In the bone marrow, CD34 is the best currently available marker of human multipotential stem cells and is downregulated upon the commitment of cells to the myeloid pathway. CD33 is the earliest marker of stem cell commitment to the myeloid lineage but is down-regulated as myeloid cells mature to granulocytes while expression is retained on monocytes, dendritic cells and macrophages. CD34 is a mucosialin and CD33 a member of the immunoglobulin superfamily. To determine the functions of CD34 and CD33 in early haematopoiesis, soluble forms of CD34 and CD33 have been constructed by PCR based construction of extracellular domain-IgGlFc (ECDFc) fusion plasmids. These chimaeric proteins have been used to define the ligand binding of CD33 and CD34 in a range of assay systems including: screening cell lines by immunofluorescence and iodination analysis; immunohistochemical staining; screening of cDNA libraries transiently expressed in COS cells by "panning" for ligands; and phosphorylation studies to assess the potential of phosphoproteins to regulate these molecules and their ligands in vitro. Iodination demonstrated that CD33 binds increasing numbers of ligands heterophilically on erythroleukemic (K562) and promonocytic (U937) cell lines at sizes ranging from 54-69Kd and 97-1 lOKd in a differentiation-dependent manner. CD33 was established to associate with several src-like kinases while Fc-CD33 precipitates phosphoproteins in the same region in KG-la, K562 and U937. Novel divalent-cation-dependent CD34 ligands of 66Kd and HOKd were isolated on HUVEC and both CD34 and Fc-CD34 precipitated phosphoproteins from HUVEC. From panning studies, Fc-CD33 demonstrated low levels of binding to ICAM-1 and a cDNA product sharing homology to the 3' end of dystrophin, termed here as apo-dystrophin-4. Apo-4 appears to give rise to two major proteins, at least one of which may provide an in vitro ligand for CD33 and contain a 3' enhancer. Models for both CD33 phosphoprotein and ligand binding behaviour are presented.

No description available.

Stem cell therapies hold great promise for diseases such as stroke, where few effective treatment options exist. Clinical translation of experimental stem cell therapies requires the ability to monitor delivery and behaviour of cells non-invasively in-vivo with clinical imaging modalities such as MRI. This thesis presents the translation of established methods for labelling and imaging stem cells with specialized MRI systems to a more clinically relevant setting. A methodology for harvesting and labelling a cell population containing stem cells with iron oxide for detection with a clinical MRI system is presented and single cell detection is demonstrated in-vitro. The feasibility of detecting iron oxide labelled stem cells intravenously delivered in a rat model of stroke is tested. Results demonstrate that while MRI is highly sensitive to the presence and distribution of iron oxide containing cells in-vivo the true origin of these cells remains ambiguous with the current methodology.

Stem cell therapies hold great promise for diseases such as stroke, where few effective treatment options exist. Clinical translation of experimental stem cell therapies requires the ability to monitor delivery and behaviour of cells non-invasively in-vivo with clinical imaging modalities such as MRI. This thesis presents the translation of established methods for labelling and imaging stem cells with specialized MRI systems to a more clinically relevant setting. A methodology for harvesting and labelling a cell population containing stem cells with iron oxide for detection with a clinical MRI system is presented and single cell detection is demonstrated in-vitro. The feasibility of detecting iron oxide labelled stem cells intravenously delivered in a rat model of stroke is tested. Results demonstrate that while MRI is highly sensitive to the presence and distribution of iron oxide containing cells in-vivo the true origin of these cells remains ambiguous with the current methodology.

