Stem cells play a vital role in the turn over of permanently self-renewing tissues (e.g. corneal epithelium, epidermis, bone marrow). Stratified cornea epithelia serve, as ideal tissue models for studying ???stemness???, as the site of cells in the different stages of differentiation are anatomically easily identifiable. Studies targeting limbal stem cell maintenance, in-vitro gene expression during storage and differentiation will benefit future therapeutic applications (e.g. corneal transplantation). Knowledge of stem cell behaviour will help explain the pathophysiology of corneal related ocular surface disorders (e.g. idiopathic limbal stem cell deficiency, pterygium and limbal dermoids), establish new treatment modalities (e.g. allogenic cellular transplants) and help promote invivo expansion of residual stem cell populations in deficiency states. The major obstacle in the progress of research on limbal stem cells is the lack of knowledge of phenotypic markers of limbal epithelial stem cells. This project first studied the limbal expression of phenotypic markers that were discovered in other human adult and embryonic stem cell populations using microarray differential expression studies. Gene expression profiles of the relatively primitive human foetal limbus were compared with that of the central cornea. Microarray was also performed to identify the differential gene expression profile of cultured primary human limbal epithelial cells, when compared to the limbal epithelial cell population isolated after 5 serial cultures. 33 genes were upregulated in the human foetal limbus and primary cultured human limbal epithelium, when compared respectively to the central foetal cornea (first experiment) and the limbal epithelial cell population after the 5th trypsin passaged culture (second experiment). Four foetal limbal and primary limbal epithelial upregulated genes (CK15, CK14, Cdh3, and Wnt4) were confirmed to be upregulated by semi quantitative RT-PCR and immunohistochemical experiments on the human foetal cornea, adult human cadaveric cornea and cultured human limbal epithelial cells. The microarray defined phenotypic profile of both the foetal limbus and cultured adult limbal epithelial cells will help identify these cells in in-vitro and in-vivo states. The expression of these 4 selected markers in the limbal dermoid and pterygium suggests that limbal epithelial cells containing a stem cell population are involved in the pathogenesis of these two disorders.

A five stage selection protocol originally applied to mouse embryonic stem cells (mESCs) was examined for the derivation of insulin producing cells from human embryonic stem cells (hESCs). Insulin gene expression was observed and insulin protein was measured by radioimmunoassay. However, the radioimmunoassay results were shown to be susceptible to false positive findings due to the presence of exogenous insulin within differentiation media and it was concluded that this particular strategy was not ideal for the derivation of insulin producing cells from hESCs. An investigation was then undertaken regarding the in vivo differentiation of cells derived from hESCs seeded within 3D scaffolds to determine if this would result in the derivation of insulin producing cells. Within scaffolds there were abundant cells which stained positively for ectoderm lineage markers including nestin. Cells which stained positively for markers of endothelial progenitors representing the mesoderm lineage were also observed and rare cells stained for endoderm markers including insulin. These investigations also demonstrated that transplanting scaffolds seeded with cells derived from hESCs between the liver lobules of immunodeficient mice could lead to the formation of teratomas. Factors that may have influence the formation of teratomas were further investigated and it was demonstrated that teratoma formation was inhibited by altering in vitro treatment of cells. An in vitro investigation was then performed to determine the extracellular matrix (ECM) producing capacity of hESCs and differentiated cells derived from hESCs because ECM proteins are required for the formation of 3D structures similar to pancreatic islets. The results from this investigation indicated that differentiated cells produced multiple ECM proteins at substantially higher levels than hESCs. The ECM producing differentiated cells could be useful in the development of surrogate islet like tissue by supplying a suitable ECM structure within a 3D scaffold environment to aid the function of ??-cell surrogates. Furthermore, these differentiated cells derived from hESCs were shown to produce an adhesive basement membrane in vitro, which is derived from human sources, and could be utilized in the derivation, propagation and differentiation of hESCs.

