The ability of the brain to continually generate new neurons throughout life is one of the most intensely researched areas of modern neuroscience. While great advancements in understanding the biochemical mechanisms of adult neurogenesis have been made, there remain significant obstacles and gaps in connecting neurogenesis with behavioural and cognitive processes such as learning and memory. The purpose of the thesis was to examine by review and laboratory experimentation the role of the dentate gyrus and of adult neurogenesis within the hippocampus in the performance of cognitive tasks dependent on the hippocampal formation and hippocampal-basal ganglia interactions. Advancement in understanding the role of neurogenesis in these processes may assist in improving treatments for common brain injury and cognitive diseases that affect this region of the brain. Mild chronic stress reduced the acquisition rate of a stimulus-response task (p=0.043), but facilitated the acquisition of a discrimination between a small and a large reward (p=0.027). In locomotor activity assays, chronic stress did not shift the dose-response to methamphetamine. Analysis of 2,5-bromodeoxyuridine incorporation showed that, overall, chronic mild stress did not effect survival of neuronal progenitors . However, learning of the tasks had a positive influence on cell survival in stressed animals (p=0.038). Microinjections of colchicine produced significant lesions of the dentate gyrus and surrounding CA1-CA3 and neocortex. Damage to these regions impaired hippocampal-dependent reference memory (p=0.054) while preserving hippocampal independent simple discrimination learning. In a delay discounting procedure, the lesions did not induce impulsive-like behaviour when delay associated with a large reward was introduced. The experiments uphold a current theory that learning acts as a buffer to mitigate the negative effects of stress on neurogenesis.

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Umbilical cord blood (CB) has been used as a source of haemopoietic stem cells in both related and unrelated bone marrow transplantation and in both settings there appears to be a reduced incidence of graft-versus-host disease (GVHD) when compared to bone marrow transplantation. The aim of this study was to perform a phenotypic and cellular analysis of both cord and adult mononuclear cells <I>in vitro</I> in order to identify any immunological characteristic that could account for the apparent reduced incidence of GVHD observed, <I>in vivo</I>. For this, the cell surface phenotype, allogeneic cellular responses, cytokine secretion and expression of co-stimulatory molecule CD40L was studied. The phenotypic analysis was carried out by determining the proportion of cells expressing the CD4, CD8, CD16, CD19, CD45RA and CD45RO markers using 3 colour flow cytometric analysis. The results showed that in cord blood mononuclear cells, there was an increased number of cells expressing CD19⁺ whereas the number of cells expressing CD8⁺ was decreased compared to adult blood mononuclear cells (ABMNCs). In contrast the number of cells expressing CD4⁺ and CD16⁺ was similar in both cord blood mononuclear cells (CBMNCs) and ABMNCs. Further phenotypic analysis confirmed that cord blood contained primarily 'unprimed' T cells expressing CD45RA, while adult peripheral blood lymphocytes express mainly the CD45R 'memory' phenotype. The primary and secondary allogeneic responses of cord and adult mononuclear cells were assessed using the standard mixed lymphocyte reaction (MLR) and the primed lymphocyte test (PLT), respectively. These results showed that there was no significant difference in primary MLR responses of CBMNCs and ABMNCs. In contrast, CBMNCs had a decreased ability to mount a secondary proliferative response compared to ABMNCs. However this impaired ability to respond could be overcome by the addition of exogenous IL-2 to the cultures. The secretion of cytokines such as IL-2, IL-4, γIFN and TNF-α and the expression of CD40L on PMA- and ionomycin-activated CBMNCs and ABMNCs was also analysed by 3 colour flow cytometry. These results showed that upon activation CBMNCs produced reduced levels of cytokines and had reduced expression of CD40L. It is likely that the reduced cytokine production and CD40L expression observed is due to the predominance of CD45RA⁺ cells in CB compared to AB. In summary, this study has shown that CB and AB have different phenotypic and functional characteristics in vitro which may well explain the reduced incidence of GVHD observed in cord blood transplantation.

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

This thesis includes two parts related to hydrogels as therapeutically useful constructs: a biomimetic hydrogel carrying stem cells for bone regeneration and an acid-sensitive hydrogel carrying drugs for cancer therapy. In tissue engineering, one of the biggest difficulties is the control of stem cell fate on scaffolds. A biodegradable and cell attachable cross-linker was synthesized by one-step Michael additional reaction, and was used to fabricate a novel hydrogel to control the stem cell fate. For anti-cancer therapy, releasing drug on tumor cells or organs while having low effects on health cells under physiological conditions is a critical requirement. Two nature polymers are modified to achieve loading anti-cancer drug while forming hydrogels which can selectively release the drug in tumor environment by acid-sensitive linkages.

Stem cells hold great promise for treating various degenerative diseases. However, the outcomes of preclinical and clinical cell therapy studies are still not close to our expectation. The unsatisfactory outcomes of cell therapy are at least partially due to: 1) insufficient homing of implanted stem cells into target organs and 2) use of heterogeneous cell populations for cell therapy. Therefore, there is a need to develop effective guiding technique for stem cells to migrate to the target organs and to isolate effective stem cell populations. In this project, I developed a microfluidics-based method for selecting chemotactic adipose-derived stem cells (ASCs) to epidermal growth factor (EGF). This method integrates cell patterning, chemotaxis and cell extraction on a single microfluidic device. Post-extraction analysis confirmed the higher chemotactic ability of the extracted cells to EGF. The extracted chemotactic ASCs shows up-regulated surface expression of EGF receptor and its downstream signaling event upon EGF stimulation.

