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.

Intestinal homeostasis is governed by fate choices of stem cells residing in the intestinal crypt base. This must involve niche-specific co-ordination of cell division to guarantee that epithelial cells divide at the right time and place. These mechanisms operate to ensure precise control of the numbers of stem and differentiated cells. Little is known about how proliferative fate decisions are regulated in intestinal crypts. Both the placement of daughter cells within a particular niche, and their decision to enter and progress through the cell cycle, contribute. This thesis investigates the spatiotemporal control of cell division in intestinal crypts to understand the relationship between cell-cycle specific fate choices and intestinal homeostasis. Firstly, I describe a novel mode of asymmetric cell division within intestinal crypts. Using high resolution microscopy of intestinal organoids, I show that a subset of mitoses produce daughters that become displaced from one another after cytokinesis. This post-mitotic separation or the ‘positional asymmetry’ of daughter cells occurs in all cycling epithelial cells. These divisions may facilitate divergent fate of daughter cells and provides a general mechanism for stochastic niche exit. Post-mitotic separation is facilitated by interkinetic nuclear migration and selective tethering to the basement membrane during mitosis. Importantly, these mechanisms are altered in tissue carrying mutations in Adenomatous polyposis coli (Apc), highlighting its importance for normal tissue homeostasis. Secondly, I aimed to understand the dynamics of cell-cycle commitment in intestinal crypt compartments by investigating the DNA Replication Licensing System. The licensing system is a master regulator of proliferative fate in all cells in adult tissue. At its core is the regulated loading of the Mcm2-7 protein complex onto origins of replication exactly once per cell cycle. Engagement of the licensing system directly indicates commitment to proliferative cell fate. A technique to visualise licensing in intestinal crypts was developed. This revealed distinct proliferation zones in intestinal crypts. Mcm licensing was most prevalent in the lower transit-amplifying compartment, the zone enriched for early TA progenitors. Licensing is inhibited in terminally differentiated cells, and not detected in the transit-amplifying cells most proximal to the differentiated zone. Strikingly, the majority of ‘active’ intestinal stem cells were found in an unlicensed state. These data suggest that licensing decisions are delayed or inhibited until late G1 phase in intestinal stem cells and explains their longer cell-cycle. We postulate that this may provide a time window for niche cues to act, either stimulating cell-cycle entry or allowing retention in a ‘shallow’ G0 state. High resolution imaging of cell-cycle phases throughout the epithelium revealed remarkable cell-cycle co-ordination. This manifested in uninterrupted ‘ribbons’ of cells in similar cell-cycle states. This was due to lineage specific cell cycle co-ordination where adjacent daughter cells progress through the cell cycle at the same rate. These field effects are the result of co-ordinated cell-cycle progression between daughter cells. These observations were validated using living organoids expressing fluorescent ubiquitination-based cell cycle indicators (FUCCI). These ribbons were occasionally interrupted by cells in other cell cycle phases suggesting the separation of sisters by daughters from another lineage. This suggests that cell-cycle coordination can facilitate post-mitotic separation, and influence stochastic niche exit.

Despite having made significant strides in the overall labor market, women continue to lag behind men at the senior and executive C-suite levels. The gap is even more striking in organizations within the fields of Science, Technology, Engineering, and Math (STEM). Although women-only leadership trainings have gained recent popularity as a strategy to address this, scholarship on the efficacy of such programs is still fairly scant. This study aimed to fill this gap in the research by looking into how graduates of such a program used the tools and knowledge gained during the training, to determine if and how it has impacted, in their view, their identity as leaders. Eighteen senior-level Information Technology (IT) leaders, graduates of the same in-house women-only leadership program were interviewed using a narrative inquiry approach. Data gathered during the interviews revealed which strategies participants put into practice, how they applied them, as well as the perceived outcomes that they derived. The narratives revealed women’s perspective of their leadership trajectory and their understanding of the training’s impact. Interviews were analyzed using content and thematic coding. Analysis of the participants’ accounts pointed to the training’s ability to facilitate leadership identity development through the following means: (1) the promotion of practical skills, (2) increased self-awareness and realization of others’ perceptions, (3) feeling a sense of belonging and connectedness, and (4) feeling recognized and empowered. It was also clear that context plays a significant role on the impact that the training can have. Three themes related to this emerged; (1) the availability of advocates, (2) executive visibility, and (3) fit with the organization’s leadership culture and ability to lead authentically. Participants’ stories also revealed the ways in which the training affected their ability to deal with a male-dominated organization, which ultimately depended on how much of an effect they believed that being a woman has on their workplace experience. Findings suggest that women-only leadership trainings can have a positive impact on senior-level leaders’ leadership identity, yet careful consideration must be paid to the contextual factors identified. Findings also provided concrete evidence pointing to the perceived effectiveness of specific program components.

No description available.

Breathing is essential for human life, yet tens of millions of people in the U.S. alone suffer from lung diseases. With each breath, lungs are exposed to the external environment. Inhaled air first passes through the trachea, bronchi, and finally the bronchioles before it reaches the alveoli where gases are exchanged. A barrier of epithelial cells protects the airways. In addition, epithelial glands also secrete protein-rich fluids onto the airway surfaces to help maintain sterility. Injury, disease, or other factors can damage these cells, and regiospecific stem cells (SCs) can divide to replace them. However, many important details about lung SCs are still unknown. For example, what processes control SC division? How do region-specific SCs differ from one another? And how does disease or injury impact SC biology? We found that some processes that regulate lung development also control adult SC division following injury. We show that SCs from airway glands give rise to surface epithelial cell types and glandular cell types. In contrast, surface SCs only generated surface cell types. Finally, we identify a type of cell in the glands that can regenerate surface cell types after severe injury. These studies provide new insights into the neighborhoods in which SCs reside in the large airways and processes that control their contribution to airway repair following injury. Overall, this research provides important new insights into adult SC biology and conditions affecting lung health.

Experts describe maker education as activities relating to the construction of artifacts that encourage learning through teamwork, problem-solving, and innovation. Teachers in recent years have been turning to maker-centered learning strategies to develop 21st century skills along with emphasizing strong content knowledge focusing on creation and creativity. Previous maker-based learning research focused primarily on the technology and tools associated with these activities; however, little research exists on the teachers'€™ involvement with these learning strategies. The purpose of this phenomenological study was to explore the experiences of teachers currently using maker-centered learning strategies as an instructional practice in grade 5-12 classrooms. Based on the idea of constructing knowledge through active learning, the conceptual framework for this research encompasses multiple learning theories including constructionism, constructivism, experiential learning, and cooperative learning. The research examined the motivation of teachers' using maker-centered learning strategies and the challenges and benefits they have experienced. Data were collected using semistructured interviews and written lived experience descriptions from seven teachers currently using maker-based learning in their classrooms and analyzed using InVivo coding. The participants described their experiences as facilitators in student-centered classrooms that focus on collaboration and learning through failure. Time and assessment are common challenges while increased student engagement and student social and academic growth are common benefits. Experts maintain that maker-centered learning improves 21st century skills and prepares students for success in college, careers, and lifelong learning opportunities.