Dissertations / Theses on the topic 'Stem cells – Research – Animal models'

The transplantation of allogeneic cells and tissues for the treatment of human disease has been a life-saving procedure for many thousands of patients worldwide. However, to date, neither solid organ transplantation nor bone marrow transplantation have reached their full clinical potential. Significant limitations to the advancement of clinical transplantation stem from our current inability to prevent the rejection of allogeneic tissues by the immune system of the host. Similarly, in patients that receive allogeneic bone marrow transplants, we cannot permanently prevent the engrafted immune system from mounting a response against the patient. This problem, termed graft versus host disease is the most prevalent cause of morbidity and mortality in recipients of allogeneic bone marrow transplants. Clinically, we rely on lifelong immunosuppression to prolong survival of allogeneic tissues within the host. Our currently available therapeutics burden patients with side-effects that range from being unpleasant to life-threatening, while in most cases offering only a temporary solution to the problem of alloimmunity. Efforts are underway to develop protocols and therapeutics that more effectively prevent the pathology associated with alloimmunity. To minimize patient risk, extensive pre-clinical studies in laboratory animals are conducted to predict clinical responses. In the case of immunologic studies, many of these pre-clinical studies are carried out in murine models. Unfortunately, studies of murine immunity often do not predict outcomes in the clinic. One approach to overcome this limitation is the development of a small animal model of the human immune system. In this dissertation, we hypothesized that NOD-scid IL2rγnull mice engrafted with human peripheral blood mononuclear cells (PBMC), termed the hu-PBMC-NOD-scid IL2rγnull model, would provide a model that more accurately reflects human immunity in vivo than other models currently available. To investigate this possibility, we first investigated whether NOD-scid IL2rγnull mice were able to support the engraftment of human PBMC. We found that NOD-scid IL2rγnull mice engraft with human PBMC at much higher levels then the previous gold standard model, the NOD-scid mouse. We then investigated the kinetics of human cell engraftment, determined the optimal cell dose, and defined the influence of injection route on engraftment levels. Even at low PBMC input, NOD-scid IL2rγnullmice reproducibly support high levels of human PBMC engraftment. In contrast to previous stocks of immunodeficient mice, we observed low intra- and interdonor variability of engraftment. We next hypothesized that the human PBMC engrafted in NOD-scid IL2rγnull mice were functional and would reject transplanted allogeneic human tissues. To test this, human islets were transplanted into the spleen of chemically diabetic NOD-scid IL2rγnull mice with or without intravenous injection of HLA-mismatched human PBMC. In the absence of allogeneic PBMC, the human islets were able to restore and maintain normoglycemia. In contrast, human islet grafts were completely rejected following injection of HLA-mismatched human PBMC as evidenced by return to hyperglycemia and loss of human C-peptide in the circulation. Thus, PBMC engrafted NOD-scid IL2rγnull mice are able to provide an in vivomodel of a functional human immune system and of human islet allograft rejection. The enhanced ability of NOD-scid IL2rγnull mice to support human cell engraftment gave rise to the possibility of creating a model of graft versus host disease mediated by a human immune system. To investigate this possibility, human PBMC were injected via the tail vein into lightly irradiated NOD-scid IL2rγnull mice. We found that in contrast to previous models of GVHD using human PBMC-injected immunodeficient mice, these mice consistently (100%) developed GVHD following injection of as few as 5x106PBMC, regardless of the PBMC donor used. We then tested the contribution of host MHC in the development of GVHD in this model. As in the human disease, the development of GVHD was highly dependent on host expression of MHC class I and class II molecules. To begin to evaluate the extent to which the PBMC-engrafted NOD-scid IL2rγnull humanized mouse model of GVHD represents the clinical disease, we tested the ability of a therapeutic in clinical trials to modulate GVHD in these mice. In agreement with the clinical experience, we found that interrupting the TNFα signaling cascade with etanercept delayed the onset and severity of disease in this model. In summary, we conclude that humanized NOD-scid IL2rγnull mice represent an important surrogate for investigating in vivo mechanisms of both human islet allograft rejection and graft versus host disease.

Advances in stem cell biology have challenged the notion that infarcted myocardium is irreparable. The pluripotent ability of stem cells to differentiate into specialized cell lines began to garner intense interest within cardiology when it was shown in animal models that intramyocardial injection of bone marrow stem cells (MSCs), or the mobilization of bone marrow stem cells with spontaneous homing to myocardium, could improve cardiac function and survival after induced myocardial infarction (MI) [1, 2]. Furthermore, the existence of stem cells in myocardium has been identified in animal heart [3, 4], and intense research is under way in an attempt to clarify their potential clinical application for patients with myocardial infarction. To date, in order to identify the best one, different kinds of stem cells have been studied; these have been derived from embryo or adult tissues (i.e. bone marrow, heart, peripheral blood etc.). Currently, three different biologic therapies for cardiovascular diseases are under investigation: cell therapy, gene therapy and the more recent “tissue-engineering” therapy . During my Ph.D. course, first I focalised my study on the isolation and characterization of Cardiac Stem Cells (CSCs) in wild-type and transgenic mice and for this purpose I attended, for more than one year, the Cardiovascular Research Institute of the New York Medical College, in Valhalla (NY, USA) under the direction of Doctor Piero Anversa. During this period I learnt different Immunohistochemical and Biomolecular techniques, useful for investigating the regenerative potential of stem cells. Then, during the next two years, I studied the new approach of cardiac regenerative medicine based on “tissue-engineering” in order to investigate a new strategy to regenerate the infracted myocardium. Tissue-engineering is a promising approach that makes possible the creation of new functional tissue to replace lost or failing tissue. This new discipline combines isolated functioning cells and biodegradable 3-dimensional (3D) polymeric scaffolds. The scaffold temporarily provides the biomechanical support for the cells until they produce their own extracellular matrix. Because tissue-engineering constructs contain living cells, they may have the potential for growth and cellular self-repair and remodeling. In the present study, I examined whether the tissue-engineering strategy within hyaluron-based scaffolds would result in the formation of alternative cardiac tissue that could replace the scar and improve cardiac function after MI in syngeneic heterotopic rat hearts. Rat hearts were explanted, subjected to left coronary descending artery occlusion, and then grafted into the abdomen (aorta-aorta anastomosis) of receiving syngeneic rat. After 2 weeks, a pouch of 3 mm2 was made in the thickness of the ventricular wall at the level of the post-infarction scar. The hyaluronic scaffold, previously engineered for 3 weeks with rat MSCs, was introduced into the pouch and the myocardial edges sutured with few stitches. Two weeks later we evaluated the cardiac function by M-Mode echocardiography and the myocardial morphology by microscope analysis. We chose bone marrow-derived mensenchymal stem cells (MSCs) because they have shown great signaling and regenerative properties when delivered to heart tissue following a myocardial infarction (MI). However, while the object of cell transplantation is to improve ventricular function, cardiac cell transplantation has had limited success because of poor graft viability and low cell retention, that’s why we decided to combine MSCs with a biopolimeric scaffold. At the end of the experiments we observed that the hyaluronan fibres had not been substantially degraded 2 weeks after heart-transplantation. Most MSCs had migrated to the surrounding infarcted area where they were especially found close to small-sized vessels. Scar tissue was moderated in the engrafted region and the thickness of the corresponding ventricular wall was comparable to that of the non-infarcted remote area. Also, the left ventricular shortening fraction, evaluated by M-Mode echocardiography, was found a little bit increased when compared to that measured just before construct transplantation. Therefore, this study suggests that post-infarction myocardial remodelling can be favourably affected by the grafting of MSCs delivered through a hyaluron-based scaffold

Els models de malalties són una eina bàsica per la comprensió de les malalties humanes. Actualment, la majoria de la informació de la que disposem de malalties humanes es basa en models animals. Tot i això, els models animals difereixen molecular i fenotípicament dels humans, i no sempre reprodueixen fidelment la malaltia humana. En les últimes dècades, les cèl·lules mare humanes s’han establert com una opció molt interessant en el camp de la modelització cel·lular. En aquest treball hem volgut caracteritzar les cèl·lules mare embrionàries com a models per a l’estudi de la inestabilitat de la repetició de trinucleotids a la distròfia miotonica tipus 1 (DM1) i la malaltia de Huntington (HD). Així mateix, hem volgut estudiar la inactivació del cromosoma X amb la intenció de fer servir linees cel·lulars com a models per l’estudi del desenvolupament embrionàri humà. A la primera part d’aquest treball, hem observant una inestabilitat de repeticions de trinucleotids significativa al locus de la malaltia DM1 de les cèl·lules mare estudiades. La diferenciació d’aquestes cèl·lules va estabilitzar el número de repeticions. L’estabilització de les repeticions va ser concomitant amb la regulació a la baixa de l’expressió de gens involucrats en els mecanismes de reparació cel·lular. Posteriorment a la publicació del nostre article, altres grups varen reproduir els nostres resultats, però en aquest cas utilitzant cèl·lules mare induïdes. Els estudis recolzen la reproductibilitat dels nostres resultats, suggerint que poden ser extrapol·lats a altres linees de cèl·lules mare arreu del mon. Referent a la mutació de HD, varem trobar que era estable en totes les condicions estudiades, en cèl·lules indiferenciades, diferenciades a progenitors d’os, teratomes i progenitors neurals. Aquests resultats estan en concordancia amb els resultats obtinguts per altres grups que descriuen un baix nombre de repeticions al locus de HD. Per altra banda, varis grups han descrit la presencia de inestabilitat de les repeticions en cèl·lules diferenciades a la linea neural. La discrepància entre els nostres resultats i aquests últims podria ser deguda a la obtenció de cèl·lules neurals menys madures en el moment del nostre estudi. A la segona part d’aquesta tesis hem estudiat la inactivació del cromosoma X en 23 línies femenines de cèl·lules mare pluripotents. Vàrem observar una ràpida progressió de les cèl·lules de dependència de XIST en la inactivació del cromosoma X cap a un estat d’adaptació al cultiu que es caracteritza per un estadi de inactivació independent de l’expressió de XIST i amb una erosió de la metilació. També describim un patró d’inactivació esbiaixat en la majoria de les línies estudiades, contrari al patró aleatori observat en cèl·lules femenines adultes. A més a més, aquest patró és independent de XIST, de l’origen del cromosoma X i d’aberracions cromosòmiques. Aquests resultats suggereixen que el patró esbiaixat observant esta dirigit provablement per l’activació o repressió d’al·lels específics que es troben en el cromosoma X i que li confereixen a la cèl·lula un avantatge respecte a les altres cèl·lules. En conclusió, les cèl·lules mare pluripotents semblen ser un bon model in vitro per a l’estudi d’ambdues malalties, DM1 i HD, ja que presenten el mateix patró d’inestabilitat de la repetició del trinucleotid que s’observa in vivo. Cal remarcar també la depencia Overall, hPSC appear to be a good in vitro model for the study of both DM1 and HD TNR instability, as the repeat follows in vitro the same patterns as found in vivo, including its dependency of the MMR machinery, particularly in the case of DM1. However, our results on the study of the X chromosome inactivation (XCI) state suggest caution when using hPSC as early human developmental research models. The eroded state of XCI found in many of the hPSC lines, and the frequency of skewed XCI patterns suggests that these cells are not a good proxy to early embryonic cells, at least what XCI is concerned. Conversely, they may still provide an interesting model to study gene function and mechanisms implicated.
Disease modelling is an essential tool for the understanding of human disease. Currently, much of the information we have on human diseases is based on animal models. However, animal models differ molecularly and phenotypically from humans, and are not always suitable to reproduce with fidelity human diseases. In the past decades, human pluripotent stem cells (hPSC) have emerged as an interesting option in the field of cellular modelling, this development recently having taken up much momentum. In this work, we aimed at characterizing hPSC as models for the study of Myotonic dystrophy type 1 (DM1) and Huntington’s disease (HD) trinucleotide repeat (TNR) instability and to investigate the status of the X-chromosome inactivation with an eye on using these cells as models for early human development. In the first part of our work, we observed a significant TNR instability for the DM1 locus in hESC, and that differentiation resulted in a stabilization of the repeat. This stabilization was concommitant with a downregulation of the mismatch repair (MMR). Our results were later replicated in hiPSC by other researchers, showing their reproducibility and suggesting they may be extrapolated to other hPSC lines worldwide. Regarding the HD repeat, we found it was very stable in all conditions studied, both in undifferentiated hESC and cells differentiated into osteogenic progenitor-like cells, teratoma cells and neural progenitors. This is in line with other studies showing that hESC show very limited TNR in the HD locus. On the other hand, some groups have now reported some instability of this locus in cells differentiated into the neuronal lineage. The instability seen in neuronal lineage in later studies, not in our study, is probably explained by the use of hPSC derived neurons more similar to the cells showing in vivo instability than the ones we were able to generate at the time of the study. In the second part of the thesis we studied the X-chromosome inactivation in 23 female hPSC lines. We found that hPSC rapidly progress from a XIST-dependent XCI state to a culture-adapted, XIST-independent XCI state with loss of repressive histone modifications and erosion of methylation. We also report a remarkably high incidence of non-random XCI patterns, and that this skewing of the methylation patterns is independent from the transition to the XIST-independent XCI state, the origin of the X chromosome or chromosomal aberrations. These results suggest that XCI skewing is possibly driven by the activation or repression of a specific allele on the X chromosome, conferring a growth or survival advantage to the cells. Overall, hPSC appear to be a good in vitro model for the study of both DM1 and HD TNR instability, as the repeat follows in vitro the same patterns as found in vivo, including its dependency of the MMR machinery, particularly in the case of DM1. However, our results on the study of the X chromosome inactivation (XCI) state suggest caution when using hPSC as early human developmental research models. The eroded state of XCI found in many of the hPSC lines, and the frequency of skewed XCI patterns suggests that these cells are not a good proxy to early embryonic cells, at least what XCI is concerned. Conversely, they may still provide an interesting model to study gene function and mechanisms implicated.

A practical consideration in the development of cellular therapy technology for the inner ear is the development of an in vitro model for assessing the optimal conditions for successful application of cells. The first part of this thesis describes the adaptation of the cochleovestibular structure harvested from P1 mouse pups for analysis of factors critical for the optimal implantation of stem cells in the inner ear. Results of these studies establish that the c17.2 neural stem cell line can be introduced into the cochleovestibular structure in vitro. Using this model, c17.2 cells demonstrated survival predominantly within the vestibule and basal spiral ganglion regions. Furthermore, the addition of the ototoxin, cisplatin and the neurotrophin, Brain Derived Neurotrophic Growth Factor (BDNF) enhanced the survival and migration/dispersion of c17.2 cells within the cochleovestibular explant. The second part of this thesis examines the hypothesis that olfactory neurosphere (ONS) and progenitor cells harvested from the olfactory epithelium represent a viable source of graft material for potential therapeutic applications in the inner ear. Olfactory epithelium represents a unique source of pluripotent cells that may serve as either homografts or autografts. The feasibility of ONSs to survive and integrate into a mammalian cochlea in vivo was assessed. The ONSs were isolated as a crude fraction from the olfactory epithelium of P1 to P3 day old swiss webster mouse pups, ubiquitously expressing the Green Fluorescent Protein (GFP) marker. The ONSs were microinjected into the cochleae of adult CD1 male mice. Four weeks following their implantation, ONS cells expressing the GFP marker and stained by Nestin were identified in all areas of the cochlea and vestibule, including the spiral ganglion. Robust survival and growth of the implanted ONS and ONS derived cells in the cochlea also included the development of ???tumor-like??? clusters, a phenomenon not observed in control animals implanted with c17.2 neural stem cells. Collectively, the results of this thesis illustrate the potential of olfactory neurosphere and progenitor cells to survive in the inner ear and expose a potential harmful effect of their transplantation.