Includes bibliographies. Aims to identify homeobox genes which may have a developmental role during early embryogenesis by the characterization of homeobox gene expression in undifferentiated ES cells, and in a range of differentiated ES cell derivatives.

Pregnancy succeeds because the fetal allograft survives in the presence of a fully functional maternal immune system. The placenta, especially its trophoblast, provides the initial barrier between the maternal and fetal environment and, due to their location, trophoblast cells could be expected to be immune-privileged. Yet in the ectopic sites tested thus far, trophoblast stem cell transplants have failed to show noticeable immune privilege and appear to lack physiological support. However in this study, portal vein injected green fluorescent protein-labeled trophoblast stem cells were able to survive for several months in the livers of allogeneic female (14/14), but not male (0/4), mice. Gonadectomy experiments revealed that this gender-dependent survival does not require the presence of ovarian hormones (4/4) but the absence of testicular factors (5/5). In contrast, similarly labeled allogeneic embryonic stem cells were reliably rejected (11/11); these same embryonic stem cells survived when mixed with unlabeled trophoblast stem cells (13/13). The protective effect offered by the trophoblast stem cells did not require any immunological similarity with the co-injected embryonic stem cells. Neither the trophoblast stem cells nor the co-injected embryonic stem cells gave rise to tumors during the study period. Thus, this study demonstrates that, provided a suitable location and hormonal context, ectopic trophoblast stem cells may exhibit and confer immune privilege. These findings suggest applications in cell and gene therapy as well as provide a new model for studying trophoblast physiology and immunology.

The selective degeneration of motoneurons in the spinal cord, the brainstem and the brain cortex is the core pathology of amyotrophic lateral sclerosis (ALS), but evidences suggest that the neighbouring non-neuronal cells are also involved in the disease progression. Beside Riluzole, only drug approved to treat this fatal neurodegenerative disease, new pharmaceutical agents or novel strategies including stem cell therapy are currently under development and evaluated preclinically in front line on mutant SOD1 rodents mimicking all hallmarks of the human disease. Current intravenously delivered drugs tested in ALS therapy assume an intact blood-brain barrier and suppose the passage across the endothelium to hit their targets in the CNS parenchyma. If BBB impairment occurs in ALS, it may lead to revision of planned pharmaceutical treatment. In the first part of the work, we have validated the mutant SOD1 rat model of ALS and we characterized properties and integrity of its BBB. We observed a significant BBB disruption at symptomatic phase of ALS, evidenced by blood protein leakage, IgG accumulation and microhemorrhage. To look for the mechanism of BBB opening, we demonstrated that the expression of key genes involved in the BBB integrity was decreased. At the ultrastructure, the morphology of endothelial cells and vascular astrocyte end-feet was altered. Our results suggest that BBB disruption is a late event in ALS disease course and appears like a consequence of the local degenerative process or neuroinflammation rather than a cause. Since a lot of extracellular oedema and swollen astrocyte end-feet were found in mutant SOD1 rats, we also looked at the expression and localization of aquaporin-4, a key protein involved in CNS water movement. We found that its expression was highly increased in the symptomatic phase of ALS course and we hypothesize that this overexpression might be related to the resolution of oedema after BBB opening. In the second part of the work, we considered an original, easy, non-invasive and safe therapeutical approach of stem cell delivery in ALS rats. Since ALS affects the motoneurons throughout the CNS, we decided to use the bloodstream to deliver neural stem cells. We studied cell homing, survival, proliferation, integration and differentiation. Interestingly, the highest efficiency of cell delivery to the CNS was found in symptomatic ALS and the lowest in healthy animals. Neural stem cells injected into ALS animals preferentially colonized the motor cortex, hippocampus and spinal cord. We detected their successful differentiation into neural lineages by the appearance of MAP2-, GFAP-positive cells and the decrease of nestin expression. One of the realistic near-term clinical goals for ALS is the transplantation of stem cells that counteract the loss of motoneurons by secreting neuroprotective factors. Accordingly, we evaluated in vitro the expression of neurotrophic factors released by stem cells after stimulation with tissue extracts from ALS rats. The aim of this paradigm was to determine whether the ALS environment triggers neuroprotective factors release from stem cells. Mesenchymal stem cells and neural stem cells were able to express a wider range of growth factors than fibroblasts. According to the stem cell population stimulated, we obtained differential expression pattern, raising the choice of cell population for appropriate clinical applications in ALS.

