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In order to understand gene regulatory networks (GRNs) in mammalian cells, it is pivotal to assess the interaction between proteins and DNA. In particular, the specific DNA binding activity of transcription factors (TFs) determines the expression of target genes and in general the overall connectivity of the GRN. However, the genomic location of TF binding cannot be predicted just from the DNA sequence, and functional assays are required to detect this interaction. The investigation of the binding of TF to DNA is usually accomplished by chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). While in the last 10 years this method enabled a better understanding of how transcription is regulated in living cells, it does have some drawbacks. In particular, the need for very highly specific antibodies and the large amount of starting material limit the ability of ChIP-seq to address biological questions when dealing with samples of small quantity. A technique called DNA Adenine Methyltransferase Identification (DamID) was developed in Drosophila as an alternative method for the detection of protein- DNA interactions and it is based on the fusion of a protein of interest (POI) with the DNA adenine methyltransferase (Dam). This fusion causes DNA methylation of adenines surrounding the sites where POI binds and the subsequent identification of the methylation sites allows mapping of the binding event without antibodies and using less cells as starting material. While this technology was successful in detecting the interaction between nuclear lamina and DNA in mammalian cells, to date little reports are present in the literature about TF DamID. This is mainly due to the different nature of TF binding compared to Lamin (punctuated instead of broad) and to the elevated intrinsic activity of Dam that makes the detection of real signal above the noise challenging. I here demonstrate a step-by-step optimization of the DamID technology coupled to next-generation sequencing (DamID-seq) that I used to map the binding of the mouse embryonic stem cell master regulator Oct4 in as few as 1,000 cells. This new technology paves the way for exciting new experiments where the number of cells is scarce such as in vitro cell state change or in vivo processes.

Corneal degenerative diseases and opacity are leading causes of corneal impairment and blindness worldwide. Like many epithelial tissues, the constant renewal of transparent corneal epithelial cells is essential for a lifelong healthy cornea and optimal vision. The limbus (the boundary between the cornea and the conjunctiva) is believed to be the site that harbours adult stem cells responsible for corneal maintenance and repair after injury, referred to as limbal epithelial stem cells (LESCs). In the basal limbal epithelium, an active LESC subset divides to yield progenitor cells that migrate centripetally into the corneal epithelium for cell renewal. This asymmetric division however, is assumed to be regulated by a balance between cell renewal and loss of cells from the corneal surface. The search for specific LESC molecular markers has been difficult and to date there are few if any candidate markers that unambiguously identify LESCs but not their immediate progeny. Consequently, LESC clonality, activity and proliferative dynamics have remained poorly understood. In addition, the nature of the regulatory molecular pathways involved during LESC activity is still an open key question. In this research project, we identified stem cells on the ocular surface of the eye, assayed their activity and demonstrated quantitively for the first time how the cornea responds to damage. The retention of DNA labelling reagents in control and wounded corneas was combined with clonal analyses of cell division and migration using mice mosaic for reporter LacZ expression. Corneal transplant in LacZ reporter transgenic mice showed migration of LacZ-positive cells into the donor corneal button, with long-term disruption of patterns of migration. Corneal epithelial scrape wounds at the periphery also showed long– term disruption. Label retention suggested a small but statistically significant proliferation response of LESCs to injury, but this was attenuated or absent in aging mice and Pax6 mutants. The Hippo signalling pathway has been shown to have promising results in regulating stem cell activity and proliferation in many organs, however, its effect on LESC proliferation is unknown. Here, we investigated the regulatory role of the Hippo−YAP signalling pathway during cell proliferation, and determined whether the label retention assay in a uniform population of dividing cells is a real indicator of slow-cycling cells in vivo. Cell-cycling kinetics, Abstract v proliferation rate, and label retention were determined in a spontaneous human corneal epithelial (HCE-S) cell line, using double-labelling IdU and EdU thymidine analogues. During homeostasis, HCE-S cells underwent approximately one cell cycle per day, however, cells in which YAP-dependent signalling was activated by 17-Allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of heat shock protein 90 (Hsp90), showed slower proliferation rate and longer cell-cycle time. In vitro label-retention assay in confluent cultures estimated number of ~3-4 cell cycles needed to dilute out the label from slow-cycling cells in vivo. The data are suggestive that the Hippo pathway has a potential regulatory role in proliferative corneal epithelium, and that label-retention assay is a real indicator of slow-cycling cells. Furthermore, this research observed the proliferative dynamics of conjunctival stem cells. Clonal analysis of patterns of cell growth in the conjunctiva were analysed following tamoxifen-induction of LacZ transgene activity. The long−term presence of coherent patches of LacZ-positive cells suggested the presence of long-lived conjunctival stem cells but that the turnover time for the bulbar conjunctival epithelium may be more than 16 weeks. The key results of this research are that the limbus is the niche for stem cells, that there is a unidirectional migration of cells from the limbus to the corneal epithelium during homeostasis, but this is disrupted, perhaps permanently, by wounding. We find only a mild response of limbal epithelial stem cells to acute corneal injury, which is reduced to no response with age and is dependent on genetic background.