Parkinson's disease (PD) is the second most common neurodegenerative disease. With the rapid global increase in population aging, neurodegenerative diseases are considered a primary threat to human health. As dopaminergic neuronal cell death and dysfunction are the main pathogenic mechanisms of PD, neural stem cell (NSC) replacement therapy has been identified as a potentially effective and indeed ideal therapeutic strategy. However, current in vitro stem cell culture methods, which require various chemical growth factors (GFs), are unsafe and relatively inefficient. To solve this problem, we developed two strategies for enhancing the proliferation and differentiation of NSCs in vitro based on extracellular nanomatrices and natural active ingredients. First, we developed novel nanomatrices comprising biomaterials used for promoting NSCs proliferation and differentiation without requiring additional GFs. We developed two types of inorganic sculptured extracellular nanomatrices comprising SiO2 (iSECnMs) which deposited by glancing angle deposition (GLAD). The physiological properties of nanomatrices mediate the activation of multiple bio-signaling pathways. Accordingly, iSECnMs, especially those sculptured in zigzag forms, can significantly promote the proliferation and specific neuronal phenotypic differentiation of NSCs without requiring additional GFs. The differentiated neurons survived well in vivo and achieved outstanding therapeutic effects in a rat model of 6-OHDA-induced parkinsonism. Second, 20(S)-protopanaxadiol (PPD) and oleanolic acid (OA), two crucial active ingredients derived from ginseng, significantly enhanced the proliferation and neuronal differentiation of NSCs through activating Wnt/GSK-3β/β-catenin pathway. This research is expected to promote significant developments in the induction of NSCs and provide insights into stem cell therapies for PD without undesirable prognoses

The following research paper was concerned with five basic objectives:(1) outlining the major theoretical and methodological approaches used in the reconstruction of early hominid social behavior/social structure as a context in which to view Richard Wrangham's and Michael Ghiglieri's phylogenetic models of stem-hominid social structure.(2) examining Wrangham's and Ghiglieri's models of stem-hominid and chimpanzee social structure.(3) indicating how theoretical and methodological aspects of structure essentially represent an extension of the theoretical and methodological approaches the same researchers applied to their models of chimpanzee social structure.(4) addressing the theoretical and methodological deficiences of Wrangham's and Ghiglieri's models of chimpanzee social structure.(5) providing suggestions for improved phylogenetic models of early hominid social structure.The first objective was achieved by: (a) reviewing Tooby and Devore's (1986) and Wrangham's (1986) evaluations of the major theoretical approaches and methodologies used in the reconstruction of hominid social behavior/structure (b) defining, classifying and evaluating Wrangham's and Ghiglieri's phylogenetic approaches within this context.The second objective was accomplished by outlining, analyzing, and comparing/contrasting Wrangham's and Ghiglieri's phylogenetic models of stem-hominid social structure (i.e.Wrangham 1986; Ghiglieri 1987, 1989) and Wrangham's and Ghiglieri's models of chimpanzee social structure (i.e. Wrangham 1975, 1979; Ghiglieri 1984, 1985, 1987, 1989).The third objective was achieved by recognizing how Wrangham and Ghiglieri used/stressed principles and concepts derived from evolutionary biology and/or behavioral ecology to develop their models of stem-hominid and chimpanzee social structure. This analysis showed that Wrangham's models of social structure were more favorably inclined toward the method of behavioral ecology than Ghiglieri's models, which favored a sociobiological paradigm. Furthermore, although neither researcher relied exclusively on the above theoretical approaches, the main thrust of their argument often centered around it. For instance, Wrangham's analysis of chimpanzee social structure (Wrangham 1975, 1979) indicated that the ultimate cause of that structure was ecological i.e., patchy food distribution leads to wide female dispersal for optimal foraging efficiency, which in turn favors a male kin breeding group that can maintain a territority that includes several individual female ranges. In contrast, Wrangham's phylogenetic model of the social structure of the stem-hominid (Wrangham, 1986) suggested that phylogenetic inertia may be partially responsible for the shared social features found among African Hominoidea. However, in the same work, Wrangham also suggested that further socioecological analysis of African apes may indicate whether food distribution and its effects on female dispersion/association may partially explain conservative African ape social features.Ghiglieri's phylogenetic model of the stem-hominid (1987, 1989), on the other hand, explained the conservative social features of bonobos, common chimpanzees, and hominids to be primarily a product of phylogenetic inertia and sexual selection. Furthermore, for Ghiglieri the most important sexual selection variable was a male communal reproductive strategy. This, according to Ghiglieri, is the ultimate cause of social structure. Notably, Ghiglieri (1984, 1985) had earlier stressed the overiding importance of a male communal reproductive strategy but was less dogmatic in his insistence that chimpanzees had essentially solved their ecological problems (e.g. that they had solved the food distribution problem by fusion-fission sociality; predators were never a real problem). Nevertheless, Ghiglieri's earlier position similarily expressed the idea that a communal reproductive strategy constituted the ultimate cause of social structure.The fourth objective was accomplished by presentation of an alternative model of chimpanzee social behavior which suggested that structure; the effect of phylogenetic inertia on social structure; chimpanzee social structure is the combined product of ecological and sexual selection forces: female optimal foraging, male mating strategies, and predator pressure. The model was considered by the author to be unique in that it integrated essential aspects of both Wrangham's and Ghiglieri's models and, in addition, provided support for Alexander's (1974) contention that predation pressure is an ultimate cause of ape social structure. The model also outlined scenarios for the evolution of chimpanzee group._ extensibility (fusion-fission sociality) and the capacity for warfare among chimpanzees.The last objective was achieved by a discussion of the implications that the author's model had for phylogenetic models of stem-hominid social structure. In this discussion the author reviewed the following issues as they related to the phylogenetic reconstruction of hominid social structure: the role of phylogeny and/or ecology in the causation of social encountered when using a phylogenetic referential model for the personal biases that enter into phylogenetic econstructions; pitfalls reconstruction of early hominid social evolution; the significance of chimpanzee models of social structure.The importance of the preceding study lay in its ability to stimulate improved conceptual models of African hominoid social structure.
Department of Anthropology

O enfisema pulmonar define-se como a destruição das paredes alveolares e consequente dispneia progressiva. Este trabalho objetivou a adequação de um modelo de transplante celular in vivo de BMMC em camundongos com enfisema pulmonar. O enfisema foi induzido por instilação nasal de elastase (4 UI por animal). O diâmetro alveolar médio para os grupos não tratados e tratados com elastase apresentou diferença estatisticamente significativa, e mudanças no padrão de expressão de metaloproteinases envolvidas no processo inflamatório foram detectadas, indicando que a instilação de uma dose de elastase promove lesão semelhante ao enfisema pulmonar. Infundiu-se 0,4ml de BMMC (7x106 céls./ml) nestes animais. No grupo tratado com células, detectou-se mudanças morfométricas e no padrão de expressão de metaloproteinases, indicando melhora na evolução da lesão pulmonar 21 dias após a infusão. Foram ainda avaliadas duas e três doses do pool de BMMC, porém os resultados das análises mostraram que não há diferenças entre estre grupos e os grupos controle.
Pulmonary emphysema is defined as the destruction of the alveolar walls and consequent progressive dyspnea. This study aimed the adequacy of a model of BMMC transplantation in vivo in mice with pulmonary emphysema. Emphysema was induced by nasal instillation of elastase (4 IU per animal). The mean linear intercept for the groups untreated and treated with elastase showed a statistically significant difference, and changes in the pattern of expression of metalloproteinases involved in inflammation were detected, indicating that the instillation of a dose of elastase promotes lung damage similar to emphysema. 0.4 ml of BMMC (7x106 céls. / ml) was infused in these animals. In the group treated with cells there were detected and morphometric changes in the pattern of expression of metalloproteinases, indicating an improvement in the evolution of lung injury 21 days after infusion. Were also evaluated two and three doses of the pool BMMC, but the results of the analysis showed no differences between experimental and the control groups.

Background: Streptozotocin (STZ) induced diabetes leads to various complications including cardiomyopathy. Recent data suggests transplanted bone marrow stem cells improve cardiac function in diabetic cardiomyopathy. However, whether modified ES, iPS cells, or factors released from these cells can inhibit apoptosis and fibrosis remains completely unknown. The present study was designed to determine the effects of transplanted ES cells overexpressing pancreatic transcription factor 1 a (Ptf1a), a pro-pancreatic endodermal transcription factor, iPS cells, or their respective conditioned media (CM) on diabetic cardiomyopathy. Methods: Experimental diabetes was induced in male Sprague Dawley rats (8-10 weeks old) by intraperitoneal STZ injections (65 mg/kg body weight for 2 consecutive days). Animals were divided into six experimental groups including control, treated with sodium citrate buffer IP, STZ, STZ + ES-Ptf1a cells, STZ + iPS cells, STZ + ES-Ptf1a CM and STZ + iPS CM. Following STZ injections, appropriate cells (1 X 106/mL/injection/day) or CM (2 mL injection/day) were given intravenously for 3 consecutive days. Animals were sacrificed and hearts were harvested at day 28. Histology, TUNEL staining, and Caspase-3 activity were used to assess apoptosis and fibrosis. ERK1/2 phosphorylation was quantified using ELISAs. M-mode echocardiography fractional shortening was used to assess cardiac function. Results: Animals transplanted with ES cells, iPS cells, or both CMs showed a significant (pless than]0.05) reduction in interstitial fibrosis, and apoptosis compared with STZ group. ERK expression was not significantly different compared with STZ. Echocardiography showed a significant (pless than]0.05) improvement in fractional shortening in cell and media transplanted groups compared with STZ. Conclusions: Our data suggest that ES cells, iPS cells, and/or CMs inhibit apoptosis, reduce fibrosis, and improve cardiac function in STZ-treated diabetic rats.
ID: 029049879; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (M.S.)--University of Central Florida, 2010.; Includes bibliographical references (p. 33-40).
M.S.
Masters
Burnett School of Biomedical Sciences
Medicine

Thesis (PhD (Physiology (Human and animal))-- University of Stellenbosch, 2007.
Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal and the ability to differentiate into multiple cell-lineages. The potential for the application of stem cells in clinical settings has had a profound effect on the future of regenerative medicine. However, to be of greater therapeutic use, selection of the most appropriate cell type, as well as optimisation of stem cell incorporation into the damaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cell population which mediate postnatal muscle growth. Following injury or in diseased conditions, these cells are activated and recruited for new muscle formation. In contrast, the potential of resident adult stem cell incorporation into the myocardium has been challenged and the response of cardiac tissue, especially to ischaemic injury, is scar formation. Following muscle damage, various growth factors and cytokines are released in the afflicted area which influences the recruitment and incorporation of stem cells into the injured tissue. Transforming Growth Factor-β (TGF-β) is a member of the TGF-β-superfamily of cytokines and has at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulation of cell growth and regeneration following activation and stimulation of receptor-signalling pathways. By improving the understanding of how TGF-β affects these processes, it is possible to gain insight into how the intercellular environment can be manipulated to improve stem cell-mediated repair following muscle injury. Therefore, the main aims of this thesis were to determine the effect of the three TGF-β isoforms on proliferation, differentiation, migration and fusion of muscle progenitor cells (skeletal and cardiac) and relate this to possible improved mechanisms for muscle repair. The effect of short- and long-term treatment with all three TGF-β isoforms were investigated on muscle progenitor cell proliferation and differentiation using the C2C12 skeletal muscle satellite and P19 multipotent embryonal carcinoma cell-lineages as in vitro model systems. Cells were treated with 5 ng/mℓ TGF-β isoforms unless where stated otherwise. In C2C12 cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, and together with total nuclear counts, used to assess the effect of TGF-β on myoblast proliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structural protein myosin heavy chain (MHC) were used as protein markers to assess early and terminal differentiation, respectively. To establish possible mechanisms by which TGF-β isoforms regulate differentiation, further analysis included determination of MyoD localisation and the rate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels of connexin-43 and MHC were analysed, together with the determination of embryoid body numbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed to analyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), as well as fusion of C2C12 cells (Chapter 8). Whereas all three isoforms of TGF-β significantly increased proliferation of C2C12 cells, differentiation results, however, indicated that especially following long-term incubation, TGF-β isoforms delayed both early and terminal differentiation of C2C12 cells into myotubes. Similarly, myocyte migration and fusion were also negatively regulated following TGF-β treatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment with TGF-β1 could potentially enhance differentiation. Further research is however required in this area, especially since migration was greatly reduced in these cells. Taken together, results demonstrated variable effects following TGF-β treatment depending on the cell type and the duration of TGF-β application. Circulating and/or treatment concentrations of this growth factor could therefore be manipulated depending on the area of injury to improve regenerative processes. Alternatively, when selecting appropriate stem or progenitor cells for therapeutic application, the effect of the immediate environment and subsequent interaction between the two should be taken into consideration for optimal beneficial results.

A Doença Pulmonar Obstrutiva Crônica (DPOC) destaca-se como uma das doenças de maior prevalência, mortalidade e incapacitação. O principal fator de risco para a DPOC vincula-se à exposição a partículas e gases nocivos, sendo o tabagismo responsável pela maioria dos casos da doença. Apesar dos avanços terapêuticos, não há até o presente uma forma de tratamento eficaz. Neste contexto, as células-tronco representam uma prática terapêutica potencialmente promissora. Foi proposto, neste estudo, o uso de um modelo animal de enfisema pulmonar que busca mimetizar as condições patológicas de pacientes tabagistas, permitindo testar, in vivo, os efeitos terapêuticos das células-tronco. Os resultados obtidos demonstraram que os animais expostos à fumaça de cigarro desenvolveram os aspectos histomorfológicos da doença. A fluorescência direta revelou que as células infundidas migraram para os pulmões, esse achado, em concomitância com a recuperação pulmonar permitem sugerir que as células-tronco atuaram na regeneração do órgão. Consolidou-se, neste estudo, um novo aparato e uma metodologia que permitem avaliar novas terapias experimentais. Além disso, demonstrou-se a potencialidade terapêutica das células-tronco no tratamento do enfisema pulmonar.
Chronic Obstructive Pulmonary Disease (COPD) stands out as one of the most prevalent diseases, mortality and disability. The main risk factor for COPD is linked to exposure to noxious particles and gases, tobacco use is responsible for most cases of the disease. Despite therapeutic advances, there is no effective treatment. In this context, stem cells represent a potentially promising therapeutic practice. It was proposed in this study, using an animal model of emphysema that seeks to mimic the pathological conditions of smokers, allowing you to test, in vivo, the therapeutic effects of stem cells. The results showed that animals exposed to cigarette smoke developed the histopathological aspects of the disease. The direct fluorescence revealed that the infused cells migrated to the lungs, this finding, in tandem with the recovering lung may suggest that the stem cells worked in organ regeneration. Consolidated, in this study, a new apparatus and methodology for assessing new experimental therapies. Furthermore, it was demonstrated that the therapeutic potential of stem cells in the treatment of pulmonary emphysema.

