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Embryogenesis entails complex signalling interactions between embryonic and extraembryonic lineages. Improved embryo culture techniques have recently allowed human embryos to be cultured in vitro past the point at which they would implant in vivo, until the internationally accepted culture limit of 14 days. Nonetheless, the reliance on surplus in vitro fertilised (IVF) embryos, along with the ethical and legal considerations associated with genetic manipulation of human embryos, present barriers to the study of postimplantation human development. However, human stem cell-based embryo models offer a system that is amenable to genetic and molecular manipulation. Within mouse development, the anterior visceral endoderm (AVE), formed from the extraembryonic endoderm, plays a crucial role in restricting primitive streak formation to the posterior epiblast, thereby establishing the anterior-posterior axis of the mouse embryo. Recent studies of human embryos cultured to postimplantation stages have alluded to the presence of a human equivalent of the mouse AVE in the anterior hypoblast, the hypoblast being the portion of the human yolk sac that lies adjacent to the epiblast. However, the functional role of such a structure within the context of human development is yet to be understood. In vitro stem cell modelling of the human yolk sac- epiblast interaction offers a system through which the role of the yolk sac in postimplantation epiblast morphogenesis can be interrogated. Consequently, the aim of this study was to derive yolk sac-like cells (YSLCs) that could be used to model human epiblast-yolk sac crosstalk in vitro. Following a screen of developmentally relevant signalling pathway activators, this project identified that the coactivation of NODAL, WNT and JAK/STAT signalling via ACTIVIN-A, CHIRON, and leukaemia inhibitory factor (LIF) (ACL) induced an endodermal fate in human pluripotent stem cells (hPSCs). Functional and transcriptional analysis of ACL-treated cell populations revealed that the pluripotent state harboured by hPSCs determines the type of endoderm that ACL treatment induces. Human embryonic stem cells (hESCs), which harbour a primed pluripotent state, were found to respond to ACL by adopting a definitive endoderm-like fate, whereas Rset hPSCs, which harbour a pluripotent state that is an intermediary between naïve and primed, gave rise to a yolk sac-like population. Accordingly, Rset hPSCs treated with ACL were coined YSLCs. YSLCs were found to express inhibitors of BMP, WNT and NODAL signalling, while YSLC conditioned medium was shown to inhibit downstream activity of BMP and WNT signalling in hESCs in both 2D and 3D culture conditions. Similarly, YSLC conditioned medium supported the coexpression of pluripotency and anterior ectoderm markers in hESCs, at the expense of posterior epiblast markers. These results indicated that YSLCs share functional characteristics with the mouse AVE, and also potentially the human anterior hypoblast. Collectively, therefore, this project proposes YSLCs as a tool for studying human epiblast-yolk sac crosstalk.

Cell transplantation of pancreatic islets has been proven to be an effective long-term treatment for Type 1 diabetes. However, the lack of sufficient cadaveric donor tissue has prevented widespread clinical adoption of cell therapy. An alternative source of cells would be hugely beneficial in overcoming this donor shortage. Cells of the islets arise during development from a shared precursor cell - the NGN3+/NEUROD1+ pancreatic endocrine progenitor. Over the past decade, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been the main focus for providing an alternative source of pancreatic cell types for transplant. While differentiation to PDX1+/SOX9+ early pancreas progenitor cells has been successful, efficient differentiation to endocrine progenitors and mature islet cells has not been achieved. This suggests that we lack the knowledge to correctly and effectively specify an endocrine fate in hES-derived pancreas cells. It was the aim of this thesis to describe simple methods for culturing authentic pancreas progenitor cells from mouse and human foetal pancreas tissue, with which we could study the biology of these progenitor cells to ultimately inform better hESC differentiation protocols, and which may also be useful for cell therapy in their own right. To achieve these aims, we have taken three approaches. First, we provide characterisation of early pancreas progenitors during human foetal pancreas development, and how expression of key markers changes over time. Second, we describe methods for isolating and expanding mouse and human foetal pancreas progenitors, producing new cellular models with which to investigate the specification of endocrine cell fate. Finally, we generated an hESC reporter line to allow for easier identification of conditions that induce hESC-derived pancreatic endocrine progenitors in vitro. We have described the precise timing of the transition from early pancreas progenitor to endocrine progenitor in the developing human and mouse foetal pancreas. Using this information, we have pinpointed the stages of foetal development when mouse and human have the maximum potential proliferative capacity. Using samples from these developmental stages, we demonstrate that PDX1+/SOX9+ human and mouse foetal progenitors can be isolated and expanded long-term in monolayer in defined conditions. Cultured mouse foetal pancreas progenitors can be induced to differentiate to endocrine progenitors in vitro. We also generated a novel NGN3- GFP/CyclinB1-Cherry hESC reporter line, which allows identification of emerging endocrine progenitor cells and their proliferative capacity. The new cellular models presented here are ideally suited for many downstream applications including high-throughput pharmacological screens and imaging assays. Studies using these systems will help improve our understanding of human pancreatic endocrine progenitor specification and harness these cells in new types of cell therapies for diabetes.

