Intestinal homeostasis is governed by fate choices of stem cells residing in the intestinal crypt base. This must involve niche-specific co-ordination of cell division to guarantee that epithelial cells divide at the right time and place. These mechanisms operate to ensure precise control of the numbers of stem and differentiated cells. Little is known about how proliferative fate decisions are regulated in intestinal crypts. Both the placement of daughter cells within a particular niche, and their decision to enter and progress through the cell cycle, contribute. This thesis investigates the spatiotemporal control of cell division in intestinal crypts to understand the relationship between cell-cycle specific fate choices and intestinal homeostasis. Firstly, I describe a novel mode of asymmetric cell division within intestinal crypts. Using high resolution microscopy of intestinal organoids, I show that a subset of mitoses produce daughters that become displaced from one another after cytokinesis. This post-mitotic separation or the ‘positional asymmetry’ of daughter cells occurs in all cycling epithelial cells. These divisions may facilitate divergent fate of daughter cells and provides a general mechanism for stochastic niche exit. Post-mitotic separation is facilitated by interkinetic nuclear migration and selective tethering to the basement membrane during mitosis. Importantly, these mechanisms are altered in tissue carrying mutations in Adenomatous polyposis coli (Apc), highlighting its importance for normal tissue homeostasis. Secondly, I aimed to understand the dynamics of cell-cycle commitment in intestinal crypt compartments by investigating the DNA Replication Licensing System. The licensing system is a master regulator of proliferative fate in all cells in adult tissue. At its core is the regulated loading of the Mcm2-7 protein complex onto origins of replication exactly once per cell cycle. Engagement of the licensing system directly indicates commitment to proliferative cell fate. A technique to visualise licensing in intestinal crypts was developed. This revealed distinct proliferation zones in intestinal crypts. Mcm licensing was most prevalent in the lower transit-amplifying compartment, the zone enriched for early TA progenitors. Licensing is inhibited in terminally differentiated cells, and not detected in the transit-amplifying cells most proximal to the differentiated zone. Strikingly, the majority of ‘active’ intestinal stem cells were found in an unlicensed state. These data suggest that licensing decisions are delayed or inhibited until late G1 phase in intestinal stem cells and explains their longer cell-cycle. We postulate that this may provide a time window for niche cues to act, either stimulating cell-cycle entry or allowing retention in a ‘shallow’ G0 state. High resolution imaging of cell-cycle phases throughout the epithelium revealed remarkable cell-cycle co-ordination. This manifested in uninterrupted ‘ribbons’ of cells in similar cell-cycle states. This was due to lineage specific cell cycle co-ordination where adjacent daughter cells progress through the cell cycle at the same rate. These field effects are the result of co-ordinated cell-cycle progression between daughter cells. These observations were validated using living organoids expressing fluorescent ubiquitination-based cell cycle indicators (FUCCI). These ribbons were occasionally interrupted by cells in other cell cycle phases suggesting the separation of sisters by daughters from another lineage. This suggests that cell-cycle coordination can facilitate post-mitotic separation, and influence stochastic niche exit.

