The recently identified DREAM complex assembles when Rb-like protein (p130, p107) recruits E2F4, DP (dimerization partner) and MuvB (multivulval complex B (Lin9, Lin37, Lin52, Lin54, and RbBp4)) during G0 and quiescence to repress cell cycle-dependent genes. DREAM assembly requires phosphorylation of the MuvB subunit Lin52 mediated by Dyrk1a, a kinase that has been linked to Down syndrome and neurodegenerative diseases. Our lab previously demonstrated an essential role for the Rb-like pocket proteins in the regulation of neural precursor population and that E2F4 is also involved in the regulation of the expression of the pluripotent gene Sox2. Here, we performed in utero electroporation experiments to overexpress the DREAM complex components and assess their roles during neurogenesis. Our results showed that the overexpression of DREAM components (Lin52 and p130) and Dyrk1a promotes commitment to differentiation at the expense of self-renewal. We also showed that Dyrk1a requires p130 or p107 to regulate neurogenesis. Furthermore, using harmine treatment which is an inhibitor of Dyrk1a the kinase that induces DREAM assembly, our results revealed that DREAM regulates the expression of self-renewal markers affecting the cell fate decision. Performing ChIP experiments, we detected a binding enrichment of the DREAM components on the promoters of not only classical cell cycle genes but on the self-renewal genes like Sox2 and EZH2. Taken together, our study confirmed that DREAM complex plays an important role in the cell fate determination during the regulation of neurogenesis through the control of the self-renewal genes.

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.

<p>The homeostatic hematopoietic stem cell compartment is comprised of quiescent long term self renewing stem cells and cycling short term stem cells with finite renewal potential. To study the molecular mechanisms governing self renewal of hematopoietic cells we must force them to enter the cell cycle and proliferate. One approach to accomplish this goal is to damage the hematopoietic compartment with ionizing radiation or cytotoxic chemotherapy. Such injuries ablate mature blood cells and drive the primitive stem cells into cycle. I have elected to use a simple model of hematopoietic damage and regeneration to study the molecular mechanisms controlling self renewal in hematopoietic stem cells. At the beginning of this project it was unclear whether the signaling pathways which homeostatically control self renewal are utilized during injury repair. In particular, there is very little understanding of the signals required for regeneration after radiation damage. We hypothesized extracellular signal transduction pathways provided by the microenvironment are critical mediators of the stem cell repair process. To address these topics and extend the previous work generated in our laboratory, I chose to pursue a candidate approach focusing on the Wnt and Notch developmental signaling pathways.</p><p>In order to examine the activation and requirement for each signaling cascade after radiation and chemotherapy damage we used a combination of loss of function and reporter mouse models. To this end, we have conducted the majority of experiments for the Wnt project in animals deficient in beta-catenin, the key transcription factor required in the pathway. Our investigations revealed the Wnt pathway is turned on within regenerating stem cells and loss of beta-catenin impairs regeneration of the stem cell compartment after both radiation and chemotherapy injury. </p><p>Using a Transgenic Notch Reporter mouse to investigate the role of Notch signaling following hematopoietic damage we determined the Notch pathway is also activated during regeneration. Furthermore, using a live imaging approach we discovered Notch activated cells change their fate choice during regeneration. To determine if Notch gain of function provides radio-protection we infected stem cells with an active form of Notch prior to radiation and then scored self renewal potential in vitro. This led us to the conclusion that Notch gain of function can provide a self renewal benefit to irradiated hematopoietic stem cells.</p> / Dissertation

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19243号 / 農博第2140号 / 新制||農||1036(附属図書館) / 学位論文||H27||N4947(農学部図書室) / 32242 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 祝前 博明, 教授 松井 徹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Colorectal tumors are hierarchically organized and governed by populations of self-renewing cancer stem cells, representing one of the deadliest types of cancers worldwide. Emergence of a cancer stem-like phenotype depends on epigenetic reprogramming, associated with profound transcriptional changes. As described for pluripotent reprogramming, epigenetic modifiers play a key role in developing and maintaining cancer stem cells by establishing embryonic stem-like transcriptional programs, thus altering the balance between self-renewal and differentiation. Through my work, I have identified overexpression of histone methyltransferase G9a as a risk factor for colorectal cancer, associated with shorter relapse-free survival. Moreover, using human transformed pluripotent cells as a surrogate model for cancer stem cells, I demonstrate that G9a activity is essential for the maintenance of an embryonic stem-like transcriptional signature that is required to promote self-renewal, tumorigenicity and an undifferentiated state. Such a role was also applicable to colorectal cancer, where inhibitors of G9a histone methyltransferase function induced intestinal differentiation while restricting tumor-initiating activity in patient-derived colorectal tumor samples. By integrating transcriptome profiling with G9a/H3K9me2 loci co-occupancy, the canonical Wnt pathway, epithelial-to-mesenchyme transition and extracellular matrix organization were identified as potential targets of such a chromatin regulation mechanism in colorectal cancer stem cells. Considering such novel insights on the role of G9a as a driver of the cancer stem cell phenotype, as well as a promoter of self-renewal, tumorigenicity and an undifferentiated state, I established and executed a multi-step drug screening pipeline to identify new repurposed drugs that selectively alter G9a functions in human CSCs. This pipeline revealed 3 new drug candidates that inhibit H3K9me2 deposition and impair human CSCs in culture. Future in-depth characterization of those candidates will represent an important step toward the development of novel CSC-targeting therapeutics.