Differentiation and cell cycle regulation in stem cell have a key function for embryonic development, organ homeostasis and tissue repair. Recent results have shown that these two mechanisms are intrinsically connected. Indeed, cell cycle machinery directly controls maintenance of pluripotency and initiation of differentiation. More precisely, the cell cycle regulator Cyclin D appears to control the transcriptional activity of Activin/Nodal signalling during progression of the cell cycle in human Embryonic Stem Cells (hESCs). As a consequence, hESCs can only differentiate into endoderm in the Early G1 phase when Cyclin Ds are expressed at low levels. These results show the mechanisms by which the cell cycle defines differentiation propensity of stem cells. However, these observations also imply the existence of interplays coordinating extra cellular signalling pathways with the epigenetic state, chromatin structure and transcriptional networks during cell cycle progression and these mechanisms remain to be fully uncovered. Here, I have utilised the FUCCI reporter system combined with ATAC-Seq to analyse chromatin dynamics during cell cycle progression in hESCs. Furthermore, I performed ChIP-Seq analyses to define the genomic location of transcriptional regulators during cell cycle progression as well as RNA-Seq to confirm variation in gene expression pattern. Integration of these data shows that the chromatin status in hESCs is highly dynamic and the core pluripotency transcription factors and epigenetic modifiers change genomic location during cell cycle progression. I also showed that hESCs in the Late G1 phase accumulate transcripts that are important for differentiation and development; therefore, indicating this phase represents a unique portion of the cell cycle for cell fate decisions. Taken together, these results uncover that transcriptional networks are unexpectedly dynamic during the progression of cell cycle in stem cells. I hypothesise that these modifications are necessary to prime hESCs for different cell fate choices allowing a diversity of differentiation that is otherwise impossible. Overall these mechanisms underline the need to study transcriptional and epigenetic mechanisms in the dynamic context of the cell cycle and have major implications for adult tissue homeostasis and disease.

The endometrium undergoes recurrent cycles of dynamic remodelling, involving breakdown and scarless repair, proliferation and differentiation, including decidualisation of the stroma, during the menstrual cycle. Extensive studies have characterised how the steroid hormones oestrogen and progesterone acting via their nuclear receptors coordinate these remarkable changes. Although a few previous studies have postulated a role for androgens the impact of androgens on endometrial function remains understudied. The studies described in this thesis aimed to 1) identify cellular processes, pathways and networks regulated by androgens in human androgen receptor-positive endometrial stromal cells (hESCs), 2) investigate the potential for regulation and determine the regulation of putative dihydrotestosterone (DHT)-regulated gene expression by androgen in hESCs, 3) investigate the expression and regulation of putative androgen-regulated genes in the human endometrium across the menstrual cycle and in early pregnancy and 4) explore the role of androgens in modulating metformin-induced gene expression associated with decidualisation of hESCs. Analysis of data from a whole genome array conducted previously in the laboratory using primary hESCs treated with DHT for 2 or 8 hours identified time dependant putative androgen-regulated mRNAs (34 and 268 genes, respectively). Thereafter, all work was completed by the author. Gene ontology and functional based bioinformatic analyses of the putative androgen-regulated gene sets revealed potential androgen regulation of a variety of cell processes, pathways and networks including those associated with gene transcription, signal transduction pathways (such as phosphatidylinositol, oestrogen receptor alpha (ERα) and Wnt signalling), cancer pathways, metabolism, cell cycle, development, apoptosis/survival. In addition, various transcription factors (e.g. AR, c-Myc, SP1, ERα, p53, E2F1, RUNX2, CREB1 and STAT3) were associated with androgen regulation in hESCs. Consensus androgen receptor binding sites were identified in the promoter sequences of 18 genes by transcription factor binding site sequence analysis. Direct DHT regulation of ten of 15 of these genes was validated in endometrial stromal cells using qRTPCR. Of these genes, RGS2, SIK1, and SNCAIP mRNAs were confirmed as DHT-regulated in hESCs by use of an AR inhibitor (flutamide) and in addition, were not found to be regulated by oestradiol. Discovery bioinformatics predicted these genes may interact in a gene network involving AR and the cAMP transduction pathway. Expression of the 15 putative androgen-regulated genes was confirmed by qRTPCR in intact human endometrial tissue (13 novel) and 9 of these genes were regulated in association with decidualisation i.e. either in the secretory phase, the time at which decidualisation begins and/or in first trimester decidua. Protein expression of RGS2, SIK1 and Synphilin-1 (encoded by SNCAIP) was confirmed by immunohistochemistry in endometrial tissues and protein expression also appeared greater in decidua. Regulation of putative androgen-regulated gene expression by decidualisation was confirmed in 4 out of 8 genes by employing a model of reduced in vivo decidualisation i.e. decidua from ectopic pregnancies. Regulation of 5 out of 7 genes was confirmed in decidualised hESCs (RGS2, SIK1, SLC6A6, SNCAIP and AXIN2) but expression of these genes was not altered by DHT inclusion during decidualisation. Finally, only a high metformin concentration enhanced hESC decidualisation and putative androgen-regulated gene expression (4 genes) in decidualised hESCs. In comparison, in the presence of DHT, a lower clinically relevant metformin concentration (100μM) did enhance decidualisation marker expression but did not alter expression of putative androgen-regulated genes. In summary, these studies have revealed new insights into androgen action in the human endometrium. Studies in hESCs 1) predicted the pathways and interacting transcription factor regulatory networks that may be androgen-dependent in this cell type, these were associated with cell differentiation, apoptosis and proliferation, 2) identified novel putative androgen-regulated genes expressed in hESCs and in endometrial tissues, 3) showed putative androgen-regulated genes are regulated by DHT (possibly via AR) in endometrial stromal cells, some of which are also regulated in association with decidualisation and 4) showed that androgens may enhance decidualisation during exposure to the commonly used drug metformin. Collectively, these new findings support a physiological role for androgens in endometrial function and provide a series of new avenues for further studies of the regulation of differentiation and proliferation.

