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The demonstration that simply by introducing four selected proteins it is possible to change mammalian somatic cells from one phenotype to another is providing important new opportunities in medicine. However, this approach has several limitations. In contrast to other methods of changing cell fate such as cloning and cell fusion it is very slow, very inefficient and it is necessary to have identified the key transcription factors. In both cloning and fusion the nucleus is exposed to the cytoplasm of the recipient cell and it is this that changes nuclear function. With the hope of creating the same effect, extracts from cells of the desired cell type have been introduced into candidate cells. This induced some changes in cell function, but did not change cells from one phenotype to another. The aim of this project was to improve methods for the introduction of extract when human skin cells were exposed to extracts of mouse pluripotent stem cells. During early studies, it was noticed that external materials could enter cells spontaneously at a specific stage of cell cycle, metaphase. When cells in metaphase were exposed to cell extracts pluripotent cell-like colonies were formed. These cells expressed markers of pluripotency such as SSEA4 and Tra-1-60 and could form embryoid bodies that would further differentiate to all three germ layers. Unfortunately this protocol was found to be unrepeatable. In subsequent studies the temperature of exposure to extract was raised from 37oC to 40oC with the aim of promoting the fluidity of the cell membrane and so enhancing uptake of extract. A new treatment regime was introduced to increase the proportion of cells exposed to extract while in metaphase. A new protease inhibitor was introduced in order to promote persistence of the extract within the cells. Also, a new TLR3 agonist was introduced to enhance chromosome modification. After these modifications were made pluripotent cell-like colonies formed within 14 days of treatment with extract and these colonies were positive for alkaline phosphatase live staining. Further research is required to complete the development of a routine procedure.

CD146 is a cell adhesion molecule that has been shown to regulate cell adhesion, migration and proliferation of different cell types. It is highly expressed in blood endothelial cells (BECs), but is only lowly expressed in lymphatic endothelial cells (LECs). The PROX1 homeobox gene is a master regulator of lymphangiogenesis and its expression is necessary and sufficient to drive venous endothelial cells into a LEC phenotype. The highly permeable nature of the lymphatic vessels may partially derive from PROX1 mediated repression of CD146 transcription. We hypothesize that PROX1 promotes lymphatic differentiation by repressing CD146 transcription. In gain of function studies, Human Umbilical Vein Endothelial Cells (HUVECs) were infected with adenoviruses encoding EGFP, wild type PROX1 (AdProx1) or a Homeo-Prospero domain deleted version of PROX1 (AdHDPD), which cannot bind DNA. In order to knockdown PROX1, LECs were transfected with PROX1 specific siRNA. When compared to EGFP infected HUVECs, AdProx1 infected HUVECs had decreased CD146 expression both at protein and mRNA levels. In contrast, AdHDPD infected HUVECs had increased levels of CD146 expression. In support of a role for PROX1 in repressing CD146, PROX1 siRNA transfected LECs express higher levels of CD146 as compared to mock transfected LECs or LECs transfected with control siRNA. Based on these results, we predict that CD146 expression is kept at basal levels by an unknown repressor bound to the CD146 promoter. By interacting with this unknown repressor, PROX1 further represses CD146 expression. On the other hand, the DNA binding-deficient ΔHDPD version of PROX1 binds the unknown repressor and sequesters it from the CD146 promoter, thereby relieving the repression of CD146 expression in ECs. Different levels of CD146 expression between BECs and LECs might reflect the structural and functional differences between blood and lymphatic vessels. Since CD146 plays a critical role in EC adhesion, regulation of CD146 expression in ECs might be one of the key factors regulating vessel permeability.

