Embryonic stem (ES) cells are maintained in an undifferentiated state by a gene regulatory network centred on the triumvirate of transcription factors Nanog, Oct4 and Sox2. Genome-wide chromatin immunoprecipitation studies indicate that in many cases target genes contain closely localised binding sites for each of these proteins, as well as additional members of the extended pluripotency transcription factor network. However, the biochemical basis of the interactions between these proteins is largely unknown, as are the mechanisms by which these interactions control ES cell identity. By purifying Nanog from ES cells and identifying co-purified proteins, we determined a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members and RNA processing factors. Validation of interactions was obtained by co-immunoprecipitation of Nanog with putative partners. Sox2 was identified as a robust interacting partner of Nanog and the interaction was investigated further. We show that the interaction is independent of DNA binding and that a region of Nanog known as tryptophan repeat, in which tryptophan is present every 5th residue is necessary and sufficient for the binding of Sox2. Furthermore, mutation of tryptophan residues within the Nanog tryptophan repeat (WR) abolishes the interaction with Sox2. A region of Sox2 known as serine rich region, a triple-repeat motif (S X T/S Y) within a stretch of 21 residues is required for the interaction with Nanog. Mutation of tyrosines to alanine within the three motifs (S X T/S Y) abrogates the Nanog–Sox2 interaction. The disruption of the Nanog-Sox2 interaction results in the alteration of expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines of the motif with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal. Together these data shed light on the extent of the interactions of Nanog with protein partners as well as the biochemical nature of the interaction between Nanog and one of the most important partners Sox2, an interaction crucial for maintaining optimal mouse ES cell self-renewal efficiency.

Cancer is a stem cell disease and populations of cancer stem cells (CSCs) are evident in many cancer types. CSCs exhibit similarities to normal embryonic and adult stem cells: they are able to self-renew and have the potential to give rise to a diverse array of differentiated progeny. CSCs are responsible for driving tumourigenesis and metastasis, and are inherently resistant to chemotherapy and radiotherapy. This cell population is enriched after treatment and, as a result of their tumourigenic capability, can re-populate tumour growth resulting in patient relapse, often with increased chemotherapeutic resistance. Increasing evidence supports that only by targeting this population of cells will a cure for cancer be possible. Hence, it is essential to identify pathways within CSC populations that can be targeted therapeutically. Cyclooxygenase-2 (COX-2) is an enzyme associated with inflammation and disease, and is upregulated in many cancers types. The COX-2 / prostaglandin E2 (PGE2) signalling pathway is associated with increased tumour growth, metastasis, immune evasion and overall worse patient prognosis. Recent evidence has identified that COX-2 is further upregulated in CSC populations isolated from cancer cell lines. Previously, we have shown that inhibition of COX-2 reduces CSC sphere-forming ability, a characteristic of stem cell self-renewal, suggesting a role for COX-2 in maintaining CSC populations. This work was carried out in both human and canine osteosarcoma cell lines with similar results. Cancer in dogs is a major health concern among an aging pet population. Many cancer types exhibit similarities between these species, suggesting that naturally occurring canine cancer may be a potential model for the human disease. The aim of this PhD project was to investigate the role of COX-2 in CSCs in a comparative cancer study. CSCs that express stem cell markers have been isolated from a panel of canine and human cancer cell lines including, mammary carcinoma and transitional cell carcinoma of the urinary bladder. CSCs over-express COX-2 compared to non-CSCs, therefore to determine the role of COX-2 in CSC biology the selective COX-2 inhibitor mavacoxib, a non-steroidal anti-inflammatory drug currently licenced for treating osteoarthritis in dogs, was utilised. Our results demonstrate that inhibiting COX-2 has a multifaceted impact on CSC biology, including reducing self-renewal capacity, clonogenicity, proliferation, migration, invasion and in vivo tumourigenicity. To confirm that mavacoxib is mediating these CSC-specific effects via inhibition of COX-2 rather than through unknown off-target effects, we generated canine specific-small interfering RNA to specifically reduce gene expression of COX-2. Our results confirm that mavacoxib exerts its anti-tumour effects via inhibition of COX-2. This project has highlighted a plethora of CSC-specific COX-2 effects, and to gain further insight we compared the global gene expression profiles of CSCs compared to non- CSCs isolated from a canine bladder carcinoma cell line. This data revealed that both mavacoxib and COX-2 specific siRNA target similar pathways within the two cell populations, confirming that mavacoxib exerts its effects in a COX-2 dependent manner. Interestingly, mavacoxib reduced the expression of a number of stemness related genes in the CSC population, including NOTCH and Wnt, suggesting that mavacoxib can inhibit CSC related pathways. Our overall results are comparable between canine and human cancer cell lines supporting the concept of naturally occurring tumours in dogs as models for the human disease. In conclusion, COX-2 plays an important role not only in maintaining CSC populations but also in their function, and targeting COX-2 in CSCs may provide therapeutic benefit.

