dc.description.abstract |
1 diabetes (T1D) is an autoimmune disease caused by the autoimmune destruction of pancreatic β-cells, leading to insufficient production of insulin. The etiology of T1D is thought to be an interplay between genetic predisposition and environmental factors. The onset of T1D generally occurs in early adolescence, and progresses through to adulthood, leading to multiple complications, such as hyperglycaemia and vascular diseases. To date, there is no cure for T1D. Owing to donor shortage of pancreases, daily injection of insulin and vigorous monitoring of glucose homeostasis are the only solutions for the majority of patients. The advancement in regenerative medicine utilizing adult stem cells has raised the hope of finding a cure for T1D. Generation of insulin-producing cells from stem cells requires differentiation by specific cell culture conditions and/or transfection with critical β-cell genes. The aim of this project was to reproduce results from previous published studies, and to explore new approaches to generate insulin-producing cells. Three types of adult stem cells were investigated in this project, two of which were derived from human peripheral blood monocytes, namely peripheral-blood monocytic cells (PBMCs) and f-macrophages. The third type was human adipose-derived stem cells (ADSCs). All three types of stem cells were reported to express stem cell properties. PBMCs and f-macrophages were cultured in Stemline haematopoietic stem cell expansion media, which had been reported to produce insulin-expressing cells. Both cell types formed large multinucleated cells in culture, and expressed several pancreatic markers, including insulin, Pdx- 1, MafA, Glut2 and HNF-6. In contrast, PBMCs cultured in a commercial pancreatic stem cell differentiation media did not differentiate into insulin-producing cells. PBMCs and fmacrophages cultured in Stemline haematopoietic stem cell expansion media expressed monocytic and antigen-presenting cell markers, including CD11c, CD14, CD31, CD68 and HLA-DR, which showed that they had not lost their monocytic character. A key question that remains is whether the insulin detected in cells in the current studies is derived from endogenous synthesis or is sequestered from the culture media. A stage-specific differentiation protocol developed by Chandra and colleagues was employed to differentiate ADSCs into insulin-expressing cells. The cells that were generated expressed insulin, but none of the other β-cell markers examined. An attempt was made to differentiate f-macrophages and ADSCs by transfection with total RNA and DNA isolated from rat RINm5f β- cells and mouse pancreas. The cells generated failed to express insulin, C-peptide and Pdx-1 despite an attempt to optimize the transfection protocol. Chandra's differentiation protocol was further modified to promote β-cell differentiation by transfecting the cells with total RNA and DNA isolated from RINm5f cells and mouse pancreas. Transfection with RNA led to high levels of insulin expression, however, none of the other β-cell markers were expressed, raising questions as to whether the insulin expression was authentic. An attempt was made to differentiate ADSCs by incubation with nucleoproteins derived from the nuclei of RINm5f cells, but no insulin-expressing cells were produced. The limited concentration of β-cell transcription factors within the nucleoprotein extract may be a key reason for the lack of cell differentiation. Carrier peptide technology offers a novel approach to introduce transcription factors into stem cells to drive lineage-specific differentiation. A novel carrier peptide, designated Xentry, developed in the laboratory was shown to be able to penetrate ADSCs. Xentry has been shown to be able to deliver siRNA, proteins and antibodies into cells, and in the present study was tested for its ability to carry the enzyme β-galactosidase into cells. Xentry conjugated to β- galactosidase was able to penetrate ADSCs cultured as a monolayer, but only penetrated the cells on the outside of large cell clusters. Xentry modified with a nuclear localization sequence was conjugated to nucleoproteins isolated from the nuclei of RINm5f cells. ADSCs treated with the Xentry-nucleoprotein conjugates failed to differentiate into insulin-producing cells. This study has trialed several novel approaches to generate insulin-producing cells, some of which have proved to be more promising than others and deserve further study. |
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