Regenerative medicine offers tremendous potential for the treatment of irreversible damage to the brain. Activation of quiescent adult neural stem cells by clinical means to regenerate tissue can improve pathological outcomes of patients afflicted by brain trauma. Control of the cell- cycle is important in activating quiescent neural stem cells for the purpose of enhancing adult neurogenesis. Here, we uncover the role of cell-cycle regulatory transcription factors E2F1 and E2F3 in adult neural stem cell activation and characterize it. We hypothesize that the Retinoblastoma-E2F pathway is crucial for neural stem cell activation. We characterized the requirement of E2Fs1/3 for adult neural stem cells activation through a combination of multiple knockout timepoints in mice and novel markers used to identify distinct neural stem cell sub- populations. The results show a marked reduction in the neurogenic capacity of the adult brain, with common markers of proliferation and different progenitor-cell lineages decreased. Additionally, the ability of quiescent neural stem cells to transition to an active state is reduced. A whole genome-analysis of RNA isolated from E2Fs1/3-knockout adult neural stem cells has shown a shift from an active identity-state to a quiescent one. In the future, E2Fs1/3 could emerge as key regulators of quiescent stem cell activation, and thus could be potential targets for therapeutic control in order to enhance neurogenesis in patients with brain pathology.

Embryonic stem (ES) cells have the remarkable ability to differentiate into all cells comprising the three germ layers of the developing embryo. It is this pluripotency that makes them attractive for use in regenerative medicine. However, in order to harness this potential, we must understand the molecular mechanisms regulating the ability of ES cells to self-renew and thereby generate identical pluripotent daughter ES cells. The Welham laboratory has previously described a requirement for PI3K signalling in maintaining self-renewal of murine ES (mES) (Paling et al., 2004; Storm et al., 2007). To identify the molecular mechanisms involved in regulating mES cell self-renewal downstream of PI3K signalling, an Affymetrix microarray screen was carried out prior to the start of this PhD. For the screen, mES cells were grown in the presence of LIF and treated with the reversible PI3K inhibitor LY294002 (LY) or a DMSO control for 24, 48 and 72 hours. A total of 646 statistically significant transcriptional changes were detected and subsequently divided into 12 clusters using k-means clustering. Experiments using pharmacological inhibitors suggest that genes within the same cluster are regulated by common mechanisms. To identify potential candidates involved in regulation of mES cell pluripotency, further analyses concentrated on transcription factors and genes with unknown functions. In our microarray data Zscan4c, a member of a SCAN-domain containing Zinc finger protein family, is one of the earliest down-regulated probe-sets. Loss-of-function experiments using siRNA approaches highlight a role for Zscan4 downstream of PI3Ks in regulation of ES cell self-renewal. Immunohistochemical staining of cells overexpressing Zscan4c showed nuclear accumulation of the protein. This, together with the fact that Zscan4c was mainly detectable in the nuclear protein fraction, strengthens a role of Zscan4c in transcriptional regulation. Potential Zscan4c protein interaction partners were identified by applying a combined immunoprecipitation (IP) - mass spectrometry strategy. Interestingly, the majority of potential Zscan4c interacting proteins identified are associated with functions related to transcriptional regulation and DNA damage response, all characteristics linked with Zscan4. Furthermore, the Class IA PI3K catalytic isoforms were genetically activated in mES cells, and liberation of the requirement for LIF was found upon over-expression of an activated p110 catalytic subunit.

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Human embryonic stem cells (hESCs) show significant therapeutic potential in treating degenerative disorders. This is in part because of their ability to produce a limitless supply of starting cells and their potential to differentiate into more than 200 different cell types. The aim of the current research was to generate a robust stage wise protocol for the differentiation of hESCs to respiratory epithelial cells. The epithelial cells could then be used either for transplantation studies or, as an in vitro model for drug toxicity testing. In order to achieve this goal, we must identify the key steps in lung development and apply these to the differentiation protocol. In this study, we maintained Shef3 hESCs in their undifferentiated pluripotent state to expand the cells prior to the differentiated towards the definitive endoderm (DE) lineage. I used a two-stage protocol based on culture with a novel glycogen synthase kinase-3 (GSK-3) inhibitor (termed 1m), along with Activin-A. We confirmed the status of the cells by a combination of immunostaining and PCR. We showed loss of the pluripotency markers (Sox2 and Oct3/4) and gain of DE markers (Sox17, FoxA2 and CXCR4). After the induction of DE from hESCs, we then treated the cells with transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) pathway inhibitors (SB431542 and Noggin respectively). This combinatorial treatment resulted in the differentiation into the anterior foregut endoderm (AFE) lineage based on expression of Pax9 and FoxA2 plus the up-regulation of Sox2. Further differentiation of AFE derivatives into more mature epithelial cells, termed lung progenitor cells (LPCs), was achieved following the treatment of AFE cells with a cocktail of trophic factors (BMP4, EGF, bFGF, FGF10, KGF and Wnt3a) yielded a population of NKX2.1-positive and FoxA2-positive cells that potentially corresponded to the lung lineage. Finally, prolonged treatment with FGF10 and FGF2 on LPC derived hESCs induced proximal (CC10, MUC5AC) and distal (SPB, SPC) airway epithelial cells. In addition, we also utilised the ectopic expression of an adenovirus expressing NKX2.1 to promote lung maturation. In conclusion, we have generated a protocol for the differentiation of hESCs into mature lung-like cells. The generation of these cells in vitro could potentially lead to a better in vitro model for toxicity testing and the development of novel therapies for promoting regeneration of lungs in patients with severe lung disorders.

