Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Lajoie, Gilles A.

2nd Supervisor

Hess, David A.

Co-Supervisor

Abstract

Novel strategies to stimulate the expansion of β-cell mass in situ are warranted for diabetes therapy. Cell-replacement therapies for the treatment of diabetes have become a focal point in recent years. Endogenous regeneration of β-cell mass has been demonstrated using human multipotent stromal cells (hMSC). However, the secretory factors responsible for initiating endogenous regeneration remain unknown. Successful large-scale proteomic applications to address these questions have been limited in part by difficulties in correctly selecting the appropriate methodologies. Thus, the goal of this thesis was a combination of assessing different proteomic workflows to facilitate investigation into hMSC biology, applying these methods to identify important factors secreted by hMSC for β-cell regeneration, as well as functionally investigating candidate proteins and refining current models of hMSC mediated β-cell regeneration.

In working towards these goals, we first assessed the advantages and disadvantages of multiple fractionation techniques to help guide future experimental designs in general proteomic workflows. By applying these methodologies, we probed the secretome of hMSC and identified candidate regulators responsible for regeneration of β-cell mass. In particular, we identified Wnt-signaling as an important contributor for islet regenerative capacity. Additionally, we recognized the clinical applicability of determining protein signatures that could be used to screen hMSC that possessed islet regenerative capacity. Therefore, a robust quantitative proteomics method was developed to screen hMSC that could be used in downstream clinical applications for β-cell regeneration. Taking cues from these proteomic screens, we demonstrate that intrapancreatic-delivery of concentrated hMSC conditioned media (CM) can independently mediate endogenous islet regeneration, without injecting cells. Therapeutic effect was augmented by increasing protein dose and by activation of Wnt-signaling during CM generation. The mechanisms of islet regeneration were multi-factorial, with evidence of glucagon+ cells emerging from the ductal niche within one day of CM injection, followed by α-β-cell conversion with NKX6.1-expression in transitioning β-cells, and augmented β-cell proliferation to generate functionally mature neoislets that respond to glucose. Altogether, these studies provide an extraordinary view of how this dynamic cell type can be used in clinical settings, to stimulate the expansion of β-cell mass and tip the balance in favor of islet regeneration versus destruction during diabetes.

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