare tumour cells. In vitro, pluripotent cells have been isolated and propagated from teratocarcinomas (EC cells), preimplantation epiblast (ES cells) and post-implantation epiblast (EpiSCs). Pluripotency is governed by a gene regulatory network centred on the triumvirate of transcription factors Oct4, Sox2 and Nanog. Interestingly, transcription factors that are important to direct pluripotent cell identity are not all equally distributed throughout the pluripotent cell population. While Oct4 levels are relatively homogeneous, other transcription factors, such as Nanog, are more heterogeneously expressed. Additionally, an increasing body of evidence indicates that extrinsic cues also play a critical role in the establishment and maintenance of pluripotency. Using biochemical and genetic tools in mouse ES cells, the role of FGF signaling and Sox2 levels on heterogeneous Nanog expression was examined. Interference with FGF or ERK activity by genetic ablation or signal inhibition, promoted high, homogenous Nanog expression and enhanced self-renewal. This is consistent with reports showing that similar manipulations reduced the ability of ES cells to commit to differentiation. Moreover, ES cells with reduced Sox2 levels displayed greater heterogeneity for Nanog expression than wild-type ES cells. Pluripotency is lost in the mouse embryo around E8.5, however, the precise timing and mechanism involved in this process has not yet been defined. Here it is shown that pluripotency is extinguished at the onset of somitogenesis, coincident with reduced expression and chromatin accessibility of Oct4 and Nanog regulatory regions. Prior to somitogenesis, the expression of both Nanog and Oct4 is regionalized. Interestingly, pluripotency tracks the in vivo level of Oct4, this correlation does not hold true for Nanog. However, Nanog expression reports on pluripotent cells. Indeed, ectopic Oct4 expression in somitogenesis-stage tissue provokes rapid reopening of Oct4 and Nanog chromatin, Nanog re-expression and resuscitation of moribund pluripotency. Competence to re-activate the pluripotency network upon enforced Oct4 expression is gradually lost with the progression of embryonic development. ES cells and EpiSCs are two distinct pluripotent populations as they show differences in their ability to undergo clonal propagation, re-colonize embryos, growth factor responsiveness, morphology and gene regulatory networks. It is possible to harness this differential growth factor responsiveness to convert ES cells into EpiSCs. Conversely, EpiSC can be reverted back to ES cell pluripotency through the overexpression of a small number of transcription factors. The inter-conversion of ES cells and EpiSCs has been documented, but detailed analyses of the changes that occur during such transitions had not been performed. The current work shows that Nanog levels are critical for the specification of the pluripotent state of the cells. Furthermore, it is shown that orphan nuclear receptor Esrrb is a potent inducer of ES cell pluripotency in EpiSC. Interestingly, Esrrb was able to restore naïve pluripotency in cells genetically depleted of Nanog.

The ground state naive pluripotency is established in the epiblast of the blastocyst and can be captured by culturing mouse embryonic stem cells (mESCs) with MEK and GSK3 inhibitors (2i). The transcription network that maintains pluripotency has been extensively studied with the indispensable core factors being Oct4, Sox2 and Nanog, together with other ancillary factors reinforcing the network. However, how this network is dissolved at the onset of differentiation is still not fully understood. To identify genes required for differentiation in an unbiased fashion, I conducted a genome-wide CRISPR-Cas9-mediated screen in Rex1GFPd2 mESCs. This cell line expresses GFP specifically in the naive state and rapidly down-regulate upon differentiation. I differentiated mutagenised mESCs for two days and sorted mutants that kept higher GFP expression. gRNA representation was subsequently analysed by sequencing. I identified 563 and 8 genes whose mutants showed delayed and accelerated differentiation, respectively, at a false discovery rate (FDR) cutoff of 10%. The majority of the previously known genes were identified in my screen, suggesting faithful representation of genes regulating differentiation. Detailed screening result analysis revealed a comprehensive picture of pathways involved in the dissolution of naive pluripotency. Amongst the genes identified are 19 mTORC1 regulators and components of the mTORC2 complex. Deficiency in the TSC and GATOR complexes resulted in mTORC1 upregulation in consistent with previous studies. However, they showed opposite phenotype during ESC differentiation: TSC complex knockout cells showed delayed differentiation, whereas GATOR1 deficiency accelerated differentiation I found that the pattern of GSK3b phosphorylation is highly correlated with differentiation phenotype. I conclude that mTORC1 is involved in pluripotency maintenance and differentiation through cross-talk with the Wnt signalling pathway. My screen has demonstrated the power of CRISPR-Cas9-mediated screen and provided further insights in biological pathways involved in regulating differentiation. It would be interesting to explore the remaining unstudied genes for better understanding of the mechanisms underlying mESC differentiation.