Breast and pancreatic cancers are among the major causes of cancer mortality in our society. There has been a significant decline in mortality from breast cancer over the last two decades, while pancreatic cancer has an exceptionally poor prognosis. Although these malignancies have very different clinical outcomes they share the common feature that metastatic disease is almost uniformly fatal. The existence of cancer stem cells has been postulated as a major factor in tumour recurrence after traditional chemo- or radio-therapy. Addressing this important clinical question requires a deeper understanding of the biology of normal and cancer stem cells and the signalling pathways involved in their regulation. The identity of the pancreatic stem cell remains elusive. However, using a murine model of haematopoietic stem cell (HSC) transplantation I have demonstrated for the first time transdifferentiation of these bone marrow derived cells into mature pancreatic acinar cells, where they appear to contribute to cell turnover ultimately forming acini and lobules. These data show that HSC have surprising developmental plasticity and provide insight into a potential stem cell niche in the pancreas. The Hedgehog, Wnt and Notch signalling pathways play a critical role in early development and in the maintenance and self-renewal of stem cells. There is also increasing evidence that dysregulation of these pathways contributes to the development of many malignancies. There is relatively little information regarding their role in breast cancer development and progression. I used immunohistochemistry for key proteins in these pathways, sonic hedgehog, beta-catenin and Notch 1 in three substantial series of human breast lesions and determined that abnormal expression of these proteins is an early event in the development in breast cancer, and is associated with particular breast cancer subtypes, Shh and beta-catenin expression is associated predominantly with the basal-like phenotype and Notch 1 with the HER2 amplified phenotype. Overexpression of Shh in particular confers a worse clinical outcome in invasive ductal carcinoma. Furthermore, increased levels of Shh in a 3D culture model of non-transformed mammary epithelial cells resulted in disorganisation of acini and the development of an abnormal discohesive phenotype. Finally the role of Shh was investigated in a mammary epithelial transplantation model, where overexpression of Shh resulted in the development of hyperplasia of the mammary ductal epithelium. Together these data confirm that the Hedgehog, Wnt and Notch developmental pathways are dysregulated in breast cancer and represent viable targets for further investigation of potential novel therapies in breast cancer.

During mammalian development a small number of pluripotent cells proliferate and differentiate to give rise to all the mature cell types of the organism. Among the earliest differentiation events is the process of gastrulation, in which pluripotent primitive ectoderm cells form the three germ lineages, mesoderm, ectoderm and endoderm under the control of complex signalling and environmental cues. This process can be modelled using embryonic stem cells, which have proven to respond to embryologically relevant signals during in vitro differentiation and promise to uncover additional insights into the process of germ lineage specification. This thesis describes the differentiation of mouse ES cells to committed cell types via a second intermediate population of pluripotent cells termed Early Primitive Ectoderm-Like (EPL) cells. The similarity of EPL cells to primitive ectoderm and the rapid acquisition of lineage specific markers and loss of pluripotent characteristics upon differentiation of EPL cells suggest they are an excellent model for the cells in the embryo that undergo germ lineage commitment. EPL cells can be differentiated as EPLEBs, which are highly enriched in mesodermal cell types and contain essentially no ectodermal derivatives and no visceral endoderm. Here it is shown that EPLEBs can be generated from EPL cells grown either adherently or in suspension culture provided the cells are reduced to a single cell suspension before reaggregation as EPLEBs. Since EPLEBs are a rich source of mesoderm and contain less non-mesodermal cell types than traditional ESEBs, they were assayed for definitive blood formation, however none was detected. Alternately, EPL cells can be differentiated in the presence of MEDII in aggregates termed EBMs, which are restricted to ectodermal cell fates. Here it is demonstrated that the switch from mesodermal to ectodermal differentiation observed in ELPEBs and EBMs relies on two variables; a mesoderm suppressing activity within MEDII and the pro-mesodermal activity of cell dissociation as undertaken during EPLEB formation. Evidence has been presented that interventions that modulate the epithelial identity of EPL cells are capable of influencing subsequent differentiation such that protection of the epithelial cell state favours ectoderm while disruption favours mesoderm. Staurosporine (SSP) is a kinase inhibitor that has been shown to induce an epithelial to mesenchymal transition in chick neural tube. Here it was added to EPL cells with the result that mesodermal differentiation was enhanced at the expense of ectoderm. DAPT is a potent inhibitor of ƴ-secretase, which cleaves a number of protein targets including the adherens junction component E-cadherin. Addition of DAPT to differentiating EPL cells has the opposite effect to SSP, with an increase in ectodermal differentiation at the expense of medoderm. It is proposed that DAPT is acting by preventing E-cadherin cleavage and thus stabilising the epithelial state. Modulation of epithelial contacts between pluripotent cells represents a novel way to control lineage induction and as such the incorporation of these findings into methodologies for directed differentiation in defined culture conditions is likely to provide improved outcomes in the production of desired cell types. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Human mesenchymal stem cells (MSCs) are being largely studied for their differentiation potential and immunological properties. In the present study, we evaluated the ability to reliably differentiate mesenchymal lineages into hepatocyte-like cells both in vitro and in vivo. For this purpose, we handled several tissue sources and compared typical MSCs from bone marrow (BM) or umbilical cord, to liver-derived mesenchymal-like cells and to fibroblasts. We observed that hepatocyte differentiation of BM-MSCs was incomplete and variable with elective expression of some specific markers. These mesenchymal-derived hepatocyte-like cells (MDHLCs) were also chimerical in their phenotype as they expressed mesenchymal markers while these were down-regulated. We therefore designed differentiation cocktails with an aim to improve MDHLC phenotype and some unexpected results were obtained with LIF cytokine whose action on stem cells for hepatocyte differentiation was not documented. Nevertheless, we observed a limitation in the acquisition yield of hepatic features. Furthermore, the hepatocytelike phenotype of MDHLCs completely disappeared when the cells were incubated into growth medium. However, we showed that hepatic functionality of these cells, as urea secretion and gluconeogenesis, could be increased under specific conditions, suggesting the potential to improve MDHLC phenotype. In vivo, MSCs were able to express hepatic markers into SCID-mice livers while their chimerical phenotype remained. In contrast, MDHLCs down-regulated their hybrid phenotype after transplantation suggesting a beneficial influence of in vitro differentiation step. MSCs were also able to engraft and even partially differentiate into wild-type mice which was a strong argument for their low immunogenicity. Surprisingly, fibroblasts showed highly similar potential than MSCs to differentiate into hepatocyte-like cells both in vitro an in vivo and these results underlined the difficulty to accurately distinguish between both cell types using current techniques. Umbilical cord-derived stem cells (UCMSCs) and adult-derived human liver stem cells (ADHLSCs) were different in nature and displayed a native hybrid phenotype while their differentiation allowed high levels of hepatocyte-like feature acquisition. Together all these data suggest the current possibility to engineer mesenchymal-derived hepatocyte-like cells owning specific features acquisition while remaining limited in their commitment. This highlights the need for further investigations to evidence the usefulness of these mesenchymal lineages for liver cell therapy.

The term “stem cell” is often broadly applied to a range of cell types that are relatively undifferentiated and have some capacity for proliferation. In this thesis, I employ a strict definition of stem cells as cells that are capable of both self-renewal and multilineage differentiation. These properties are tested in single precursor cells from the forebrain and its derivative, the retina, using clonal assays. Poor survival is a common problem in single cell cultures, and I show that low oxygen dramatically improves viability in neural stem cells clonally derived from mouse embryonic stem cells, as well as in cultured forebrain neural stem cells. Caspase-dependent and apoptosis-inducing factor-dependent cell death pathways were found to be differentially influenced in low oxygen culture of early, primitive and later, definitive neural stem cells. I isolate precursors from 2 separate regions of the adult mouse forebrain, the lateral ventricle and the hippocampus and argue that only cells resident in the lateral ventricle can be classified as stem cells while the hippocampus contains restricted progenitor cells. Unlike neural stem cells, the very existence of retinal precursors in the adult mammal is controversial. I investigate methods to prospectively identify a rare stem cell population in the pigmented ciliary epithelium of the adult mouse eye and show that, although this population intrinsically gives rise to all retinal cell types, cells can be directed specifically towards a photoreceptor fate by the addition of exogenous factors to the culture media. Pigmentation of retinal stem cells is used as a convenient marker to isolate a retinal stem cell from human embryonic stem cells differentiating under conditions known to promote neural differentiation. Retinal stem cells derived from human embryonic stem cells have highly similar properties to those directly isolated from the eye, and their progeny can similarly be driven to differentiate into photoreceptors. The findings presented in this thesis help to define intrinsic properties of adult neural and retinal precursors and provide a basis for manipulating these cells, potentially for future use in clinical applications.