The purpose of this mixed-methods study was to evaluate the effectiveness of 14 Mathematics, Science, Technology Teacher Preparation (MSTTP) Academies located across the state of Texas. The aim of the academies was to increase the number of highly qualified mathematics, science, and technology teachers, while also improving the quality of certified teachers in these areas by focusing on seven established goals. The researcher examined best practices for professional development and teacher preparation utilized by the academies, as well as strengths and weaknesses. Additionally, the extent to which the participants perceived the academy had improved their content knowledge and pedagogical skills was examined. Finally, the extent to which the seven goals were associated with participants’ perceived content knowledge and pedagogical knowledge was analyzed. The study used secondary data from a larger evaluation of the MSTTP Academies. A mixed-methods design utilizing triangulation to analyze both quantitative and qualitative data was employed for the study. The results of the current study revealed that the14 MSTTP academies demonstrated the following key strengths: (a) a focus on strengthening content knowledge; (b) a willingness for developing professionally committed teachers; and (c) providing funding for participants. In regard to weaknesses, the degree of program effectiveness revealed that none of the academies had fully implemented all seven goals. All 14 academies, however, struggled to accomplish two of the goals: (a) the integration of the areas of science technology and mathematics; and (b) the infusion of technology into curriculum. Additionally, the findings indicate that participants felt as though the academies had improved their content knowledge and pedagogical skills. The findings also reveal that all academies exhibited three features of effective professional development: (a) a focus on content; (b) active learning opportunities; and (c) intensive and sustained over time. Only one academy exhibited the remaining two features, collective participation and coherence. Finally, the study revealed that only the goal of strengthening content knowledge was a good predictor for participants’ content qualifications, while strengthening content knowledge and strengthening pedagogical skills were good predictors of participants’ pedagogical qualifications. This research study contributes to the to fields of teacher preparation and professional development.

Stem cells are cells that have a unique ability to divide for an indefinite period. Additionally, they can give rise to a plethora of specialized cell types. The advent of high-throughput technologies made it possible to investigate gene expression on a large scale. This enabled scientists to perform comprehensive gene profiling studies of stem cells. Several authors have suggested that there might be a common set of genes that control the stemness of stem cells. In this study, we suggest that ”stemness” genes that are related to ”stemness” characteristics show a statistically significant down-regulation between undifferentiated and differentiated cells. For this we have analyzed microarray data from five different cell lines and compared their global expression profiles. Common down-regulated transcripts among those data sets were de- rived by using a well-established gene expression analysis procedure called Significance Analysis of Microarrays. Since all three data sets were provided by Cellartis AB, the derived list of common transcripts was subsequently compared with an external study. Moreover, we also performed a comparison with down-regulated genes derived from mouse embryonic stem cells. This was done to determine if there is a common set of stemness genes even across distinct species. Re- sults were further evaluated using a comprehensive data-set from a study by Skottman et al. (2005). All results where compared uti- lizing using a range of false discovery rate threshold values and the results were subsequently used for gene ontology term enrichment. GO terms where utilized to functionally annotate and classify those embryonic stem cell transcripts, that were found to be common in all data-sets and identify over-represented biological processes related to those genes.