The family of runt related transcription factors (RUNX/Cbfa/AML/PEBP2) are essential for cellular differentiation and fetal development. RUNX factors are distributed throughout the nucleus in punctate foci that are associated with the nuclear matrix/scaffold and generally correspond with sites of active transcription. Truncations of RUNX proteins that eliminate the C-terminus including a 31-amino acid segment designated the nuclear matrix targeting signal (NMTS) lose nuclear matrix association and result in lethal hematopoietic (RUNX1) and skeletal (RUNX2) phenotypes in mice. These findings suggest that the targeting of RUNX factors to subnuclear foci may mediate the formation of multimeric regulatory complexes and contribute to transcriptional control. In this study, we hypothesized that RUNX transcription factors may dynamically move through the nucleus and associate with subnuclear domains in a C-terminal dependent mechanism to regulate transcription. Therefore, we investigated the subnuclear distribution and mobility of RUNX transcription factors in living cells using enhanced green fluorescent protein (EGFP) fused to RUNX proteins. The RUNX C-terminus was demonstrated to be necessary for the dynamic association of RUNX with stable subnuclear domains. Time-lapse fluorescence microscopy showed that RUNX1 and RUNX2 localize to punctate foci that remain stationary in the nuclear space in living cells. By measuring fluorescence recovery after photobleaching, both RUNX1 and RUNX2 were found to dynamically and rapidly associate with these subnuclear foci with a half-time of recovery in the ten-second time scale. A large immobile fraction of RUNX1 and RUNX2 proteins was observed in the photobleaching experiments, which suggests that this fraction of RUNX1 and RUNX2 proteins are immobilized through the C-terminal domain by interacting with the nuclear architecture. Truncation of the C-terminus of RUNX2, which removes the NMTS as well as several co-regulatory protein interaction domains, increases the mobility of RUNX2 by at least an order of magnitude, resulting in a half-time of recovery equivalent to that of EGFP alone. Contributions of the NMTS sequence to the subnuclear distribution and mobility of RUNX2 were further assessed by creating point mutations in the NMTS of RUNX2 fused to EGFP. The results show that these point mutations decrease, but do not abolish, association with the nuclear matrix compared to wild-type EGFP-RUNX2. Three patterns of subnuclear distribution were similarly observed in living cells for both NMTS mutants and wild-type RUNX2. Furthermore, the NMTS mutations showed no measurable effect on the mobility of RUNX2. However, the mobility of RUNX proteins in each of the different subnuclear distributions observed in living cells were significantly different from each other. The punctate distribution appears to correlate with higher fluorescence intensity, suggesting that the protein concentration in the cell may have an effect on the formation or size of the foci. These findings suggest that the entire NMTS and/or the co-regulatory protein interaction domains may be necessary to immobilize RUNX2 proteins. Because RUNX factors contain a conserved intranuclear targeting signal, we examined whether RUNX1 and RUNX2 are targeted to common subnuclear domains. The results show that RUNX1 and RUNX2 colocalized in common subnuclear foci. Furthermore, RUNX subnuclear foci contain the co-regulatory protein CBFβ, which heterodimerizes with RUNX factors, and nascent transcripts as shown by BrUTP incorporation. These results suggest that RUNX subnuclear foci may represent sites of transcription containing multi-subunit transcription factor complexes. RUNX2 transcription factors induce expression of the osteocalcin promoter during osteoblast differentiation and to study both RUNX2 and osteocalcin function, it would be helpful to have transgenic mice in which OC expression could be easily evaluated. Therefore, to assess the in vivo regulation of osteocalcin by RUNX protein, we generated transgenic mice expressing EGFP controlled by the osteocalcin promoter. Our results show that EGFP is expressed from the OC promoter in a cultured osteosarcoma cell line, but not in a kidney cell line, and is induced by vitamin D3. Furthermore, the OC-EGFP transgenic mice specifically express EGFP in osteoblasts and osteocytes in bone tissues. Moreover, EGFP is expressed in mineralized bone nodules of differentiated bone marrow derived from transgenic mice. Thus, these mice produce a good model for studying the in vivo effects of RUNX-mediated osteocalcin regulation and for developing potential drug therapies for bone diseases. Taken together, our results in living cells support the conclusion that RUNX transcription factors dynamically associate with stationary subnuclear foci in a C-terminal dependent mechanism to regulate gene expression. Moreover, RUNX subnuclear foci represent transcription sites containing nascent transcripts and co-regulatory interacting proteins. These conclusions provide a mechanism for how RUNX transcription factors may associate with subnuclear foci to regulate gene expression. Furthermore, the OC-EGFP transgenic mice now provide a useful tool for studying the in vivo function and regulation of osteocalcin by RUNX proteins during osteoblast differentiation and possibly for developing therapeutic drugs for treatment of bone diseases in the future.

Tolerance-based stem cell transplantation using sub-lethal conditioning is being considered for the treatment of human disease, but safety and efficacy remain to be established. In order to study these two issues, we first established that mouse bone marrow recipients treated with sub-lethal irradiation plus transient blockade of the CD40-CD154 costimulatory pathway develop permanent hematopoietic chimerism across allogeneic barriers. Our conditioning regimen of 6 Gy irradiation, a short course of anti-CD154 mAb and 25 million fully allogeneic BALB/c bone marrow cells consistently produced long-term, stable, and multilineage chimerism in C57BL/6 recipients. Furthermore, chimeric mice displayed donor-specific transplantation tolerance, as BALB/c skin allografts were permanently accepted while third-party CBA/JCr skin allografts were promptly rejected. We next determined both the safety and efficacy of this protocol by infecting chimeric mice with lymphocytic choriomeningitis virus (LCMV) either at the time of transplantation or at several time points afterwards. Infection with LCMV at the time of transplantation prevented engraftment of allogeneic, but not syngeneic, bone marrow in similarly treated mice. Surprisingly, infected allograft recipients also failed to clear the virus and died. Post-mortem study revealed hypoplastic bone marrow and spleens. Hypoplasia and death in these mice required the combination of 6 Gy irradiation, LCMV infection on the day of transplantation, and an allogeneic bone marrow transplant but did not require the presence of anti-CDl54 mAb. Allochimeric mice infected with LCMV 15 days after transplantation were able to survive and maintain their bone marrow graft, indicating that the deleterious effects of LCMV infection on host and graft survival are confined to a narrow window of time during the tolerization and transplantation process. The final section of this thesis studied the mechanisms of graft rejection and death in sublethally irradiated recipients of allogeneic bone marrow and infection with LCMV at the time of bone marrow transplantation. Infection of interferon-α/β receptor knockout mice at the time of transplantation prevented the engraftment of allogeneic bone marrow, but the mice survived. Therefore, IFN-αβ is involved in the development of marrow hypoplasia and death, whereas a second mechanism is involved in blocking the development of chimerism in these mice. Through the use of depleting mAb's and knockout mice we demonstrate that three types of recipients survived and became chimeric after being given sublethal irradiation, anti-CD154 mAb, an allogeneic bone marrow transplant and a day 0 LCMV infection: mice depleted of CD8+ T cells, CD8 knockout mice, and TCR-αβ knockout mice. Our data indicate that the mediator of bone marrow allograft destruction in LCMV-infected mice treated with costimulatory blockade is a radioresistant CD8+ NK1.1- TCRαβ+ T cell. We conclude that a non-cytopathic viral infection at the time of transplantation can prevent engraftment of allogeneic bone marrow and result in the death of sub-lethally irradiated mice treated with costimulation blockade. The abrogation of allogeneic bone marrow engraftment is mediated by a population of CD8+ NK1.1- TCRαβ+ T cells and the mediator of hypoplasia and death is viral induction of IFN-αβ.

Ovarian cancer is the fifth leading cause of cancer death in women between the ages of 35 and 74. With 22 thousand new cases and 15 thousand deaths annually ovarian cancer is among the most deadly cancers with a death to incidence ratio of 68%. With 70% of cases High Grade Serous Ovarian Carcinoma (HGSOC) is the most common type of ovarian cancer and causes 90% of ovarian cancer deaths. 80% of patients have reoccurrence within five years and only 15-30% of patients with recurrent metastatic ovarian cancer respond to current therapies, chemotherapy and surgery. One reason for the high reoccurrence rate is thought to be linked to the heterogeneity of tumors: there is evidence that, among tumor cells, a subpopulation is cancer stem cells (CSCs). Since CSCs are frequently drug resistant, when the patient undergoes chemotherapy many of the cells may die but the CSCs are left behind and the tumors can therefore regrow. CSCs are also more likely to undergo epithelial-mesenchymal transition which gives these cells the ability to more readily migrate and invade through the extracellular matrix, leaving the primary tumor to form metastases. One key inducer of EMT and therefore possibly of metastasis of particular interest in this project is SNAI1 (Snail). It is therefore the goal of this project to understand the growth, makeup and metastatic ability of HGSOC cell lines to test possible strategies to decrease growth of cancer and prevent metastasis. In this thesis project the phenotype, CSC population make up, and functionality of various HGSOC cell lines was examined. The cell lines assessed were A2780, Kuramochi, OVSAHO, COV318, SKOV3 and OVCAR8. A Snail knockdown OVCAR8 cell line was also assessed as described above and in a xenograft model. It was determined that the cell lines show varying phenotype from epithelial like to mesenchymal like morphology and the cell lines have varying concentrations of cancer stem cells. It was also determined that the CSC population of the HGSOC cell lines were positive for both epithelial and mesenchymal markers in the same cells. OVCAR8 stood out as a hybrid line with both epithelial and mesenchymal characteristics and was therefore chosen for the Snail knockdown model. In the Snail knockdown we observed that CSC markers were reduced, however no change between control and knockdown was seen in the in vitro functional experiments. There was a difference seen between Snail knockdown and control in the in vivo mouse xenograft model. Snail knockdown showed a trend for decreasing tumor burden in both primary and metastatic tumors and showed a significant decrease in growth of metastatic tumor at day 43. Based on these results Snail may be an important target for cancer therapy.

Approximately 45% of men will develop cancer during their lifetime; some of which will be of reproductive age (Canadian Cancer Society, 2008). Current advances in treatment regimens such as radiotherapy have significantly lowered cancer-related mortality rates; however, one major quality-of-life issue in cancer survivors is the ability to produce healthy offspring. Exposure to ionizing radiation (IR) leads to genomic instability in the germline, and further to transgeneration genome instability in unexposed offspring of preconceptionally exposed parents. The results presented in this thesis define, in part, the molecular consequences of direct and indirect irradiation for the male germline. Direct exposure results in a significant accumulation of DNA damage, altered levels of global DNA methylation and microRNAome dysregulation of testis tissue. Localized cranial irradiation results in a significant accumulation of unrepaired DNA lesions and loss of global DNA methylation in the rodent (rat) germline. Biological consequences of the changes observed are discussed.
xii, 121 leaves : ill. ; 29 cm.

Introdução: As células-tronco apresentam capacidade de proliferação, autorrenovação, potencial de diferenciação e já foram descritas na hipófise estando envolvidas na renovação celular e regulação homeostática, porém pouco se sabe sobre o seu perfil de expressão nos quadros de hipopituitarismo. Dentre os marcadores de células-tronco descritos previamente na hipófise, destacam-se os genes Sox2, Nanog, Nestina, Cd44 e Oct4. Outro marcador, o gene Nr2e1 (Tlx), encontrado em células-tronco neuronais, apresenta-se elevado durante a embriogênese e na vida adulta no cérebro de camundongos, mas, até o momento, não foi caracterizado na hipófise. Objetivo: Analisar a imunolocalização do SOX2 e o padrão de expressão de marcadores de células-tronco/progenitoras, fatores de transcrição precoce, marcadores de apoptose e proliferação celular na hipófise de três linhagens de camundongos com hipopituitarismo de causa genética por alteração em fatores precoces de diferenciação glandular, as linhagens Ames (Prop1) e Snell (Pou1f1), e por fator tardio de conjugação dos hormônios glicoproteicos, a linhagem alfaGSU, nocaute do gene Cga. Material e Métodos: Foram coletadas hipófises nos tempos P0 (ao nascimento), P7 (final da primeira onda de crescimento glandular), 4 semanas (4S-período da puberdade) e 8 semanas (8S-vida adulta). Nas três linhagens de animais, realizou-se imuno-histoquímica com SOX2 e RT-qPCR com os marcadores de células-tronco/progenitoras Sox2, Nanog, Nestina, Cd44, Oct4 e Nr2e1, fatores de transcrição precoces (Hesx1, Hes1 e Otx2), fator de proliferação celular (Ki67), fatores de diferenciação celular (S100beta e Sox9) e marcadores de apoptose (Caspases 3 e 7). A quantificação relativa dos genes-alvo nos animais mutantes teve como calibrador os seus respectivos selvagens. Resultados: A imunolocalização do SOX2 foi observada na zona que circunda a fenda de Rathke (camada marginal) e em nichos difusos pela glândula nas três linhagens estudadas. Na linhagem alfaGSU, evidenciou-se uma redução de Nanog, Nr2e1, Oct4, e Hesx1 em 4S e de Nestina em 8S. Na linhagem Snell, observou-se aumento na expressão de Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e aumento de Sox2, Cd44, Hesx1, Otx2 e Sox9 em 8S, associado à redução de Ki67 em ambos os períodos. Na linhagem Ames, evidenciou-se aumento de Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e 8S. O gene Nr2e1 esteve hiperexpresso em todos os tempos. Houve redução do Ki67 em 4S. As caspases 3 e 7 não se apresentaram alteradas em nenhuma linhagem e/ou tempo. Discussão e conclusão: O padrão de imunolocalização de SOX2 encontrado nas três linhagens estudadas foi semelhante ao descrito em animais sem hipopituitarismo. A evidência da presença do Nr2e1 o coloca como um novo marcador de células-tronco/progenitoras na hipófise. A expressão elevada dos marcadores de células-tronco/progenitoras nas linhagens Ames e Snell sugere que a ausência dos fatores de transcrição precoces não permitiria que a célula tronco/progenitora iniciasse o processo de diferenciação celular, enquanto o oposto ocorreria na linhagem alfaGSU. Adicionalmente, estes achados justificam a hipoplasia hipofisária observada em animais com defeitos em fatores de transcrição expressos no início da diferenciação hipofisária, nos quais o acúmulo de células-tronco pode ser um indicador da indiferenciação hipofisária
Introduction: The role of stem cells, with their capacity for proliferation, self-renewal, and differentiation, has already been described in the cell turnover and homeostatic regulation of the pituitary gland. However, little is known about the expression profiles of these markers in hypopituitarism. Among the stem cell markers previously described in the pituitary include the genes for Sox2, Nanog, nestin, CD44 and Oct4. Another gene marker, Nr2e1 (Tlx), found in neural stem cells, is highly expressed during embryogenesis and adulthood, but so far has not been characterized in the pituitary. Objective: To analyze the immunohistochemical profile of SOX2, as well as the pattern of expression of various markers of stem/progenitor cells, early transcription factors, apoptosis factors and cell proliferation in three pituitary strains of mice with a genetic cause of hypopituitarism. Strains studied with hypopituitarism due to changes in factors of precocious glandular differentiation, include the Ames (Prop1) and Snell (Pou1f1) lineages; hypopituitarism due to the delayed conjugation of glycoprotein hormones include the alfaGSU strain, which is caused by the knockout of the Cga gene. Material and Methods: We collected pituitaries at four time points including P0 (birth), P7 (considered the end of the first wave of growth glandular), 4 weeks (4S - puberty period) and 8 weeks (8S - adulthood). All three strains were subjected to immunohistochemical analysis of SOX2 and RT-qPCR of markers of stem/progenitor cells Sox2, Nanog, Nestin, Cd44, Oct4 and Nr2e1, early transcription factors (Hesx1, Otx2 and Hes1), cell proliferation (Ki67), cell differentiation factors (S100beta and Sox9) and apoptosis (caspases 3 and 7) markers. Relative quantification of target genes in mutant animals was normalized to their respective wild type littermate. Results: The immunolocalization of SOX2 was observed in the area surrounding the Rathke cleft (marginal layer), as well as in diffuse niches throughout the gland in all three strains studied. The alfaGSU strain showed a reduction of Nanog, Nr2e1, Oct4 and Hesx1 at 4S, and Nestin at 8S. The Snell mice exhibited an increase of expression in Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta and Sox9 in at 4S and increased Sox2, Cd44, Hesx1, Otx2 and Sox9 at 8S, associated with the reduction of Ki67 in both periods. The Ames strain showed an increase of Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta and Sox9 at 4S and 8S; the gene Nr2e1 was over expressed at all times; and there was reduction in Ki67 at 4S. Caspases 3 and 7 had not changed in any strain, at any time. Discussion and Conclusion: The pattern of immunolocalization of SOX2 found in the three strains studied was similar to that described in animals without hypopituitarism. The presence of Nr2e1 in our study suggests it as a new marker of stem/progenitor cells in the pituitary. The high expression of markers of stem/progenitor cells in the Ames and Snell strains suggests that the absence of early transcription factors Prop1 and Pou1f1 do not allow the stem/ progenitors cells to start the process of cell differentiation, while the opposite occurs in the alfaGSU lineage. Additionally, these findings explain the pituitary hypoplasia observed in animals with defects in early transcription factors, as indicated by the accumulation of stem cells in the Snell and Ames lineages, preventing the initiation of pituitary differentiation

