published_or_final_version / abstract / Medicine / Master / Master of Philosophy

published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Activation of Ras/mitogen-activated protein kinase (ERK MAPK) signalling controls the differentiation of mouse embryonic stem (ES) cells. An established modulator of the ERK MAPK pathway is the IRS-1 (Insulin Receptor Substrate 1) family adaptor protein Gab1 (Grb2-associated binder 1). Gab1 is ubiquitously expressed and is activated by a wide range of cell surface receptors, mediating growth factor, cell-cell and cell-substratum interactions. The N-terminal region of Gab1 contains a pleckstrin homology (PH) domain required for membrane binding and a nuclear localisation sequence (NLS) that facilitates nuclear translocation. Undifferentiated mouse ES cells preferentially express high levels of a novel form of Gab1 (Gab1β) lacking the N-terminal region. Based on its novel structure and abundance, Gab1β may act in a dominant negative manner by binding and mislocalising downstream effectors. Alternatively, it may have a deregulated function unrestrained by the PH or NLS domains. Data presented here shows that Gab1β is tyrosine phosphorylated in response to the self-renewal factor Leukemia Inhibitory Factor (LIF) and/or Foetal Bovine Serum (FBS) stimulation. This then leads to the formation of complexes with Shp2 and the p85 subunit of PI3K. Experiments comparing the responses of wild-type and Gab1β knock-out ES cells indicate that Gab1β enhances ERK and potentially AKT phosphorylation in response to LIF. In contrast, Gab1β has a negative effect on ERK and AKT phosphorylation in response to IGF-1 (Insulin Growth Factor 1). These results suggest that the contribution of Gab1β to signalling activity is receptor specific and may imply that the response of ES cells to ERK activation is context specific. By reintroducing fluorescently tagged Gab1 proteins into Gab1β knockout ES cells, I investigated the localisation of Gab1β in ES cells. Gab1β localised at the cell membrane as well as in a perinuclear body. I next investigated the potential role of Gab1β in the differentiation of ES cells into neural precursors. A monolayer differentiation protocol was used to differentiate Gab1β wild-type and knock-out cells into neural precursors. Furthermore, the effect of insulin on the emergence of neural precursors from Gab1β-targeted cells was also explored.

Embryonic stem (ES) cells were used as a model system to understand the signalling events in pancreas development. ES cells were differentiated through known precursor stages towards the tissue of interest in order to recapitulate development in vitro. Thus, protocols directing differentiation of mouse ES cells towards definitive endoderm and pancreatic β-cells were developed. A combination of activin A and bone morphogenic protein 4 resulted in a population of enriched cells expressing genetic markers of definitive endoderm. In vitro differentiation of ES cells into functional pancreatic β-cells has only been partially successful, as it results in cells that are not fully differentiated or functional. This might be due to a lack of cues emanating from surrounding cells present in the developing pancreas. Conditioned media from the mouse MIN6 β-cell line were used on the basis that differentiated β- cells might send out signals affecting the differentiation of the surrounding islet cells. Mouse ES cells were enriched in definitive endoderm and then treated with MIN6 conditioned medium. Gene expression of the β-cell markers Insulin1, Insulin2, and Glucose transporter 2 was significantly increased relative to the untreated control group after 10 days of treatment with conditioned medium. This result was specific for conditioned medium from MIN6 cells as conditioned medium from a kidney-, a neuronal-, and an exocrine pancreatic cell line had no effect. In order to characterise the secreted factor(s) the conditioned medium was subjected to protein precipitation. The pancreatic differentiation factor was present in a protein fraction, suggesting that the factor(s) was proteinaceous. The protein in question was neither proinsulin nor insulin. This knowledge will support the efficient generation of insulin-secreting cells for diabetes therapy.

Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes.  Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic beta-cell development.  Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse ES cell.  By activating an exogenous Pdx1VP16 protein in populations of cells enriched in definitive endoderm we show a distinct lineage-dependent requirement for this transcription factor’s activity.  mimicking the natural biphasic pattern of Pdx1 expression was necessary to induce an endocrine pancreas-like cell phenotype, in which 30% of the cells were beta-cell-like.  Cell markers consistent with the different beta-cell differentiation stages appeared in a sequential order following the natural pattern of pancreatic development.  Furthermore, the differential beta-like cells secreted C-peptide (insulin) in response to KC1 and IBMX, suggesting that following a natural path of development in vitro represents the best approach to generate functional pancreatic cells.  Together these results reveal for the first time a significant effect of the timed expression of Pdx1 on the non-beta cells in the developing endocrine pancreas.  Collectively, we show that this method of <i>in vitro</i> differentiation provides a template for inducing and studying ES cell differentiation into insulin-secreting cells.

Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is divided into 4 subtypes based on different genomic alterations and gene expression profiles. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example, expression of the transcription factor, Orthodenticle homeobox2 (OTX2) is frequently upregulated in multiple MB variants; however, its role may be subtype-specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs), but not their normal counterparts (hENs), resemble Groups 3 and 4 MBs. These trans-hENs also have >10-fold expression of OTX2. Therefore, we hypothesize that OTX2 has cell context-dependent functions in MB and using both normal and trans-hENs, we can delineate its specific roles in MB progression. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and sonic hedgehog (SHH) MB cells by regulating growth, self-renewal and migration in vitro and tumor growth in vivo. Overexpression of OTX2 was accompanied by a decrease in expression of pluripotent genes such as SOX2. This was supported by exogenous introduction of SOX2 in OTX2+ SHH MB and hENs that rescued the OTX2 induced cellular deficits including self-renewal and cell migration. In contrast, OTX2 is oncogenic and promotes self-renewal of trans-hENs and Group 3 and 4 MBs by modulating expression of genes related to neurodevelopment and axonal guidance. OTX2 may play a central role in regulating the balance between self-renewal and differentiation in these aggressive MB cells. Our studies underscore the value of hESC derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression. Using the neural derivatives of hESCs, we have demonstrated a novel role for OTX2 in self-renewal and migration of hENs and MB cells. Moreover, our results reveal a cell context-dependent link between OTX2 and pluripotent genes. The association between OTX2 and axonal guidance genes is important for its oncogenic role and may potentially be exploited for managing drug resistant stem cell and highly motile cellular populations in the most aggressive Group 3 and 4 MB subtypes. / February 2017

Human Artificial Chromosomes (HAC) are fascinating extrachromosomal molecules that stay independently from the host genome and are capable of segregating as efficiently as endogenous chromosomes. It has been proven that HAC are potential tools for both basic chromosome behavioural research and agents for gene therapy purposes. My DPhil project is divided into two main themes. The first theme was to develop a novel artificial chromosome in mouse embryonic stem cells. The second theme was to understand the factors affecting chromosome stability which may also affect the efficiency of artificial chromosome formation. so that our protocol for better HAC preparation can be refined. There are six results chapters in my thesis. The first three chapters described how I developed human artificial chromosomes in mouse embryonic stem cells. Initially, vectors containing a long stretch of human alphoid DNA were delivered to mouse cells using the Herpes Simplex Virus-I (HSV-l) amplicon system but the efficiency was low. Next, mouse pericentromeric and centromeric DNAs were employed for mouse artificial chromosome (MAC) via HSV-l system. However, the efficiency remained the same. Finally, I used the Microcell-Mediated Chromosome Transfer (MMCT) system to transfer HAC from HTl 080 cells into mouse ES cells and successfully established HAC in ES which were highly stable. The results obtained in this first part of my thesis suggested that to increase HAC formation efficiency it would be necessary to improve the techniques of HSV-I delivery and MMCT. Moreover, it would also be important to better characterize factors affecting chromosome behaviour. The last three results chapters focus on factors affecting chromosomes stability and improving the HSV -1 delivery system and MMCT. I undertook an in vivo study of whole cell fusion experiments with the aid of live cell irnaging system, and found that histone H2B proteins underwent a dynamic assembly/disassembly processes. Live cell imaging of MMCT suggested that the microcell delivery is a very slow process and the results may lead to a refinement of the MMCT protocol. I found it is possible to generate a single HAC using two HSV-l amplicons containing two different constructs, potentially doubling the HSV-l HAC capacity from 150 kb to 300 kb. The last chapter illustrated how the expression of non- coding centromeric satellites impaired chromosome stability in both human cultured and human embryonic stem cells. The findings revealed that non-coding centromeric RNA plays an important role on chromosome stability that might be important for artificial chromosome development.