Mammalian neural stem cell (NSC) lines provide a useful experimental model for basic and applied research across stem cell and developmental biology, regenerative medicine and neuroscience. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with functional genetic analyses. However, targeted genetic manipulations have not been reported for mammalian NSC lines. Here, we deploy the CRISPR/Cas9 technology and demonstrate a variety of diverse targeted genetic modifications in both mouse and human NSC lines such as: targeted transgene insertion at safe harbour loci; biallelic knockout of neurodevelopmental genes; knock-in of epitope tags and fluorescent reporters; and engineering of glioma driver mutations at endogenous proto-oncogenes. Leveraging these new optimised methods, we explored gene editing to model the earliest stages of paediatric gliomagenesis in primary human NSCs. Our data indicate that oncogenic mutations in histone H3.3 play a role in NSC transformation and may operate through suppression of replication induced senescence. By comparing cellular responses of NSC cultures from different compartments of the developing brain, we also identify differences in susceptibility to distinct H3.3 mutations that mirror the disease etiology. Altogether, our findings indicate that CRISPR/Cas9-assisted genome editing in NSC lines is a versatile tool to explore gene function in CNS development and cancer biology. Our project resulted in the creation of valuable human cellular models of paediatric gliomagenesis, which will allow us to further our understanding of the disease and carry out cell based drug discovery projects.

Pluripotent cells have the dual abilities to self-renewal and to differentiate into all three germ layers. Pluripotent cells can be isolated from two different stages of mouse embryogenesis. Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of the pre-implantation embryo and are considered to be in a naïve state. On the other hand, cells isolated from epiblast of the post-implantation embryo are referred as epiblast stem cells (EpiSC) and are representative of primed pluripotency. ESCs and EpiSCs are distinct from each other in terms of the morphology, the gene regulatory network and the signalling pathways regulating self-renewal. Under certain conditions, ESCs and EpiSCs can be transitioned into each other. However, the mechanism that regulates this transition from naïve to primed pluripotent state remains to be solved. Nanog, Oct4 and Sox2 form the core gene regulatory network of pluripotency. Additionally, the Id protein family is also important in the maintenance of pluripotency in ESCs. Id proteins function by inhibiting the activity of pro-differentiation factors. Tcf15 is identified as one of the targets of Id inhibition in ESCs. Moreover, Tcf15 has been identified as a repression target of Nanog. In this study, to understand the function of Tcf15, the expression of Tcf15 was characterized in differentiating ESCs. The transient upregulation of Tcf15 mRNA and protein was detected at early stages of differentiation before lineage commitment. Furthermore, Tcf15 protein was heterogeneously expressed in differentiating cells. Mutually exclusive expression of Nanog and Tcf15 proteins were demonstrated in both self-renewing and differentiating ESCs. Further characterization of the effect of Nanog on Tcf15 transcription showed that Tcf15 pre-mRNA was downregulated within 20 minute of Nanog induction. A Nanog binding site was identified at +32kb relative to the Tcf15 transcription start site (TSS). Initially, Nanog binding at this region was confirmed by performing ChIP-PCR experiments. Then, this Nanog binding region was further analysed for its enhancer activity related to the Tcf15 gene. Deletion of the Nanog binding region using CRISPR-Cas9 confirmed