Transcription factors GATA-1, Fli-1 and FOG-1 are essential proteins for normal megakaryopoiesis, however, the detailed analyses of their functions within developmental stages of megakaryopoiesis are lacking. In my thesis, over expression of gene in target cells was adopted as the main strategy to study the biological functions of these proteins, therefore, an efficient gene delivery method was first developed by using retrovirus.This approach was then utilized to over express GATA-1, Fli-1 and FOG-1 in murine leukemia M1 cells and mouse hematopoietic stem cells (HSCs), and their effects on different developmental stages of megakaryopoiesis were investigated. In the transduced M1 cells, enforced expression of GATA-1 and Fli-1 was found to induce the megakaryocytic development, which was associated with the formation of megakaryocyte (Mk) and the increased expression of Mk specific genes c-Mpl and GPIX. In the transduced mouse HSCs, it was found that the expression of endogenous GATA-1, Fli-1 and FOG-1 was up-regulated throughout Mk differentiation; enforced expression of these transcription factors led to the significantly enhanced Mk development. Megakaryocytes over expressing GATA-1, Fli-1 and FOG-1 were characterized by the increased expression of various Mk-specific genes including GPIX, c-Mpl, platelet factor 4 (PF4), acetylcholinesterase (AChE) and NF-E2, an important transcription factor for terminal megakaryopoiesis; however, GATA-1, Fli-1 and FOG-1 displayed the different abilities in promoting the proliferation of hematopoietic cells and MK differentiation, as well as regulating other transcription factors involved in hematopoiesis. To further elucidate the role of the functional domains of Fli-1, various mutants of Fli-1 were also over expressed in mouse HSCs. The results demonstrated that first, the combination of the activation domain of Fli-1 and its Ets domain is required for early megakaryopoiesis but not sufficient for terminal megakaryopoiesis; second, DNA binding of Fli-1 was not the only requirement for early Mk enhancement, moreover, the interaction between Fli-1 and GATA-1 through the Ets domain and the resultant transcriptional synergy was the essential determinant for Fli?1 ability in Mk development. Taken together, the studies presented in this thesis provided strong in vitro evidence that GATA-1, Fli-1 and FOG-1 indeed play the critical roles in normal megakaryopoiesis.

Indiana University-Purdue University Indianapolis (IUPUI)
To cope with the high metabolic demands of the body during pregnancy, the maternal liver adapts by increasing its mass and size. This increase is proportional to the increase in total body weight during the course of gestation. The pregnancy-induced maternal liver growth is a result of both hepatocyte hypertrophy and hyperplasia. Microarray analysis of pregnant maternal livers shows markedly different gene expression profiles when compared to a non-pregnant state. Most interesting was the 2,500-fold up-regulation in the mRNA expression of Ascl1, a transcription factor responsible for the differentiation of neural progenitor cells into various neuronal types, during the second half of pregnancy. Our investigation aimed at (1) characterizing the identity of maternal hepatic Ascl1-expressing cells and (2) tracing the fate of Ascl1-expressing cells in the maternal liver during pregnancy. Timed pregnancies were generated and non-pregnant (NP) and pregnant maternal livers were harvested and analysed. To identify the maternal hepatic Ascl1-expressing cells we used the Ascl1GFP/+ reporter mouse line. NP and gestation day 15 (D15) maternal livers were immunostained for green fluorescent protein (GFP). The result shows that GFP-positive, Ascl1-expressing cells are hepatocyte-like cells, which are present in D15 maternal livers, but absent in NP livers. The Rosa26floxstopLacZ/ floxstopLacZ;Ascl1CreERT2/+ mouse line was used to trace the fate of Ascl1-expressing cells during pregnancy. LacZ staining of gestation day 13 (D13) and 18 (D18) maternal livers demonstrates that D13 hepatic Ascl1-expressing cells (labeled with LacZ) undergo hyperplasia to repopulate a large portion of D18 maternal livers. Furthermore, LacZ and HNF4α co-staining of D13 and D18 maternal livers shows the presence of two populations of LacZ-expressing cells: HNF4α+ population and HNF4α- population. HNF4α+ LacZ-expressing cells represent hepatocyte lineage cells that are derived from Ascl1-expressing cells. We observe that, towards the end of pregnancy, a considerable portion of the maternal liver is comprised of hepatocytes derived from Ascl1-expressing cells. Taken together, our preliminary study suggests that pregnancy induces maternal liver turnover via Ascl1-expressing cells.

Indiana University-Purdue University Indianapolis (IUPUI)
Fetal alcohol spectrum disorders (FASD) is the leading neurodevelopment deficit in children born to women who drink alcohol during pregnancy. The hippocampus and cortex are among brain regions vulnerable to alcohol-induced neurotoxicity, and are key regions underlying the cognitive impairment, learning and memory deficits shown in FASD individuals. Hippocampal and cortical neuronal differentiation and maturation are highly influenced by both intrinsic transcriptional signaling and extracellular cues. Epigenetic mechanisms, primarily DNA methylation and histone modifications, are hypothesized to be involved in regulating key neural development events, and are subject to alcohol exposure. Alcohol is shown to modify DNA methylation and histone modifications through altering methyl donor metabolisms. Recent studies in our laboratory have shown that alcohol disrupted genome-wide DNA methylation and delayed early embryonic development. However, how alcohol affects DNA methylation in fetal hippocampal and cortical development remains elusive, therefore, will be the theme of this study. We reported that, in a dietary alcohol-intake model of FASD, prenatal alcohol exposure retarded the development of fetal hippocampus and cortex, accompanied by a delayed cellular DNA methylation program. We identified a programed 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC) cellular and chromatic re-organization that was associated with neuronal differentiation and maturation spatiotemporally, and this process was hindered by prenatal alcohol exposure. Furthermore, we showed that alcohol disrupted locus-specific DNA methylation on neural specification genes and reduced neurogenic properties of neural stem cells, which might contribute to the aberration in neurogenesis of FASD individuals. The work of this dissertation suggested an important role of DNA methylation in neural development and elucidated a potential epigenetic mechanism in the alcohol teratogenesis.

Indiana University-Purdue University Indianapolis (IUPUI)
The hematopoietic system is highly proliferative, making hematopoietic stem and progenitor cells (HSPC) sensitive to radiation damage. Total body irradiation and chemotherapy, as well as the risk of radiation accident, create a need for countermeasures that promote recovery of hematopoiesis. Substantive damage to the bone marrow from radiation exposure results in the hematopoietic syndrome of the acute radiation syndrome (HS-ARS), which includes life-threatening neutropenia, lymphocytopenia, thrombocytopenia, and possible death due to infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow HSPC may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E2 (PGE2) is known to have pleiotropic effects on hematopoiesis, inhibiting apoptosis and promoting self-renewal of hematopoietic stem cells (HSC), while inhibiting hematopoietic progenitor cell (HPC) proliferation. We assessed the radiomitigation potential of modulating PGE2 signaling in a mouse model of HS-ARS. Treatment with the PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) at 24 hours post-irradiation resulted in increased survival of irradiated mice compared to vehicle control, with greater recovery in HPC number and colony-forming potential measured at 30 days post-irradiation. In a sublethal mouse model of irradiation, dmPGE2-treatment at 24 hours post-irradiation is associated with enhanced recovery of HSPC populations compared to vehicle-treated mice. Furthermore, dmPGE2-treatment may also act to promote recovery of the HSC niche through enhancement of osteoblast-supporting megakaryocyte (MK) migration to the endosteal surface of bone. A 2-fold increase in MKs within 40 um of the endosteum of cortical bone was seen at 48 hours post-irradiation in mice treated with dmPGE2 compared to mice treated with vehicle control. Treatment with the non-steroidal anti-inflammatory drug (NSAID) meloxicam abrogated this effect, suggesting an important role for PGE2 signaling in MK migration. In vitro assays support this data, showing that treatment with dmPGE2 increases MK expression of the chemokine receptor CXCR4 and enhances migration to its ligand SDF-1, which is produced by osteoblasts. Our results demonstrate the ability of dmPGE2 to act as an effective radiomitigative agent, promoting recovery of HSPC number and enhancing migration of MKs to the endosteum where they play a valuable role in niche restoration.

Indiana University-Purdue University Indianapolis (IUPUI)
The non-hematopoietic, stationary stromal cells located inside and surrounding skin-draining lymph nodes play a key role in regulating immune responses. We studied distinct populations of lymph node stromal cells from both human subjects and animal models in order to describe their phenotype and function. In the mouse model, we studied two distinct populations: an endothelial cell population expressing Ly51 and MHC-II, and an epithelial cell population expressing the epithelial adhesion molecule EpCAM. Analysis of intra-nodal and extra-nodal lymph node (CD45-) stromal cells through flow cytometry and qPCR provides a general phenotypic profile of the distinct populations. My research focused on the EpCAM+ epithelial cell population located in the fat pad surrounding the skin draining lymph nodes. The EpCAM+ population has been characterized by surface marker phenotype, anatomic location, and gene expression profile. This population demonstrates the ability to inhibit the activation and proliferation of both CD4 and CD8 T cells. This population may play a role in suppressing overactive inflammation and auto-reactive T cells that escaped thymic deletion. The other major arm of my project consisted of identifying a novel endothelial cell population in human lymph nodes. Freshly resected lymph nodes were processed into single cell suspensions and selected for non-hematopoietic CD45- stromal cells. The unique endothelial population expressing CD34 HLA-DR was then characterized and analyzed for anatomic position, surface marker expression, and gene profiles. Overall, these studies emphasize the importance of stationary lymph node stromal cells to our functioning immune systems, and may have clinical relevance to autoimmune diseases, inflammation, and bone marrow transplantation.

Indiana University-Purdue University Indianapolis (IUPUI)
In the US, more than 1 million burn injuries are reported annually. About 45,000 injuries due to fires and burns result in hospitalization and ten percent of these result in death every year. Advances in burn treatment have led to a reduction in mortality rate over the last decades. Since more patients are surviving the initial resuscitation phase even with very large areas of skin being burned away, wound care has become increasingly important to ensure continued patient survival and improvement. While currently a common treatment for third degree burn wounds, skin grafts have several drawbacks. The availability of donor sites for autografts may be limited, especially in incidences of extensive skin loss. The rejection associated with the use of allografts and xenografts may render them inadequate or undesirable. Even if a suitable graft is found, poor retention due to infection, hematoma, and low vascularity at the recipient site are other drawbacks associated with the use of skin grafts as a primary treatment for severe burn wounds. As such, research has been done into alternative treatments, which include but are not limited to artificial skin, cell therapy, and growth factor application. We propose the delivery of adipose derived stem cells (ASC) in combination with endothelial progenitor cells (EC) via Integra Dermal Regenerative Template (DRT) to promote faster graft vascularization and thus faster healing of wounds. Integra DRT is an acellular skin substitute that consists of a dermal layer composed of bovine collagen and chondroitin-6-sulfate glycosaminoglycan, and an "epidermal" layer, which consists of silicone polymer. This silicone layer is removed after the collagen matrix is adequately vascularized (usually takes 2-3 weeks), and then a thin layer autograft is applied to the top of the neo-dermis. ASC are derived from the stromal-vascular fraction (SVF) of adipose tissue and are a readily available, pluripotent, mesenchymal cell known to promote angiogenesis. They are being explored as a treatment for a myriad of diseases and conditions, including wound healing. In combination with ECs, they form stable microvessel networks in vitro and in vivo. In our work, we found that ASC+EC form stable microvessel networks when cultured on Integra DRT. Also, ASC and ASC+EC conditioned media promoted both survival and migration of human epidermal keratinocytes compared to control medium. In a full thickness wound healing model, using healthy NSG mice, the ASC+EC case showed a significantly higher rate of wound closure compared to control. Based on best linear unbiased estimates (BLUE), the difference between the healing rates of ASC alone treatment and the Control treatment group is -0.45 +/- 0.22 mm²/day (p=0.041), which is not less than 0.025 and thus not statistically significant (Bonferroni Adjusted). However, the BLUE for the difference between the ASC+EC group and the Control group healing rates is -0.55 +/- 0.28 mm²/day (p = 0.017<0.025, Bonferroni Adjusted), which is statistically significant. Histology revealed a significantly higher number of vessels compared to control in both ASC alone and ASC+EC case. CD31 staining revealed the presence of human vessels in ASC+EC treatment scaffolds. We conclude that the combination of ASC and EC can be used to accelerate healing of full-thickness wounds when delivered to site of the wound via Integra. This result is especially compelling due to the fact that the mice used were all healthy. Thus our treatment shows an improvement in healing rate even compared to normal wound healing.

Mice with human immune systems, generated by transplanting human CD34+ cells into immunodeficient mice, are essential tools for studying phenomena unique to the human immune system or poorly reproduced in existing mouse models. Human immune tolerance induction, function and autoimmunity have been poorly modeled in conventional murine models, which often have poor predictive value for preclinical development. Models that allow the study of human immune cells with the reproducibility and flexibility of small animal models are required. In our lab, humanized mouse models have been used to study preclinical protocols for human xenotolerance induction and to better understand the immunological underpinnings of human autoimmunity. These are each areas of critical unmet medical need. Xenotolerance-inducing protocols may be necessary to allow long-term survival of a transplanted pig organ in a human patient, and, with more than 113,000 Americans currently waiting for a life-saving organ, the need to expand the pool available for transplantation is urgent. Additionally, clinical options for patients with autoimmune diseases are limited. Currently, most patients with autoimmunity are only diagnosed after significant immune damage of target organs. Predicting who will develop autoimmunity – and who will not – before damage occurs would be very useful but is currently very difficult. Small animal models that can better help us understand how human autoimmunity develops could help us develop protocols for early detection and even prevention. We have developed a personalized immune model to study the development of an individual patient’s immune system in a transplanted mice to better understand immune abnormalities that underlie autoimmunity. We have used existing humanized mouse models to answer important questions related to human xenotolerance induction and autoimmunity, but in the studies described here we have worked to extend our capacity to use these models to study human T cell development and peripheral function. We would like to be able to study both the initial selection of T cell receptors (TCRs) in the thymus based on their ability to recognize antigen in the context of presenting MHC without reacting unduly to self-antigen, as well as in the periphery, where T cells interact with peripheral antigen-presenting cells (APCs) to maintain homeostasis and respond to antigen. First, we have incorporated TCR transgenesis into our humanized mouse models to allow greater precision in studying thymic selection in our humanized mice. Developing a system for this would allow us to study in greater detail mechanisms of human xenotolerance induction, including confirming that a swine thymus can support positive selection of T cells with human-restricted TCRs to allow a future xenotransplantation patient to maintain immune competence, while also robustly tolerizing human T cells expressing pig-reactive TCRs. We will also expand this system to study the thymic selection of human T cells with autoreactive TCRs to better understand mechanisms of central tolerance and understand how they fail in autoimmunity. Finally, while processes of thymic selection are critical for human T cell development and function, peripheral interactions also have a large impact on human T cell function and homeostasis and may contribute to the development of autoimmunity. For these interactions to occur appropriately requires robust engraftment and reconstitution of APCs, especially of myeloid and B cell lineages, in transplanted immunodeficient mice. APC reconstitution tends to be suboptimal in humanized mice and is even more so in mice transplanted with patient-derived CD34+ cells. Better characterization of human APC populations and their progenitors could allow us to develop approaches to improve long-term human APC reconstitution in patient-derived humanized mice, allowing us to more fully model patient peripheral T cell function.

Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
Machado Joseph disease (MJD), or spinocerebellar ataxia type 3, is a neurodegenerative disease caused by a cytosine-adenine-guanine (CAG) trinucleotide expansion in the coding region of the ATXN3 gene, which is translated into the mutant ataxin-3 protein. The mutated protein is prone to misfold and aggregate as neuronal cytoplasmic and intranuclear inclusions, leading to defects in motor neuron activity mainly in the cerebellum, brainstem, and spinocerebellar tracts, which results in progressive impairment of motor coordination in patients. Despite current efforts and advances, MJD remains an incurable fatal disease, with no disease-modifying treatment available, so far. This gap may be explained by the inexistence of consistent and reliable disease models, and consequently, the lack of knowledge of the underlying pathological mechanisms of MJD and reliable tools for pre-clinical therapies development. Thus, the generation of more suitable and accurate disease models is an urgent need. Human-induced pluripotent stem cells (iPSCs) hold great potential for modeling MJD, representing accurate in vitro models of human patient-specific neurons, while providing evidence on the disease’s natural history progression. Moreover, mouse models are consensually preferred for the study of MJD, as they share major anatomical and physiological features of the disease in humans. Among them, YACMJD transgenic mouse model has, integrated in its genome, the full gene of human ATXN3, and thus stands out for better recapitulating protein expression and neurological motor phenotype of MJD. The present study aimed to characterize the phenotype of 1) iPSCs-derived neuronal cultures obtained from MJD patients and control individuals, and 2) the YACMJD transgenic mice model. In this work, several markers of the pathological mechanisms of MJD were analyzed through immunocytochemistry and western blot. Our data demonstrated that iPSCs-derived cells were successfully differentiated into heterogeneous cultures of neural and glial cells, while YACMJD homozygous mice displayed an early progressive neurological deterioration. In summary, the results suggest that both models can be used as powerful tools for a deep understanding of MJD pathogenesis, discovery of disease biomarkers, and pre-clinical studies for the development of effective therapies.
A doença de Machado Joseph (DMJ), ou ataxia espinocerebelosa tipo 3, é uma doença neurodegenerativa causada pela expansão do trinucleótido citosina-adenina-guanina (CAG) na região codificadora do gene ATXN3, que leva à formação da proteína mutada ataxina-3. A proteína mutada tende a adquirir uma conformação incorreta, e agregar-se sob a forma de inclusões neuronais citoplasmáticas e intranucleares, causando uma disfunção na atividade dos neurónios motores, principalmente no cerebelo, tronco cerebral e tratos espinocerebelosos, o que resulta num comprometimento progressivo da coordenação motora nos doentes.Apesar dos esforços e avanços até à data, a DMJ continua a ser uma doença fatal e sem cura, não dispondo de nenhum tratamento. Esta lacuna pode ser explicada devido à falta de modelos de doença consistentes e confiáveis e, consequentemente, à falta de conhecimento dos mecanismos patológicos subjacentes à DMJ, bem como de boas plataformas para o desenvolvimento de estudos pré-clínicos. Assim, a geração de modelos de doença mais adequados e precisos é de urgente necessidade. As células estaminais humanas pluripotentes induzidas (iPSCs) têm um grande potencial para modelar a DMJ, como modelos in vitro de neurónios específicos derivados de doentes humanos, permitindo estudar a progressão da história natural da doença. Além disso, os modelos animais de ratinho são preferidos, de forma consensual, para o estudo da DMJ, pois compartilham as principais características anatómicas e fisiológicas da doença em humanos. Entre eles, o modelo de ratinho transgénico YACMJD tem, integrado em seu genoma, o gene completo da ATXN3 humana e, por este motivo destaca-se por recapitular mais fielmente a expressão da proteína mutada e o fenótipo neurológico motor da DMJ.O presente estudo teve como objetivo caracterizar o fenótipo de 1) culturas neuronais derivadas de iPSCs obtidas de doentes e indivíduos controle, e 2) o modelo de ratinho transgénico YACMJD. Neste trabalho, diversos marcadores de mecanismos patológicos da DMJ foram analisados pelas técnicas de imunocitoquímica e western blot. Os nossos dados demonstraram que as células derivadas de iPSCs foram diferenciadas com sucesso em culturas heterogéneas de células neurais e da glia, enquanto que os ratinhos homozigóticos YACMJD exibem uma deterioração neurológica progressiva precoce.Em suma, os resultados sugerem que ambos os modelos podem ser usados como poderosas ferramentas para a compreensão profunda dos mecanismos de patogénese da DMJ, a descoberta de biomarcadores de doença, e em estudos pré-clínicos para o desenvolvimento de terapias eficazes.
Outro - This work was funded by the ERDF through the Regional Operational Program Center 2020, Competitiveness Factors Operational Program (COMPETE 2020, POCI) and National Funds through FCT (Foundation for Science and Technology) - BrainHealth2020 projects (CENTRO-01-0145-FEDER-000008), UID/NEU/04539/2019, ViraVector (CENTRO-01-0145-FEDER-022095), CortaCAGs (PTDC/NEU-NMC/0084/2014|POCI-01-0145-FEDER-016719), SpreadSilencing POCI-01-0145-FEDER-029716, Imagene POCI-01-0145-FEDER-016807, CancelStem POCI-01-0145-FEDER-016390, POCI-01-0145-FEDER-030737, POCI-01-0145-FEDER-032309, as well as SynSpread, ESMI and ModelPolyQ under the EU Joint Program - Neurodegenerative Disease Research (JPND), the last two co-funded by the European Union H2020 program, GA No.643417; by National Ataxia Foundation (USA), the American Portuguese Biomedical Research Fund (APBRF) and the Richard Chin and Lily Lock Machado-Joseph Disease Research Fund.

Law, Sau Kwan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 111-117).
Abstract also in Chinese.
Thesis Committee --- p.i
Acknowledgements --- p.ii
Contents --- p.iii
Abstract --- p.vii
論文摘要 --- p.x
Abbreviations --- p.xi
List of Figures --- p.xiii
List of Tables --- p.xxiii
Chapter CHAPTER ONE --- INTRODUCTION
Chapter 1.1 --- Embryonic Stem (ES) Cells
Chapter 1.1.1 --- Characteristics of ES Cells l
Chapter 1.1.2 --- Therapeutic Potential of ES Cells --- p.3
Chapter 1.1.3 --- Myocardial Infarction and ES cells-derived Cardiomyocytes --- p.4
Chapter 1.1.4 --- Current Hurdles of Using ES cells-derived Cardiomyocytes for Research and Therapeutic Purposes --- p.6
Chapter 1.2 --- Transcription Factors for Cardiac Development
Chapter 1.2.1 --- GATA-binding Protein 4 (GATA-4) --- p.8
Chapter 1.2.2 --- Myocyte Enhancer Factor 2C (MEF2C) --- p.10
Chapter 1.2.3 --- "NK2 Transcription Factor Related, Locus 5 (Nkx2.5)" --- p.11
Chapter 1.2.4 --- Heart and Neural Crest Derivatives Expressed 1 /2 (HANDI/2) --- p.11
Chapter 1.2.5 --- T-box Protein 5 (Tbx5) --- p.13
Chapter 1.2.6 --- Serum Response Factor (SRF) --- p.14
Chapter 1.2.7 --- Specificity Protein 1 (Spl) --- p.15
Chapter 1.2.8 --- Activator Protein 1 (AP-1) --- p.16
Chapter 1.3 --- Reactive Oxygen Species (ROS)
Chapter 1.3.1 --- Cellular Production of ROS --- p.18
Chapter 1.3.2 --- Maintenance of Redox balance --- p.18
Chapter 1.3.3 --- Redox Signaling --- p.19
Chapter 1.4 --- Nitric Oxide (NO) and NO Signaling --- p.20
Chapter 1.5 --- Aims of the Study --- p.22
Chapter CHAPTER TWO --- MATERIALS AND METHODS
Chapter 2.1 --- Mouse Embryonic Fibroblast (MEF) Culture
Chapter 2.1.1 --- Derivation of MEF --- p.23
Chapter 2.1.2 --- Maintenance of MEF Culture --- p.24
Chapter 2.1.3 --- Irradiation of MEF --- p.25
Chapter 2.2 --- Mouse ES Cell Culture
Chapter 2.2.1 --- Maintenance of Undifferentiated Mouse ES Cell Culture --- p.26
Chapter 2.2.2 --- Differentiation of Mouse ES Cells --- p.26
Chapter 2.2.3 --- Exogenous addition of hydrogen peroxide (H2O2) and NO --- p.27
Chapter 2.3 --- ROS Localization Study
Chapter 2.3.1 --- Frozen Sectioning --- p.28
Chapter 2.3.2 --- Confocal microscopy for ROS detection --- p.28
Chapter 2.4 --- Intracellular ROS Measurement
Chapter 2.4.1 --- "Chemistry of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA)" --- p.29
Chapter 2.4.2 --- Flow Cytometry for ROS Measurement --- p.29
Chapter 2.5 --- Gene Expression Study
Chapter 2.5.1 --- Primer Design --- p.30
Chapter 2.5.2 --- RNA Extraction --- p.31
Chapter 2.5.3 --- DNase Treatment --- p.32
Chapter 2.5.4 --- Reverse Transcription --- p.32
Chapter 2.5.5 --- Quantitative Real Time PCR --- p.33
Chapter 2.5.6 --- Quantification of mRNA Expression --- p.34
Chapter 2.6 --- Protein Expression Study
Chapter 2.6.1 --- Total Protein Extraction --- p.34
Chapter 2.6.2 --- Nuclear and Cytosolic Protein Extraction --- p.35
Chapter 2.6.3 --- Measurement of Protein Concentration --- p.36
Chapter 2.6.4 --- De-sumoylation Assay --- p.36
Chapter 2.6.5 --- De-phosphorylation Assay --- p.37
Chapter 2.6.6 --- De-glycosylation Assay --- p.38
Chapter 2.6.7 --- Western Blot --- p.39
Chapter 2.7 --- Statistical Analysis --- p.41
Chapter CHAPTER THREE --- RESULTS
Chapter 3.1 --- Study of Endogenous ROS
Chapter 3.1.1 --- Level and Distribution of Endogenous ROS --- p.47
Chapter 3.1.2 --- Quantification of intracellular ROS --- p.48
Chapter 3.2 --- Effect of Exogenous Addition of Nitric Oxide (NO) on Cardiac Differentiation
Chapter 3.2.1 --- Beating Profile of NO-treated Embryoid Bodies (EBs) --- p.50
Chapter 3.3 --- Effect of Exogenous Addition of H2O2 on Cardiac Differentiation
Chapter 3.3.1 --- Beating Profile of H2O2-treated EBs --- p.51
Chapter 3.3.2 --- mRNA Expression of Cardiac Structural Genes --- p.52
Chapter 3.3.3 --- Protein Expression of Cardiac Structural Genes --- p.54
Chapter 3.3.4 --- mRNA Expression of Cardiac Transcription Factors --- p.58
Chapter 3.3.5 --- Protein Expression of Cardiac Transcription Factors --- p.67
Chapter 3.3.6 --- Post-translational Modifications of Cardiac Transcription Factors --- p.74
Chapter 3.3.7 --- Translocation of Cardiac Transcription Factors --- p.89
Chapter CHAPTER FOUR --- DISCUSSION
Chapter 4.1 --- Changes in the Level of Endogenous ROS During Cardiac Differentiation of Mouse ES Cells --- p.96
Chapter 4.2 --- H2O2 and NO Have Opposite Effects Towards Cardiac Differentiation --- p.97
Chapter 4.3 --- Exogenous Addition of H2O2 Advances Differentiation of Mouse ES Cells into Cardiac Lineage --- p.99
Chapter 4.4 --- Possible Role of H2O2 in Mediating Cardiac Differentiation of Mouse ES Cells --- p.103
Chapter 4.5 --- Future Directions --- p.108
Conclusions --- p.110
References --- p.111