Fluid mechanics driven bioreactor design has entered a new phase of development in the last 15 years: the scalable expansion and differentiation of human pluripotent stem cells (hPSCs) for translational therapies, drug screening and discovery, developmental biology research, and disease pathology. Demand for hPSC-derived resources is expected to increase due to the evolving global human health initiatives. Bioreactors are a suitable alternative to tissue culture dishes for the purposes of mass production. Stirred spinner flasks are a common bench-scale bioreactor for testing hPSC culture outcomes including the efficient production of undifferentiated cells, intermediate progenitor cells and terminally differentiated end-products. To continuously supply nutrients and oxygen, and homogeneously distribute cellular metabolic waste, culture medium in spinner flasks is stirred; however, this generates heterogenous fluidic shear stress on hPSCs due to the laws of fluid mechanics. As hPSCs are sensitive to physiochemical and bioenergetic cues, this poses a unique challenge. Exposure to shear stress or other forces has been known to kill the cells, or counterproductively direct them to an undesired phenotype. For these reasons, fluidic shear stress is often avoided in bioreactor design and parameterization, but much is unknown about the nuanced effect of fluidic shear on hPSCs. It is unclear if appropriate application of fluidic shear could productively direct lineage specification, maintain pluripotency, or enhance differentiation efficiency or functional maturity. Thus, the development of novel devices and methods capable of integrating refined engineering control of the bulk hydrodynamic conditions and the fluidic microenvironments in which the cells are grown is warranted. This work seeks to uncover the role of fluidic shear in dynamic suspension in bioreactors. This was achieved in part by observing the cellular and molecular expression of hPSC aggregates cultured in stirred spinner flasks compared to monolayer colonies and aggregates grown in static, or stationary, fluid culture medium. Culture outcomes were observed for their cell counts and lineage potentials, and finally assessed with bulk RNAseq. The results of these assays showed that hPSC aggregates exposed to shear in spinner flasks form a gene signature that suggest fluidic agitation may promote de-differentiation to a more naïve-like pluripotency state and promote lineage specification resulting in a dual population. To test these types of shear effects in hPSC cultures in the future, an operating manual for a novel concentric cylinder rotating wall vessel bioreactor (CRWV) is described in this work. The CRWV can impose fluidic shear over a narrow distribution based on circular Couette flow and was shown to support hPSC survival for 24-hrs post-inoculation under certain conditions.

Genome architecture has emerged as a key factor of gene regulation and is tightly coordinated with the temporal order of DNA replication (Replication Timing – RT). RT and 3D genome organization change dynamically throughout development in correlation with the establishment of cell type specific gene expression patterns. The first organ to develop during embryonic development is the heart. The heart is a post-mitotic, terminally differentiated organ which, compared to other organs such as the liver, lacks the capacity to regenerate upon injury such as myocardial infarction. Instead, it forms fibrotic tissue that maintains organ integrity but undermines pump function, often leading to congestive heart failure and premature death. Deciphering the 3D genome organization in cardiac differentiation may help our understanding of gene regulation during cardiac development. DNA replication timing (RT) is a very informative functional readout of large-scale chromatin organization across distinct cell types and its regulation during development. RT is cell type-specific, highly conserved and changes in RT affect approximately half the genome during development and differentiation. I hypothesize that changes in the 3D genome organization play a critical role in gene regulation and cell function in cardiac differentiation. In this work, I will differentiate human embryonic stem cells (hESCs) towards cardiomyocytes and use a multiomics approach (RT, transcriptome and 3D genome organization) to identify the dynamic changes in genome architecture, RT and gene expression during normal cardiac development. This will allow us, in the future, to construct an integrative model of nuclear function in cardiac cells that can be leveraged as a framework to identify cellular alterations associated with congenital cardiovascular diseases.

Systematic identification of regulatory factors that control human embryonic stem cell (hESC) differentiation can provide valuable insights into early development and disease. The discovery of the CRISPR/Cas gene editing system and its subsequent development in mammalian cells has revolutionized functional genomic studies. However, despite these advances, effective modulation of gene expression during multi-lineage differentiation of stem cells has proven technically challenging. Here, we report the first described hESC lines to repress (via CRISPR interference) or activate (via CRISPR activation) transcription during differentiation into all three germ layers. We subsequently performed an unbiased, genome-wide, FACS-based CRISPRi screen targeting long non-coding RNAs (lncRNAs) during definitive endoderm differentiation. Our screen yielded dozens of hits, including both known and novel lncRNA loci required for proper differentiation. Analysis of common features revealed that hits were more conserved, closer to enhancers, and significantly enriched for endoderm-specific GWAS SNPs compared to non-hits. Surprisingly, expression was not predictive of enrichment scores, supporting the need for functional screening instead of relying solely on gene expression analysis. Further downstream analysis of two hits, RP11-222K16.2 and FOXD3-AS1, revealed two lncRNA loci required for differentiation and pluripotency, respectively. Taken together, the cell lines and screen methodology described herein can be adapted to discover novel regulators of differentiation into any lineage.

Asthma is a major public health epidemic that presents a heavy burden on those who suffer from the disease. Little is currently understood about the genetic signature that distinguishes one type of asthma from another. Recently, the single nucleotide polymorphism (SNP) rs968567 was found to have a high degree of association in asthmatic patients (Sharma et al., 2014). This particular SNP is in the promoter region of the FADS2 gene that synthesizes the enzyme delta-6-desaturase (D6D). D6D mediates the formation of pro-inflammatory factors that lead to exacerbation of asthmatic symptoms. We engineered a novel, customized CRISPR-Cas9 construct to induce the SNP rs968567 in the HUES9 human embryonic stem cell (hESC) line. Our results show success in generating the custom CRISPR-Cas9 construct for use in stem cells, while efficiency in expressing the desired mutation in our cell line is currently being optimized. Disease modeling in the genomic era of medicine provides an opportunity for the development of personalized medical treatment. Future projects aim to differentiate stem cell lines edited with our CRISPR-Cas9 construct to lung progenitor cells to study the cellular phenotype of this mutation in context of asthma pathogenesis.

Understanding how neuronal diversity is achieved within the cerebral cortex remains a major challenge in neuroscience. The surge of human embryonic stem cells (hESCs) as a novel model system provides a unique opportunity to study human corticogenesis in vitro and€ identify the mechanisms that promote neuronal differentiation to achieve neuronal diversity in the human brain. Here, we demonstrate the derivation of corticofugal neurons from a genetically engineered Fezf2-YFP hESC reporter line, and uncover two distinct Fezf2 subpopulations that are reminiscent of the 2 Fezf2-expressing neuronal subtypes in the developing mouse brain. Fezf2 is a transcription factor that is both necessary and sufficient for the specification of subcerebral projection neurons in mouse. Its role in human corticogenesis is still unknown. However, the high conservation of FEZF2 protein between mouse and human suggest that Fezf2 is a specific marker of human cerebral neurons as well. Two hypotheses drove our research: 1) Extrinsic factors that modulate in vivo developmental signaling pathways play a critical role in the differentiation of hESCs to a corticofugal fate in vitro; 2) Human embryonic stem cell-derived neurons can survive in vivo and extend axonal projections to specific targets in the rodent brain. Our research shows that hESCs-derived corticofugal neurons are an effective model system to investigate the molecular pathways that regulate human cortical differentiation, axon extension and survival, an endeavor that was until now proscribed due to the manipulation of human embryos.