Improving the efficiency of induced pluripotent cell (IPS) generation in the dog

The overall aim of this project is to develop a non-integrating, non-viral system to cause reprogramming in canine cells and to improve the efficiency of IPS generation in this species. Further, we aim to characterize these lines at a molecular level, and explore the mechanisms that maintain pluripotency. In part 1, we will ask whether canine IPS cells be generated efficiently using synthetically modified mRNA Briefly, we will synthesize canine-specific modified mRNA that will be used for reprogramming experiments. Initially we will synthesize modified RNA encoding GFP to demonstrate penetrant expression in a broad range of canine cells. Subsequently we will derive modified RNA molecules for the classical 4 factors (Oct-4, KLF-4, Sox-2 and cMyc and/or Lin-28). We will examine transfection efficiency and protein expression using immunofluourescence and flow cytometry. Subsequently, we will test the ability of modified mRNA to produce IPS cells in culture. Cells will be characterized using microscopy/morphology and the expression of specific proteins such as alkaline phosphatase. To further characterize the IPS cells we will map mRNA and microRNA expression during initiation, maturation and stabilization of IPS cells in both mesenchymal and epithelial cells. Ultimately, we will examine the potential of generated cells to produce teratomas in NOD-SCID mice. We will finally look at the potential for these derived cells to terminally differentiate into a specific somatic cell type. Subsequently, will compare the generation of IPS cells from either mesenchymal or epithelial cells, focusing on MET signaling to explore the effects of BMP proteins, TGF-b, and P53 and inhibitors of ARF signaling on IPS generation. In parallel, we will use mouse ES cells to fuse with canine B cells in a multi-well tissue culture system to produce Heterokaryons. We will use a range of media and inhibitors to derive the most suitable media conditions for stem cell culture for this species. If successful this could be applied to other species in an attempt to accelerate stem cell derivation in farm and companion animal species.

In this project we aim to derive stem cells from adult tissues in dogs. We do this by taking normal cells and adding proteins to the cells which allows them to revert a stem cell (a cell that has the capacity to turn into different cell types that make up the body tissues). If we can achieve this, we can develop cell culture systems to be able to reduce the number of animals required for drug testing. This process is very inefficient and this study aims to improve the efficiency.

1) Have been able to derive canine IPS cells
2) Canine IPS cells have been characterised
3) We have dissected the culture conditions required to maintain canine pluripotency