Conventional or “primed” human embryonic stem cells (hESCs) rely on FGF and TGFβ signalling for self-renewal, and occupy a developmentally advanced state of pluripotency comparable to mouse EpiSCs. Recent reports demonstrate that a naïve state of human pluripotency can be consistently derived either through transient histone deacetylase inhibition mediated resetting of conventional hESCs or via isolation of the inner cell mass. Long-term propagation of this state can be achieved using a cocktail of MEK, GSK3 and PKC inhibition in conjunction with leukaemia inhibitory factor (LIF) supplementation (t2iLGö) and a feeder layer of inactivated mouse embryonic fibroblasts. However, the way in which this signalling environment is interpreted in order to maintain naïve pluripotency remains unclear. I demonstrate a substrate consisting of a high concentration of tissue-derived laminin in combination with t2iLGö is sufficient to replace the feeder layer. Cultures maintained under these conditions are karyotypically normal, maintain a naive pluripotent transcriptional profile and exhibit reduced aberrant expression of mesodermal and endodermal lineage markers. I utilise the increased stringency of this culture system in combination with small molecule inhibitors to examine the roles of FGF, Activin/Nodal and JAK/STAT signalling in human naïve pluripotency. Naïve hESCs proliferate and maintain pluripotency marker expression in the presence of FGF receptor inhibition. In contrast, TGFβ signalling inhibition leads to rapid downregulation of human specific naïve pluripotency marker, KLF17, followed by the eventual collapse of the naïve transcription factor circuitry. Naïve hESCs self-renew in both the absence of LIF and presence of JAK/STAT inhibitors. However, further investigation of JAK/STAT signalling identified the increased potency of Interleukin 6 (IL-6) over LIF to activate the JAK/STAT pathway. Supplemental IL-6 improves colony-forming capacity under self-renewing conditions and attenuates differentiation following inhibitor withdrawal. Furthermore, prolonged activation of IL-6 signalling suppresses expression of GATA2 and GATA3 and upregulates KLF4 transcripts. Finally, I investigate whether ablation of PKCι is sufficient to replace the activity of the PKC inhibitor, Gö6983. Established naïve cultures that are PKCι null continue to express naïve markers and suppress upregulation of lineage makers following withdrawal of Gö6983. Furthermore, ablation of PKCι in conventional ESCs enables the maintenance of NANOG expression and the emergence of KLF17 expression in the absence of Gö6983 during histone deactylase mediated resetting.

No description available.

The Oct-4 transcription factor is believed to co-regulate early embryonic development of mammals due to the correlation of its presence with the maintenance of pluripotency. It is commonly used as a marker for the identification of embryonic stem (ES) cells for this reason. Until 1999, Oct-4 studies were limited to in vivo-produced embryos; equine embryos have not been studied for their Oct-4 expression patterns. In addition, equine stem-like cells (defined by marker expression, induced differentiation, passage survival, and morphology) have recently been isolated from in vivo-produced embryos, but no work has been performed in horses to isolate ES cells from in vitroproduced embryos. This study investigated the expression of Oct-4 transcription factor using immunocytochemistry in 42 in vitro-produced embryos aged 1-10 days and in 5 in vivoproduced blastocysts aged 7-10 days. Effective conditions for rapid establishment of a feeder layer of equine fetal fibroblasts were established, and this feeder layer was used to grow isolated equine inner cell mass (ICM) cells from in vitro-produced embryos. The expression of Oct-4 was examined in resultant cell growths. In vitro-produced embryos less than 6 days of age showed variable staining within blastomeres of the same embryo, and the peak of variability correlated with maternal-zygotic transition. After Oct-4 staining of in vitro-produced blastocysts, no cells could be identified as an ICM based on a difference in fluorescent intensity from the other cells of the blasyocysts. However, in vitro-produced blastocysts that were subsequently cultured in vivo contained a presumptive ICM, visible based on greater fluorescent intensity of Oct-4 stain. The trophoblast of all blastocysts also stained positively for Oct-4 protein. Fibroblasts were successfully isolated from equine feti. Treatment with 20 µg/ml of Mitomycin C arrested cell growth without causing excessive death. Fibroblasts were inactivated and frozen, then thawed as needed to establish a confluent monolayer for ICM isolation overnight. ICMs from in vitro-produced embryos formed outgrowths, but none that could be identified morphologically as ES cells. Outgrowth cells contained about 20% Oct-4 expressing cells in sporadic groupings. Assuming appropriate binding of the Oct-4 antibody, Oct-4 expressing cells (potentially indicating pluripotency) are found throughout the embryo in early development and in the feeder layer after co-culture.

“The scientist, by the very nature of his commitment, creates more and more questions, never fewer. Indeed the measure of our intellectual maturity, one philosopher suggests, is our capacity to feel less and less satisfied with our answers to better problems.” ~G.W. Allport, Becoming, 1955 It will be interesting to look back at this thesis in a few decades and reflect on how the questions and interpretation of data in the field of developmental biology have changed. Indeed, a biologist currently in their twilight years might reflect on their youth, before the discovery of hereditary material, and compare that bookend with the range of genome sequences and related knowledge currently available. How long will it take before this thesis reads like a debate about whether the male or female contributed the ‘homunculus,’ a miniature preformed human to the embryo that grows into an adult? In this thesis I asked three related questions: whether the role of Oct4 during embryogenesis provides insight into its contribution to pluripotency; how surfaceome changes contribute to functional maturation of neural stem cells and to what extent the murine genome is imprinted. Our data indicate that Oct4 is required for posterior expansion. We propose that the function of the protein is conserved, but that its expression has been coopted to yield different cell types based on its combination with different factors. We show that fundamental aspects of cell biology are altered during the maturation from pluripotent populations to neural stem cells, and identify mediators of proliferation, survival and adhesion that distinguish neural stem cell regulation from their precursors. Finally, we validated discovery of a dozen novel imprinted transcripts using a genomic approach. These discoveries will contribute to a holistic view of the causes and consequences of imprinting, but do not support a paradigm shift in the scale and consequences of imprinting.

“The scientist, by the very nature of his commitment, creates more and more questions, never fewer. Indeed the measure of our intellectual maturity, one philosopher suggests, is our capacity to feel less and less satisfied with our answers to better problems.” ~G.W. Allport, Becoming, 1955 It will be interesting to look back at this thesis in a few decades and reflect on how the questions and interpretation of data in the field of developmental biology have changed. Indeed, a biologist currently in their twilight years might reflect on their youth, before the discovery of hereditary material, and compare that bookend with the range of genome sequences and related knowledge currently available. How long will it take before this thesis reads like a debate about whether the male or female contributed the ‘homunculus,’ a miniature preformed human to the embryo that grows into an adult? In this thesis I asked three related questions: whether the role of Oct4 during embryogenesis provides insight into its contribution to pluripotency; how surfaceome changes contribute to functional maturation of neural stem cells and to what extent the murine genome is imprinted. Our data indicate that Oct4 is required for posterior expansion. We propose that the function of the protein is conserved, but that its expression has been coopted to yield different cell types based on its combination with different factors. We show that fundamental aspects of cell biology are altered during the maturation from pluripotent populations to neural stem cells, and identify mediators of proliferation, survival and adhesion that distinguish neural stem cell regulation from their precursors. Finally, we validated discovery of a dozen novel imprinted transcripts using a genomic approach. These discoveries will contribute to a holistic view of the causes and consequences of imprinting, but do not support a paradigm shift in the scale and consequences of imprinting.

This thesis presents the development of microfluidic devices designed to facilitate research into mouse embryonic stem cells (ESCs). ESCs are a well-studied cell, largely due to their pluripotent nature, meaning they are able to differentiate into all cell types of the body and may self-renew indefinitely in appropriate culture conditions. ESCs, along with many other lines of biological enquiry, are increasingly studied with the use of micro uidic technology which enables fine tuning of physical and chemical environments unachievable on the macro scale. Two varieties of microfluidic technology are presented in this thesis, one for high- resolution mechanical phenotyping of ESCs and the second as a novel in-chip culturing platform to study cellular transitions. Chapter 1 presents a broad introduction to ESCs and biological enquiry with microfluidics, aimed to underpin the following Chapters. Chapters 2 and 3 present self-contained projects, thus each include a motivation and introduction section more specific than that presented in Chapter 1. These Chapters also contain their own methods, results and conclusion sections. Finally, Chapter 4 presents a summary of the work performed along with an outlook of upcoming investigations. In Chapter 2, I present a microfluidic device developed and utilised in collaboration with Christophe Verstreken (Department of Physics, University of Cambridge), which has been used to apply a mechanical stress to live cells enabling measurement of their nuclear deformability. The device facilitates detection of both nucleus and cytoplasm which can then be analysed with a custom-written MATLAB code. Quantitative measurements of nuclear sizes and strains of ESCs indicated a negative Poisson ratio for nuclei of cells cultured in specific medium conditions. Furthermore, we demonstrate that the device can be used to physically phenotype at high-throughput by detecting changes in the nuclear response after treatment with actin depolymerising and chromatin decondensing agents. Finally, we show the device can be used for biologically relevant high-resolution confocal imaging of cells under compression. The work from this chapter is presented in Hodgson et al. [1]. In Chapter 3, I present a novel microfluidic platform developed in collaboration with Prof. Austin Smith and Dr Carla Mulas (Centre for Stem Cell Research, Cambridge). The developed platform enables individual ESCs to be cultured under continued observation as they exit their pluripotent stem cell state. Each cell within the device may be extracted from the chip at any time for further investigation without disturbing other cells. Assessing the transition from the stem cell state in individual cells is paramount if we are to understand the mechanisms of pluripotency.

Stem cell pluripotency and self-renewal are two important attributes of human embryonic stem cells which have led to enhanced interest in stem cell research. Understanding the mechanisms that underlie the regulation and maintenance of these properties is imperative to the clinical application of stem cells. Pluripotency and self-renewal are regulated by different genes, transcription factors and other co-factors such as FoxD3 and Klf4. Oct4, Nanog and Sox2 are central to the stem cell regulatory circuitry. They form interactions with co-factors to promote cell proliferation and inhibit differentiation by negatively regulating differentiation markers. However, there are other novel pluripotency associated factors yet to be studied. In this study, bioinformatics and functional analyses were employed to identify a potential pluripotency gene called YY1AP1 from our lab's pre-existing microarray data. YY1AP1, a transcription regulatory gene, showed consistent down-regulation with induced cell differentiation. It was further investigated. First, its co-localization with Oct4 in both hESCs and iPSCs was confirmed by immunofluorescence staining. Knockdown experiments were then performed on this gene to investigate effects of knocking it down on gene expression in hESCs. Knocked-down cells were characterized for markers of pluripotency and differentiation at the transcript level. Results showed a down-regulation of pluripotency genes with no specific promotion of any of the germ layer markers. Gene expression at the protein level in knocked down cells was then assessed for YY1AP1, and its binding partner YY1, and pluripotency markers. Results showed that proteins of YY1AP1, YY1, Oct4, Nanog and CTCF were down regulated while the tumour suppressor gene protein, p53, was up-regulated in YY1AP1 deficient stem cells. Protein to protein interaction studies showed that YY1AP1, YY1, Nanog and CTCF proteins directly interacted with each other. Differentiation of YY1AP1deficient cells into EBs led to an almost complete shutdown of all gene expression, an indication that the cells did not form 'real' EBs. Differentiation of YY1AP1 ablated cells did not support any lineage promotion either. These results suggest a potentially new role for YY1AP1 in proliferation and self-renewal of stem cells through its possible direct binding to CTCF or its indirect binding to CTCF in complex with YY1.

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.