The term “stem cell” is often broadly applied to a range of cell types that are relatively undifferentiated and have some capacity for proliferation. In this thesis, I employ a strict definition of stem cells as cells that are capable of both self-renewal and multilineage differentiation. These properties are tested in single precursor cells from the forebrain and its derivative, the retina, using clonal assays. Poor survival is a common problem in single cell cultures, and I show that low oxygen dramatically improves viability in neural stem cells clonally derived from mouse embryonic stem cells, as well as in cultured forebrain neural stem cells. Caspase-dependent and apoptosis-inducing factor-dependent cell death pathways were found to be differentially influenced in low oxygen culture of early, primitive and later, definitive neural stem cells. I isolate precursors from 2 separate regions of the adult mouse forebrain, the lateral ventricle and the hippocampus and argue that only cells resident in the lateral ventricle can be classified as stem cells while the hippocampus contains restricted progenitor cells. Unlike neural stem cells, the very existence of retinal precursors in the adult mammal is controversial. I investigate methods to prospectively identify a rare stem cell population in the pigmented ciliary epithelium of the adult mouse eye and show that, although this population intrinsically gives rise to all retinal cell types, cells can be directed specifically towards a photoreceptor fate by the addition of exogenous factors to the culture media. Pigmentation of retinal stem cells is used as a convenient marker to isolate a retinal stem cell from human embryonic stem cells differentiating under conditions known to promote neural differentiation. Retinal stem cells derived from human embryonic stem cells have highly similar properties to those directly isolated from the eye, and their progeny can similarly be driven to differentiate into photoreceptors. The findings presented in this thesis help to define intrinsic properties of adult neural and retinal precursors and provide a basis for manipulating these cells, potentially for future use in clinical applications.

This dissertation presents three separate studies designed to provide structure and evidence-based insight into the impact of religious beliefs on public perceptions and U.S. government policies regarding embryonic stem cell research. First, a systematic literature review of nine (n=9) empirical studies that examined individuals' religious beliefs and perceptions/utilization of genetic technologies/services will be presented. Based on the finding from the review, there was an equal balance between studies that found that religion was a factor positively affecting intention to submit to genetic testing and those that illustrated a negative association. Secondly, a qualitative examination of college students' from various racial/ethnic and religious backgrounds exploring the definition, interpretation, and conceptualization of the influence of religious beliefs on perceptions regarding embryonic stem cell research will be offered. Employing an emergent design, the data collection process encompassed thirty-seven in-depth interviews. The majority of participants in this study believed that ESCR should be conducted and federally funding in the United States, regardless of their religious beliefs. Lastly, the findings from the analysis of congressional records from the U.S. Congress for areas of convergence and divergence between discussions, voting, and legislation regarding stem cell research with the official stances of the major religious groups in the United States accessing the influence of religious rhetoric on political discourse regarding embryonic stem cell research will be discussed. Findings from this study suggest that religious rhetoric has a substantial influence on political rhetoric regarding ESCR.