A complex network of transcription factors, which are regulated by signalling molecules, is responsible in coordinating the formation of differentiated skeletal and cardiac myocytes from undifferentiated stem cells. The present study aims to understand and compare the transcriptional regulation of skeletal and/or cardiac muscle development in the absence of Sox7 or in the presence of a collagen-based matrix in P19 embyonal carcinoma (EC) and mouse embryonic stem (ES) cells. First, knock-down of Sox7 , by shRNA, in muscle inducing conditions (+DMSO) and in the absence of RA (-RA), decreased muscle progenitor transcription factor and myogenic regulatory factor (MRF) levels, suggesting that Sox7 is necessary for myogenesis. However, knock-down of Sox7 in the presence of RA (+RA) and DMSO increased expression of muscle progenitor markers and MRFs, suggesting that Sox7 is inhibitory for myogenesis +RA. Furthermore, Sox7 overexpression enhanced myogenesis -RA, but inhibited myogenesis and enhanced neurogenesis +RA. These results suggest an important interplay between RA signalling and Sox7 function during P19 differentiation. Second, Q-PCR analysis showed that compared to the mouse ES cells differentiated on the regular TC plates, differentiation on the collagen matrices had a higher expression of skeletal and cardiac precursors, MRFs and terminal differentiation markers. Collagen alone enhanced myotube formation. The enhanced collagen matrix, containing the oligosaccharide sialyl LewisX (sLeX), specifically enhanced cardiomyogenesis. These studies have added to our understanding of the transcriptional regulation of premyogenic mesoderm factors and the role of Sox7 in this process. In addition these studies provide a vision for possible use of biomaterials in directed differentiation of stem cells for the purpose of cell therapy.

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes and chronic liver disease. Various attempts have been made to produce cells that can serve as precursors for pancreas and liver. By using all-trans-retinoic acid, basic fibroblast growth factor, dibutyryl cAMP, and cyclopamine, an attempt has been made to produce definitive endoderm and subsequently cells that can serve as pancreatic and hepatocyte precursors from mouse embryonic stem cells. By using retinoic acid and basic-FGF, in the absence of embryoid body formation, mouse embryonic stem cells were differentiated at different culture periods. Four protocols of varying lengths of culture and reagents and their cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, pancreas and hepatocytes. Inclusion of DBcAMP and extension of culture time resulted in cells that display features of definitive endoderm by expression of Sox 17 and FOXA2 and minimal expression of primitive endoderm and other germ cell layers such as ectoderm and mesoderm. These cells produced insulin and C-peptide and secreted insulin in a glucose responsive manner. However, they seem to lack mature insulin secretion mechanism. There was a production of hepatocyte markers (AFP-2 and transthyretin) but there was insufficient data to assess for convincing production of hepatocytes. In summary, one of the protocols produced cells that displayed characteristics of definitive endoderm and they may serve as pancreatic endocrine precursors.

Telomeres are the repetitive DNA sequence and associated proteins found at the ends of linear chromosomes. They have a role in biological processes including meiosis and aging as well as implications in a number of genomic instability disorders and cancers. Telomeres maintain genomic stability by protecting chromosome ends from terminal fusions and misidentification as DNA damage sites. Their wide range of functions has resulted in an increased interest in developing tools to study the dynamics of telomeres in live cells. To do this, current studies use the ubiquitously expressed protein Telomere Repeat Factor 1 (TRF1) tagged with a fluorescent protein. TRF1 is a negative regulator of telomere length that binds exclusively to telomere repeats. Over-expression of the fluorescent protein fused to TRF1 has been a useful tool to track telomere movement. The foci formed by the tagged TRF1 protein accurately represent the number of telomeres expected in the cells and the localization is maintained throughout the cell cycle. A caveat with this system is that over-expression of TRF1 leads to accelerated telomere shortening, as well as replication defects that can stall telomere replication. These caveats make it difficult to draw conclusions about telomere dynamics based solely on observations of cells over-expressing fluorescently tagged TRF1. To eliminate problems associated with protein over-expression, I have tried to develop knock-in embryonic stem (ES) cells expressing fluorescently tagged TRF1 from the endogenous Trf1 promoter. To do this, I have used a recombineering technique using Bacterial Artificial Chromosomes (BACs). BAC recombineering allows for the direct knock-in of a fluorescent tag into the mouse Trf1gene locus. Genetic constructs with the correct sequence inserts have been obtained and have been used for transfection of ES cells. While no correctly targeted ES cells have been identified so far, the expectation is that ES cell lines with correctly targeted fluorescently tagged TRF1 will be obtained in the near future. Such lines will be used to study telomere dynamics in ES cells, differentiated cells generated from ES cells, as well as to generate mice.

Thesis (MAppSc (Medical Laboratory Science)) --University of South Australia, 1993