No description available.

<p>Human pluripotent stem cells (hPSCs) exist as a heterogeneous population within a dynamic niche, which governs their ability to self-renew and differentiate. Evidence modeled after mouse embryonic stem cells (mESCs) reveals the existence of a developmentally primitive, or homogeneous, state through chemically defined culture methods that is modulated by NANOG, a core pluripotent regulator. However, the differentiation potential and transcription factor control of the homogeneous state in human pluripotent stem cells remains elusive. Previous work suggests that bFGF/ACTIVIN extrinsic regulation provides the heterogeneous nature of hiPSCs with ability to differentiate into several multilineage lineage progenitors. Here, we illustrate that altering the extrinsic environment of hiPSCs with LIF and inhibitors of GSK3b and MAPK/ERK1/2 pathways (LIF/2i), rewires the intrinsic pluripotent regulation of OCT4 and NANOG, which ultimately prevents the <em>in vitro</em> hematopoietic differentiation potential. Upon conversion of hiPSCs to a primitive state of pluripotency with LIF/2i, this study reveals that prolonged culture of hiPSCs with LIF/2i erases the hematopoietic differentiation potential through retained expression of the POU domain pluripotent transcription factor, OCT4. Interestingly, shRNA mediated knockdown of <em>OCT4</em> recovers the restricted differentiation potential in LIF/2i cultured hiPSCs, while knockdown of <em>NANOG</em>, does not. This study identifies a distorted differentiation potential of hPSCs cultured in mouse ESC conditions, despite comparable gene expression profiles and signaling pathway dependence. In efforts to simplify culture methods of human pluripotent stem cells, we identify that alteration of the extrinsic environment highlights explicit differences between human and mouse intrinsic pluripotent regulation, which ultimately controls differentiation efficiency.</p> / Master of Science (MSc)

The relationship between cellular proliferation and differentiation is a major topic in cell biology. What we know comes from models of somatic cell differentiation, where it is widely viewed that cycling and differentiation are coupled, antagonistic phenomena linked at the G1 phase. The extension of this view to stem cells, however, is unclear. One potential possibility is that stem cells also tightly link their G1 phase with their differentiation, indicating a similarity between the differentiation of stem cells and the differentiation of more mature somatic cells. On the other hand, stem cells may utilize different mechanisms or adaptations that confer on them some aspect of uniqueness or "stemness." In this case, stem cells will not exhibit the same coupling with the cell cycle as in many somatic cell models. In this thesis, we examined mouse embryonic stem cells (mESCs), a stem cell that is pluripotent and rapidly cycling with a highly condensed G1 phase. Direct extension of the somatic view posits that elongation of their G1 phase to somatic lengths by cyclin-dependent kinase (CDK) activity inhibition should induce or increase differentiation of these stem cells. Evidence supporting this claim has been contradictory. We show that elongation of the cell cycle and elongation of G1 to somatic lengths is fully compatible with the pluripotent state of mESCs. Multiple methods that lengthen the cell cycle and that target CDK activity or that trigger putative downstream mechanisms (i.e. Rb and E2F activity) all fail to induce differentiation on their own or even to facilitate differentiation. These results indicates that the model of linkage between the G1 phase and differentiation in mESCs is incorrect and leads us to propose that "stemness" may have a physiological basis in the decoupling of cell cycling and differentiation. In summary, we provide evidence that there is a resistance of mESCs to differentiation induced by lengthening G1 and/or the cell cycle. This could allow for separate control of these events and provide new opportunities for investigation and application.

The embryonic stem cell (ESC) microenvironment contains various localized physical and biochemical cues to direct cell fate. Current approaches for microenvironmental regulation rely on restricting cell behaviour to control endogenous signals such as secreted ligands. This report presents a microfluidic device that can directly manipulate the removal of autoregulatory ligands from culture and control the activation of Signal Transducer and Activator of Transcription-3 (Stat3) in ESCs. Specifically, the response of Stat3 was measured under diffusive and convective mass transfer regimes. A Brownian dynamics algorithm was also developed to simulate ligand transport and predict cellular response under these conditions. Stat3 activation under perfusion culture was found to depend on flow rate and axial distance in the flow direction. Long-term perfusion also allowed for the formation of a sustained gradient of Stat3 activation that led to selective loss of ESC pluripotency. These results demonstrate the utility of microfluidic culture for stem cell bioengineering applications.

The embryonic stem cell (ESC) microenvironment contains various localized physical and biochemical cues to direct cell fate. Current approaches for microenvironmental regulation rely on restricting cell behaviour to control endogenous signals such as secreted ligands. This report presents a microfluidic device that can directly manipulate the removal of autoregulatory ligands from culture and control the activation of Signal Transducer and Activator of Transcription-3 (Stat3) in ESCs. Specifically, the response of Stat3 was measured under diffusive and convective mass transfer regimes. A Brownian dynamics algorithm was also developed to simulate ligand transport and predict cellular response under these conditions. Stat3 activation under perfusion culture was found to depend on flow rate and axial distance in the flow direction. Long-term perfusion also allowed for the formation of a sustained gradient of Stat3 activation that led to selective loss of ESC pluripotency. These results demonstrate the utility of microfluidic culture for stem cell bioengineering applications.