that this region acts as Tcf15 enhancer; it was shown that this region was required for the activation of Tcf15 transcription during differentiation. Tcf15 induction experiments were performed in order to the check whether Tcf15 affects Nanog transcription. The results indicate that Nanog is not a direct target of Tcf15, but Tcf15 contributes indirectly to the repression of Nanog. Additional analysis with the Tcf15 enhancer deletion cells showed that Tcf15 is not required for efficient downregulation of naïve markers and the upregulation of primed markers. However, the genes related to the regulation of adhesion properties of cells such as Zyc, Itga3 were induced with lower efficiency in the absence of Tcf15 compared to the wild type cells. In summary, I investigated the reciprocal regulation of Tcf15 and Nanog and the role of Tcf15 for the differentiation. My results suggest that Tcf15 is expressed in the cells that have initiated differentiation but are not lineage-committed. Additionally, Tcf15 can contribute to the regulation of adhesion related genes in order to help the epithelisation of the cells required during the differentiation from naïve to the primed pluripotent state. As a conclusion, Nanog is proposed to help to prevent certain aspects of ESCs differentiation by repressing Tcf15.

The production of fully functional haematopoietic stem cells (HSCs) for clinical transplantation is a highly sought after goal in the field of regenerative medicine. Given their capacity for extensive self-renewal and differentiation into any cell type, human pluripotent stem cells (hPSCs) provide a potentially limitless source of haematopoietic cells in vitro for clinical application. However, to date, fully functional HSCs have not been produced from hPSCs without the overexpression of transcription factors. In this study I first investigated the production of HSCs and haematopoietic progenitor cells (HPCs) in an established clinical-grade haematopoietic differentiation protocol. I demonstrated the efficient and reproducible production of HPCs but showed that the strategy did not produce fully functional HSCs that could repopulate the haematopoietic system of immune-deficient mice. Modification of the protocol by manipulation of the hedgehog signalling pathway and co-aggregation with OP9 stromal cells did not provide any significant enhancement of HPC production. To gain the required knowledge with which to improve our current protocol, I therefore switched my focus towards studying the development of HSCs in the early human embryo. It has been shown that HSCs first emerge from the ventral wall of the dorsal aorta in the aorta-gonad-mesonephros (AGM) region of the human embryo but the precise location and the mechanisms underpinning this process remain unknown. In this study, I established a culture system to map the spatio-temporal distribution of HSCs and to investigate the presence of HSC precursors. I showed that embryonic HSCs emerge predominantly around and above the vitelline artery entry point in the dorsal aorta and can be maintained in our explant culture system. I then performed RNA-sequencing of cells derived from AGM sub-regions, and this identified molecular signatures which could potentially underlie the ventral polarity of HSC emergence in the AGM. To elucidate the role of the stromal compartment in this unique haematopoietic niche, I derived stromal cell lines from the human AGM region and showed they were capable of supporting haematopoiesis in vitro. This work has provided some important insights into the mechanisms regulating HSC development in the human AGM region and identified interesting candidate molecules for future testing in differentiation protocols. This knowledge brings us a step closer to the successful in vitro production of HSCs for clinical use.

