Improving the Efficiency of Induced Pluripotent Cell (IPS) Generation in the Dog

Stem cells have recently generated more public and professional interest than almost any subject in biology. These cells have captured the imagination of scientists as a result of the promise offered for a greater understanding of cellular biology and their potential for revealing pathways towards better treatment for chronic illnesses. In veterinary medicine, there is the added advantage of developing a series of reagents that can be used to reduce the number of animals in research. It has recently been demonstrated in humans and mice that adult cells in the body can be 'reprogrammed' to become stem cells by the introduction of four genes or 'factors'. These reprogrammed cells are called Induced Pluripotent Stem Cells (iPS cells) and are of incredible importance because it means that stem cells can be made without the need for deriving them from embryos. Recently, we demonstrated that this can be achieved in the dog using a viral gene delivery approach and we consider that IPS generation will have major beneficial effects, including: 1) We consider that an understanding of stem cell biology will help to understand chronic diseases of the dog. 2) This project will provide the raw materials for us to be able to explore the potential of regenerating diseased tissues. 3) This project will provide the raw materials to be able to test pharmaceutical drugs without the need for experimental animals. 4) For veterinary medicine to advance significantly, these cells and reagents are vital for providing the foundation and building blocks for the next generation of medicines. 5) Ultimately, efficient generation of IPS cells could be applied to generate disease-specific IPS cells that could be applied to better understand the basic mechanisms of disease. However, the current methodology to make iPS cells includes the use of viruses to deliver the factors or genes and is considered to be an inefficient process. For this project we consider that Canine iPS cells can be generated using synthetically modified mRNA delivery (possible combined with small molecule inhibitors) or recombinant proteins instead of viral vectors. Further, this project will analyse the reprogramming events during this process that will help us develop more efficient means of generating canine iPS cells rapidly. The overall aim of this project is to improve the efficiency of iPS generation in the dog, characterize these lines at a molecular level, and explore the mechanisms that maintain pluripotency. We believe that this project will provide the reagents and tools to form the basis of 21st century advances in veterinary medicine. Further, it will provide reagents to help reduce the number of experimental animals used in research.

The aim of this project is to develop a non-integrating, non-viral system for reprogramming canine cells and to improve the efficiency of iPSC generation. 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.

In addition to researchers in cell biology, cellular reprogramming, and veterinary basic sciences, there are a number of other key beneficiaries: 1) Commercial private sector: This is an LINK programme and data arising from this proposal could have an impact on commercial companies developing products based upon cellular reprogramming. It is anticipated that we will generate a toolbox of reagents that can be applied to research using the dog as a model system. This includes the development of reagents (In particular IPS cells) that could be used to develop in vitro systems for drug and toxicity testing. This could have a major impact on reducing, refining and replacing animals in research. 2) This research may have an impact on government and policy makers with regards the value of comparative studies between species. As an example, it may highlight the importance of large animal models to understanding both human and animal physiology and pathophysiology. 3) The Veterinary Profession will benefit with a greater understanding of reprogramming mechanisms in health and disease. This could have major implications for biomarker discovery in domestic animals and also further understanding normal and disease progression in veterinary species. Ultimately, the efficient generation of IPS cells in the dog could be applied to a system for rapidly generating animal disease-specific IPS cells. Combining this technology with the publication of the canine genome project will allow a greater understanding of the genetic basis of disease. 4) The wider animal-owning population will ultimately benefit with a greater understanding of normal health and disease of their animals. Considering the UK pet population and the amount of money that is spent of veterinary care, this could, long term, have major economic benefits to the UK. 5) The research will help develop UK veterinary research on an international level and will thus have an economic impact on gaining further national and international funding. 6) The staff working on this project will further enhance their research skills. in addition they will enhance their skills in communication through publication preparation and oral and poster presentations to a wider international audience. We also now encourage all of our research staff to take part in the Edinburgh Beltane Beacon programme to have specific training and experience in Public Engagement of Science.