The orphan nuclear receptor EAR-2 (NR2F6) is a gene that I previously found to be expressed at a higher level in clonogenic leukemia single cells than in leukemia cells that can not divide. For this thesis I undertook to perform the first investigations of the roles EAR-2 may play in normal haematopoiesis and in the pathogenesis of acute myelogenous leukaemia. Here, I show that EAR-2 is overexpressed in the bone marrow of patients with MDS, AML and CMML compared to healthy controls and that EAR-2 is a gatekeeper to hematopoietic differentiation. Over-expression of EAR-2 prevents the differentiation of cell lines, while knock down induces their spontaneous differentiation. In vitro, primary bone marrow cells that over-express EAR-2 do not differentiate into granulocytes in suspension culture, but have greatly extended replating capacity in colony assays. In vivo, overexpression of EAR-2 in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome characterised by hypercellular bone marrow, an increase in blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice. Furthermore, animals that are transplanted with grafts of sorted bone marrow develop a rapidly fatal leukemia that is characterized by pancytopenia, enlargement of the spleen, infiltration of blasts into the spleen, liver and peripheral blood. Interestingly, development of leukemia is preceded by expansion of the stem cell compartment. Overexpression of EAR-2 increases the maintenance of KSL primitive bone marrow cells in ex vivo suspension culture, while knockdown of EAR-2 induces rapid differentiation of KSL cells into granulocytes. These data establish that EAR-2 is a novel oncogene that regulates hematopoietic cell differentiation. Furthermore, I show that EAR-2 is also a novel negative regulator of T-cell lymphopoiesis, and demonstrate that down-regulation of EAR-2 is important for the survival, proliferation and differentiation of T-cell progenitors. Overall, this work establishes that expression of EAR-2 is an important determinant of cell fate decisions in the hematopoietic system.

SirT1 is a class III histone deacetylase that has beneficial roles in various diseases related to aging such as cancer, diabetes and neurodegenerative disease. Resveratrol is a natural compound that mimics most of the beneficial effects attributed to SirT1. Resveratrol has toxicity towards cancer cells and has been reported to be a direct activator of SirT1. Interestingly, SirT1 over-expression has also been reported to be toxic. We set out to determine if resveratrol toxicity is mediated through activation of SirT1. We have assessed resveratrol toxicity in embryonic stem cells and mouse embryonic fibroblast (MEFs) across different SirT1 genotypes. Our data indicates that SirT1 is not implicated in resveratrol toxicity in either normal or transformed MEFs. Thus, resveratrol toxicity does not appear to be mediated by SirT1.

The in-vitro culture of human embryonic stem and keratinocyte cells has great potential to revolutionise the therapeutics industry. Indeed it is hoped that these cells will provide a superior alternative to current tissue and organ transplantation. However, both of these cell types require animal and/or donor products for their successful maintenance in-vitro. This requirement results in a significant risk of cross contamination from the animal or donor products to either the primary keratinocyte or hES cells. These potentially transplantable cells therefore need to be cultured in an environment free from animal or donor products to remove the risk of contamination to the patient. The ideal growth conditions must comprise of two attributes; firstly they must be free from animal or donor products, and secondly the culture system must be fully defined. Recently, it was discovered that an extra-cellular matrix protein, vitronectin, could be used in conjunction with growth factors and growth factor-binding proteins (VN:GF combination), to promote enhanced cell migration and growth through the co-activation of integrin and growth factor receptors. Given that growth factors and serum are clearly important in supporting the in-vitro cultivation of mammalian cells, and that vitronectin is an abundant protein in serum, I hypothesised that these VN:GF combinations could be translated into a serum-free medium that would support the serial propagation and self renewal of primary keratinocytes and hES cells. As reported in this thesis I have developed a defined, serum-free media for the culture of these cells that incorporates the VN:GF combinations. While the two media differ slightly in their compositions, both support the serial, undifferentiated expansion of their respective cells types. Together, this represents a significant advance that will ultimately facilitate the therapeutic use of these cells. However, the in-vitro expansion of these cells in these new media still required the presence of a feeder cell layer. In view of this I aimed to explore the in-vitro micro-environment of primary keratinocytes using a novel proteomic approach in an attempt to find candidate factors that could be used in conjunction with the VN:GF media to replace both serum and the feeder cells. The proteomic approach adopted examined the secretion of proteins into the defined, minimal protein content VN:GF media when the feeder cells were cultured alone, as well as in co-culture with primary keratinocytes. This strategy allowed assessment of proteins/factors that are secreted in response to both autocrine and paracrine cellular interactions and revealed a number of candidate factors that warrant further investigation. Ultimately this proteomic information and the associated new insights into the keratinocyte in-vitro culture microenvironment may lead to the development of a culture system for these cells that is not reliant on either a feeder cell layer or serum for their successful propagation. Moreover, it is likely that this will also be relevant to the feeder cell-free propagation of hES cells. This has obvious advantages for the culture of primary keratinocytes and hES cells in that it will allow a safe defined culture system for the undifferentiated propagation of these cells. This will facilitate the generation of cells and tissues free from xenogeneic and allogeneic contaminants, thus ensuring any therapeutics developed from these cell types are approved for therapeutic applications and importantly, will minimise risks to patients.

