Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Physiology and Pharmacology

Supervisor

Hess, David A.

Abstract

Human bone marrow-derived multipotent stromal cells expanded under Wnt pathway stimulation secrete beta cell-regenerative factors collected as conditioned media (Wnt+ CdM). We used the cytokeratin 19 (CK19)-CreERT Rosa26-mTomato lineage tracing mouse to observe CK19+ cell conversion to insulin+ beta cells following intra-pancreatic injection of Wnt+ CdM. Tamoxifen treatment in mice induced labelling of CK19+ ductal and acinar cells with tdTomato and streptozotocin (50 mg/kg/day x 5 days) induced hyperglycemia. Injection of Wnt+ CdM preserved beta cell mass, reduced non-fasted blood glucose levels, and improved glucose tolerance over a 28-day period compared to controls. Insulin+/tdTomato+ cells in mice given Wnt+ CdM were increased, suggesting a small percentage (<5%) of regenerated beta cells may have originated from CK19+ ductal or acinar cells.

Summary for Lay Audience

Diabetes mellitus is characterized by a loss of insulin-producing beta cells that reside in the pancreatic islets of Langerhans resulting in high blood glucose levels or hyperglycemia. Residual beta cell function has been found in patients living with diabetes for over 50 years. Thus, the induction of beta cell regeneration may be a promising approach to replenish lost beta cells in individuals with diabetes. Multipotent stromal cells (MSC), isolated from human bone marrow, secrete a mixture of proteins that can aid in repairing and regenerating damaged cells. Pro-regenerative factors released by MSC in the lab can be collected as conditioned media and can be used to regenerate beta cells that were destroyed during diabetes. Our lab has previously shown that direct injection of conditioned media collected from MSC into the pancreas of hyperglycemic mice induced beta cell regeneration and lowered blood glucose levels. The mechanism by which MSC conditioned media stimulated beta cell regeneration was unclear, although new beta cells formed adjacent to pancreatic ductal structures. We hypothesized that pancreatic ducts contain precursor or parent cells that can be activated to form new beta cells. Thus, the goal of this project was to track the offspring of pancreatic ductal cells during beta cell regeneration stimulated by MSC conditioned media. To achieve this goal, we used a ‘lineage tracing’ mouse model that permanently labelled ductal cells and their offspring with a red fluorescent tag. Hyperglycemic mice that were intrapancreatic-injected with MSC conditioned media showed reduced blood glucose levels and an improved ability to regulate a glucose meal compared to control mice not given conditioned media. The pancreas of mice treated with MSC conditioned media had an increased number of beta cells compared to controls, indicating islet regeneration had occurred. A small percentage of these beta cells were labelled with the red tag, suggesting some newly formed beta cells may have originated from a ductal cell. By documenting the role of pancreatic ductal cells during beta cell regeneration, the work outlined in this thesis has contributed to the development of cell-free and protein-based regenerative therapies for diabetes.

Available for download on Monday, May 01, 2023

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