Injuries to connective tissues such as ligaments and tendons are common, and rather than healing, repair typically results in fibrosis, or the formation of mechanically inferior and disorganized scar tissue. This fibrotic repair response is due in part to inflammation, during which the injury site is invaded by a number of cell types, including macrophages, neighboring fibroblasts, and homed stem cells or progenitor cells. Activation of macrophages is believed to be modulated by communications with fibroblasts and stem cells, prompting either a pro-fibrotic or a pro-regenerative response. Beyond changes to the cellular microenvironment, fibrosis also results in changes to the organization and mechanical properties of the matrix microenvironment. For healthy fibrous connective tissues, the matrix is comprised of aligned collagen fibers, while scar tissue is disorganized and exhibits weaker mechanical properties than healthy tissue. To date, the nature of the cell-cell and cell-matrix interactions and their relevance in tissue healing or repair remain understudied. To better understand the cellular and matrix-based cues that direct scar formation versus tissue regeneration, and using anterior cruciate ligament (ACL) injuries as a model, Aim 1 of this thesis tests the hypothesis that in vitro models of cellular communications between fibroblasts, macrophages, and mesenchymal stem cells (MSC) can be used to determine the effects of cellular interactions on macrophage activation and fibrosis. In Aim 2, the contribution of matrix-based cues (alignment and mechanical properties) to the inflammatory and fibrotic response, as well as their modulation of cellular interactions, were examined. Findings from these two aims reveal that 1) communications between native tissue fibroblasts and macrophages drive inflammation and fibrosis, while stem cells modulate the repair process through a combination of trophic signaling and immunomodulatory roles, and 2) matrix alignment and mechanical properties exert combined regulation on cell response during inflammation. From a clinical application perspective, stem cells delivered in conjunction with an engineered matrix that provides the critical cues for driving stem cell immunomodulation and trophic signaling will be essential for promoting tissue regeneration and minimizing fibrosis. In particular, an aligned matrix with an elastic modulus similar to that of developing connective tissue may serve to further minimize inflammation and scar formation, and activate stem cell-guided regeneration of mechanically functional connective tissue.

Une tumeur est composée de plusieurs sous populations cellulaires. L’une d’entre elles, celle des cellules souches tumorales, est à l’origine du développement des tumeurs. Une des propriétés majeures de ces cellules est la capacité d’entrer dans un état de quiescence. De ce fait, elles sont résistantes aux thérapies anticancéreuses conventionnelles qui visent les cellules cyclantes et peuvent ainsi persister pendant de nombreuses années. Ce phénomène est appelé dormance tumorale. L’activation de ces cellules souches tumorales quiescentes conduit à la récidive de la maladie. Le passage de l’état quiescent à l’état activé serait réversible, cependant les mécanismes responsables ne sont pas encore connus. Notre hypothèse est que les facteurs inflammatoires stimulent la transition des cellules de l’état quiescent à l’état activé. Dans ce but, nous avons étudié les effets de la principale cytokine pro-inflammatoire, le TNF, sur le compartiment des cellules souches de mélanome et leur activation. Pour cela, nous avons utilisé un système d’expression, inductible par la tétracycline, qui nous a permis d’identifier et d’étudier les cellules quiescentes H2B-GFP positives et cela dans les modèles in vitro des mélanosphères et des équivalents de peaux humaines reconstruites, afin de se rapprocher de l’organisation tumorale in vivo. Grâce à des tests fonctionnels, comme la formation de mélanosphères et de colonies, et diverses techniques telles que la cytométrie en flux, la microscopie à fluorescence et l’analyse de l’expression de gènes au niveau protéique, nous avons mis en évidence que les cellules H2B-GFP positives (« label retaining cells ») au sein des mélanosphères montrent un enrichissement en marqueurs de cellules souches du mélanome (ABCB5, VEGFR). De plus, nous avons montré que le TNF agit sur le compartiment des cellules souches. En effet, un traitement au TNF augmente le pourcentage de cellules exprimant des marqueurs de cellules souches de mélanome, inhibe la différenciation des cellules de mélanome (inhibition de l’expression de Melan-A dans les mélanosphères et diminution de la pigmentation des équivalents de peau), active les cellules souches quiescentes et induit des effets qui perdurent après le retrait du TNF. Notre étude a montré que ces effets seraient causés par une activation des voies PI3K/Akt et NFκB par le TNF. Un grand nombre de données suggérant qu’une sous-population de cellules cancéreuses est capable d’entrer en quiescence en réponse à une thérapie anticancéreuse, nous avons également étudié les effets de la première