CD146 is a cell adhesion molecule that has been shown to regulate cell adhesion, migration and proliferation of different cell types. It is highly expressed in blood endothelial cells (BECs), but is only lowly expressed in lymphatic endothelial cells (LECs). The PROX1 homeobox gene is a master regulator of lymphangiogenesis and its expression is necessary and sufficient to drive venous endothelial cells into a LEC phenotype. The highly permeable nature of the lymphatic vessels may partially derive from PROX1 mediated repression of CD146 transcription. We hypothesize that PROX1 promotes lymphatic differentiation by repressing CD146 transcription. In gain of function studies, Human Umbilical Vein Endothelial Cells (HUVECs) were infected with adenoviruses encoding EGFP, wild type PROX1 (AdProx1) or a Homeo-Prospero domain deleted version of PROX1 (AdHDPD), which cannot bind DNA. In order to knockdown PROX1, LECs were transfected with PROX1 specific siRNA. When compared to EGFP infected HUVECs, AdProx1 infected HUVECs had decreased CD146 expression both at protein and mRNA levels. In contrast, AdHDPD infected HUVECs had increased levels of CD146 expression. In support of a role for PROX1 in repressing CD146, PROX1 siRNA transfected LECs express higher levels of CD146 as compared to mock transfected LECs or LECs transfected with control siRNA. Based on these results, we predict that CD146 expression is kept at basal levels by an unknown repressor bound to the CD146 promoter. By interacting with this unknown repressor, PROX1 further represses CD146 expression. On the other hand, the DNA binding-deficient ΔHDPD version of PROX1 binds the unknown repressor and sequesters it from the CD146 promoter, thereby relieving the repression of CD146 expression in ECs. Different levels of CD146 expression between BECs and LECs might reflect the structural and functional differences between blood and lymphatic vessels. Since CD146 plays a critical role in EC adhesion, regulation of CD146 expression in ECs might be one of the key factors regulating vessel permeability.

Drugs that alter microtubule dynamics are often used in chemotherapy regimes in combination with other agents in order to treat various cancers. Despite the success over many years, there remain problems in toxicity, resistance and predictability to the drugs. In order to overcome these problems, there is a need to gain an understanding of how these drugs kill cancer cells in cell culture. As microtubule function is particularly important for chromosome movement in mitosis, cells treated with these agents cause a mitotic arrest through activation of the spindle assembly checkpoint. Following induction of a mitotic arrest, cells can escape this arrest (mitotic slippage) or undergo mitotic death, determined in part by the response of the apoptotic network. Levels of an anti-apoptotic protein, Mcl-1, are often lost over time in mitosis. Using time-lapse analysis on a cell line unable to escape the mitotic arrest, this thesis shows that Mcl-1 protein contributes to cell death both in mitosis and the subsequent interphase in response to microtubule toxin, taxol. This suggests that inhibiting Mcl-1 may increase the efficacy of anti-mitotic agents. In addition, mitotic cell lines prone to mitotic slippage were found to have higher levels anti-apoptotic protein, Bcl-xL, in comparison to Mcl-1, indicating one way in which these cells can cope with loss of Mcl-1 during mitosis. Secondly, an evaluation of the contribution of the previously identified interaction between Mcl-1 and mitotic E3 ligase complex, the APC/C-Cdc20, to the rate of mitotic death and mitotic slippage was assessed. Inhibition of APC/C-Cdc20 activity or mutation of a Mcl-1 motif (RxxL) thought to engage with the APC/C-Cdc20 complex did not have a substantial effect on Mcl-1 degradation or mitotic death, thereby questioning the functional significance of this interaction. However, it appears that Mcl-1 protein levels can influence the rate of mitotic slippage and this influence was affected via Mcl-1’s RxxL motif within Mcl-1. This suggests that Mcl-1 protein may delay mitotic slippage via substrate competition for the APC/C-Cdc20 complex with Cyclin B1, whose degradation is required for mitotic exit. Further analysis of this effect showed that this interaction may not be a universal effect. This together with the specific functional effect on mitotic slippage rather than mitotic death, suggests that this is an indirect effect caused by network interference between the components of the death and slippage pathways.

