Date of Award

12-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

Martine LaBerge

Committee Member

Xuejun Wen

Committee Member

Ning Zhang

Committee Member

Ken Webb

Abstract

Diabetes mellitus, the third most common disease in the world, is a chronic metabolic disorder caused by a failure of insulin production and/or an inability to respond to insulin. Specifically, type 1 diabetes is a disorder characterized by targeted autoimmune-directed destruction of a patient's beta-cell population within the pancreatic islets of Langerhans. The current primary treatment for type 1 diabetes is daily multiple insulin injections. However, this treatment cannot provide sustained physiological release, and the insulin amount is not finely tuned to glycemia. Pancreatic transplants or islet transplants would be the preferred treatment method but the lack of donor tissue and immunoincompatibility has been shown to be a roadblock to their widespread use. The objective of this project is to develop an effective strategy for the treatment of type 1 diabetes using beta-cells based replacement therapy. To improve the viability of transplanted beta-cells, one novel approach is to transplant optimal size range of beta-cell spheroids rather than cell suspension. Uniform sized multicellular spheroids can be coated with a thin layer of non-degradable hydrogel for immunoisolation. In addition, the survival of spheroids of optimized size can be further improved with a novel coating of multiple layers of human mesenchymal stem cells (hMSCs), a cell type that has profound immunoregulatory effect, to prevent graft rejection. To prevent hMSC migrate away from spheroids, another layer of non-degradable hydrogel can be added. To further improve the viability and suppress the immune rejection, spheroids will be encapsulated with nanoparticles loaded with angiogenic and immune regulatory molecules. By this means the spheroid will passively evade the complications of stressors in addition to actively modulating the immune microenvironment for regulatory tolerance and long-term engraftment. Firstly, through optimizing our hydrogel systems based on poly (ethylene glycol) (PEG), we have created specific niche for beta-cells to form artificial islets in vitro. We have found that the optimal condition is the concentration of PEG at 5% and the ratios of 4-arm thiolated PEG to 4-arm PEG acrylate at 1:2. Conjugated with adhesive peptides, especially, RGD at 0.2 mM, can significantly promote the glucose stimulated insulin secretion of encapsulated beta-cells. Secondly, we have fabricated different sizes of uniformed beta-cells spheroids through our designed high-throughput automatic spheroids maker. Beta-cells in the spheroids of 200 μm exhibited largest insulin secretion based on glucose stimulus when compared to others with sizes of 100, 300, 400 and 500 μm. The novel core-shell structured spheroids-hMSCs complex was successfully achieved. Methylcellulose hydrogel was applied as physical barrier on the surface of beta-cells spheroids to inhibit invasion of hMSCs. Human MSCs prevented apoptosis of beta-cells spheroids and benefited insulin secretion when exposed to pro-inflammatory cytokines. Thirdly, immune regulatory molecules [leukemia inhibitory factor (LIF) and interleukin 10 (IL-10)] and angiogenic molecule [vascular endothelial growth factor (VEGF)] loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles have been successfully fabricated through solvent extraction/evaporation technique. These growth factors can be controlled release about 6 weeks. The bioactivity of released VEGF has been confirmed by the in vitro HAEC proliferation assay. Finally, beta-cells spheroids were transplanted under the kidney capsule to treat diabetic mice. Beta-cells spheroids kept the glucose level of diabetic mice constant. Co-transplanted hMSCs suppressed the host inflammation response, activated the regulatory T cells and also promoted angiogenesis at the transplantation site. The beta-cells spheroids/hMSCs/hydrogel complex initiated a mild inflammatory response. The LIF and IL-10, and VEGF loaded complex can further inhibited this response and promoted blood vessel network formation at the transplantation site. Our approach holds a great potential to treat type 1 diabetes.

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