thérapie ciblée du mélanome : le vemurafenib, sur le compartiment des cellules souches. Nos résultats ont montré que le vemurafenib augmente le compartiment des cellules souches de mélanome (augmentation du nombre de mélanosphères formées et du pourcentage de cellules exprimant un marqueur de cellules souches de mélanome : ABCB5) et induit leur quiescence (augmentation du pourcentage de cellules H2B-GFP+ et en phase GO du cycle cellulaire). Nous avons également montré que le vemurafenib stimule l’activation de protéines régulant la quiescence des cellules souches.Nous espérons que nos recherches apporteront de nouvelles connaissances sur les mécanismes qui contrôlent l’activation des cellules souches cancéreuses quiescentes et offrir de nouvelles perspectives pour le traitement du cancer. / Accumulating data suggest that both cancer development and recurrence depend on the ability of resistant tumor cells to adopt a quiescent or dormant phenotype following treatment. These dormant cells reside in various tissues of patients in complete remission without any clinical manifestation until they reactivate and cause tumor recurrence. Mechanisms that control the activation of quiescent tumor cells remain poorly understood, however, the tumor microenvironment, cellular interactions and various diffusible factors appear essential. Herein, our goal is to decipher whether a major pro-inflammatory cytokine, Tumor Necrosis Factor (TNF) contributes to the quiescence/activation phenotypic switch in melanoma. For this purpose, we used a 3D melanosphere and the in vivo-like skin equivalent models in which to reconstitute the in vivo-relevant cellular heterogeneity and tumor organization and an inducible histone 2B coupled to the GFP (H2B-GFP) expression system to identify the quiescent cell compartment and to monitor the TNF-induced changes. Our results suggest that TNF increases the proportion of H2B-GFP-positive, label retaining cells (LRC) in melanospheres. The LRCs were enriched in melanoma stem cell markers, ABCB5 and VEGFR and this was upregulated by TNF. Furthermore, TNF increases the number of melanospheres, and in skin equivalents, the presence of TNF seems to inhibit the differentiation of melanoma cells and increase the stem cell compartment. This effect appears to be governed by the activation of the PI3K / Akt pathway. In conclusion, these data show that inflammatory environment induced by TNF, activates melanoma quiescent stem cells and increases the compartment of stem cells in skin equivalents by preventing their differentiation. Therefore, the control of inflammation and signaling pathways involved in the maintenance of tumor dormancy during the treatment of the original tumor would be a good therapeutic strategy in the fight against cancer recurrences.A lot of data suggest that a cancer cell subpopulation is able to enter quiescence in response to cancer therapy, therefore we have also studied the effects of the first targeted therapy of melanoma: vemurafenib, on the stem cell compartment. Our results show that vemurafenib increases the number of melanospheres and the percentage of ABCB5+ cells. So vemurafenib increases the melanoma stem cell compartment. Vemurafenib increases also the percentage of H2B-GFP + cells and the percentage of cells in the GO phase of the cell cycle, so induces quiescence of melanoma cells. We also showed that vemurafenib stimulates activation of proteins regulating quiescence of stem cells.We hope that our research will provide new knowledge about the mechanisms that control the activation of quiescent cancer stem cells and provide new perspectives for the treatment of cancer.

Genomic integrity in mouse embryonic and induced pluripotent stem cells can be compromised by factors such as extended time in culture and cellular reprogramming. Surprising, only a few studies have thus far examined the accumulation of chromosomal imbalances in mouse pluripotent populations upon prolonged propagation in vitro. It is presumed that specific recurring genetic changes can confer selective growth advantage and resistance to apoptosis and/or differentiation to the affected cells, although the genes that drive these processes remain elusive. The presence of these changes in published studies can confound the analysis of the data and hinder the reproducibility of the results. At the transcriptional level, aneuploidy manifests as large chromosomal regions of aberrant gene expression. This thesis presents a method to identify these regions in large-scale datasets and interrogate for recurrent patterns. The present analysis shows that over half of the 315 mouse pluripotent samples examined carry whole or partial-chromosome spanning clusters of aberrant transcription. Furthermore, there are common gene expression changes across samples with any type of predicted aneuploidy and samples with chromosome-specific aberrations. These transcriptional signatures have been used to train classification models which can predict aneuploid samples with over 90% accuracy. This is an important step towards the development of a low-cost and reliable transcriptional validation assay for the presence of aneuploidy.