The acquisition of renogenic competence (the ability to give rise to kidney) during embryonic development is not yet fully understood. Clarifying the temporal and molecular aspects of this process is equally essential for understanding excretory system development and for devising methods for successful differentiation of embryonic stem cells (ESCs) to renal cells for disease modeling, toxicology screening and potential cell replacement therapies. In embryo development, the metanephric (permanent) kidney arises as a result of inductive interactions between two embryonic structures that arise in the intermediate mesoderm - the ureteric bud (UB, a diverticulum of the Wolffian duct) and the metanephric mesenchyme (MM). The UB develops into the collecting duct system and the MM undergoes an epithelial-to-mesenchymal transition to form the secretory units of the kidney - the nephrons. In this thesis, I used a tissue disaggregation-reaggregation method that allows the reconstruction of mouse organotypic kidney rudiments to place different embryonic cells in the environment of a developing kidney and assess their potential to integrate into kidney epithelia and differentiate to renal cells. First, the suitability of this method was evaluated and a quantitative assay for evaluating the numbers of test cells integrating in various renal compartments was developed. Second, the reaggregation method was used to characterise the renogenic potential of undifferentiated mouse ESCs, ESC-derived cells after Notch inhibition, and cells derived from the presumptive nephrogenic regions of embryos at various stages of development. ESCs are isolated from the inner cell mass of an embryo and have the potential to differentiate to any tissue of the body when injected into mouse blastocysts. Strategies have successfully been devised for ESC differentiation to many lineages, but very few studies reported any success with the differentiation of ESCs to a renal lineage. Undifferentiated ESCs showed a very good ability to form chimeric structures with developing kidney tubules (both nephrons and extending UBs). Nevertheless, the resulting structures were morphologically different from renal epithelia in most cases and integrated ESC-derived cells were not positive for several combinations of kidney markers. These results suggested that the influence of the niche was not sufficient for a successful ESC differentiation to renal cells. Treatment of ESC with an inhibitor of the Notch pathway to increase the proportion of mesodermal cells did not improve this outcome. On the basis of these results, it was speculated that the earliest lineage to which embryonic stem cells must be differentiated in order to become competent to make renal cells should first be identified. I addressed this by determining the developmental stage at which cells able to contribute to the formation of metanephric epithelia first appear in mouse embryo development. When mixed in embryonic kidney reaggregates labelled cells isolated from the nephrogenic regions of E9.5 embryos integrated into various renal compartments. These cells were seen in UBs, nephrons, glomeruli and the condensing mesenchyme. Marker expression studies showed that the exogenous E9.5 cells expressed an array of kidney markers - Pax2 in renal epithelia and the condensing mesenchyme, Wt1 in glomeruli and Six2 in the condensing mesenchyme. Furthermore, exogenous E9.5 cells also co-expressed Pax2/Wt1 in the condensing mesenchyme, Megalin/Ecadherin in the proximal tubule and Pax2/E-cadherin in renal epithelia. This provides evidence that challenges the existing model and suggests that some cells from the intermediate mesoderm at a stage where the metanephric blastema is yet formed are competent to contribute to kidney structures. Furthermore, experiments with E8.5 embryos showed that such a renocompetence could be acquired even before the specification of intermediate mesoderm. These findings contribute to our knowledge about kidney cell specification and provide valuable information to guide future attempts to develop an efficient method for deriving renal cells from ESCs. Furthermore, the reported ability of ESC-derived non-kidney cells to form chimeric structures with renal tubules provides a proof-of-principle that it might be possible to use exogenous types of cells for physiological support to injured kidney tubules, thus offering a possible novel approach for cell replacement therapies.