In Form einer Zusammenfassung aus zwei Publikationen und bisher unveröffentlichtem Material stellt die vorliegende Arbeit die Frage, welche der Zellfraktionen (HUCB-MNC, CD45+/CD34+ und CD45+/CD133+) innerhalb des heterogenen HUCB die am effektivsten wirksame in Hinblick auf die Neuroprotektion nach einem experimentellen Schlaganfall ist und welche Mechanismen dabei zum Tragen kommen. Für die Untersuchung der molekularen Mechanismen des Zusammenwirkens zwischen den genannten HUCB Fraktionen und neuronalen Zellen bzw. Gewebe, kamen ein selbst etabliertes Zellkulturmodel neuronaler Hypoxie bzw. OGD geschädigte hippocampale Schnittkulturen zur Anwendung. Die zu untersuchenden HUCB Zellfraktionen wurden direkt oder indirekt appliziert und ihr Effekt wurde über drei aufeinanderfolgende Tage hinweg untersucht. Das molekulare Mikromilieu, welches durch die hypoxisch geschädigten neuronalen Zellen produziert wurde, stimulierte alle untersuchten HUCB Fraktionen zur Sekretion neurotrophischer Faktoren und/oder immunologisch aktiver Mediatoren, die die neuronale Apoptose günstig beeinflussten. HUCB-MNC zeigten, sowohl in der direkten als auch in der indirekten Kokultur mit geschädigten neuronalen Zellen eine sehr überzeugende Fähigkeit zur Neuroproduktion. In den direkten Kokulturen reduzierten sie die Apoptose der neuronalen Zellen sogar bis auf das Niveau der Kontrollen. Dies kann auf den deutlichen Anstieg der von den HUCB-MNC produzierten Chemokine CCL5; CCL3 und CXCL10 zurückgeführt werden. Weiterhin war zu beobachten, dass HUCB-MNC aktiv zu geschädigten neuronalen Zellen migrierten und sich, vorzugsweise unter Ausbildung von direkten Zell-Zellkontakten, an Axonen und Somata anlagerten. Überraschenderweise zeigte die CD45+/CD133- Zellfraktion ein ähnliches Potential wie HUCB-MNC. Für diese nahezu stammzellfreie Fraktion konnten in den indirekten Kokulturen hohe Konzentrationen an CCL3 und neuroprotektiven G-CSF nachgewiesen werden, wobei letzteres für die Aufrechterhaltung des neuronalen Phänotyps verantwortlich gemacht werden kann. CD45+/CD133+ Stammzellen, die aus der HUCB-MNC Fraktion isoliert wurden, konnten die neuronale Apoptose in direkten Kokulturen signifikant reduzieren. Die Konzentration an löslichen Faktoren, die von den Stammzellen produziert wurde, lag dabei unterhalb der Nachweisbarkeitsgrenze. Die Ergebnisse aus den Untersuchungen der hippocampalen Schnittkulturen zeigen, dass HUCB-MNC direkt neuroprotektiv wirken. Dies gilt insbesondere, wenn sie direkt und in ausreichender Konzentration (12.5x104 Zellen pro Schnitt) appliziert werden. In Kokulturen mit der CD45+/CD34+ Stammzellfraktion fand sich eine verringerte Sekretion an Nervenwachstumsfaktor und damit verbunden eine geringere Anzahl degenerierter Pyramidenzellen. In Kokulturen in welchen die stammzellfreie CD45+/CD34- Fraktion verwendet wurde, trat dieser Effekt nicht auf. Die Resultate, die in den beiden hier verwendeten in vitro Modellen gefunden wurden, legen nahe, dass der Einsatz von HUCB-MNC eine stabile Neuroprotektion hervorruft. Im Vergleich der verwendeten Modelle lieferten die Applikationen von verschiedenen Stammzellfraktion keine einheitlichen Ergebnisse. Damit wird eine starke Systemabhängigkeit induziert. Speziell im Hinblick auf den klinischen Einsatz scheint es keinen deutlich überlegenen Vorteil durch die Verwendung reiner, aus der HUCB-MNC Fraktion gewonnener Stammzellfraktionen zu geben, der den Aufwand rechtfertigt eine zahlenmäßig so geringe Zellfraktion aus der HUCB-MNC Fraktion zu separieren.

