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Braam, Mitchell James Saddy

University of British Columbia

Diabetes is a debilitating disorder that affects nearly 500 million people worldwide and is characterized by chronic hyperglycemia caused by pancreatic β-cell dysfunction or death. Those that are affected need to frequently monitor their blood glucose levels and administer exogenous insulin, but this strategy does not match the tight regulation maintained by healthy β-cells. Progress in the field has demonstrated an effective clinical path to treat type 1 diabetes through the success of islet transplants. More recently, groups have demonstrated that human pluripotent stem cells, differentiated into pancreatic progenitors or beyond, can prevent or reverse diabetes in rodents and have shown promising results in human clinical trials. Here I develop a differentiation protocol to further differentiate pancreatic progenitors generated with a commercially available kit into endocrine cells. I identified FGF7 signalling as being important for aggregate survival and Notch-inhibition improved endocrine commitment at the expense of ductal cells. Combining in vitro β-cell differentiation strategies with newly developed precision gene editing techniques allows for disease modelling in a dish and transgene elements can be introduced into cells to provide new functionalities. To this end, I used a novel gene editing technique to correct a single-base mutation in pluripotent stem cells derived from an individual living with a severe form of neonatal diabetes. Using stem cell-derived cell therapies does not come without risk, given the potential for teratoma outgrowth, inappropriate differentiation and function, or an immune response to the graft. Here, I generated cell lines containing an inducible safety-switch, which was shown to eliminate pluripotent and pancreatic progenitor cells in vitro and teratomas in vivo. Finally, I targeted the B2M locus for silencing using an inducible CRISPRi platform, preventing HLA-ABC formation in a reversible manner. This strategy could both allow cells to avoid immune detection as well as act as a safety-switch by deactivating CRISPRi and re-expressing the B2M gene. Overall, the projects described in this dissertation demonstrate the combined potential of pluripotent stem cells, gene editing, and cell differentiation with the goal of advancing the cell therapy field, particularly for diabetes.

Text

2023-04-19

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/84322

Genome Science and Technology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/