Thesis (M. S.)--Computer Science, Georgia Institute of Technology, 2009. / Committee Chair: Dr. Samuel Dudley; Committee Member: Dr. Micheal Davis; Committee Member: Dr. Robert Butera

Thesis (Ph. D.)--Universität Mannheim, 2004/2005. / Includes bibliographical references (p. [313]-344).

Thesis (Ph.D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 153-173). Also available on the Internet.

Thesis (Ph.D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 153-173). Also available on the Internet.

Both cDNA tag-based and DNA chip hybridization assays have revealed widespread transcriptional activity across mammalian genomes, providing a rich source of novel protein-coding and non-coding transcripts. Annotation and functional evaluation of this undefined transcriptome space represents a major step towards the comprehensive definition of biomolecules regulating the properties of living cells, including embryonic stem cells (ESCs) and their derivatives. In this study I analysed 87 rare mRNA transcripts from human ESCs that mapped uniquely to the human genome, in regions lacking evidence for known genes or transcripts. In addition, the transcripts appeared enriched in the hESC transcriptome as enumerated by serial analysis of gene expression (SAGE). Full-length transcripts corresponding to twelve novel LongSAGE tags were recovered and evaluated with respect to gene structure, protein-coding potential, and gene regulatory features. In addition, transcript abundance was compared between RNA isolated from undifferentiated hESCs and differentiated cells. Analysis of full-length transcripts revealed that the novel ORFs did not exceed a size of 129 amino acids and no matches were observed to well characterized protein domains. Interesting protein level predictions included small disulfide-bonded proteins, known members of which are important in a variety of biological processes. Transcripts evaluated for differential expression by real-time RT-qPCR (Reverse Transcription followed by real-time quantitative Polymerase Chain Reaction) were found to be variably expressed (0.2- to 4.5-fold) in Day-2 or Day-4 retinoic acid-induced differentiation cultures compared to undifferentiated hESCs. Relative quantitation using a universal reference RNA (derived from pooled adult tissues) showed large differences in novel transcript levels (0.002- to 35-fold) compared to hESCs. Collectively, these results provide a detailed analysis of a set of novel hESC transcripts and their abundance in early and adult differentiated cell types, both of which may advance our understanding of the transcriptional events governing stem cell behavior. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Haematopoiesis is the process to produce haematopoietic stem cells (HSCs), haematopoietic progenitors (HPCs) and terminally differentiated cell types. In the adult, HSCs resided in bone marrow while in the embryo, haematopoiesis occurred sequentially in several niches including yolk sac, aorta-gonad-mesonephros (AGM) region, placenta and fetal liver. The AGM region is the first place where HSCs arise in vivo and therefore should provide important factors to induce haematopoiesis. The mouse embryonic stem cells (mESC) system is a powerful platform to mimic the development process in vitro and is widely utilized to study the underlying mechanisms because they are pluripotent and can be genetically manipulated. A novel co-culture system has been established by culturing differentiating mESCs with primary E10.5 AGM explants and a panel of clonal stromal cell lines derived from dorsal aorta and surrounding mesenchyme (AM) in AGM region. Results of these co-culture studies suggested that the AM-derived stromal cell lines could be a potent resource of signals to enhance haematopoiesis. Molecular mechanism involved in haematopoiesis is a key research direction for understanding the regulation network of haematopoiesis and for further clinical research. A series of studies have demonstrated involvement of the Notch signalling pathway in haematopoiesis during development but with controversial conclusions because of the difference of models concerning various time windows and manipulating populations. This project aimed to investigate the role of Notch signalling pathway during haematopoiesis in the AGM environment. We analyzed the expression of Notch ligands in AGM-derived stromal cells with or without haematopoietic enhancing ability. No correlation was observed between ligand expression and haematopoietic enhancing ability in stromal cell lines or between Notch activity in EBs and haematopoietic enhancing ability. We demonstrated that inhibition of the Notch signalling pathway using the γ-secretase inhibitor could abrogate Notch activity in both mES-derived cells and the haematopoietic enhancing AM stromal cell line. To better understand the involvement of the Notch signalling pathway in a more specific spatial-temporal environment, we established a co-culture system of haemangioblast like cells (Flk1+) with one of AM region derived stromal cell lines with haematopoietic enhancing ability . We found that the AM stromal cell line could enhance Flk1+ derived haematopoiesis as assessed by haematopoietic colony formation activity and production of CD41+cKit+ progenitor cells. Based on the issue that the inhibitor could potentially affect both the ES cells and stromal cells, we carried out genetic approaches to overexpress or knock down Notch signalling pathway in this Flk1+/AM co-culture system. Interestingly, it was found that when Notch activity was enhanced in Flk1+ cells, the production of haematopoietic progenitors was inhibited and the number of cells expressing the pan-haematopoietic marker CD45 was reduced. By using the inducible dominant negative MAML1 system to knock down Notch activity, it was found that the haematopoiesis in the Flk1+/AM co-culture system was not affected, which could be accounted for the low Notch activity in this system. These results supported the hypothesis that the Notch signalling pathway plays a role in modulating Flk1+ derived haematopoietic differentiation within the AGM microenvironment.

