Poster Session 1 - R11
1,2Amanda Fantin, 2Jennifer Whiteley, 1,2,3Ian Rogers
1 Department of Physiology, University of Toronto, ON, Canada; 2 Women’s and Infant’s Health, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, ON, Canada; 3 Department of Obstetrics and Gynaecology, Mount Sinai Hospital and the University of Toronto, ON, Canada
Type 1 Diabetes Mellitus (T1DM) is an autoimmune disorder that destroys the body’s β-cells. These β-cells are located within clusters of endocrine cells in the pancreas called islets. Replacement of the lost β-cells through islet transplantation has emerged as a treatment in recent years. It has shown promise, especially for those T1DM patients with severe intractable hypoglycemia. However, issues with cadaveric islet quality and compatibility, as well as access to sufficient quantities, continues to limit this treatment option. To address these issues, the use of induced pluripotent stem cells (iPSC) to derive mature islets has been investigated. Issues persist, however, as the maturation process takes months in vivo and does not occur efficiently in vitro. To address these issues, it is clear that the factors affecting maturation and differentiation in pancreatic development must be further explored.
The extracellular matrix (ECM) is known to possess structural and biological properties important for the differentiation, migration and proliferation of cells during development. Furthermore, these cues have been shown to persist into adulthood where they are used in repair mechanisms. Despite this, traditional cell culture methods lack this cell- ECM interaction. It has been shown in multiple different organs, including the kidney and lungs, that adult ECM is capable of driving the maturation of progenitor cells in vitro. This project aims to investigate the role of murine adult pancreatic ECM in the maintenance and maturation of murine islets.
This project examined three different protocols for obtaining murine pancreatic ECM using a process called decellularization. This process involves the removal of cells and cellular contents from a tissue, leaving only the extracellular matrix and associated proteins. In order to maintain the structural and biological properties of the ECM, the decellularization process must be optimized for each tissue. Each of the methods used for decellularization affects the composition of the ECM and it’s proteins differently. Optimization of this process involves ensuring adequate cellular removal with the maximal retention of ECM proteins. Detergents are commonly used in decellularization protocols; however, they can vary greatly in strength and effect. To determine the ideal one for murine pancreas decellularization, three detergents were tested: SDS, CHAPS or Triton X-100. To deliver the solutions, whole-organ profusion was utilized. Immunohistochemistry (IHC) testing for the retention of key ECM proteins, as well as the removal of nuclear material, was performed for each protocol.
Upon inspection, all three detergent options produced visibly decellularized ECMs. IHC results for the CHAPS and SDS protocols are presently being analysed. For the Triton X-100 protocol, IHC results showed retention of the ECM proteins laminin, fibronectin and collagen IV. Additionally, results showed the removal of nuclear material under DAPI staining. While further work is needed, results are promising that murine pancreatic decellularization can be accomplished using SDS, CHAPS or Triton X-100. The examination of differences in the retention of ECM proteins following these decellularizations may allow for the optimization of the process. Once optimized, decellularization may allow for the attainment of ECM capable of mimicking cell-ECM interactions seen in pancreatic development. Such interactions may one day lead to improvements in the in vitro culture of islets through the use of ECM in a co-culture system.