Remarkable progress has been made in our ability to design and produce synthetic DNA binding domains (TALE or Cas9-based), which can be further functionalized into synthetic transcription factors (sTFs). This technology is revolutionizing our ability to modulate expression of endogenous mammalian genes. Forced expression of cDNAs encoding transcription factors (TFs) is widely used to drive lineage conversions. However, this process is often inefficient and unreliable. Multiplex delivery of sTFs pool to activate endogenous master regulators and extinguish the expression profile of the host cell type could be a potential solution to this problem. We have developed a novel, simple TALE assembly method that enabled us to produce and screen large numbers of TAL effectors and compare their activity to dCas9-based TFs. During this process, we constructed many new functionally validated sTFs. Our ultimate goal is to test whether combining synthetic transcriptional activators and repressors can efficiently reprogram fibroblasts to NS cells or alternatively ‘program’ NS cell differentiation to neurons. We performed analyses of the transcriptome and chromatin accessibility of both fibroblasts and neural stem cells to unravel their core TF networks and their epigenetic state. This will allow us in the future the targeted design of sTFs and synthetic chromatin modifiers for specifically changing cell identity.

Red blood cell (RBC) transfusion is the major treatment for patients suffering from trauma or severe anaemias, and life-long transfusion may be needed to alleviate symptoms and maintain body functioning. However, with a relatively low portion of people are donating, shortage in blood supply is becoming a life-threatening issue in the aging society. Among attempts to identify novel sources for transfusion medicine, human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are currently the most promising candidate, which is capable of producing donor-independent, pathogen-free and immunologically compatible RBCs. Currently, hESC-derived erythropoiesis in vitro is considered to mimic the very primitive yolk sac haematopoiesis, indicated by a low or absent level of β globin production and incomplete enucleation. Thus these cells are not mature enough to be used in transfusion medicine. The aim of this PhD project was to overexpress a key erythroid transcription factor, Erythroid Krüppel-like factor (EKLF or KLF1) in an inducible manner to improve the maturation of hESC-derived erythroid cells. EKLF is a member of the Krüppel-like factor family, which is characterized by three C2H2 type zinc finger motifs. EKLF expression in vivo is highly restricted to erythroid cells in yolk sac, fetal liver, spleen and the bone marrow, although recently a low-level of expression was found in haematopoietic precursors. Published reports demonstrate that EKLF can 1) activate β globin expression by binding to the CACCC box in its promoter or by altering β-like globin locus chromatin structure; 2) exert a role in MEP (common progenitor for erythrocytes and megakaryocytes) stage by favouring erythroid differentiation against megakaryocyte differentiation; 3) promote enucleation by affecting the DNase II-alpha expression in the central macrophage of a fetal liver erythroblastic island; 4) act as an instructive factor for lineage commitment towards erythroid fate in HSCs. In this project, 1) We tested and evaluated a feeder-free, serum-free differentiation system for deriving erythroid cells from hESCs; 2) We constructed constitutive and inducible EKLF expression vectors and validated them in K562 cells; 3) We generated hESC lines carrying these EKLF expression vectors and assessed their effects on erythrocyte production and maturation. We found that our differentiation system was capable of generating haematopoietic progenitors (HPCs) and erythroid cells at high efficiency. Using this differentiation system, we concluded that enhanced expression of EKLF upregulated adult β globin expression selectively, without altering expressions of other globins. This finding provides hints for the development of novel approaches to “reprogramme” hESCs towards a certain fate and overexpression of EKLF in this differentiation system may be beneficial for resolving issues in future transfusion medicine.