Ng, Sze Ying.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 110-124).
Abstract also in Chinese.
Thesis Committee --- p.i
Acknowledgements --- p.ii
Contents --- p.iii
Abstract --- p.vii
論文摘要 --- p.x
Abbreviations --- p.xi
List of Figures --- p.xiii
List of Tables --- p.xvii
Chapter CHAPTER ONE --- INTRODUCTION --- p.1
Chapter 1.1 --- Embryonic Stem Cells (ESCs) --- p.1
Chapter 1.1.1 --- What are ESCs and the characteristics of ESCs --- p.1
Chapter 1.1.1.1 --- Pluripotent markers --- p.2
Chapter 1.1.1.2 --- Germ layers' markers --- p.3
Chapter 1.1.2 --- Mouse ESCs (mESCs) --- p.4
Chapter 1.1.2.1 --- mESCs co-culture with mitotically inactivated mouse embryonic fibroblast (MEF) feeder layers --- p.4
Chapter 1.1.2.2 --- Feeder free mESCs --- p.4
Chapter 1.1.3 --- Promising uses of ESCs and their shortcomings --- p.5
Chapter 1.1.4 --- Characteristics of ESC-derived cardiomyocytes (ESC-CMs) --- p.6
Chapter 1.2 --- Cardiovascular diseases (CVD) --- p.7
Chapter 1.2.1 --- Background --- p.7
Chapter 1.2.2 --- Current treatments --- p.8
Chapter 1.2.3 --- Potential uses of ESC-CMs for basic science research and therapeutic purposes --- p.9
Chapter 1.2.4 --- Current hurdles in application of ESC-CMs for clinical uses --- p.10
Chapter 1.3 --- Cardiac gene markers --- p.13
Chapter 1.3.1 --- Atrial-specific --- p.13
Chapter 1.3.2 --- Ventricular-specific --- p.19
Chapter 1.4 --- Lentiviral vector-mediated gene transfer --- p.27
Chapter 1.5 --- Cell cycle in ESCs --- p.29
Chapter 1.5.1 --- Cell cycle --- p.29
Chapter 1.5.2 --- Characteristics of cell cycle in ESCs --- p.30
Chapter 1.6 --- Potassium (K+) channels --- p.31
Chapter 1.6.1 --- Voltage gated potassium (Kv) channels --- p.32
Chapter 1.6.2 --- Role of Kv channels in maintenance of membrane potential --- p.32
Chapter 1.7 --- Objectives and significances --- p.33
Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.35
Chapter 2.1 --- Mouse embryonic fibroblast (MEF) culture --- p.35
Chapter 2.1.1 --- Derivation of MEF --- p.3 5
Chapter 2.1.2 --- MEF culture --- p.37
Chapter 2.1.3 --- Irradiation of MEF --- p.37
Chapter 2.2 --- mESC culture and their differentiation --- p.38
Chapter 2.2.1 --- mESC culture --- p.38
Chapter 2.2.2 --- Differentiation of mESCs --- p.39
Chapter 2.3 --- Subcloning --- p.40
Chapter 2.3.1 --- Amplification of Irx4 --- p.40
Chapter 2.3.2 --- Purification of DNA products --- p.41
Chapter 2.3.3 --- Restriction enzyme digestion --- p.42
Chapter 2.3.4 --- Ligation of Irx4 with iDuet101A vector --- p.43
Chapter 2.3.5 --- Transformation of ligation product into competent cells --- p.43
Chapter 2.3.6 --- Small scale preparation of bacterial plasmid DNA --- p.44
Chapter 2.3.7 --- Confirmation of positive clones by restriction enzyme digestion --- p.45
Chapter 2.3.8 --- DNA sequencing of the cloned plasmid DNA --- p.45
Chapter 2.3.9 --- Large scale preparation of target recombinant expression vector --- p.45
Chapter 2.4 --- Lentiviral vector-mediated gene transfer to mESCs --- p.47
Chapter 2.4.1 --- Lentivirus packaging --- p.47
Chapter 2.4.2 --- Lentivirus titering --- p.48
Chapter 2.4.3 --- Multiple transduction to mESCs --- p.48
Chapter 2.4.4 --- Hygromycin selection on mESCs --- p.49
Chapter 2.5 --- Selection of stable clone --- p.49
Chapter 2.5.1 --- Monoclonal establishment and clone selection --- p.49
Chapter 2.6 --- Differentiation of cell lines after selection --- p.50
Chapter 2.7 --- Gene expression study on control and Irx4-overexpressed mESC lines --- p.50
Chapter 2.8 --- Analysis of mESCs at different phases of the cell cycle --- p.55
Chapter 2.8.1 --- Go/Gi and S phase synchronization --- p.55
Chapter 2.8.2 --- Cell cycle analysis by propidium iodide (PI) staining followed by flow cytometric analysis --- p.55
Chapter 2.8.3 --- Gene expression study by qPCR of Kv channel isoforms --- p.56
Chapter 2.8.4 --- Membrane potential measurement by membrane potential-sensitive dye followed by flow cytometry --- p.57
Chapter 2.9 --- Apoptotic study --- p.58
Chapter 2.10 --- Determination of pluripotent characteristic of mESCs --- p.59
Chapter 2.10.1 --- Expression of germ layers' markers by qPCR --- p.59
Chapter 2.10.2 --- Differentiation by hanging drop method and suspension method --- p.61
Chapter CHAPTER THREE --- RESULTS --- p.62
Chapter 3.1 --- mESC culture --- p.62
Chapter 3.1.1 --- Cell colony morphology of feeder free mESCs --- p.62
Chapter 3.2 --- Subcloning --- p.63
Chapter 3.2.1 --- PCR cloning of Irx4 --- p.63
Chapter 3.2.2 --- Restriction digestion on iDuet101A --- p.64
Chapter 3.2.3 --- Ligation of Irx4 to iDuet101A backbone --- p.66
Chapter 3.2.4 --- Confirmation of successful ligation --- p.67
Chapter 3.3 --- Lentivirus packaging --- p.68
Chapter 3.3.1 --- Transfection --- p.68
Chapter 3.4 --- Multiple transduction of mESCs and hygromycin selection of positively-transduced cells --- p.69
Chapter 3.5 --- FACS --- p.70
Chapter 3.6 --- Irx4 and iduet clone selection --- p.71
Chapter 3.7 --- Characte rization of mESCs after clone selection --- p.74
Chapter 3.7.1 --- Immunostaining of pluripotent and differentiation markers --- p.74
Chapter 3.8 --- Differentiation of cell lines after selection --- p.77
Chapter 3.8.1 --- Size of EBs of the cell lines during differentiation --- p.77
Chapter 3.9 --- Gene expression study by qPCR --- p.79
Chapter 3.10 --- Kv channel expression and membrane potential of mESCs at Go/Gi phase and S phases --- p.84
Chapter 3.10.1 --- Expression of Kv channels subunits at G0/Gi phase and S phase --- p.86
Chapter 3.10.2 --- Membrane potential at Go/Gi phase and S phase --- p.87
Chapter 3.11 --- Effects of TEA+ on feeder free mESCs --- p.89
Chapter 3.11.1 --- Apoptotic study --- p.89
Chapter 3.11.2 --- Expression of germ layers´ة markers --- p.91
Chapter 3.11.3 --- Embryo id bodies (EBs) measurement after differentiation --- p.92
Chapter CHAPTER FOUR --- DISCUSSION --- p.95
Chapter 4.1 --- Effect of overexpression of Irx4 on the cardiogenic potential of mESCs --- p.95
Chapter 4.2 --- Role of Kv channels in maintaining the chacteristics of mESCs --- p.99
Chapter 4.2.1 --- Inhibition of Kv channels led to a redistribution of the proportion of cells in different phases of the cell cycle: importance of Kv channels in cell cycle progression in native ESCs --- p.99
Chapter 4.2.2 --- Inhibition of Kv channels led to a loss of pluripotency at molecular and functional levels: importance of Kv channels in the fate determination of mESCs --- p.102
Chapter 4.3 --- Insights from the present investigation on the future uses of ESCs --- p.105
Conclusions --- p.108
References --- p.110

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the embryonic neural development and adult neurogenesis. But the effects of 5-HT on stem cells are not fully known. In this study, the effects and underlying signal pathways of 5- HT on proliferation and neural differentiation of mouse embryonic stem (ES) cells, neural progenitor (NP) cell line C 17.2 and embryonic neural stem (NS) cells were explored. Molecular analysis, immunostaining and western blotting revealed that NP/NB cells expressed the rate-limiting enzyme tryptophan hydroxylase (TPH) and produced endogenous 5-HT. While mouse ES cells showed no expression of TPH. Quantitative PCR demonstrated that ES cells and NPINS cells expressed majority of 5-HT receptor sUbtypes. In serum free propagation culture, WST1, BrdU incorporation and neural colony forming cell assay demonstrated that 5-HT enhanced proliferation of ES cells and NPINS cells in a dose-dependent manner. Tryptophan hydroxylase (TPH) inhibitor para-chlorophenylalanine (PCPA) which can inhibit biosynthesis of endogenous 5-HT decreased viability of mouse NP/NS cells. Mouse ES cells derived embryoid bodies (EB) and NS/NP cells were subjected to neural induction in serum-free medium with and without 5-HT or PCPA. On day 8 of EB cultures, immunofluorescence staining displayed a less percentage of SSEA-1+ cells derived from cultures supplemented with 5-HT. Nestin positivity are comparable. Quantitative PCR analysis suggested that supplement of 5-HT in EB culture inhibit neural differentiation of ES cells and induce mesodermal commitment. On day 21 of ES cells neural induction, compared to cultures without 5-HT treatment, a significantly less number of ß-tubulin III+ neurons, GEAP+ astrocytes and GaIC+ oligodendrocytes were noted in 5-HT -supplemented cultures. For NS/NP cells, the inhibitory effects of 5-HT on neuronal and oligodendrocytic commitment were also observed. And the application of PCPA exerted a promoting effect on neural differentiation of NS cells. Manipulating 5-HT level can affect the expression level of key genes which involved in 5-HT metabolism. ES and NS/NP cells treated with 5-HT showed decreased production of endogenous reactive oxygen species (ROS). 5-HT demonstrated a significant anti-apoptotic effect on NP cells and this antiapoptotic effect may be mediated by up-regulated expression of anti-apoptotic gene Bel- 2. Whole genome cDNA microarray analysis and quantitative RT-PCR revealed that notch signal pathway was involved in mediating the biological effects of 5-HT. Western blotting further confirmed that 5-HT treatment up-regulated the protein level of NICD and notch downstream effectors Hes-l and Hes-5. Finally, the therapeutic effects of ES cell-derived neural cells were testified in a mouse model of global ischemia. Two weeks post-transplantation, BrdU labeled ES cell-derived neural cells survived and migrated throughout brain parenchyma. A majority of transplanted cells remained nestin positive. The cognitive functions of cell transplanted groups showed significant recovery compared with untransplanted arms, but no significant difference was observed between transplanted groups treated with and without 5-HT. Taken together, data of this study indicated 5-HT play an important role in neural development and ES cell-derived neural cells might be applicable in the treatment of stroke.
Li, Jin.
"November 2011."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 195-241).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
Abstracts in English.
ACKNOWLEDGEMENTS --- p.i
LIST OF PUBLICATIONS --- p.ii
ABSTRACT --- p.iii
ABSTRACT [in Chinese] --- p.v
TABLE OF CONTENT --- p.vi
LISTS OF FLOWCHARTS --- p.xii
LISTS OF FIGURES --- p.xiii
LIST OF TABLES --- p.xvi
LIST OF EQUIPMENTS --- p.xvii
LIST OF ABBREVATIONS --- p.xvii
Chapter Chapter1 --- Introduction --- p.1
Chapter 1.1 --- Central nervous system disorder --- p.1
Chapter 1.1.1 --- Stroke --- p.1
Chapter 1.1.2 --- Spinal cord injuries --- p.4
Chapter 1.1.3 --- Parkinson's disease --- p.6
Chapter 1.1.4 --- Amyotrophic Lateral Sclerosis --- p.8
Chapter 1.2 --- Stem cell therapy --- p.10
Chapter 1.2.1 --- General considerations in stem cell therapy --- p.11
Chapter 1.2.2 --- Stem cell therapy for stroke --- p.11
Chapter 1.2.3 --- Stem cell therapy for spinal cord injury --- p.15
Chapter 1.2.4 --- Stem cell therapy for Parkinson's disease --- p.16
Chapter 1.2.5 --- Stem cell therapy for ALS --- p.18
Chapter 1.3 --- Stem cells --- p.20
Chapter 1.3.1 --- Embryonic stem cells --- p.21
Chapter 1.3.1.1 --- Derivation and characterization --- p.21
Chapter 1.3.1.2 --- Biology of ES cells --- p.21
Chapter 1.3.1.2.1 --- Pluripotency of ES cells --- p.21
Chapter 1.3.1.2.2 --- Differentiation of ES cells to multiple lineages --- p.24
Chapter 1.3.1.2.2.1 --- Ectodermal differentiation --- p.25
Chapter 1.3.1.2.2.2 --- Mesodermal differentiation --- p.27
Chapter 1.3.1.2.2.3 --- Endodermal differentiation --- p.28
Chapter 1.3.2 --- Neural stem cells --- p.30
Chapter 1.3.2.1 --- Derivation and characterization --- p.30
Chapter 1.3.2.2 --- Biology of NS cells --- p.32
Chapter 1.3.3 --- Induced pluripotent stem cells --- p.34
Chapter 1.3.4 --- Mesenchymal stem cells --- p.35
Chapter 1.4 --- Serotonin (5-HT) --- p.36
Chapter 1.4.1 --- Distribution --- p.37
Chapter 1.4.2 --- Metabolism --- p.37
Chapter 1.4.3 --- Biological effects of 5-HT --- p.38
Chapter 1.4.4 --- Serotonin receptor subtypes and receptor signal transduction pathways --- p.40
Chapter Chapter2 --- Aim --- p.43
Chapter 2.1 --- Hypothesis and study objectives --- p.43
Chapter Chapter3 --- Materials and Methods --- p.49
Chapter 3.1 --- Chemicals and Reagents --- p.49
Chapter 3.1.1 --- Cell culture --- p.49
Chapter 3.1.2 --- Serotonin, serotonin receptor subtypes specific agonists/antagonists and drugs that regulate serotonin metabolism --- p.51
Chapter 3.1.3 --- Cell proliferation assay --- p.52
Chapter 3.1.4 --- Cell apoptosis assay --- p.52
Chapter 3.1.5 --- Immunohistochemistry and staining --- p.52
Chapter 3.1.6 --- Western blotting --- p.55
Chapter 3.1.7 --- Molecular biology --- p.56
Chapter 3.1.8 --- Whole genome cDNA micro array --- p.58
Chapter 3.1.9 --- MAO activity assay --- p.58
Chapter 3.1.10 --- Endogenous ROS production assay --- p.58
Chapter 3.2 --- Consumable --- p.58
Chapter 3.3 --- Cells --- p.60
Chapter 3.3.1 --- Feeder cell --- p.60
Chapter 3.3.1.1 --- Mouse embryonic fibroblasts --- p.60
Chapter 3.3.2 --- ES cells --- p.61
Chapter 3.3.2.1 --- ES cell D3 --- p.61
Chapter 3.3.2.2 --- ES cell-E14TG2a --- p.61
Chapter 3.3.3 --- NS cells --- p.61
Chapter 3.3.3.1 --- Neural progenitor cells line C172 --- p.61
Chapter 3.3.3.2 --- Mouse embryonic neural stem cells --- p.61
Chapter 3.4 --- In-house prepared solutions --- p.62
Chapter 3.4.1 --- Stock solution ofInsulin, Transferrin, Selentine (ITS) Supplement --- p.63
Chapter 3.4.2 --- Gelatin solution 01% --- p.62
Chapter 3.4.3 --- Paraformaldehyde solution 4% (PFA) --- p.62
Chapter 3.4.4 --- Tritox X-lOO solution 03% --- p.63
Chapter 3.4.5 --- Popidium iodide solution 1 ug/ml (PI) --- p.63
Chapter 3.4.6 --- Poly-L-ornithine solution --- p.63
Chapter 3.4.7 --- Laminin solution --- p.64
Chapter 3.4.7 --- MEF Maintenance medium --- p.64
Chapter 3.4.9 --- Cryopreservation Media for MEF and C172 (2X) --- p.64
Chapter 3.4.10 --- Cryopreservation Media for mouse ES cell (2X) --- p.65
Chapter 3.4.11 --- Cryopreservation Media for mouse NS cell (2X) --- p.65
Chapter 3.4.12 --- Serum based maintenance medium for C172 --- p.65
Chapter 3.4.13 --- Serum free maintenance medium for C172 --- p.66
Chapter 3.4.14 --- Serum-based propagation medium for ES cells --- p.66
Chapter 3.4.15 --- Serum-free propagation medium forES cells --- p.67
Chapter 3.4.16 --- Serum-free induction medium for ES cells --- p.67
Chapter 3.4.16.1 --- Serum-free induction medium I --- p.67
Chapter 3.4.16.2 --- Serum-free induction medium II --- p.68
Chapter 3.4.16.3 --- Serum-free induction medium III --- p.68
Chapter 3.4.17 --- Tris-HCl (1 M), pH 74 --- p.68
Chapter 3.4.18 --- Tris-HCl (1 M), pH 87 --- p.69
Chapter 3.4.19 --- Tris-HCI (1 M), pH 69 --- p.69
Chapter 3.4.20 --- APS 10% (wt/vol) --- p.69
Chapter 3.4.21 --- Protease inhibitor (10X) --- p.70
Chapter 3.4.22 --- RIPA --- p.70
Chapter 3.4.23 --- Resolving buffer (8X) --- p.70
Chapter 3.4.24 --- Stacking buffer (4X) --- p.71
Chapter 3.4.25 --- Protein running buffer (lOX) --- p.71
Chapter 3.4.26 --- Transfer buffer (10X) --- p.72
Chapter 3.4.27 --- Transfer buffer (IX) --- p.72
Chapter 3.4.28 --- Blocking buffer (lOX) --- p.72
Chapter 3.4.29 --- TBS (10X) --- p.73
Chapter 3.4.30 --- TBS-T (IX) --- p.73
Chapter 3.4.31 --- Stacking gel --- p.73
Chapter 3.4.32 --- Resolving gel --- p.74
Chapter 3.5 --- Methods --- p.75
Chapter 3.5.1 --- Cell culture --- p.75
Chapter 3.5.1.1 --- Preparation of acid washed cover slips --- p.75
Chapter 3.5.1.2 --- Preparation of gelatinized culture wares --- p.75
Chapter 3.5.1.3 --- Poly-L-omithine and laminin coating --- p.76
Chapter 3.5.1.4 --- Thawing cryopreserved cells --- p.76
Chapter 3.5.1.5 --- Passage of culture --- p.77
Chapter 3.5.1.5 --- 6 Cell count --- p.78
Chapter 3.5.1.7 --- Cytospin --- p.78
Chapter 3.5.1.8 --- Trypan blue dye exclusion test --- p.78
Chapter 3.5.1.9 --- Cryopreservation --- p.79
Chapter 3.5.1.10 --- Derivation and culture of mouse embryonic fibroblasts (MEF) --- p.79
Chapter 3.5.1.11 --- Propagation of ES cells in serum-based/free medium --- p.81
Chapter 3.5.1.12 --- Neural differentiation ofES cells --- p.83
Chapter 3.5.1.13 --- Propagation ofNP cell C172 in serum-based or serum-free medium --- p.84
Chapter 3.5.1.14 --- Neural differentiation ofC172 --- p.85
Chapter 3.5.1.15 --- Derivation and propagation of embryonic NS cells --- p.85
Chapter 3.5.1.13 --- Neural differentiation of embryonic NS cells --- p.86
Chapter 3.5.1.17 --- BrdU labeling of the ES cells derived products --- p.87
Chapter 3.5.2 --- Cell proliferation assay --- p.87
Chapter 3.5.2.1 --- Cell morphology --- p.87
Chapter 3.5.2.2 --- WST-1 assay --- p.88
Chapter 3.5.2.3 --- BrdU incorporation assay --- p.88
Chapter 3.5.2.4 --- NCFC assay --- p.89
Chapter 3.5.3 --- Conventional and quantitative RT-PCR --- p.89
Chapter 3.5.3.1 --- RNA extraction --- p.89
Chapter 3.5.3.2 --- RNA quantitation --- p.90
Chapter 3.5.3.3 --- Reverse Transcription ofthe First Strand complementary DNA --- p.90
Chapter 3.5.3.4 --- Polymerase chain reaction --- p.91
Chapter 3.5.3.5 --- RNA Integrity Check --- p.91
Chapter 3.5.3.6 --- Electrophoresis and visualization of gene products --- p.91
Chapter 3.5.3.7 --- Real-time quantitative PCR --- p.92
Chapter 3.5.4 --- Microarray --- p.94
Chapter 3.5.5 --- Immunofluoresent staining --- p.94
Chapter 3.5.6 --- Western blot --- p.95
Chapter 3.5.6.1 --- Harvesting samples --- p.95
Chapter 3.5.6.2 --- Protein extraction --- p.96
Chapter 3.5.6.3 --- Protein quantification --- p.96
Chapter 3.5.6.4 --- SDS-PAGE --- p.97
Chapter 3.5.6.5 --- Wet transfer of protein to PVDF membrane --- p.97
Chapter 3.5.6.6 --- Blocking the membrane --- p.97
Chapter 3.5.6.7 --- Immunoblotting --- p.97
Chapter 3.5.6.8 --- Signal detection --- p.98
Chapter 3.5.7 --- Cell apoptosis assay --- p.98
Chapter 3.5.7.1 --- ANNEXINV-FITC apoptosis detection --- p.98
Chapter 3.5.7.2 --- TUNEL --- p.99
Chapter 3.5.8 --- Endogenous ROS assay --- p.100
Chapter 3.5.9 --- In vivo studies --- p.101
Chapter 3.5.9.1 --- Induction of cerebral ischemia in mice --- p.101
Chapter 3.5.9.2 --- Transplantation --- p.101
Chapter 3.5.9.3 --- Assessment of learning ability and memory --- p.102
Chapter 3.5.10 --- Histological analysis --- p.103
Chapter 3.5.10.1 --- Animal sacrifice for brain harvest --- p.103
Chapter 3.5.10.2 --- Cryosectioning --- p.103
Chapter 3.5.10.3 --- Haematoxylin and eosin staining --- p.104
Chapter 3.6 --- Data analysis --- p.104
Chapter Chapter4 --- Results --- p.113
Chapter 4.1 --- Expression profile of 5-HT receptors and metablism of endogenous 5-HT --- p.113
Chapter 4.1.1 --- Expression profiles of 5-HT receptors in stem cells --- p.113
Chapter 4.1.2 --- Biosynthesis of endogenous 5-HT --- p.115
Chapter 4.2 --- Effects of 5-HT on proliferation of mouse ES cells and NS cells --- p.115
Chapter 4.2.1 --- Effects of 5-HT on proliferation ofES cells --- p.115
Chapter 4.2.2 --- Effects of 5-HT on proliferation ofNP and NS cells --- p.117
Chapter 4.3 --- Effects of 5-HT on differentiation of mouse ES cells and NS cells --- p.119
Chapter 4.3.1 --- Neural differentiation ofES cells --- p.119
Chapter 4.3.2 --- Effects of 5-HT on differentiation ofES cells --- p.119
Chapter 4.3.3 --- Neural differentiation ofNP and NS cells --- p.120
Chapter 4.3.4 --- Effects of 5-HT on differentiation ofNP and NS cells --- p.121
Chapter 4.4 --- 5-HT metabolism in mouse ES cells and NS cells --- p.122
Chapter 4.4.1 --- Expression of key 5-HT metablic genes in stem cells --- p.122
Chapter 4.4.2 --- Detection ofROS generation in mouse NS cells --- p.123
Chapter 4.4.3 --- Effects of 5-HT on expression level of MAO-A, MAO-B and SERT --- p.123
Chapter 4.5 --- Anti-apoptotic effect of 5-HT on NP and NS cells in neural induction --- p.127
Chapter 4.6 --- Potential signaling pathways mediated by 5-HT --- p.130
Chapter 4.7 --- Therapeutic effects of 5-HT treated mouse ES cell-derived cells in a stoke model --- p.130
Chapter 4.7.1 --- Induction of global ischemia by transient BCCAO --- p.130
Chapter 4.7.1.1 --- HE staining of post ischemic brain --- p.131
Chapter 4.7.1.2 --- TUNEL analysis of cell apoptosis at post ischemia day 3 --- p.132
Chapter 4.7.2 --- Cell labelling --- p.132
Chapter 4.7.3 --- Cognition monitoring post transplantation --- p.133
Chapter 4.7.4 --- Survival, migration and differentiation of transplanted neural cells --- p.135
Chapter Chapter5 --- Discussion --- p.180
Chapter Chapter6 --- Conclusions --- p.192
References --- p.195