Embryonic stem cells (ESCs) represent a promising source of cells for cell replacement therapy in the context of muscle diseases; however, before ESC-based cell therapy can be translated to the clinic, we must learn to modulate cell-fate decisions in order to maximize the yield of myocytes from this systems. In order to gain a better understanding of the myogenic cell fate, we sought to define the molecular mechanisms underlying the specification and differentiation of ESCs into cardiac and skeletal muscle. More specifically, the central hypothesis of the thesis is that myogenic signalling cascades modulate cell fate via regulation of transcription factors. Retinoic acid (RA) is known to promote skeletal myogenesis, however the molecular basis for this remains unknown. We showed that RA expands the premyogenic progenitor population in mouse stem cells by directly activating pro-myogenic transcription factors such as Pax3 and Meox1. RA also acts indirectly by activating the pro-myogenic Wnt signalling cascade while simultaneously inhibiting the anti-myogenic influence of BMP4. This ultimately resulted in a significant enhancement of skeletal myogenesis. Furthermore, we showed that this effect was conserved in human embryonic stem cells, with implications for directed differentiation and cell therapy. The regulation of cardiomyogenesis by the Wnt pathway was also investigated. We identified a novel interaction between the cardiomyogenic transcription factor Nkx2.5 and the myosin phosphatase (MP) enzyme complex. Interaction with MP resulted in exclusion of Nkx2.5 from the nucleus and inhibition of its transcriptional activity. Finally, we showed that this interaction was modulated by phosphorylation of the Mypt1 subunit of MP by ROCK, downstream of Wnt3a. Treatment of differentiating mouse ESCs with Wnt3a resulted in exclusion of Nkx2.5 from the nucleus and a subsequent failure to undergo terminal differentiation into cardiomyocytes. This likely represents part of the molecular basis for Wnt-mediated inhibition of terminal differentiation of cardiomyocytes. Taken together, our results provide novel insight into the relationship between myogenic signalling cascades and downstream transcription factors and into how they function together to orchestrate the myogenic cell fate in stem cells.

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.

The decisions stem cells make impact both the development of adult vertebrates and systems within the body that require cellular replenishment to sustain life. Regardless whether a stem cell remains quiescent, divides, differentiates, or undergoes apoptosis—these processes are precisely controlled by internal gene regulatory networks that are instructed by external stimuli. The exact mechanisms governing stem cell fate are not completely understood. These studies explore new ways in which cell fate is mediated. Through a study of mitochondrial content in human embryonic stem cells (hESCs) and their differentiated progeny, we discovered differences in mitochondrial morphologies. Mitochondria began as elongated and networked structures in self-renewing conditions and changed their shape after differentiation. The addition of external growth factors that direct hESCs toward the definitive endoderm (DE) lineage promoted mitochondrial fragmentation, which was mediated by the mitochondrial fission machinery. Globular, punctate mitochondria were observed prior to the induction of the DE-specific transcriptional program. Differentiation of hESCs to other lineages did not result in any mitochondrial shape changes. Thus, mitochondrial fission in differentiating hESCs, an internal cellular process, is induced by DE-inducing external stimuli, an effect that was lineage specific. In a second study, we investigated the role of the splenic environment in the development of the blood system—during hematopoiesis. The spleen made a distinct contribution to hematopoiesis, a process predominantly attributed to the bone marrow. We discovered a previously unidentified population of cells, uniquely represented in the mouse spleen that could develop into erythrocytes, monocytes, granulocytes, and platelets. These multipotent progenitors of the spleen (MPPS) expressed higher levels of the transcription factor, NR4A1 compared to their bone marrow counterparts and relied on NR4A1 expression to direct their cell fate. The activation of NR4A1 in MPPS biased their production of monocytes and granulocytes in vitro whereas NR4A1-deficient MPPS over-produced erythroid lineage cells in vivo. Together, these data suggest the splenic niche supports distinct myeloid differentiation programs of multi-lineage progenitors cells. Both studies identify new mechanisms by which external stimuli regulate internal mechanisms of cell fate. These insights provide a better understanding of stem and progenitor cell differentiation that have the potential to impact cellular replacement therapies.

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.