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A transcription factor guided approach to the generation of pancreatic endocrine-like cells from stem cells

University of British Columbia

Diabetes mellitus results in elevated blood glucose levels due to an insufficiency of the glucose lowering hormone insulin. In type 1 diabetes, insulin loss is due to an autoimmune destruction of the insulin producing pancreatic β-cells. One treatment for type 1 diabetes is the transplantation of cadaveric islets, although this process is limited by a lack of donor tissue. We and others are examining factors that influence the generation of new β-cells from stem cells. Specifically, the aims of this thesis were to examine the role of transcription factors in the formation of endocrine cells from stem cells. To do this, we developed a high content screening approach in human amniotic fluid stem cells to assess the effect of six pancreatic transcription factors on insulin expression. From this screen and subsequent studies, we observed that while transcription factor overexpression was capable of driving insulin expression, the resulting cells were unable to reverse diabetic hyperglycemia upon transplantation. Given the well-established developmental capacity of human embryonic stem cells (hESCs), we next characterized a novel hESC line (CA1S), which is amenable to high throughput screening and pancreatic differentiation. Using these cells, we found that the number of cells seeded into a culture system had a significant effect on the formation of endodermal, pancreatic progenitor and pancreatic endocrine cells. This effect correlated with hESC cell cycle status and resulted in the formation cells co-expressing insulin, glucagon and somatostatin. We next examined the effects of the transcription factors PAX4 and ARX on pancreatic endocrine specification. We revealed that increased PAX4 expression reduced ARX and glucagon expression leaving insulin positive cells. Reduced ARX expression by genomic editing resulted in fewer glucagon positive cells and increased PAX4 levels. Loss of ARX was also associated with an abundance of somatostatin positive cells and a partial reduction in insulin, which was rescued with re-expression of ARX adenoviral gene delivery methods. Collectively, the data presented in this thesis emphasise the role of transcription factor expression as a primary control point for the possible generation of β-cells from stem cells, which represents a potential cellular therapy for type 1 diabetes.

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2014-08-07

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/49970

Cell and Developmental Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/