Liver cirrhosis places an increasing burden on healthcare worldwide. Currently the only treatment is liver transplantation. Whilst liver transplant has a relatively good five-year survival, donor organ shortage costs many lives every year and results in lifelong immunosuppression. Alternative treatments are thus urgently needed. It is with this background that there is understandable interest for the development of stem cell therapies for liver regeneration. The identification of putative liver stem cells has brought closer the previously separate fields of liver ontology, regeneration, and carcinogenesis. Significant overlaps in the regulation of these processes are now being described. For example, studies in embryonic liver development have already provided the basis for directed differentiation of human embryonic stem cells and induced pluripotent stem cells into hepatocyte-like cells. As a result, the understanding of the cell biology of proliferation and differentiation in the liver has been improved. This knowledge can be used to improve the function of hepatocyte-like cells for drug testing, bio-artificial livers, and transplantation. In parallel, the mechanisms regulating cancer cell biology are now clearer, providing fertile soil for novel therapeutic approaches. Recognition of the relationships between development, regeneration, and carcinogenesis, and the increasing evidence for the role of stem cells in all of these areas, has sparked fresh enthusiasm in understanding the underlying molecular mechanisms and has led to new targeted therapies for liver cirrhosis and primary liver cancers. Human liver progenitor cells (LPCs) have therapeutic potential but their in vitro culture results in inadequate differentiation, function, and phenotypic instability reflecting an incomplete understanding of in vivo processes. LPCs can be robustly isolated from second trimester human foetal livers by immunoselection for EpCAM+/CD29+/CD49d+/CD49e–/CD235a–/CD45– cells. Expression profiling of mRNA and microRNA in human foetal LPCs was performed and compared with mature human hepatocytes and human embryonic stem cells undergoing hepatocytic differentiation. Foetal LPCs exhibit a distinct transcriptome profile consistent with a stem cell signature, cell division, and some liver-specific functions. Bioinformatic integration of microRNA and mRNA datasets revealed that microRNAs up-regulated in LPCs targeted genes involved in metabolic processes implying repression of the mature hepatocyte phenotype. Control of LPC gene expression therefore occurs at both transcriptional and, via microRNAs, post-transcriptional levels. Furthermore, transcription factor binding site analyses revealed enriched E2F1 motif in gene and microRNA promoters suggesting feedback control in determining LPC fate. Foetal LPCs were capable of differentiation to a hepatocytic phenotype in the presence of appropriate paracrine signals provided by EpCAM– non-parenchymal cells (NPCs), which consist mainly of endothelial cells and hepatic stellate cells. Fibronectin, despite being produced in abundance by EpCAM– NPCs, had no effect on LPC synthetic function in vitro. The expression of fibronectin in the perisinusoidal space suggests its potential role of modulating cross-talk between hepatoblasts/hepatocytes, liver sinusoidal endothelial cells, and hepatic stellate cells. Fibronectin expression in the portal vein mesenchyme and laminin α5 expression along the ductal plate suggest that both matrix molecules, located in close proximity to LPCs, may be important in supporting the LPC niche. Findings in this work provide insight into the regulation of the human foetal LPC functional phenotype, bringing stem cell-based therapies for liver disease one step closer.

In this thesis we have investigated permutation and monomial progenitors of the form p^*n:N (p=2,3,5,...) and p^*n:_m N (p=3,5,7,...) respectively. We have discovered new symmetric presentations of several finite nonabelian simple groups including linear groups, unitary groups, orthogonal groups, and sporadic groups. We have constructed interesting groups found using the technique of double coset enumeration and found the isomorphic types of the numerous groups that appeared as homorphic images. These include the sympletic group, S_4(5) and the Janko groups, J_2 and J_1 which were found using a variety of different control groups over finite fields.