Cardiac disease is the leading cause of death in the United States, despite the continuing efforts contributed to scientific research and disease management in the past few decades. However many advances have been made in cardiovascular research in recent decades and one of the advances is the development of human induced pluripotent stem cell(iPSC)-based disease models. The human iPSC-based disease models are derived from the somatic cells of patients with cardiac diseases and capture the genotypes of the original patients, which make them more ideal for mimicking human diseases compared with conventional rodent models. So far, the iPSC-based disease models have been used to model several types of cardiac diseases, one of which is the focus of this work-Timothy syndrome. Timothy syndrome is caused by the missense mutations in the CACNA1C gene encoding the voltage-gated calcium channel CaV1.2, which plays an essential role in cardiac function. The disease is a multisystem disorder that is featured by long QT syndrome and syndactyly. Timothy syndrome patients are treated clinically with beta-adrenergic blockers, calcium channel blockers, and sodium channel blockers. However, these regimens are insufficient to prevent lethal arrhythmias in Timothy syndrome patients, especially infants with Timothy syndrome. Therefore, new therapeutics to prevent the lethal arrhythmias in Timothy syndrome patients are needed until the age when an implantable defibrillator is available. The iPSC-based model of Timothy syndrome was first reported in 2011. The previous report showed that the Timothy syndrome iPSC-derived cardiomyocytes demonstrated several cellular phenotypes including abnormal contractions, abnormal electrophysiological properties and abnormal calcium handling, which were consistent with the clinical features of the patients that the iPSCs were derived from. In addition, the authors demonstrated that Roscovitine, a cyclin-dependent kinase (CDK) inhibitor, could rescue the cellular phenotypes in Timothy syndrome cardiomyocytes. However, the mechanisms underlying the beneficial effects of Roscovitine on Timothy syndrome cardiomyocytes were not fully elucidated. This work will employ the iPSC-based model of Timothy syndrome to investigate the mechanisms underlying the beneficial effects of Roscovitine on Timothy syndrome cardiomyocytes and search for additional therapeutic compounds and targets for Timothy syndrome. In chapter 1 of this work, we presented new methods to generate iPSCs from human skin fibroblasts or hair keratinocytes, and to differentiate iPSCs into cardiomyocytes in a monolayer format. The major advantage of the two new methods is that they are technically simple and generally applicable for samples from healthy control donors and patients with cardiac diseases. The new methods enabled us to generate a sufficient amount of Timothy syndrome cardiomyocytes from iPSCs derived from the skin fibroblasts of Timothy syndrome patients, which became the foundation for the subsequent mechanistic study. Chapter 2 presents the identification of CDK5 as a new therapeutic target for Timothy syndrome. As introduced above, the previous report demonstrated that Roscovitine, a CDK inhibitor, could rescue the cellular phenotypes in Timothy syndrome cardiomyocytes. However, the mechanisms underlying the beneficial effects of Roscovitine on Timothy syndrome cardiomyocytes were not fully elucidated. To identify additional therapeutic compounds for Timothy syndrome and investigate the mechanisms underlying the therapeutic effects of Roscovitine on Timothy syndrome cardiomyocytes, we conducted a phenotypic screen using Timothy syndrome cardiomyocytes to screen through twenty Roscovitine analogs and four CDK inhibitors with different specificities for different CDKs. Four positive compounds were identified from the screen. When we summarized the CDK targets of the four positive compounds and the lead compound Roscovitine, it was found that four out of the five positive compounds shared a common CDK target, which is CDK5, indicating that CDK5 could be involved in the pathogenesis of Timothy syndrome as a therapeutic target. We next validated CDK5 as a new therapeutic target for Timothy syndrome using two independent approaches. The two approaches are expressing a dominant negative mutant of CDK5 and expressing short hairpin RNAs targeting CDK5 in Timothy syndrome cardiomyocytes using lentiviruses. Both approaches led to CDK5 inhibition in Timothy syndrome cardiomyocytes and we examined the changes in the cellular phenotypes in Timothy syndrome cardiomyocytes with CDK5 inhibition. The results indicated that CDK5 inhibition alleviated all the previously-reported phenotypes in Timothy syndrome cardiomyocytes. To investigate the mechanisms underlying the beneficial effects of CDK5 inhibition on Timothy syndrome cardiomyocytes, we examined the expression of CDK5 activator p35 and the activity of CDK5 in Timothy syndrome cardiomyocytes. We found that Timothy syndrome cardiomyocytes showed a higher expression of CDK5 activator p35 and a higher activity of CDK5 compared with control cardiomyocytes. When we over-expressed CDK5 in control cardiomyocytes, we found that CDK5 over-expression caused a change in the function of CaV1.2 channels in control cardiomyocytes that resembled the phenotype in Timothy syndrome cardiomyocytes. In summary of the results, we propose that in Timothy syndrome cardiomyocytes, the increased expression of CDK5 activator p35 causes CDK5 hyper-activation, which enhances the abnormal function of the mutant CaV1.2 channels, leading to more severe phenotypes. Thus, CDK5 inhibition alleviates the phenotypes in Timothy syndrome cardiomyocytes. The results in this chapter reveal that CDK5 is a new therapeutic target for Timothy syndrome and CDK5-specific inhibitors can potentially be developed into new therapeutics for Timothy syndrome. However, we found that the currently-available chemical inhibitors for CDK5 are not highly-selective and have several significant side effects that make them not ideal candidates to be developed into new therapeutics for cardiac diseases. Therefore new therapeutic compounds and targets are still needed for Timothy syndrome. Chapter 3 presents the identification of the sigma-1 receptor as a new therapeutic target for Timothy syndrome. Due to the side effects associated with the currently-available chemical inhibitors for CDK5, we made an effort to search for an additional therapeutic target and therapeutic compounds for Timothy syndrome. We reasoned that instead of directly inhibiting CDK5, we could potentially alleviate the phenotypes in Timothy syndrome cardiomyocytes by affecting the CDK5 activator p35 and this idea led us to the sigma-1 receptor. After we looked into the sigma-1 receptor, we found that in addition to being reported to modulate p35 protein level, the sigma-1 receptor had also been reported to modulate calcium homeostasis, which is another favorable effect for Timothy syndrome cardiomyocytes. Therefore we hypothesized that the activation of the sigma-1 receptor could be beneficial for Timothy syndrome cardiomyocytes, which feature an increased expression of p35 and a dysregulation of calcium homeostasis. To test this hypothesis, we examined the effects of two sigma-1 receptor agonists, one of which is a FDA-approved drug, on the phenotypes in Timothy syndrome cardiomyocytes. The results demonstrated that the treatment of the two sigma-1 receptor agonists alleviated the previously-reported phenotypes in Timothy syndrome cardiomyocytes. We also examined the effects of the two sigma-1 receptor agonists on the functions of control cardiomyocytes and found that the treatment of the two sigma-1 receptor agonists did not have significant side effects on the regular contractions and normal calcium transients in control cardiomyocytes. Overall, the results reveal that the sigma-1 receptor is a new therapeutic target for Timothy syndrome. The results also demonstrate that the two sigma-1 receptor agonists that we tested are promising lead compounds that can developed into novel therapeutics for Timothy syndrome in the future. Since one of the sigma-1 receptor agonists that we tested is a FDA-approved drug, this drug could potentially be used directly in Timothy syndrome patients for treating the cardiac arrhythmias in the near future. In summary, this work is a proof of concept that the iPSC-based models of cardiac diseases can be used to generate novel insights into disease pathogenesis, and to identify new therapeutic targets and compounds for cardiac diseases, and in particular for Timothy syndrome. The therapeutic targets and compounds that we have identified in this work would be helpful for the development of novel therapeutics for treating the lethal arrhythmias in Timothy syndrome patients in the future.