Triple‐negative breast cancer (TNBC) is the most refractory subtype of breast cancer to current treatments and accounts disproportionately for the majority of breast cancer‐related deaths. Research has not yet identified specific therapies for TNBC and chemotherapy remains the conventional therapy in the clinic. While conventional chemotherapy regimens have demonstrated success at reducing bulk tumor burden, they have been shown to enrich cancer stem cells (CSCs). CSCs promote chemoresistance, metastasis, heterogeneous tumor regeneration and disease relapse. Owing to tumor plasticity and the conversion between CSC and non-CSC subpopulations development of a strategy capable of inhibiting both non-CSC and CSC subpopulations is crucial for TNBC therapy. In this compilation of my main research projects, several new approaches for the treatment of TNBC were identified which target not only the bulk tumor population but also the CSC populations residing within the tumor: 1. Co-suppression of Wnt, HDAC, and ESR1 using clinically relevant low‐dose inhibitors effectively repressed both bulk and CSC subpopulations and converted CSCs to non‐CSCs in TNBC cells. 2. Co-inhibition of mTORC1, HDAC, and ESR1 was capable of reducing both bulk and CSC subpopulations as well as the conversion of fractionated non-CSC to CSCs in in a human TNBC xenograft model and hampered tumorigenesis following treatment. 3. Inhibition of Wnt and YAP retarded tumor growth of TNBC cells in either epithelial or mesenchymal states, and both CD44high/CD24low and ALDH+ CSC subpopulations were diminished in a human xenograft model reducing tumorigenicity following treatment.

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Stem cells play a vital role in the turn over of permanently self-renewing tissues (e.g. corneal epithelium, epidermis, bone marrow). Stratified cornea epithelia serve, as ideal tissue models for studying ???stemness???, as the site of cells in the different stages of differentiation are anatomically easily identifiable. Studies targeting limbal stem cell maintenance, in-vitro gene expression during storage and differentiation will benefit future therapeutic applications (e.g. corneal transplantation). Knowledge of stem cell behaviour will help explain the pathophysiology of corneal related ocular surface disorders (e.g. idiopathic limbal stem cell deficiency, pterygium and limbal dermoids), establish new treatment modalities (e.g. allogenic cellular transplants) and help promote invivo expansion of residual stem cell populations in deficiency states. The major obstacle in the progress of research on limbal stem cells is the lack of knowledge of phenotypic markers of limbal epithelial stem cells. This project first studied the limbal expression of phenotypic markers that were discovered in other human adult and embryonic stem cell populations using microarray differential expression studies. Gene expression profiles of the relatively primitive human foetal limbus were compared with that of the central cornea. Microarray was also performed to identify the differential gene expression profile of cultured primary human limbal epithelial cells, when compared to the limbal epithelial cell population isolated after 5 serial cultures. 33 genes were upregulated in the human foetal limbus and primary cultured human limbal epithelium, when compared respectively to the central foetal cornea (first experiment) and the limbal epithelial cell population after the 5th trypsin passaged culture (second experiment). Four foetal limbal and primary limbal epithelial upregulated genes (CK15, CK14, Cdh3, and Wnt4) were confirmed to be upregulated by semi quantitative RT-PCR and immunohistochemical experiments on the human foetal cornea, adult human cadaveric cornea and cultured human limbal epithelial cells. The microarray defined phenotypic profile of both the foetal limbus and cultured adult limbal epithelial cells will help identify these cells in in-vitro and in-vivo states. The expression of these 4 selected markers in the limbal dermoid and pterygium suggests that limbal epithelial cells containing a stem cell population are involved in the pathogenesis of these two disorders.

A five stage selection protocol originally applied to mouse embryonic stem cells (mESCs) was examined for the derivation of insulin producing cells from human embryonic stem cells (hESCs). Insulin gene expression was observed and insulin protein was measured by radioimmunoassay. However, the radioimmunoassay results were shown to be susceptible to false positive findings due to the presence of exogenous insulin within differentiation media and it was concluded that this particular strategy was not ideal for the derivation of insulin producing cells from hESCs. An investigation was then undertaken regarding the in vivo differentiation of cells derived from hESCs seeded within 3D scaffolds to determine if this would result in the derivation of insulin producing cells. Within scaffolds there were abundant cells which stained positively for ectoderm lineage markers including nestin. Cells which stained positively for markers of endothelial progenitors representing the mesoderm lineage were also observed and rare cells stained for endoderm markers including insulin. These investigations also demonstrated that transplanting scaffolds seeded with cells derived from hESCs between the liver lobules of immunodeficient mice could lead to the formation of teratomas. Factors that may have influence the formation of teratomas were further investigated and it was demonstrated that teratoma formation was inhibited by altering in vitro treatment of cells. An in vitro investigation was then performed to determine the extracellular matrix (ECM) producing capacity of hESCs and differentiated cells derived from hESCs because ECM proteins are required for the formation of 3D structures similar to pancreatic islets. The results from this investigation indicated that differentiated cells produced multiple ECM proteins at substantially higher levels than hESCs. The ECM producing differentiated cells could be useful in the development of surrogate islet like tissue by supplying a suitable ECM structure within a 3D scaffold environment to aid the function of ??-cell surrogates. Furthermore, these differentiated cells derived from hESCs were shown to produce an adhesive basement membrane in vitro, which is derived from human sources, and could be utilized in the derivation, propagation and differentiation of hESCs.

Includes bibliographies. Aims to identify homeobox genes which may have a developmental role during early embryogenesis by the characterization of homeobox gene expression in undifferentiated ES cells, and in a range of differentiated ES cell derivatives.