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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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Allogeneic haematopoietic stem cell transplantation (HSCT) has been used successfully to treat children and adults with high-risk or relapsed hematopoietic malignancies, marrow failure syndromes, and hereditary immunodeficiency disorders. When initially developed, allogeneic HSCT was conceived as a method of rescuing patients from the toxic side effects of dose-intensive chemoradiotherapy. Due to transplant-related toxicities, the application of myeloablative allogeneic HSCT has been limited to younger patients without organ dysfunctions. Since the early 1990s many groups of investigators have explored strategies using less intensive preparative regimens that would allow engraftment of hematopoietic progenitor cells from either identical or non-identical donors. These reduced-intensity conditioning (RIC) regimens result in less tissue damage, less inflammatory cytokine secretion, and possibly lower rates of graft-versus-host disease (GVHD) and non-relapse mortality (NRM). Such non-myeloablative approach, or "mini-transplant", has been suggested to benefit older patients as well as in conditions in which traditional myeloablative conditioning regimens are associated with high rates of non-relapse mortality. / Allogeneic HSCT is the only curative therapy for many patients with myeloid malignancies or myelodysplastic syndrome (MDS). The development of reduced-intensity preparative regimens may allow the extension of this form of treatment to older and patients with coexisting medical illness. On the other hand, relapse after transplantation remains the most important cause of treatment failure in patients with refractory acute myeloid leukemia (AML) or MDS, and is associated with poor survival. Evaluation of prognostic factors may help to improve the results of myeloablative and RIC allogeneic HSCT in this group of patients. Furthermore, the impact of comorbidities on outcomes of RIC allogeneic HSCT in this group of patients with refractory AML or MDS needs to be defined. / The application of embryonic and adult stem cells in regenerative and reparative therapies of non-hematopoietic diseases is emerging rapidly. Human umbilical cord blood (UCB) is a rich source of hematopoietic stem cells and mesenchymal progenitor cells. Although clinical experience to date with UCB has focused on hematological application, early preclinical studies support the hypothesis that multipotential stem cells derived from UCB exhibit functional characteristics similar to that observed in adult marrow-derived stem cells in mediating vascular and organ regenerative capabilities. However, the application of these preclinical findings in clinical setting needs to be further studied. Mini-transplant of human UCB may be an effective approach to repair organ damage in patients with non-hematological diseases. / Wong Siu Ming Raymond. / Adviser: Joseph J.Y. Sung. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 187-223). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

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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.

1 / Connor MacCrimmon

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