published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Stem cell therapy is a promising approach for tissue regeneration but there exists a problem of low engraftment rate to the injury site. Our laboratory has shown that hMSCs that were capable to penetrate through collagen barrier have higher migration capacity and engraftment efficiency than those remained inside the collagen matrix and those in traditional 2D culture. These cells capable of penetrating through collagen barrier might be hopeful candidate for improving engraftment efficacy. Major processes of engraftment, such as transmigration through basement membrane and invasion to the site of injury, involve cell-extracellular matrix-interacting proteins. As matrix metalloproteinases (MMP) and integrins are the key players in these processes, MMP and integrin profiles of the hMSCs were studied In this study, we demonstrated that hMSCs that were capable of penetrating through the collagen barrier have distinctive MMP profile to traditional 2D culture. These cells secrete significantly higher amount of MMP-1 than 2D culture, but the amount of MMP-2 secreted is comparable to traditional 2D culture. On the other hand, MMP-9 and MMP-13 were below detection limit by ELISA in both groups. Moreover, we have investigated the subcellular localization of MMPs and integrins. The cells were seeded on dishes with or without ECM coatings. It was demonstrated that hMSCs capable of penetrating through collagen barrier exhibit higher amount of subcellular MT1-MMP and integrin 6271 on ECM coated dish. Moreover, these cells exhibit a prominent feature of perinuclear localization of MT1-MMP., whereas the level of subcellular MMP-2, integrin 65 and 6v73 is comparable to that in 2D culture. We have also investigated the stem properties of hMSCs penetrated through collagen barrier. These properties include proliferation capacity, self-renewal capacity and differentiation potential towards chondrogenic, osteogenic and adipogenic lineages. It has been demonstrated that these properties are not compromised for superior migratory activities. / published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 99-107) Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 1996. / Includes bibliographical references (leaf 135-164).

Thesis (M. Phil.)--University of Hong Kong, 1994. / Includes bibliographical references (leaves 129-157).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 68-79).

Abstract The purpose of the study was to evaluate model systems of cancer development and compare some of their critical features with cancer development in vivo. Ovarian and endometrial cancers in man were used as correlates. Tumor development in experimental animals, exposed to carcinogens and UV irradiation, showed the entire spectrum of tumor development as compared to spontaneous carcinomas: hyperplasia, dysplasia, benign papillomas and malignant squamous cell carcinomas. For short-term analysis of differentiated homogenous cell populations, the transplant model proved most useful. For long term analysis of effects of extraneous agents, the skin carcinogenesis model is probably the most rewarding. Analysis of proliferation markers in human tumor samples as studied by immunohistochemistry, showed that an increased expression of PCNA and Ki-67 was associated with poor prognosis in ovarian neoplasms. Analysis of cell proliferation in model tumors showed that the transplant model has a better sensitivity when compared to the animal skin model and the subcutaneous injection model, in that effect of changes in cell-host interaction on the location and extent of the proliferating cell population can be studied therein. The expression of some growth factors, their receptors, oncogenes and suppressor genes were studied in ovarian and endometrial carcinomas and in skin cancer model system in mouse exposed to carcinogens and UV irradiation. Variability in expression and methodological problems precluded detailed analysis of these markers in different models. The expression of TGFβ1, TGFβ2 and TGFβ3 was determined in normal human keratinocytes, and in 7 immortalized and ras-transfected benign and malignant keratinocyte cell lines, maintained as transplants and as subcutaneous tumors in nude mice. By differential immunohistochemical localization of TGFβ isoforms, we demonstrated that each isoform may serve specific roles in tumor development and progression. The complex nature of TGFβ expression prevented detailed analysis of isozymes in different models, the results in this study, however, indicated a similar pattern in the models analyzed. Morphological methods were used to determine relationship between epithelial growth and formation and deposition of collagens in the extracellular matrix in experimental models and human tumors. The composition of the mesenchyme differed in tumors originating from different cell lines reflecting functional interaction between epithelial cells and the mesenchyme in neoplastic development. Tumor-stroma interaction was distinct in human, comparable alterations were observed in experimental models, more so in transplants, less in subcutaneous tumors, affecting tumor growth and differentiation in the different models.

Hemopoiesis is thought to be regulated in part by specific, but as yet undefined, interactions between primitive hemopoietic cells and fixed, non-hemopoietic marrow elements collectively referred to as the stroma. Recently, a marrow culture system has been described that allows the maintenance of primitive human hemopoietic progenitor cells for many weeks in the absence of exogenously added hemopoietic growth factors. The formation of a heterogeneous adherent layer in which many stromal elements are found appears to be important to the maintenance of hemopoiesis in this system. As part of the overall goal of delineating the cellular and molecular interactions involved, my first objective was to develop an experimental system for assessing the hemopoiesis-sustaining function of the adherent layer of long-term human marrow cultures. This required the identification of a suitable procedure for separating the hemopoietic and non-hemopoietic regulatory components so that the former could be used to quantitate the function of the latter. This was achieved using irradiation to selectively inactivate residual hemopoietic cells in long-term culture adherent layers, and using a medium containing cis-4-hydroxy-L-proline to selectively inactivate stromal cells and their precursors present in suspensions of unseparated human marrow which were then added back in co-culture experiments. My second objective was to develop a strategy for obtaining purified populations of cells corresponding to the various mesenchymal cell types in long-term adherent layers. I therefore prepared a high titre SV-40 virus stock and used it to establish permanent, cloned lines from human marrow "fibroblast" colonies, long-term culture adherent layers, and umbilical cord endothelial cells. Characterization of the transformants generated showed that they were all positive for SV-40, and in general expressed the phenotypic characteristics of the cells originally infected. Functional studies showed that these transformants, like their normal counterparts, respond to activation by producing two types of hemopoietic growth factors. These studies suggest that marrow mesenchymal cells may regulate the growth and maintenance of primitive hemopoietic cells by producing hemopoietic growth factors in response to appropriate perturbation. The availability of permanent cloned lines of human marrow stromal cells should facilitate future analysis of these events at the molecular level. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate