During the past decade clinical islet transplantation has become a viable strategy for curing type 1 diabetes. The limited supply of organs, together with the requirement for islets from multiple donors to achieve insulin independence, has greatly limited the application of this approach. The islets are infused into the liver via the portal vein, and once exposed to the blood, the grafted tissue has been shown to be damaged by the instant blood-mediated inflammatory reaction (IBMIR), which is characterized by coagulation and complement activation as well as leukocyte infiltration into the islets. Islet revascularization is a subsequent critical step for the long-term function of the transplanted graft, which may partially be impeded by the IBMIR. In this thesis, we have explored novel strategies for circumventing the effects of the IBMIR and facilitating islet revascularization. Systemic inhibitors of the IBMIR are typically associated with an increased risk of bleeding. We therefore evaluated alternative strategies for modulating the islets prior to transplantation. We demonstrated, using an adenoviral vector, that a high level of expression and secretion of the anticoagulant hirudin could be induced in human islets. An alternative approach to limiting the IBMIR was developed in which anticoagulant macromolecular heparin complexes were conjugated to the islet surface. This technique proved effective in limiting the IBMIR in both an in vitro blood loop model and an allogeneic porcine model of islet transplantation. An increased adhesion of endothelial cells to the heparin-coated islet surface was demonstrated, as was the capacity of the heparin conjugate to bind the angiogenic factors VEGF and FGF; these results have important implications for the revascularization process. The outcome of the work in this thesis suggests that modulation of the islet surface is an attractive alternative to systemic therapy as a strategy for preventing the IBMIR. Moreover, the same techniques can be employed to induce revascularization and improve the engraftment of the transplanted islets. Ultimately, improved islet viability and engraftment will make islet transplantation a more effective procedure and increase the number of patients whose diabetes can be cured.

Angiogenesis, the growth of new blood vessels, is a complex process involving several cell types and molecular signals. Excessive vascular growth is a problem in tumors, and insufficient vascularization hampers the function of transplanted insulin-producing pancreatic islets. Understanding the mechanisms behind blood vessel growth generates increased means to control angiogenesis. In this thesis a model of pancreatic islet transplantation to muscle has been used to study the involvement of leukocytes in the development of new vasculature. Transplantation of isolated islets of Langerhans into mouse muscle promoted revascularization of the grafts to a level comparable to native islets in the pancreas. The complete and functional vascular restoration resulted in improved blood glucose control compared to the clinical standard implantation site, the liver. This proved muscle as a transplantation site to be a clinically relevant option for the treatment of type 1 diabetes. The rapid islet revascularization process was found to be dependent on a distinct subset of neutrophils characterized by high expression of the chemokine receptor CXCR4 and the enzyme matrix metalloproteinase 9 (MMP-9). These cells were recruited to recently transplanted and hypoxic grafts by islet-secreted vascular endothelial growth factor A (VEGF-A). Leukocyte migration and interactions in the engraftment area were monitored using a high-speed confocal microscope followed by software tracking. New software was developed to visualize migration statistics. This tool revealed areas around the islet graft where neutrophil gathering coincided with sites of angiogenesis. Macrophages in the engraftment area positioned themselves close to the newly formed vasculature and were shown to have a stabilizing effect on the vessels. When macrophages were removed, no pericytes were recruited to the forming vasculature. The perivascular macrophages also began to express a pericyte marker when in the graft, suggesting a close relationship between these cell types or macrophage plasticity. In conclusion, this thesis presents muscle as a proangiogenic transplantation site for pancreatic islets for the treatment of type 1 diabetes, where the revascularization of the grafts was dependent on the recruitment and actions of specialized immune cells.