The biochemical and molecular processes that maintain the stem cell pool, and govern the proliferation and differentiation of haemopoietic stem/progenitor cells (HSPCs) have been widely investigated but are incompletely understood. The purpose of this study was to identify and characterise novel genes that may play a part in regulating the mechanisms that control the proliferation, differentiation and self-renewal of human HSPCs. Reverse transcription differential display polymerase chain reaction (dd-PCR) was used to identify differences in gene expression between a HSPC population defined by expression of the CD34 phenotype, and the more mature CD34 depleted populations. A total of 6 differentially expressed complementary deoxyribonucleic acid (cDNA) sequences were identified. Four of these transcripts were homologous to well characterised genes, while two (band 1 and band 20) were homologous to unknown and uncharacterised partial gene sequences on the GenBank database and were thus chosen for further investigation. The partial cDNA sequences for band 1 and band 20 were designated ORP-3 and MERP-1 (respectively) due to homologies with other well-characterised gene families. Differential expression of the ORP-3 and MERP-1 genes was confirmed using Taqman™ real-time polymerase chain reaction (PCR) with 3 - 4-fold and 4-10 -fold higher levels in the CD34+ fractions of haemopoietic cells compared to CD34- populations respectively. Additionally, expression of both these genes was down regulated with proliferation and differentiation of CD34+ cells further confirming higher expression in a less differentiated subset of haemopoietic cells. The full coding sequences of ORP-3 and MERP-1 were elucidated using bioinformatics, rapid amplification of cDNA ends (RACE) and PCR amplification. The MERP-1 cDNA is 2600 nucleotides (nt) long, and localizes by bioinformatics to chromosome 7.. It consists of three exons and 2 introns spanning an entire length of 31.4 kilobases (kb). The MERP-1 open reading frame (ORF) codes for a putative 344 amino acid (aa) type II transmembrane protein with an extracellular C-terminal ependymin like-domain and an intracellular N-terminal sequence with significant homology to the cytoplasmic domains of members of the protocadherin family of transmembrane glycoproteins. Ependymins and protocadherins are well-characterised calcium-dependant cell adhesion glycoproteins. Although the function of MERP-1 remains to be elucidated, it is possible that MERP-1 like its homologues plays a role in calcium dependent cell adhesion. Differential expression of the MERP-1 gene in haemopoietic cells suggests a role in haemopoietic stem cell proliferation and differentiation, however, its broad tissue distribution implies that it may also play a role in many cell types. Characterization of the MERP-1 protein is required to elucidate these possible roles. The ORP-3 cDNA is 6631nt long, and localizes by bioinformatics to chromosome 7pl5-p21. It consists of 23 exons and 22 introns spanning an entire length of 183.5kb. The ORP-3 ORF codes for a putative 887aa protein which displays the consensus sequence for a highly conserved oxysterol-binding domain. Other well-characterised proteins expressing these domains have been demonstrated to bind oxysterols (OS) in a dose dependant fashion. OS are hydroxylated derivatives of cholesterol Their biological activities include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, including haemopoietic cells. Differential expression of the ORP-3 gene in haemopoietic cells suggests a possible role in the transduction of OS effects on haemopoietic cells, however, its broad tissue distribution implies that it may also play a role in many cell types. Further investigation of ORP-3 gene expression demonstrates a significant correlation with CD34+ sample purity, and 2-fold higher expression in a population of haemopoietic cells defined by the CD34+38- phenotype compared to more mature CD34+38+ cells. This finding, taken together with the previous observation of down-regulation of ORP-3 expression with proliferation and differentiation of CD34+ cells, indicates that ORP-3 expression may be higher in a less differentiated subset of cells with a higher proliferative capacity. This hypothesis is supported by the observation that expression of the ORP-3 gene is approximately 2-fold lower in differentiated HL60 promyelocytic cells compared to control, undifferentiated cells. ORP-3 expression in HL60 cells during normal culture conditions was also found to vary with expression positively correlated with cell number. This indicates a possible cell cycle effect on ORP-3 gene expression with levels highest when cell density, and therefore the percentage of cells in G(0)/G(1) phase of the cell cycle is highest. This observation also correlates with the observation of higher ORP-3 expression in CD34+38-cells, and in CD34+ and HL60 cells undergoing OS induced and camptothecin induced apoptosis that is preceded by cell cycle arrest at G(0)/G(1). Expression of the ORP-3 gene in CD34+ HSPCs from UCB was significantly decreased to approximately half the levels observed in control cells after 24 hours incubation in transforming growth factor beta-1 (TGFâl). As ≥90% of these cells are stimulated into cell cycle entry by TGFâl, this observation further supports the hypothesis that ORP-3 expression is highest when cells reside in the G(0)/G(1) phase of the cell cycle. Data obtained from investigation of ORP-3 gene expression in synchronised HL60 cells however does not support nor disprove this hypothesis. Culture of CD34+ enriched HSPCs and HL60 cells with 25-OHC significantly increased ORP-3 gene expression to approximately 1.5 times control levels. However, as 25-OHC treatment also increased the percentage of apoptotic cells in these experiments, it is not valid to make any conclusions regarding the regulation of ORP-3 gene expression by OS. Indeed, the observation that camptothecin induced apoptosis also increased ORP-3 gene expression in HL60 cells raises the possibility that up-regulation of ORP-3 gene expression is also associated with apoptosis, Taken together, expression of the ORP-3 gene appears to be regulated by differentiation and apoptosis of haemopoietic progenitors, and may also be positively associated with proliferative and G(0)/G(1) cell cycle status indicating a possible role in all of these processes. Given the important regulatory role of apoptosis in haemopoiesis and differential expression of the ORP-3 gene in haemopoietic progenitors, final investigations were conducted to examine the effects OS on human HSPCs. Granulocyte/macrophage colony forming units (CFU-GM) generated from human bone marrow (ABM) and umbilical cord blood (UCB) were grown in the presence of varying concentrations of three different OS - 7keto-cholesterol (7K-C), 7beta-hydroxycholesterol (7p-OHC) and 25-hydroxycholesterol (25-OHC). Similarly, the effect of OS on HL60 and CD34+ cells was investigated using annexin-V staining and flow cytometry to measure apoptosis. Reduction of nitroblue tetrazolium (NBT) was used to assess differentiative status of HL60 cells. CFU-GM from ABM and HL60 growth was inhibited by all three OS tested, with 25-OHC being the most potent. 25-OHC inhibited ≥50% of bone marrow CFU-GM and ≥95% of HL60 cell growth at a level of 1 ug/ml. Compared to UCB, CFU-GM derived from ABM were more sensitive to the effects of all OS tested. Only 25-OHC and 7(5-OHC significantly inhibited growth of UCB derived CFU-GM. OS treatment increased the number of annexin-V CD34+ cells and NBT positive HL60 cells indicating that OS inhibition of CFU-GM and HL60 cell growth can be attributed to induction of apoptosis and differentiation. From these studies, it can be concluded that dd-PCR is an excellent tool for the discovery of novel genes expressed in human HSPCs. Characterisation of the proteins encoded by the novel genes ORP-3 and MERP-1 may reveal a regulatory role for these genes in haemopoiesis. Finally, investigations into the effects of OS on haemopoietic progenitor cells has revealed that OS are a new class of inhibitors of HSPC proliferation of potential relevance in vivo and in vitro.

Bone marrow-derived progenitor cell populations have been implicated in tissue regeneration and also in human disease pathology. This thesis investigated the hypothesis that Clara Cell Secretory Protein positive (CCSP+) epithelial-like progenitor cells and circulating fibrocyte numbers are altered in human lung disease and injury, and aimed to determine the predictive value of these cell profiles. It was found that cystic fibrosis patients have an increased number of CCSP+ cells in their bone marrow and peripheral blood, while patients with bronchiolitis obliterans syndrome (BOS) have a decreased number. In addition, BOS and pulmonary fibrosis patients have increased circulating fibrocytes. In response to ischemia reperfusion injury, an increase in CCSP+ cells in the peripheral blood was found at 24 hrs following lung transplant. Lastly, in patients studied greater than 1-year from transplant, those diagnosed with BOS had a higher number of fibrocytes and a loss of CCSP+ peripheral blood cells when compared to patients with stable lung function, with increased fibrocytes being associated with time post-transplant. In these patients, the ratio of fibrocytes-to-CCSP+ cells was predictive of lung function. Multiplex protein arrays were used to investigate corresponding patient plasma, aiming to elucidate key mediators of progenitor cell recruitment. While differences in various cytokines were found between end-stage diseases, a specific relationship between Stem Cell Growth Factor- and CCSP+ cells was identified and between Monocyte Chemotactic Protein-1 and fibrocytes. Conversely, response of CCSP+ cells following transplant appears to be mediated by known mobilizing factors SDF-1 and GM-CSF. Interestingly, in patients followed long-term after transplant, MCP-1 was associated with the number of CCSP+ cells, while SDF-1 correlated with fibrocyte numbers. These observations suggest common pathways acting on both populations that may be altered by the microenvironment, and may further suggest a common origin. This work contributes important information regarding changes in lung progenitor cells and their association with human disease and tissue repair, which could ultimately support future directions that directly advance therapy and improve patient care.