Thesis (PhD in AppSc)--University of South Australia, 1996

Title page, contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The growth plate is a cartilaginous structure located at the proximal and distal ends of immature long bones, which contributes to longitudinal growth through the process of endochondral ossification. Cartilage has a limited ability to regenerate and in children, injury to the the growth plate can result in limb length discrepancies and angular deformity, due to formation of a bone bridge at the damaged site which disturbs structure and function of the growth plate. Current treatments of the abnormalities arising from growth plate arrest involve surgical correction once the deformities have manifested. To date, there is no biological based therapy for the repair of injured/damaged growth plate cartilage. Mesenchymal stem cells (MSC) are self renewable mulitpotential progenitor cells with the capacity to differentiate toward the chondrogenic lineage. Since their discovery, significant interest has been generated in the potential application of these cells for cartilage regeneration. In this study, the ability of autologous bone marrow mesenchymal stem cells to regenerate growth plate cartilage in a sheep model was examined. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1330837 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Transcription factors GATA-1, Fli-1 and FOG-1 are essential proteins for normal megakaryopoiesis, however, the detailed analyses of their functions within developmental stages of megakaryopoiesis are lacking. In my thesis, over expression of gene in target cells was adopted as the main strategy to study the biological functions of these proteins, therefore, an efficient gene delivery method was first developed by using retrovirus.This approach was then utilized to over express GATA-1, Fli-1 and FOG-1 in murine leukemia M1 cells and mouse hematopoietic stem cells (HSCs), and their effects on different developmental stages of megakaryopoiesis were investigated. In the transduced M1 cells, enforced expression of GATA-1 and Fli-1 was found to induce the megakaryocytic development, which was associated with the formation of megakaryocyte (Mk) and the increased expression of Mk specific genes c-Mpl and GPIX. In the transduced mouse HSCs, it was found that the expression of endogenous GATA-1, Fli-1 and FOG-1 was up-regulated throughout Mk differentiation; enforced expression of these transcription factors led to the significantly enhanced Mk development. Megakaryocytes over expressing GATA-1, Fli-1 and FOG-1 were characterized by the increased expression of various Mk-specific genes including GPIX, c-Mpl, platelet factor 4 (PF4), acetylcholinesterase (AChE) and NF-E2, an important transcription factor for terminal megakaryopoiesis; however, GATA-1, Fli-1 and FOG-1 displayed the different abilities in promoting the proliferation of hematopoietic cells and MK differentiation, as well as regulating other transcription factors involved in hematopoiesis. To further elucidate the role of the functional domains of Fli-1, various mutants of Fli-1 were also over expressed in mouse HSCs. The results demonstrated that first, the combination of the activation domain of Fli-1 and its Ets domain is required for early megakaryopoiesis but not sufficient for terminal megakaryopoiesis; second, DNA binding of Fli-1 was not the only requirement for early Mk enhancement, moreover, the interaction between Fli-1 and GATA-1 through the Ets domain and the resultant transcriptional synergy was the essential determinant for Fli?1 ability in Mk development. Taken together, the studies presented in this thesis provided strong in vitro evidence that GATA-1, Fli-1 and FOG-1 indeed play the critical roles in normal megakaryopoiesis.