Live cell imaging can be used to study dynamic cellular systems at single cell resolution. In heterogeneous cell populations, analyzing cell properties at the single cell level reduces the generalization of results caused by population-based assays. This thesis details the implementation of live cell imaging and single cell tracking to characterize heterogeneous cell systems undergoing dynamic processes over multiple generations. This approach enables the consideration of both spatial and temporal variables as well as the mapping of cell phenotype trajectories along their generational lineages. Cell-, lineage-, and colony-level properties are used as descriptors of the underlying molecular mechanisms that they are produced by. These may be unexpected, emergent properties that can not be predicted or completely characterized at the molecular level. Analysis of these properties can reveal and characterize the properties and processes of dynamic, heterogeneous cell systems. Live cell imaging culture strategies were developed to enable characterization of both two- and three-dimensional cell systems. Computational modeling was performed to evaluate the conditions imposed by a confined imaging chamber that enables single cell resolution imaging of monolayer and multilayer cell systems. Imaging chamber dimensions and cell colony/aggregate sizes were calculated that would prevent the introduction of metabolite transport limitations and allow for stable, long term imaging. Methods for single cell tracking and analysis were also developed, which produces a database detailing the tracked, observed and extracted properties of every cell and colony, while maintaining the lineage structure of the data. Visualizations such as lineages, histograms and scatter plots were implemented to enable interactive data analysis and querying. These methods were used to characterize heterogeneity in two separate cell systems: human islet of Langerhans-derived progenitor cells, and human embryonic stem cells. Islet-derived progenitors are an expandable source of cells with potential for treatment of diabetes. Here, it was shown that there is an unequal contribution of islets to the progenitor derivation process. Islet-derived progenitors consist of two distinct sub-populations of cells that were distinguished by morphological identification during live cell image analysis. These sub-populations possess unique proliferation profiles and appear to exist in a dynamic state with each other. Three-dimensional tracking of islet progenitor derivation was implemented, but suffered from a lack of resolution to capture the dynamic nature of the transformation process. However, entire islets were imaged and tracked successfully under maintenance conditions, suggesting that this system may be useful for other cell types. These results highlight that live cell imaging and cell tracking may not be suitable for all cell systems and that inclusion of other analytical information, such as immunocytochemistry, would improve the power of cell tracking analysis. Human embryonic stem cell cultures were studied using live cell imaging to identify the mechanisms by which they differentiate to produce supportive niche cells. Cell tracking, morphology scoring and lineage analysis revealed a previously unappreciated level of heterogeneity within human embryonic stem cell colonies. The results show that a sub-population of human embryonic stem cells exist that are precursors to niche cell differentiation. However, these cells exist in a dynamic equilibrium with self-renewing stem cells, which is dependant on the presence of existing local niche cells. Sub-optimal niche conditions leads to the production of niche differentiation-competent cells and, significantly, considerable cell death. The effect of cell death is the clonal selection of self-renewing cells that contribute to colony expansion. Overall, these results highlight the importance of the co-transfer of existing niche cells and the dynamic balance that regulates human embryonic stem cell self-renewal and differentiation. This thesis displays the utility of live cell imaging, cell tracking and cell, colony and lineage analysis for studying dynamic heterogeneous systems. Furthermore, it highlight the fact that cell-, lineage- and colony-level analysis can uncover previously unappreciated heterogeneity and unknown sub-populations of cells. The system does not rely on characterization at the molecular level, but uses higher order measures to generalize them. However, future incorporation of cell, lineage and colony information with molecular-level information may results in analytical power not possible from either level alone. Such systems will be valuable tools in the growing fields of stem cell biology and systems biology.

Live cell imaging can be used to study dynamic cellular systems at single cell resolution. In heterogeneous cell populations, analyzing cell properties at the single cell level reduces the generalization of results caused by population-based assays. This thesis details the implementation of live cell imaging and single cell tracking to characterize heterogeneous cell systems undergoing dynamic processes over multiple generations. This approach enables the consideration of both spatial and temporal variables as well as the mapping of cell phenotype trajectories along their generational lineages. Cell-, lineage-, and colony-level properties are used as descriptors of the underlying molecular mechanisms that they are produced by. These may be unexpected, emergent properties that can not be predicted or completely characterized at the molecular level. Analysis of these properties can reveal and characterize the properties and processes of dynamic, heterogeneous cell systems. Live cell imaging culture strategies were developed to enable characterization of both two- and three-dimensional cell systems. Computational modeling was performed to evaluate the conditions imposed by a confined imaging chamber that enables single cell resolution imaging of monolayer and multilayer cell systems. Imaging chamber dimensions and cell colony/aggregate sizes were calculated that would prevent the introduction of metabolite transport limitations and allow for stable, long term imaging. Methods for single cell tracking and analysis were also developed, which produces a database detailing the tracked, observed and extracted properties of every cell and colony, while maintaining the lineage structure of the data. Visualizations such as lineages, histograms and scatter plots were implemented to enable interactive data analysis and querying. These methods were used to characterize heterogeneity in two separate cell systems: human islet of Langerhans-derived progenitor cells, and human embryonic stem cells. Islet-derived progenitors are an expandable source of cells with potential for treatment of diabetes. Here, it was shown that there is an unequal contribution of islets to the progenitor derivation process. Islet-derived progenitors consist of two distinct sub-populations of cells that were distinguished by morphological identification during live cell image analysis. These sub-populations possess unique proliferation profiles and appear to exist in a dynamic state with each other. Three-dimensional tracking of islet progenitor derivation was implemented, but suffered from a lack of resolution to capture the dynamic nature of the transformation process. However, entire islets were imaged and tracked successfully under maintenance conditions, suggesting that this system may be useful for other cell types. These results highlight that live cell imaging and cell tracking may not be suitable for all cell systems and that inclusion of other analytical information, such as immunocytochemistry, would improve the power of cell tracking analysis. Human embryonic stem cell cultures were studied using live cell imaging to identify the mechanisms by which they differentiate to produce supportive niche cells. Cell tracking, morphology scoring and lineage analysis revealed a previously unappreciated level of heterogeneity within human embryonic stem cell colonies. The results show that a sub-population of human embryonic stem cells exist that are precursors to niche cell differentiation. However, these cells exist in a dynamic equilibrium with self-renewing stem cells, which is dependant on the presence of existing local niche cells. Sub-optimal niche conditions leads to the production of niche differentiation-competent cells and, significantly, considerable cell death. The effect of cell death is the clonal selection of self-renewing cells that contribute to colony expansion. Overall, these results highlight the importance of the co-transfer of existing niche cells and the dynamic balance that regulates human embryonic stem cell self-renewal and differentiation. This thesis displays the utility of live cell imaging, cell tracking and cell, colony and lineage analysis for studying dynamic heterogeneous systems. Furthermore, it highlight the fact that cell-, lineage- and colony-level analysis can uncover previously unappreciated heterogeneity and unknown sub-populations of cells. The system does not rely on characterization at the molecular level, but uses higher order measures to generalize them. However, future incorporation of cell, lineage and colony information with molecular-level information may results in analytical power not possible from either level alone. Such systems will be valuable tools in the growing fields of stem cell biology and systems biology.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "December 2003." Typescript. Vita. Includes bibliographical references (leaves 146-172). Also issued on the Internet.