The interplay between extrinsic and intrinsic processes as they influence a cell’s behaviour is a perennial question in both cellular and developmental biology. In this thesis these two issues are examined in the context of adult stem cells, a somatic stem cell present in the adult murine brain and a germline stem cell present in the adult Drosophila melanogaster ovary. I find that both of these distinct cell types exhibit patterns of non-random chromatid segregation in which the stem cells retain chromosomes carrying the older DNA strands. This unusual behaviour seems to exclusively occur in the context of differentiation, when one cell remains a stem cell and the other goes on to differentiate. Following these studies, the effects of extrinsic processes are tested in adult murine stem cells. It is determined that such cells can only produce neural progeny regardless of their association with foreign environments. These results argue against the phenomenon of stem cell plasticity which is proposed in several other systems and seem to support a primarily intrinsic-centered view of stem cell behaviour. However, the role of adhesion mediating proteins is next studied in such cells to determine their requirement for specific environments. The results of these experiments suggest that adult murine neural stem cells require association with support cells expressing E-Cadherin. Because the loss of such association results in a loss of stem cell number, these data show that intrinsic processes are insufficient to account for all stem cell behaviour. Indeed, based on these data and the results of other studies, it is hypothesized that the extrinsic association of stem cells in these diverse systems determines their polarity and subsequent intrinsic processes that enable these to divide asymmetrically. The implications of this theory are discussed with a view to general biological issues, the proximate mechanisms underlying these phenomena and the ultimate reasons these occur.

The interplay between extrinsic and intrinsic processes as they influence a cell’s behaviour is a perennial question in both cellular and developmental biology. In this thesis these two issues are examined in the context of adult stem cells, a somatic stem cell present in the adult murine brain and a germline stem cell present in the adult Drosophila melanogaster ovary. I find that both of these distinct cell types exhibit patterns of non-random chromatid segregation in which the stem cells retain chromosomes carrying the older DNA strands. This unusual behaviour seems to exclusively occur in the context of differentiation, when one cell remains a stem cell and the other goes on to differentiate. Following these studies, the effects of extrinsic processes are tested in adult murine stem cells. It is determined that such cells can only produce neural progeny regardless of their association with foreign environments. These results argue against the phenomenon of stem cell plasticity which is proposed in several other systems and seem to support a primarily intrinsic-centered view of stem cell behaviour. However, the role of adhesion mediating proteins is next studied in such cells to determine their requirement for specific environments. The results of these experiments suggest that adult murine neural stem cells require association with support cells expressing E-Cadherin. Because the loss of such association results in a loss of stem cell number, these data show that intrinsic processes are insufficient to account for all stem cell behaviour. Indeed, based on these data and the results of other studies, it is hypothesized that the extrinsic association of stem cells in these diverse systems determines their polarity and subsequent intrinsic processes that enable these to divide asymmetrically. The implications of this theory are discussed with a view to general biological issues, the proximate mechanisms underlying these phenomena and the ultimate reasons these occur.

Pregnancy succeeds because the fetal allograft survives in the presence of a fully functional maternal immune system. The placenta, especially its trophoblast, provides the initial barrier between the maternal and fetal environment and, due to their location, trophoblast cells could be expected to be immune-privileged. Yet in the ectopic sites tested thus far, trophoblast stem cell transplants have failed to show noticeable immune privilege and appear to lack physiological support. However in this study, portal vein injected green fluorescent protein-labeled trophoblast stem cells were able to survive for several months in the livers of allogeneic female (14/14), but not male (0/4), mice. Gonadectomy experiments revealed that this gender-dependent survival does not require the presence of ovarian hormones (4/4) but the absence of testicular factors (5/5). In contrast, similarly labeled allogeneic embryonic stem cells were reliably rejected (11/11); these same embryonic stem cells survived when mixed with unlabeled trophoblast stem cells (13/13). The protective effect offered by the trophoblast stem cells did not require any immunological similarity with the co-injected embryonic stem cells. Neither the trophoblast stem cells nor the co-injected embryonic stem cells gave rise to tumors during the study period. Thus, this study demonstrates that, provided a suitable location and hormonal context, ectopic trophoblast stem cells may exhibit and confer immune privilege. These findings suggest applications in cell and gene therapy as well as provide a new model for studying trophoblast physiology and immunology.