The use of both multipotent progenitors and fully differentiated cells has been demonstrated to be effective for cell-based immunotherapy. The goal of this thesis was to establish an in vitro hematopoietic differentiation system to generate hematopoietic progenitor cells (HPCs) and functional T cells from pluripotent stem cells. Generation of progenitor T cells by co-culturing stem cells on Notch ligand-expressing OP9 stromal cells (OP9-DL1) had been successfully employed previously. However, further differentiation of these cells in vitro into mature, antigen-specific, functional T cells, without retroviral transduction of T cell receptors (TcRs), had not been achieved. In the thymic niche, differentiation of T cells to a state of antigen specificity is controlled by the interaction of their developing TcRs with the Major Histocompatibility Complex (MHC) on thymic stromal cells. We hypothesized that, by providing exogenous antigen-specific MHC/TcR signals, stem and progenitor cells could be engineered into functional effector T cells specific for the same antigen. In Chapter 3 and 4, we demonstrate that both thymus-derived double positive (DP: CD4+CD8+) immature T cells and mouse Embryonic Stem (ES) cells can be efficiently differentiated into antigen-specific CD8+ T cells using either MHC tetramers or peptide-loaded stromal cells. DP cells, following MHC/TcR signaling, retained elevated RAG1 levels, suggesting continuing TcR gene rearrangement. Both DP and ES cell-derived CD8+ T cells showed significant Cytotoxic T Lymphocyte (CTL) activity against antigen-loaded target cells, indicating that these cells are functional. This directed differentiation strategy could provide an efficient method for generating functional, antigen-specific CTLs from stem cells for potential use in adoptive T cell therapies. The use of ES cells in the clinic has been hindered by the unavailability of patient-specific ES cells and the ethical issues surrounding the use of human embryos. Induced pluripotent stem (iPS) cells offer great hope to regenerative medicine as their use can circumvent both the patient-specific and ethical issues associated with ES cells. In Chapter 5, we have developed a feeder cell-free suspension culture system supplemented with OP9-DL1 secretary factors to efficiently generated HPCs from iPS and ES cells. The differentiation potential of these HPCs was demonstrated by generation of DCs in the presence of GM-CSF and IL-3. The DCs express the activation molecules, CD86 and CD80 in response to LPS stimulation and are able to stimulate T cell proliferation in a mixed lymphocyte reaction. We employed extensive quantitative RT-PCR analysis to identify a number of differentially expressed genes in HPCs generated from the feeder-free culture. / text

Loss of polarity is a defining characteristic of epithelial cancers. The cytoskeletal proteins, HUGL1 and HUGL2, mediate polarity in epithelial cells through diversified roles in defining membrane identity and trafficking to the basolateral membrane. Importantly, an ortholog of these molecules can inhibit tumor growth in Drosophila, although the mechanisms of their tumor suppressive functions in mammary epithelial cells are unknown. Here, we show nonredundant tumor protective roles for HUGL1 and HUGL2 in human mammary epithelial cells. Using a three dimensional culture system, we report that loss of HUGL1 or HUGL2 causes loss of apicobasal polarity, aberrant growth of multilayered epithelium, nuclear enlargement, loss of membrane identity, and cellular overgrowth. Experiments on plastic also revealed that HUGL1 or HUGL2 loss results in induction of a phenotypic EMT in breast epithelial cells and overexpression of HUGL1 in breast cancer cells reduces proliferation.In a Drosophila model of cancer driven by loss of lgl, we have discovered the consistent dysregulation of a number of miRNAs and mRNAs including the loss of let-7 and miR-9a, which are implicated in breast cancer and associated with the suppression of stem cells. Cross comparisons revealed a set of mRNAs that are both dysregulated in vivo and represent putative targets of the miRNAs changed in lgl mutants. Among these, Thrombospondin, a component of the extracellular matrix was found to be misexpressed in both flies and human cells lacking Lgl. Moreover, genetic interaction experiments showed miR-9a to be a functional effector of lgl in controlling proliferation in the wing. Taken together, the findings reported in this dissertation suggest that HUGL1 and HUGL2 function as tumor suppressors through their roles in polarity and miRNA regulation. These two proteins, functioning as modulators of cell plasticity and promoters of differentiation, are potentially able to control the transition between a differentiated epithelial cell and a cancer stem cell. This research offers new insight into the role of HUGL1 and HUGL2 in breast cancer and reveals novel targets downstream of polarity proteins for therapeutic intervention.