La réussite de la thérapie cellulaire à l'aide des cellules souches embryonnaires, nécessite une bonne compréhension des mécanismes moléculaires qui contrôlent leur différenciation. Une des voies de signalisation, impliquée dans le contrôle de la différenciation des cellules souches, est la voie p38MAPK. Dans le but de comprendre les mécanismes moléculaires impliqués dans le contrôle de la différenciation précoce des cellules ES, nous avons réalisé un criblage sur puces à ADN. Les résultats du criblage ont montré que certains gènes régulés différentiellement, comme le gène Bcl2, sont des cibles communes de p38MAPK et de p53. En plus de son rôle de répresseur de tumeur, p53 a été impliqué dans le développement embryonnaire et dans la biologie des cellules ES. Ainsi, il est connu que p53 agit comme un répresseur du gène de pluripotence, Nanog. Il est également connu que p53 interfère avec le processus de reprogrammation des cellules adultes ; et que son activité transcriptionnelle augmente avec l'entrée des cellules ES en différenciation. En revanche, son rôle dans la différenciation et dans la formation des différents lignages issus des cellules ES, reste inconnu. Nous avons trouvé que le traitement des cellules ES sauvages avec l'inhibiteur spécifique de p53, la pifithrine-α, durant le processus de différenciation, inhibe les lignages mésodermiques, et à l'inverse, stimule la neurogenèse. De plus, la transfection transitoire des cellules ES avec des siRNAs spécifiques, dirigés contre p53 ainsi que l'utilisation des cellules ES déficientes pour le gène p53, montrent que l'absence de p53, affecte les lignages cardiaques, du muscle lisse et du muscle squelettique. / Embryonic stem cells (ESCs) differentiate in vitro into all cell lineages. We previously found that p38MAPK controls two independent successive steps during the early mesodermal commitment of ESCs. The first one is Brachyury dependent, a master gene of mesoderm formation whereas the second one is not. In order to understand the molecular mechanism implicated in the second step, we treated ESCs with the p38 specific Inhibitor PD169316 and performed microarray experiments on mRNAs extracted from treated versus untreated cells. Our results show that many regulated genes are common targets of p38MAPK and p53 transcription factor. In addition to its role as a tumor suppressor and cell cycle checkpoint control, p53 has been involved in embryonic development, but its role in ESC differentiation is still unknown. We found that treatment of wild type ESCs with the p53 specific inhibitor pifithrin α during the differentiation process inhibits mesodermal lineages and, by contrast, stimulates neurogenesis. Likewise, ESCs Transfected with p53 siRNAs and p53 KO ESCs show an inhibition of cardiac, endothelial, smooth muscle and skeletal muscle lineage formation. Furthermore, p38MAPK inhibition by PD169316 for 24h induces a strong decrease of p53 protein level. Our results suggest that p53 mediates the p38MAPK control of the commitment of ESCs towards mesodermal lineages. The involvement of the various p53 isoforms in this process will be discussed.

Gene expression is regulated at various levels, such as transcription, RNA transport and translation. Additionally, it has been shown that chromatin structure, location and dynamics also have an important role in gene expression control. While active gene regions are strongly associated with an open chromatin structure at the surface of the chromosome territory (CT) and a location in the nuclear interior, inactive gene regions seem to be related with a closed structure within the CT and a position at the nuclear periphery. However, it is still unclear how these features are regulated. Importantly, malfunction of gene regulation can impact on health and longevity. Therefore, the aim of this project was to investigate the correlation of gene expression and chromatin organisation both in single gene loci and the MHC gene cluster. The MHC locus has the highest gene density in mammalian cells and contains genes that can be reprogrammed by pro-inflammatory cytokines. The original goal of this project was to label the MHC locus by the Lac operator/repressor (LacO/LacI) approach in order to study chromatin dynamics in living cells using labelled CTs as reference for genome mobility. The thymidine analogue EdU, that can be used to label CTs, was analysed for its effects on cell cycle progression and survival, and revealed to have a strong negative impact on the cells' well-being. In the end, the LacO/LacI-recognition system for live-cell imaging did not succeed, thus FISH analyses were carried out to study chromatin dynamics in snap-shots. The location and structure of the hybridised gene regions were analysed in response to gene activation and inactivation during ESC differentiation to neuroepithelial progenitors (NPs). Single-gene focused experiments were performed using the cell line specific genes, Oct4 and Sox1, together with Gapdh as a housekeeping gene. Even though, the results showed less changes between the days of differentiation on the Gapdh locus, the gene expression profiles for the cell line specific genes did not match with the hypothesised chromatin organisation (see above). However, investigations on the gene-dense MHC locus showed structural chromatin changes that correlated with the activation of genes in this region. Interestingly, ESC treated with TNFalpha were unable to activate NF-kappaB signalling, probably due to the lack of a functional IKK complex. In summary, this project was focussing on the regulation of gene expression by the chromatin architecture and revealed complex chromatin dynamics that are likely to be affected by the sum of genes in a genome region, rather than a single gene.