Beta Cell Development and Regeneration Program
Characterisation of Stem Cell Sources
Dr Fang-Xu Jiang; Emma Jamieson; Ben Dwyer
Liver progenitor oval cells stained with an oval cell marker (green) and our new monoclonal antibodies (red). Panel A shows that this antibody stains a separate cell population in the liver. The antibody in panel B co-stains the oval cells, defined by the green marker (co-staining cells appear orange in this combined image).
The current focus of Ben's project is exploring the use of liver embryonic stem (ES) cells as a source of β cells. To date, he has generated ES cells which fluoresce yellow when insulin is produced, allowing him to see when the ES cells have become insulin-producing cells. Our aim is to differentiate ES cells in the laboratory by mimicking the processes that occurs in the body.
Another promising stem cell population is liver stem cells, also called "oval cells". We have generated antibodies which recognise these oval cells in injured liver. Some of these antibodies also recognise molecules on cells within the developing pancreas. Our recent experiments suggest that this antibody highlights oval-like cells within the liver and, as they also stain pancreas cells, may be useful for identifying a group of oval cells capable of becoming β cells. This process is termed "transdifferentiation". The antibodies we have developed will be useful in studies of both liver and pancreatic stem cells.
Development of Beta Cells in Tissue Culture
Foetal pancreas cultures that received injections of MIP-GFP liver stem cells. Different stem cell lines were injected into the pancreas culture on the left and the right. Upper panel: normal light. Lower panel: under UV light to view the Green Fluorescent Protein produced by cells that are making insulin. The cells injected into the right pancreas culture have developed so that they can express the insulin gene. The cells in the left pancreas do not have this ability.
In collaboration with Professor George Yeoh (UWA) we have generated five clones of liver stem cells from the foetal liver of mice (MIP-GFP). These MIP-GFP mice show a green fluorescent protein (GFP) in any cells that make insulin. Therefore, appearance of fluorescence in these MIP-GFP clones is an indication that they transdifferentiated into β cells. When placed with foetal pancreas, one of the five clones expresses fluorescence, telling us that it expresses the insulin gene.
This indicates that we have established a system in which pancreatic cells may encourage liver cells to transdifferentiate into β cells. Other research groups have had to used artificial expression of β cell genes to drive this transdifferentiation.
Beta Cell Development within the Pancreas
Cells that produce antibodies (Hybridomas) have been generated in collaboration with the Monoclonal Antibody Facility. Fourteen hybridomas produce antibodies that react with foetal pancreas and also our embryonic stem cell line. This suggests that these antibodies react with a stem cell marker and provide a "handle", allowing us to select and isolate stem cells from pancreatic or other tissues.
One of the major issues in this area is to determine whether the insulin-secreting β cells present in the adult pancreas are derived from self-duplication, or from differentiation of the pancreatic stem cells (pancreatic progenitor/stem cells (PPSCs)). Studies from Canada, have shown that insulin-independence can be achieved for as long as seven years in some patients after pancreatic islet transplantation. Because the life span of individual β cells is only about 30-60 days, new insulin-secreting β cells must come from somewhere - either self-duplication or differentiation of PPSCs. We will employ state-of-the-art resources and technologies to address this difficult question. The lack of an excellent flow cytometry facility in WA has been an obstacle to our progress in this area thus far.
Another area of active research is to further characterise PPSCs during β cell development, a project Fang-Xu initiated several years ago while at WEHI. In addition to using the above resources and technologies, high-throughput microarray analysis of PPSCs will be undertaken to understand their genes. Understanding the mechanisms of PPSC development could provide an inexhaustible source of β cells in the laboratory from either human PPSCs or ES cells. In order to develop a cure for T1D, we will need to generate an unlimited number of β cells for transplantation therapy.