At embryonic day 3.5 (E3.5), inner cell mass (ICM) cells co-express the transcription factors NANOG and GATA6. Between E3.5 and E4.5, cells of the ICM differentiate into epiblast (Epi) and primitive endoderm (PrE). These two lineages are distinguished by the differential expression of the previously coexpressed transcription factors; Epi cells express NANOG while PrE cells express GATA6. FGF/ERK signalling is responsible for Epi and PrE differentiation but it does not explain the initial co-expression of both factors and how the mutually exclusive expression arises. β-catenin is the downstream effector of Wnt signalling, and it is also found in the membrane forming a complex with E-cadherin. Depending on it subcellular location, β-catenin has been associated with pluripotency and differentiation of mESCs, whose origin is the mouse embryo. My hypothesis was that changes in both cellular pools of β-catenin are involved in ICM differentiation. To characterize Wnt/β-catenin role during preimplantation development, I applied quantitative immunofluorescence analysis (QIF) together with chemical and classical genetics in in vitro and in vivo models. I found that high membrane β-catenin levels are associated with Epi cells from E4.0 stage, while nuclear β-catenin levels are higher in co-expressing cells at E3.5 and PrE precursors at E4.0. My results indicate that increases in nuclear β-catenin levels allow the ICM cells to be specified earlier, determined by an earlier appearance of mutually exclusive expression of GATA6 and NANOG in vitro and ex vivo. Moreover, increased β-catenin levels promote specification towards PrE fate, observed by the presence of higher percentages of PrE cells. Conversely, a decrease in β-catenin levels result in slower ICM specification into Epi and PrE. Finally, modulation of FGF/ERK signalling in mouse embryos, which is the main pathway in this cell fate choice, led to changes in β-catenin subcellular location and levels. Altogether, my results are consistent with a role for Wnt/β-catenin signalling facilitating PrE fate acquisition concomitantly with FGF/ERK signalling.

The role that each of the Notch receptors play in controlling alveolar development and cell fate determination in the mouse mammary gland has remained unclear. By utilizing a cre-conditional constitutively active intracellular Notch1 knock-in I define, in vivo, that ectopic Notch1 activation is sufficient to inhibit ductal outgrowth, cause the formation of alveolar-like cell accumulations, and promote Elf5+/ER- cell fate, at the expense of ER+ cell fate, in the mammary gland of pubescent mice. Furthermore, ectopic Notch1 in the pregnant mammary gland is sufficient to promote the formation of pregnancy/lactation-dependent lactating adenomas. These lactating adenomas consist of differentiated secretory cells and normally regress during involution but progress into non-regressing tumours after multiple pregnancies. These lactating adenomas exhibit decapitation secretions characteristic of apocrine differentiation. Together these results suggest that Notch1 may function to promote Elf5+/ER- cell fate and may be misregulated in pregnancy-associated masses and apocrine-carcinoma of the breast in humans.

The role that each of the Notch receptors play in controlling alveolar development and cell fate determination in the mouse mammary gland has remained unclear. By utilizing a cre-conditional constitutively active intracellular Notch1 knock-in I define, in vivo, that ectopic Notch1 activation is sufficient to inhibit ductal outgrowth, cause the formation of alveolar-like cell accumulations, and promote Elf5+/ER- cell fate, at the expense of ER+ cell fate, in the mammary gland of pubescent mice. Furthermore, ectopic Notch1 in the pregnant mammary gland is sufficient to promote the formation of pregnancy/lactation-dependent lactating adenomas. These lactating adenomas consist of differentiated secretory cells and normally regress during involution but progress into non-regressing tumours after multiple pregnancies. These lactating adenomas exhibit decapitation secretions characteristic of apocrine differentiation. Together these results suggest that Notch1 may function to promote Elf5+/ER- cell fate and may be misregulated in pregnancy-associated masses and apocrine-carcinoma of the breast in humans.

The tumor suppressor p53 has a close relation with reactive oxygen species (ROS). As an indispensable component of the cellular redox system, ROS not only have been established to be involved in p53-dependent apoptosis, but also regulate p53 activity. Recent studies revealed several novel actions of p53, such as transactivation of antioxidative proteins, mitochondria translocation and inhibition of glycolysis. The fate of cells where p53 signaling pathways are initiated is either survival or death. In this review, we examine the hypothesis that ROS regulate cell fate through p53, in a way that physiological ROS levels trigger the protective pathways, while p53 behaves more like a cell killer under cytotoxic oxidative stress.