Islet plasticity has proven to be an important platform for the engineering of alternative islet tissue for transplantation. In vitro studies have shown the ability of islets to transdifferentiate into duct-like epithelial structures (DLS) thought to possess progenitor cells capable of replenishing damaged tissue within the pancreas. The aim of this study was to investigate the natural history of human derived duct-like epithelial structures transplanted into nude mice. / Human islet derived duct-like structures from three cadaver pancreases were subcutaneously transplanted into 6-8 week old male HSD athymic nude-Foxn1 mice. Six mice were sacrificed at day 3, 7, 14 and 21 from each time period. DLS were also placed in matrigel for in-vitro control samples. DLS were processed for immunohistochemistry for endocrine markers, epithelial markers, cell death and proliferation markers, islet maturation markers and angiogenic factors. / Our results show that as DLS are transplanted, there is an increase in cell death and proliferation. This increase in cell death and proliferation causes an increase in PDX-1 expression as well as VEGF, an angiogenic factor. But over time, transplanted DLS do not show an increase in cell death and show a small decrease in cell proliferation from pre-transplanted DLS. At day 3 of engraftment, DLS show a significant expression of PDX-1. We see a small increase in endocrine tissue after 3 days of transplantation, then an increase in endocrine cell death, which returns the percentage of endocrine cells back to pre-transplantation levels at day 21. DLS were shown to express VEGF, and once transplanted into an initial hypoxic environment there is a substantial increase in expression, followed by a recruitment of microvessels. Although there is a dynamic change in expression of cell markers throughout engraftment, there is no significant change in DLS size, nuclei per DLS or cell morphology over time. / DLS have been shown to survive subcutaneous transplantation and possess an initial increase in cell proliferation leading to increases in PDX-1 and VEGF expression. Transplanted DLS have shown to possess significant angiogenic properties with the recruitment of microvessels into subcutaneous DLS grafts. Subcutaneous DLS transplantation could be used in combination with islet transplantation to alleviate current problems with islet transplantation such as islet cell death and insufficient blood supply.

Suspensions of pancreatic islet cells from ob/ob-mice were incubated with Trypan Blue. Microscope photometry showed that apparently viable cells excluded the dye completely, whereas the nuclei of non-viable cells accumulated Trypan Blue by a saturable process. Alloxan rapidly increased the permeability of the plasma membrane in mouse 3-cells; the exclusion of Trypan Blue is a valid and useful measure of islet cell viability following alloxan exposure. The diabetogenic action of alloxan may be mediated by hydroxyl radicals. In several biological systems hydroxyl radicals are formed by an iron-catalyzed reaction between superoxide anion radicals and hydrogen peroxide. To test whether this applies to alloxan diabetogenicity, the effects of superoxide dismutase, catalase, scavengers of hydroxyl radicals, and metal ion chelators were tested (a) in a cell-free radical-generating system and (b) on islets and islet-cells exposed to alloxan In vitro. The effect of longtime-circulating superoxide dismutase injected prior to alloxan was tested on mice in vivo. Luminol chemiluminescence was used to monitor alloxan-dependent radical production. Accumulation of 8^Rb+ and exclusion of Trypan Blue were used as cell viability criteria in isolated mouse islets and islet-cells. Blood glucose was determined to monitor the development of diabetes in living animals. Superoxide dismutase, catalase, scavengers of hydroxyl radicals, and metal ion chelators inhibited the alloxan-dependent chemiluminescence and decreased the toxic effects on Rb+ accumulation or Trypan Blue exclusion in islets and islet-cells. Superoxide dismutase, linked to polyethylene glycol and injected 12 hours before alloxan, largely prevented the development of alloxan diabetes. Alloxan toxicity _in vitro and in vivo seems to depend on the formation of superoxide radicals and hydrogen peroxide which in turn form the noxious hydroxyl radical via an iron-catalyzed Haber-Weiss reaction. As free radicals and hydrogen peroxide can be formed by other chemicals and during inflammation, and inflammation may accompany the outbreak of human diabetes, studies on the beneficiary effects of superoxide dismutase and other scavengers of free radicals in other forms of diabetes seem warranted. / <p>S. 1-38: sammanfattning, s. 39-74: Härtill 6 uppsatser</p> / digitalisering@umu

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 3 uppsatser.