Bone marrow-derived progenitor cell populations have been implicated in tissue regeneration and also in human disease pathology. This thesis investigated the hypothesis that Clara Cell Secretory Protein positive (CCSP+) epithelial-like progenitor cells and circulating fibrocyte numbers are altered in human lung disease and injury, and aimed to determine the predictive value of these cell profiles. It was found that cystic fibrosis patients have an increased number of CCSP+ cells in their bone marrow and peripheral blood, while patients with bronchiolitis obliterans syndrome (BOS) have a decreased number. In addition, BOS and pulmonary fibrosis patients have increased circulating fibrocytes. In response to ischemia reperfusion injury, an increase in CCSP+ cells in the peripheral blood was found at 24 hrs following lung transplant. Lastly, in patients studied greater than 1-year from transplant, those diagnosed with BOS had a higher number of fibrocytes and a loss of CCSP+ peripheral blood cells when compared to patients with stable lung function, with increased fibrocytes being associated with time post-transplant. In these patients, the ratio of fibrocytes-to-CCSP+ cells was predictive of lung function. Multiplex protein arrays were used to investigate corresponding patient plasma, aiming to elucidate key mediators of progenitor cell recruitment. While differences in various cytokines were found between end-stage diseases, a specific relationship between Stem Cell Growth Factor- and CCSP+ cells was identified and between Monocyte Chemotactic Protein-1 and fibrocytes. Conversely, response of CCSP+ cells following transplant appears to be mediated by known mobilizing factors SDF-1 and GM-CSF. Interestingly, in patients followed long-term after transplant, MCP-1 was associated with the number of CCSP+ cells, while SDF-1 correlated with fibrocyte numbers. These observations suggest common pathways acting on both populations that may be altered by the microenvironment, and may further suggest a common origin. This work contributes important information regarding changes in lung progenitor cells and their association with human disease and tissue repair, which could ultimately support future directions that directly advance therapy and improve patient care.

Upper airways are lined with a pseudostratified mucociliary epithelium maintained by basal cells. To investigate functional and phenotypic heterogeneity within the human basal cell compartment, we used a combination of limiting dilution assays and surface marker profiling on primary cultures of basal cells with verified progenitor activity. The limiting dilution assay suggested functional heterogeneity in the ability of basal cells to repopulate a filter and maintain a barrier at ALI. The frequency of cells with this activity varied between patient strains and ranged from 0.08%-1% of basal cells. Validation of large-scale comprehensive surface marker profiling on basal cells led to identification of 74 antigens demarking consistent subpopulations. Preliminary functional analyses suggest differences in differentiation potential of some subpopulations. This work supports the idea that the basal cell compartment may be functionally heterogeneous, and provides new molecular tools for interrogation of human basal cells.

Upper airways are lined with a pseudostratified mucociliary epithelium maintained by basal cells. To investigate functional and phenotypic heterogeneity within the human basal cell compartment, we used a combination of limiting dilution assays and surface marker profiling on primary cultures of basal cells with verified progenitor activity. The limiting dilution assay suggested functional heterogeneity in the ability of basal cells to repopulate a filter and maintain a barrier at ALI. The frequency of cells with this activity varied between patient strains and ranged from 0.08%-1% of basal cells. Validation of large-scale comprehensive surface marker profiling on basal cells led to identification of 74 antigens demarking consistent subpopulations. Preliminary functional analyses suggest differences in differentiation potential of some subpopulations. This work supports the idea that the basal cell compartment may be functionally heterogeneous, and provides new molecular tools for interrogation of human basal cells.

Recent studies have uncovered de novo mutations of the gene encoding the chromatin remodelling protein Snf2l in patients with schizophrenia, Rett-like syndrome and intellectual disability. Snf2l and its closely related protein, Snf2h, play a critical role in embryonic and post-natal brain development. Murine models lacking functional Snf2h or Snf2l point to complementary activities of these remodelers; Snf2h cKO mice present with a significantly reduced cerebellum, while Snf2l Ex6DEL (exon 6 deleted) cerebella are larger than their wild-type counterparts. Granule neuron progenitors (GNPs) isolated from Ex6DEL cerebella display delayed cell cycle exit and hindered terminal differentiation compared to wild-type controls. Moreover, loss of Snf2l activity results in widespread transcriptome shifts which underlie the Ex6DEL GNP differentiation phenotype. In particular, key transcription factors are differentially expressed without Snf2l remodelling activity. We confirm that ERK pathway activation is misregulated in Ex6DEL GNPs, possibly in response to elevated fibroblast growth factor 8 (Fgf8) expression in these cultures. We find that Snf2l activity maintains the chromatin landscape throughout GNP differentiation, as Ex6DEL cultures have a global increase in chromatin accessibility. We suggest that Snf2l-mediated chromatin condensation is responsible for proper regulation of gene expression programs in GNP differentiation.

Adipose tissue (AT) expands through hypertrophy and hyperplasia. Hyperplasic AT expansion requires an adequate number of adipose progenitor cells. This study investigates the influence of depot origin on the susceptibility of adipose progenitors to cell death, and measures the effect of macrophage-secreted factors on adipose progenitor survival. Using serum deprivation alone or in the presence of TNFα, omental (OM) versus subcutaneous (SC) adipose progenitors, obtained from human AT, displayed a 3- and 1.7-fold-increase in apoptosis, respectively, as assessed by Hoechst staining, (p<0.05). Similar results were observed with cell enumeration. The ratio of OM/SC cell death from serum deprivation positively correlated with body mass index (BMI). The depot-specific difference in cell death was lost when TNFα and cycloheximide (CHX) were used. Monocyte-derived macrophages (MD-macrophages), isolated from human blood, did not have an effect on apoptosis. Depot-related differences in adipose progenitor apoptosis may influence AT remodeling and alter metabolic functionality in obesity.

Cultures of peripheral blood mononuclear cells (MNCs) give rise to at least two different variants of endothelial progenitor cells (EPCs), early and late outgrowth EPCs. We investigated whether microRNAs in early and late EPCs could serve as markers of internal processes that can be exploited to distinguish cell identity and functional capacity. We hypothesized that as MNCs give rise to early and late EPCs, there is a gradual change in total microRNA profile, reflecting a total change in processes within the predominant cell population. Using a candidate microRNA array, early and late EPCs showed vastly different microRNA expression profiles. MiR-146a expression increased progressively as early EPCs emerged around 5-7 days (p<0.05). Through targeting TRAF6 and IRAK1, miR-146a conferred inflammatory tolerance in early EPCs, likely contributing to their purported ability to suppress inflammation. MiR-146a knock down (KD) in endotoxin-stimulated early EPCs reduced anti-inflammatory cytokine IL-1RA (p<0.001), and increased expression of pro-inflammatory cytokines IL-1 (p<0.001) and IL-8 (p<0.01). Interestingly, the microRNA expression profile of late EPCs was highly congruent to mature endothelial cells, with 100-fold greater miR-126 expression than monocytes and early EPCs (p<0.01). MiR-126KD in late EPCs abolished matrigel-network formation (p<0.05); while overexpression (OE) in early EPC augmented network formation (p<0.05) and chemotactic migration (p<0.001). We also found that the melanoma cell adhesion molecule or MCAM (CD146) identified late EPC precursors. Only MCAM+MNCs from adult blood (<5% of total MNCs) yielded late EPC-like colonies. Robust miR-126 expression in these cells predicted the generation of late EPCs. Overall, our results suggest that miR-146a in early EPCs likely contributes to repair by suppressing inflammation during cardiovascular injury; while in late EPCs, miR-126 directly promotes angiogenesis and vascular repair. Finally, we highlight a unique method for the efficient generation of late EPCs by using MCAM selection and screening for miR-126.