In adults blood stem cells, called haematopoietic stem cells (HSC), give rise to all blood cells throughout life. The origin and biology of HSCs during embryo development has been an intensely studied topic. Definitive HSCs are generated intra-embryonically in the aorta-gonad-mesonephros (AGM) region of the mid-gestation embryo. Recent research revealed that HSCs emerge through multistep maturation of precursors: proHSC → preHSC I → preHSC II → definitive HSC (dHSC). A hallmark of the HSC emergence is the appearance of intra-aortic haematopoietic clusters that are considered to be sites of haematopoiesis. It was shown in vitro that the E11.5 HSCs are slowly cycling compared to progenitor cells. However, cell cycle status and its role during early HSC development remain unclear. Here I used Fucci transgenic mice that enable in vivo visualisation of the cell cycle. Functional and phenotypic analysis showed that in the early embryo the proHSC precursors cycle slowly, whereas committed progenitors are actively cycling. Meanwhile the preHSC I precursors arising in the E10.5 AGM region become more rapidly cycling. They are located closer to the luminal cavity of the dorsal aorta, while their ancestors, the proHSCs, are slowly cycling and are located at base of the clusters. Furthermore, in the mid-gestation embryo the preHSC I become slowly cycling and are closer to the endothelial lining of the aorta, while they give rise to the actively cycling preHSC II that are located to the luminal area of the artery. Finally, definitive HSCs are mainly slowly cycling at this stage like their foetal liver counterparts. As expected, HSCs in adult bone marrow are mainly dormant. The data suggest that transition from one precursor type to another is accompanied by distinct changes in cell cycle profile and that HSCs become progressively quiescent during development. To test the role of cell cycle in HSC maturation, we used inhibitors against signalling pathways known to play important roles in HSC development. Notch inhibitor affected the cell cycle status of haematopoietic precursors, by possibly promoting them to rapidly proliferate and potentially blocking the maturation from preHSC I to preHSC II precursors. Shh antagonist had the opposite effect and enhanced the HSC activity from the preHSC I precursors. Altogether these results suggest that the cell cycle status plays an important role in the HSC development. A better understanding of the molecules that control this process will allow us to optimize the culture condition for generation of functional HSCs in the laboratory.

The generation of induced pluripotent stem cells (iPSCs) presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. While several intermediate populations have been described, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPSCs. The rapid expansion of a minor population of reprogrammed cells can also obscure investigation of relevant processes. Understanding of the biological mechanisms essential for successful iPSC generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that reprogramming follows an orderly sequence of stage transitions marked by changes in cell surface markers CD44 and ICAM1, and a Nanog-GFP reporter. RNA-sequencing (RNA-seq) analysis of these populations demonstrates two waves of pluripotency gene up-regulation, and unexpectedly, transient up-regulation of multiple epidermis-related genes, demonstrating that reprogramming is not simply the reversal of normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and this improved understanding of the reprogramming process will lead to novel reprogramming strategies.

Embryonic stem (ES) cells are cell lines isolated from the embryo at a time just prior to implantation into the uterus. In the right cocktail of medium and cytokines, these cell lines can be maintained indefinitely in vitro in a self-renewing state. Initially it was assumed that these cells represented a homogeneous population however, more recently it has been shown that there are a great number of genes that are expressed heterogeneously. ES cell cultures are therefore a mix of different subpopulations, some of which have distinct functional properties including a bias or ‘lineage priming’ towards a particular cell fate. These populations are also dynamic in nature, converting from one state to another with fairly rapid kinetics. The main focus of this thesis was to gain a more in depth understanding of the mechanisms regulating heterogeneity and lineage priming in murine ES cells by asking which signalling pathways play a role in this phenomenon and how the switch between states is regulated at a transcriptional level. These questions were asked using an ES cell line containing a sensitive reporter for the endoderm marker Hex. This reporter, developed by a previous lab member, allowed the identification and separation of a population of ES cells primed towards a primitive endoderm fate. Primarily, I assessed the effect of a defined culture system (2i) on the Hex-expressing population. This culture system contains inhibitors that block FGF signalling and the Wnt pathway component GSK3. Culturing ES cells in 2i has been suggested to generate a more homogeneous culture. Here, I have shown that culturing ES cells or pre-implantation embryos in 2i did not eliminate heterogeneity but maintained them in an early state prior to lineage segregation. When ES cells were cultured in standard serum-containing medium, Hex was expressed in a mutually exclusive manner with the embryonic marker NANOG, while in 2i a subpopulation of cells coexpressed both Hex and NANOG. This population was functionally primed towards extraembryonic endoderm and trophoblast. Furthermore, these ES cells could efficiently contribute to 2-cell embryos in chimaera assays. LIF signalling promoted this population through the JAK/STAT pathway. I then asked how transcription was regulated during the switch between unprimed ES cells to those primed towards a primitive endoderm fate, as well as how regulation changes during further differentiation. To ask this, Hex positive (primed) and negative (unprimed) ES cell populations were sorted as well as a Hex positive differentiated sample. These samples were analysed by GRO-seq to determine the location, density and orientation of RNA-polymerase throughout the genome. Changes in gene expression between primed and unprimed states were regulated primarily through elongation whereas genes upregulated during differentiation were regulated at the point of de novo initiation.

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.

Heterozygous mutations in the transcription factor, hepatocyte nuclear factor 1B (HNF1B), result in multisystem disease including diabetes due to beta-cell dysfunction and pancreatic hypoplasia. However, the mechanisms that underlie development of diabetes in HNF1B mutation carriers are still not fully understood due to lack of an appropriate model system. Human induced pluripotent stem cells (hiPSCs), which are capable of self-renewal and can differentiate into any cell type, provide an advantageous alternative to model human developmental diseases. The aim of this project was to develop a hiPSC based model system to determine the molecular mechanisms by which HNF1B mutations cause pancreatic hypoplasia and diabetes. HNF1B mutant hiPSC lines were produced using CRISPR-Cas9 genome editing. Isogenic HNF1B wild-type, homozygous and heterozygous mutant hiPSC lines were directed to differentiate along the pancreatic lineage and cells were phenotyped at each stage of the differentiation process to check for appropriate expression of lineage markers. The normal expression pattern of HNF1B in human pancreas development was analysed and showed up-regulation of HNF1B at the foregut stage, and during pancreas specification. Homozygous knockout of HNF1B resulted in failure of foregut and pancreatic progenitor development, while heterozygous knockout of HNF1B resulted in impairment of pancreatic progenitor and endocrine cell differentiation as well as impaired insulin secretion upon glucose stimulation. Cell proliferation analyses showed a significant decrease in the proliferation rate in HNF1B heterozygous and homozygous mutant cells compared with wild-type cells at the foregut stage while no change in the apoptosis rate could be detected. RNA-sequencing and ATAC-sequencing, were used to further define the molecular mechanisms controlled by HNF1B and the effect HNF1B on modulation of chromatin accessibility during pancreas development. These results provide further insights into the molecular mechanisms by which HNF1B regulates human pancreas development and function, revealing that HNF1B haploinsufficiency impairs the expansion and maintenance of pancreatic progenitor cells in vitro. In vivo, this would likely result in reduced beta cell numbers at birth and diabetes later in life in patients with HNF1B-associated disease. These mechanisms suggest that the capacity to produce pancreatic progenitor cells during embryonic life could determine individual susceptibility to diabetes.

The formation of neurospheres was important in demonstrating that neurogenesis in the adult brain may be fuelled by a stem cell population. The olfactory mucosa is another site of neurogenesis which, in humans, has been observed to contain a stem cell population through the formation of neurospheres (Murrell et al., 2005). Stem cells can be defined as cells capable of self-renewal and multipotency. The aim of this study was to investigate the potential of rat olfactory stem cells growing as neurospheres. The hypothesis is that the rat olfactory mucosa contains a 'true' stem cell population that can be cultured as neurospheres and that will demonstrate multipotency by differentiating into 'non-olfactory' cell types and possess the capacity for self-renewal, if provided with the appropriate environmental niche. Here it was found that adult rat olfactory mucosa is capable of generating neurospheres when cultured in EGF and bFGF. Evidence of self-renewal was provided by the formation of six generations of neurospheres, the formation of neurospheres from single cells and the expression of markers associated with self-renewal by neurosphere cells. The multipotency of olfactory neurosphere cells was demonstrated through manipulation of the stem cell niche. In defined culture conditions, extracellular matrix molecules and growth factors were able to induce the differentiation of neurosphere cells down the dopaminergic lineage pathway. When co-cultured with differentiating cells, neonatal myoblasts and 3T3-L1 cells, olfactory neurosphere cells were able to differentiate and incorporate into a skeletal muscle myotube and differentiate into adipocytes, respectively. In conclusion it was found that the adult rat olfactory mucosa is capable of generating neurospheres. When presented with an appropriate niche neurosphere cells are able to self-renew and demonstrate multipotency.