Baicalin, which is a flavonoid, was previously shown to exert neuroprotective effects against ischemic injury and oxidative insults. In this study, baicalin was found to induce neuronal differentiation on both C17.2 NSC and primary mouse NSC originated from hippocampuses of E14.5 mouse embryos. The baicalin-mediated differentiation of C17.2 NSC was noted in dose- and time-dependent manners. Baicalin-treated NSC displayed long processes of neurites. The gene expression of neuronal markers, NF-L, TUBB3 and MAP2 was also significantly increased after treated with 20 to 50 muM baicalin on C17.2 NSC. Treating C17.2 NSC with baicalin significantly increased the number of TUBB3 positive cells by 300%. A significant increase in the gene expression of TUBB3 was also observed on primary NSC upon baicalin treatment at 5 to 10 muM. The number of TUBB3 positive cells was increased by 100% after treating with 10 muM baicalin. C17.2 NSC treated with baicalin also increased the gene expression of GABAergic and serotonergic neuronal subtype specific enzymes GAD1 and TPH1.
Nature provides a vast pool of natural compounds with neuroprotection and neurotrophism. A few of these compounds can induce the differentiation of neural stem cells (NSC). There are ample opportunities to discover more natural compounds with differentiation inducing effect on NSC. One of the objectives of this project is to look for novel natural compounds showing neurogenic effect on NSC. This project has established a platform for screening medicinal materials and natural compounds with neural differentiation promoting effect on C17.2 mouse neural stem cell line. Screening results identified total Sanqi saponins, total Renshen saponins, Huangqin extracts and baicalin as potent candidates for inducing this differentiation of NSC.
This project also aims at characterizing the mechanisms involved in the neuronal differentiation effect of baicalin on NSC. Annotation from microarray analysis indicated that baicalin treatment on C17.2 NSC is related to development of tissue and nervous system. qPCR study attested the increased gene expression of nerve growth factor-beta, neurotrophin-3, pro-neural transcriptional factors Ngn1, Ngn2 and NeuroD2. Western blotting showed that baicalin activated ERK1/2 MAP kinase but not JNK and p38 MAP kinases.
This project demonstrated the neurogenic potential of natural resources on NSC. A novel neuronal induction effect of baicalin on NSC was also demonstrated with its mechanisms characterized. This project also revealed that baicalin can be used for promoting functional recovery of post-ischemia animals.
This study showed for the first time that baicalin exerts neuronal differentiation inducing effect on NSC. Another objective of this project is to study whether baicalin can promote functional recovery of animals with ischemia brain injury. Mice having undergone transient occlusion of the bilateral common carotid arteries with blood-reperfusion to induce global cerebral ischemia were treated with baicalin and/or EGFP-NSC. Ischemia animals received implantation of EGFP-NSC into the caudate putamen and/or intravenous injection of baicalin on alternate days for two-week on day seven post-ischemia displayed significant improvement of the cognitive function in terms of the incident of error and escape time in the water T-maze task compared to the control arm of ischemia mice. Data of the study suggested that the therapeutic effect of baicalin would be comparable to that of neural stem cell transplant in improving the cognitive function in a mouse ischemic stroke model.
Li, Ming.
Adviser: P. C. Shaw.
Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 199-232).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

胰臟內分泌細胞分化的調控事件的研究揭示了胰島素分泌細胞的形成。這一原理既有利於體外誘導用於移植的胰島素分泌細胞，又可應用于糖尿病病人自體胰島素分泌細胞的再生。正在發育的組織和器官中，發現了腎素血管緊張素（RAS）成員，揭示了他們在發育過程中的潛在調控作用。另外，對 RAS 信號系統做出應答的活性氧化物質（ROS），被認為是第二信使，通過對轉錄調控因子的氧化還原的修飾促進分化。作為 ROS 的主要來源，NADPH 已被證實在各類細胞和組織中參與了祖細胞的分化。儘管如此，依賴於 NADPH 氧化酶的 ROS對于胰腺內分泌細胞分化的調控作用仍不清楚。基於這個背景，本研究致力於揭示 RAS 和 NADPH 氧化酶依賴性 ROS 在胰腺內分泌細胞分化中的作用。本實驗將在小鼠胰臟原基培養物和尿鏈黴素（STZ）誘導的新生大鼠上進行。 結果顯示，經典 RAS 成員中的血管緊張素 2 型受體（AT₂R）分佈於內分泌祖細胞的細胞核，之後穿梭定位於胰島素分泌細胞的細胞質。阻斷 AT₂R 功能抑制了Ngn3，胰島素的表達以及 β 細胞的增值。在不同的胚胎期 ROS 的水平發生了改變。對于培養的胰臟原基施加適當的外源 ROS，刺激了內分泌細胞的分化。同時，ROS 清除劑減弱了胰島細胞分化和成熟的標記基因的表達。NOX4 以及其相關的亞基 p22phox 是 NADPH 氧化酶成員，其在胰臟發育過程中的變化同 ROS 水平的變化相似，並且持續表達與內分泌細胞系統。在 NGN3 高表達的胚胎期15.5 天，它們定位于表達 NGN3 的細胞；在 NGN3 表達下調，且胰島素表達升高的胚胎期 17.5 天，它們分佈於胰島素表達細胞。而且，NADPH 氧化酶的抑製劑 DPI 削弱了胰臟培養物中的內分泌祖細胞的分化, 外源 H₂O₂ 的加入扭轉了這一現象。
另一方面，在 STZ 誘導的新生大鼠的研究中，DPI 負調節 β 細胞的再生。血糖失調，胰島結構毀壞以及血清胰島素匱乏的現象發生在了 DPI 處理組。另外，DPI 減弱了 NGN3 的表達而並非 Ki67, 顯示 β 細胞的分化而並非增值對於 ROS 的刺激進行了應答。在體內和體外的實驗中，DPI 也抑制了 NGN3 的轉綠調控因子 SOX9 在胰腺祖細胞中的表達。有趣的是，過表達 SOX9 可以恢復 DPI 引發的對於 NGN3 的抑制 。結合以上數據，本研究顯示 NADPH 氧化酶依賴性ROS 誘導的信號通路參與了胰腺祖細胞到胰島素分泌細胞的分化。
Investigations into the regulatory events that modulate pancreatic endocrine cell differentiation shed light on the generation of sufficient insulin-producing cells in vitro for transplantation or regeneration of β cells in patients with diabetes. The expression of renin-angiotensin system (RAS) components has been detected in development tissue and organs, implicating their regulatory role in developmental processes. On the other hand, reactive oxygen species (ROS) are responsive to RAS signaling pathways and act as second messengers to promote differentiation through redox modification of transcriptional factors essential for differentiation. As a major source of ROS, NADPH oxidase has been shown to participate in the progenitor differentiation in a variety of cells and tissues. Despite this finding, the role of NADPH oxidase-dependent ROS in regulating pancreatic endocrine cell differentiation remains ambiguous. Against this background, the study was aimed at elucidating the roles of RAS components and NADPH oxidase-derived ROS during differentiation of pancreatic endocrine cells using mouse pancreatic rudiments and streptozotocin-treated neonatal rats.
Results showed that angiotensin II type 2 receptor (AT₂R), a major component of the classical RAS, was localized within the nuclei of endocrine progenitors in the cultured pancreatic rudiments; following the differentiation of endocrine progenitors into insulin producing cells, it translocated to cytoplasm. Blockade of AT₂R impeded the expression of Ngn3 and insulin as well as proliferation of β-cells. In addition, the dynamic changes of ROS levels were found in mouse pancreata at different embryonic days, concomitant with induction of endocrine cell differentiation induced by modest exogenous ROS in pancreatic rudiment cultures. Moreover, scavenger of ROS diminished the expression of islet cell markers for differentiation and maturation. NOX4 and its associated subunit p22phox, which are the member of NADPH oxidase, exhibited similar changes of expression to that of ROS levels during pancreas development and persisted in the endocrine lineage; they were located in NGN3⁺ cells at E15.5 during the burst of NGN3 expression and then distributed in insulin⁺ cell at E17.5, the latter being the phase that has a decline in NGN3 expression with an increase of insulin. Furthermore, administration of NADPH oxidase inhibitor, diphenylene iodonium (DPI) attenuated the differentiation of endocrine progenitors in rudiment cultures, while exogenous ROS reversed this effect.
On the other hand, studies performed in streptozotocin-induced neonatal rats showed that β cell regeneration was negatively affected by DPI treatments; consistently, impaired blood glucose control, disturbed islet architecture and deficient serum insulin were observed in DPI-treated groups. In addition, DPI treatments blunted NGN3 expression, but not Ki67-labeling beta-cells, suggesting that differentiation beyond proliferation of β-cells was accountable in response to ROS stimulation. Administration of DPI also suppressed the levels of SOX9, a transcriptional regulator of NGN3, in pancreatic progenitor cells, as evidenced by both in vivo and in vitro studies. Interestingly, over-expression of SOX9 could restore the repression of NGN3 induced by DPI. Taken all these data together, our results indicate that NADPH oxidase-dependent ROS-induced signaling pathway is involved in the differentiation of pancreatic endocrine progenitors into insulin-producing β cells.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Liang, Juan.
Thesis (Ph.D.) Chinese University of Hong Kong, 2014.
Includes bibliographical references (leaves 171-205).
Abstracts also in Chinese.

博士
南台科技大學
電機工程系
102
According to the latest annual statistics report of the Ministry of Interior, as of 2013, there is an estimated 382,000 people with physical disability. This figure belongs to the largest percentage (34%) of the disabled population. Among them, about 40,000 people are patients with spinal cord injury (SCI), and annually, a continuous increase of around a thousand people is expected. In general, regardless of the field of experimental medical research, most studies are done through experiments on animals. The analysis of the motor behavior among animals is also often used in comparing experimental studies. Studies related to spinal injuries mostly use male rats of the Sprague-Dawley strains (SD) as the object of animal experiments. The Basso-Beattie-Bresnahan (BBB) open field locomotor scale test is used as the criterion to evaluate hind limb motor function. In investigating the relationship between cellular and molecular motor functions, this study firstly attempted to assess and compare the protective effect on hind limb functional recovery after human umbilical cord blood cells (HUCBC) or DMEM medium (without cell culture, as a vehicle) which is given through purified hematopoietic stem cells (CD34+), secreting cells or substances called conditioned medium (CM) cultured from human umbilical cord blood-derived CD34+ cells, combined CM and 17β-estradiol medium were given intravenously immediately after spinal cord injury. In this study, the following conditions for recovery after spinal cord injury were designed: (1) the increase in number and activity of apoptotic protease (caspase-3) of DNA terminal deoxynucleotidyl transferase-mediated dUTP DNA labeled cells (TUNEL - positive cell is evidence) is used as evidence in determining apoptosis; (2) the increase in performance of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α as support) and the activity of myeloperoxidase (MPO) are evidences of activated inflammatory response; (3) the increase in nitric oxide (NO), 2,3-dihydroxybenzoic acid, and malondialdehyde (MDA as evidence) and the reduction of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) are evidences of increased oxidative stress; (4) the amount of glial fibrillary acidic protein-positive cells (GFAP-positive cells) shows evidence of increased astrogliosis. The entire study found out that rats given conditioned medium, 17β-estradiol, and combined CM and 17β-estradiol medium immediately after SCI can effectively improve their situation on the 7th day of injury. Also, injured spinal cord is neuroprotected, and this effectively improves hind limb motor function. Subsequently, in treating SCI, combined CM and 17β-estradiol medium is better than a single CM or 17β-estradiol. These results illustrate that 17β-estradiol can strengthen the cultivation of CD34+ stem cell-conditioned medium derived from human umbilical cord blood cells, increasing the therapeutic effect on injured spinal cord of experimental rats. In coordination with the above-mentioned work, this study also attempted to establish a set of computer-assisted quantitative assessment system for animal behavior analysis on the basis of imaging technology in order to test the hind limb behavior of observed rats after spinal cord injury. The system mainly conducted image segmentation and coding of animal behavior through images taken using a high-speed camera. Parameters such as variation of joint angles, variances of gait cycle, stride length, and shift in center of gravity when walking, etc. were quantized through a pre-designed image analysis software program. Afterwards, the correlation between quantization parameters and traditional BBB scores was studied. This study also found that the variation of joint angle and the variance of the shift in center of gravity when walking have a good correlation with traditional assessment methods of BBB scores. Lastly, it is anticipated that the quantitative assessment system developed could provide an objective and reliable secondary assessment basis for future BBB score-related tests.

Indiana University-Purdue University Indianapolis (IUPUI)
The transcriptional repressor BCL6 has been shown to be essential for the differentiation of germinal center (GC) B cells and follicular T helper (TFH) cells. The interaction of TFH and GC B cells is necessary for the development of high affinity antibodies specific for an invading pathogen. Germline BCL6-deficient mouse models limit our ability to study BCL6 function in T cells due to the strong inflammatory responses seen in these mice. To overcome this, our lab has developed a new BCL6 conditional knockout (cKO) mouse using the cre/lox system, wherein the zinc finger region of the BCL6 gene is flanked by loxP sites. Mating to a CD4-Cre mouse allowed us to study the effects of BCL6 loss specifically in T cells, without the confounding effects seen in germline knockout models. Using this cKO model, we have reaffirmed the necessity of BCL6 for TFH differentiation, including its role in sustained CXCR5 surface expression, a signature marker for TFH cells. This model also allowed us to recognize the role of BCL6 in promoting the expression of PD-1, another key surface marker for TFH cells. Without BCL6, CD4+ T cells cannot express PD-1 at the high levels seen on TFH cells. Our discovery of DNMT3b as a target for BCL6 suggests BCL6-deficient T cells have increased DNA methyltransferase activity at the PD-1 promoter. This data establishes a novel pathway for explaining how BCL6, a transcriptional repressor, can activate genes. Experiments with the BCL6 cKO model have also established a role for BCL6 in naïve CD4+ T cell activation. Furthermore, we did not observe increased differentiation of other helper T cell subsets, in contrast to what has been reported elsewhere with germline BCL6-deficient models. Unexpectedly, we found decreased T helper type 2 (Th2) cells, whereas mouse models with a germline mutation of BCL6 have increased Th2 cells. These results indicate that BCL6 activity in non-T cells is critical for controlling T cell differentiation. Finally, using an HIV-1 gp120 immunization model, we have, for the first time, shown BCL6-dependent GCs to be limiting for antibody development and affinity maturation in a prime-boost vaccine scheme.

Indiana University-Purdue University Indianapolis (IUPUI)
Neurofibromatosis Type 1 (NF1) is a genetic disorder resulting from mutations in the NF1 tumor suppressor gene. Neurofibromin is the protein product of NF1 and functions as a negative regulator of Ras activity in both hematopoietic and vascular wall cells, which are critical for maintaining blood vessel homeostasis. NF1 patients are predisposed to chronic inflammation and premature cardiovascular disease, including development of large arterial aneurysms, which may result in sudden death secondary to their rupture. However, the molecular pathogenesis of NF1 aneurysm formation is completely unknown. Utilizing a novel model of Nf1 murine aneurysm formation, we demonstrate that heterozygous inactivation of Nf1 (Nf1+/-) results in enhanced aneurysm formation with myeloid cell infiltration and increased reactive oxygen species in the vessel wall. Using cell lineage-restricted transgenic mice, we show that loss of a single Nf1 allele in myeloid cells is sufficient to recapitulate the Nf1+/- aneurysm phenotype in vivo. Additionally, oral administration of simvastatin, a statin with antioxidant and anti-inflammatory effects, significantly reduced aneurysm formation in Nf1+/- mice. Finally, the antioxidant apocynin was administered orally and also resulted in a significant reduction of Nf1+/- aneurysms. These data provide genetic and pharmacologic evidence that neurofibromin-deficient myeloid cells are the central cellular triggers for aneurysm formation in a novel model of NF1 vascular disease, implicated oxidative stress as the key biochemical mechanisms of NF1 aneurysm formation and provide a potential therapeutic target for NF1 vasculopathy.

Indiana University-Purdue University Indianapolis (IUPUI)
Solid organ transplantation is limited by available donor allografts. Pig to human transplantation, xenotransplantation, could potentially solve this problem if physiologic and immunologic incompatibilities are overcome. Genetic modifications of pigs have proven valuable in the study of xenotransplantation by improving pig to human compatibility. More genetic targets must be identified for clinical success. First, this study examines platelet homeostasis incompatibilities leading to acute thrombocytopenia in liver xenotransplantation. Mechanisms for xenogeneic thrombocytopenia were evaluated using liver macrophages, Kupffer cells, leading to identification of CD18, beta-2 integrin, as a potential target for modification. When disruption of CD18 was accomplished, human platelet binding and clearance by pig Kupffer cells was inhibited. Further, human and pig platelet surface carbohydrates were examined demonstrating significant differences in carbohydrates known to be involved with platelet homeostasis. Carbohydrate recognition domains of receptors responsible for platelet clearance Macrophage antigen complex-1 (CD11b/CD18) and Asialoglycoprotein receptor 1 in pigs were found to be different from those in humans, further supporting the involvement of platelet surface carbohydrate differences in xenogeneic thrombocytopenia. Second, immunologic incompatibilities due to antibody recognition of antigens resulting in antibody-mediated rejection were studied. Identification of relevant targets was systematically approached through evaluation of a known xenoantigenic protein fibronectin from genetically modified pigs. N-Glycolylneuraminic acid, a sialic acid not found in humans, was expressed on pig fibronectin and was identified as an antigenic epitope recognized by human IgG. These studies have provided further insight into xenogeneic thrombocytopenia and antibody-mediated rejection, and have identified potential targets to improve pig to human transplant compatibility.

A population of stromal cells that retains osteogenic capacity in adult bone (adult bone stromal cells or aBSCs) exists and is under intense investigation in relation to osteogenesis and relevant pathology. aBSCs may be different from their embryonic or neonatal counterparts, and are influenced by species-/age-specific and other factors. Mice heterozygous for a null allele of prkar1a (Prkar1a+/-, a gene encoding for cyclic adenosine mono-phosphate (cAMP)-dependent regulatory subunit of protein kinase A (PKA), developed bone lesions that resembled fibrous dysplasia (FD) originated from cAMP-responsive osteogenic cells. Prkar1a +/- mice were crossed with mice heterozygous for catalytic subunit Calpha (Prkaca+/-), the main PKA activity-mediating molecule and generated mouse model with double heterozygosity for prkar1a and prkaca (Prkar1a +/-Prkaca+/-). Unexpectedly, Prkar1a+/-Prkaca+/- mice developed a large number of osseous lesions starting at 2--3 months of age that varied from the rare chondromas in the long bones and the ubiquitous osteochondrodysplasia of tail vertebral bodies to the occasional sarcoma in older animals. Cells from these lesions were fibroblast- and FD-like, and almost always originated from an area proximal to the growth plate and adjacent to endosteal surface of the periosteum; they expanded gradually in the bone marrow space. These cells expressed osteogenic cell markers, showed higher PKA activity that was mostly type II (PKA-II) and display an alternate pattern of catalytic subunit expression, and surprisingly possessed higher cAMP levels. In addition, markers of bone synthesis and lysis were increased. Gene expression profiling not only confirmed an early (progenitor) osteoblastic nature for these cells but also showed a signature that was indicative of mesenchymal-to-epithelial (MET) transition and increased Wnt signaling, particularly the brachyury expression. These studies show that a specific subpopulation of aBSCs can be stimulated in adult bone by PKA-II and altered Calpha activity, generating the only available germline mutant mouse model of a disorder that has similarities to human FD. Along with previous data, these studies also suggest that the effects of cAMP signaling on osteogenesis and stromal cell maintenance and proliferation in mice are age-, bone-, site- but also PKA-type and catalytic subunit-specific.
Parts of the work have been published in Proceedings of the National Academy of Sciences of the United States of America 2010; 107(19):8683--8.
Tsang, Kit Man.
Advisers: Constantine A. Stratakas; Kwak-Pui Fung.
Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 144-183).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Indiana University-Purdue University Indianapolis (IUPUI)
Type 2 diabetes manifests from peripheral insulin resistance and a loss of functional beta cell mass due to decreased beta cell function, survival, and/or proliferation. Beta cell stressors impair each of these factors by activating stress response mechanisms, including endoplasmic reticulum (ER) stress. The glucolipotoxic environment of the diabetic milieu also activates a stress response in beta cells, resulting in death and decreased survival. Whereas the cell cycle machinery (comprised of cyclins, kinases, and inhibitors) regulates proliferation, its involvement during beta cell stress in the development of diabetes is not well understood. Interestingly, in a screen of multiple cell cycle inhibitors, p21 was dramatically upregulated in INS-1-derived 832/13 cells and rodent islets by two independent pharmacologic inducers of beta cell stress - dexamethasone and thapsigargin. In addition, glucolipotoxic stress mimicking the diabetic milieu also induced p21. To further investigate p21’s role in the beta cell, p21 was adenovirally overexpressed in 832/13 cells and rat islets. As expected given p21’s role as a cell cycle inhibitor, p21 overexpression decreased [3H]-thymidine incorporation and blocked the G1/S and G2/M transitions as quantified by flow cytometry. Interestingly, p21 overexpression activated apoptosis, demonstrated by increased annexin- and propidium iodide-double-positive cells and cleaved caspase-3 protein. p21-mediated caspase-3 cleavage was inhibited by either overexpression of the anti-apoptotic mitochondrial protein Bcl-2 or siRNA-mediated suppression of the pro-apoptotic proteins Bax and Bak. Therefore, the intrinsic apoptotic pathway is central for p21-mediated cell death. Like glucolipotoxicity, p21 overexpression inhibited the insulin cell survival signaling pathway while also impairing glucose-stimulated insulin secretion, an index of beta cell function. Under both conditions, phosphorylation of insulin receptor substrate-1, Akt, and Forkhead box protein-O1 was reduced. p21 overexpression increased Bim and c-Jun N-terminal Kinase, however, siRNA-mediated reduction or inhibition of either protein, respectively, did not alter p21-mediated cell death. Importantly, islets of p21-knockout mice treated with the ER stress inducer thapsigargin displayed a blunted apoptotic response. In summary, our findings indicate that p21 decreases proliferation, activates apoptosis, and impairs beta cell function, thus being a potential target to inhibit for the protection of functional beta cell mass.

Indiana University-Purdue University Indianapolis (IUPUI)
Signal Transducer and Activator of Transcription 3 (STAT3) is known to be related to bone metabolism. Mutation of STAT3 causes a rare disorder in which serum levels of IgE are elevated. This causes various skeletal problems similar to osteoporosis. To examine the effect of STAT3 in the osteoclast, we obtained two osteoclast specific STAT3 knockout mouse models: one using the CTSK promoter to drive Cre recombinase and another using a TRAP promoter. Examination of these mice at 8 weeks of age revealed a decreased trabecular bone volume in CTSK specific STAT3 knockout mice along with a slight decrease in osteoclast number in both CTSK and TRAP specific STAT3 knockout females. We also noticed changes in bone mineral density and bone mechanical strength in females. These data suggest that STAT3 plays a part in the function of the osteoclast.