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

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description We have refined techniques for producing canine induced pluripotent stem cells. These are cells that are derived from adult tissue but can be used in a similar fashion to stem cells.

Since our last submission to research fish we have now completed the project. We have successfully derived canine induced pluripotent stem cells from somatic cells. We have characterised these and we have demonstrated that they are able to form primitive tumours (teratomas) in rodent species. This is one of the hallmarks of pluripotency. We have generated a number of cell lines, some of which are not true pluripotent cells but develop highly primitive tumours (non teratomas) in rodents (for which we are further investigating). Currently we are writing the key manuscripts for publication.
Exploitation Route Developing in vitro assays for drug testing and reducing the number of animals required for research
Sectors Agriculture

Food and Drink

Healthcare

Pharmaceuticals and Medical Biotechnology

 
Description The non academic impact of this project is the closer association we have developed with our industrial partner which is helping to develop new programmes of research. The generation of more robust canine IPS cells is now leading to a programme looking at CRISPR technology in canine cells and further industry funding opportunities
First Year Of Impact 2019
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Merk Animal Health 
Organisation Merck
Country Germany 
Sector Private 
PI Contribution This is an industrial Link grant that began with Intervet BV and then was taken over by MERCK animal health. We have developed the techniques for IPS cell production in the dog
Collaborator Contribution Provision of: 1) Intellectual input and study design 2) Development of Reagents and cell lines
Impact Combined research has led to improved IPS production as per objectives. This will eventually produce publications
Start Year 2012
 
Description support to generate commercial canine IPS cells 
Organisation ReproCELL Europe Ltd
Country United Kingdom 
Sector Private 
PI Contribution Joint discussions on best methodology to commercially produce canine IPS cells
Collaborator Contribution Joint discussions on best methodology to commercially produce canine IPS cells
Impact none as yet
Start Year 2017
 
Description Arthur Willis Distinguished Speaker Lecture entitled Can comparative biology inform rational cancer drug development? 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Sparked discussion

Development of early stage collaborations in Canada
Year(s) Of Engagement Activity 2013
 
Description British Society of Veterinary Pathology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Sparked discussion and debate

Requests for information about our work
Year(s) Of Engagement Activity 2014
 
Description International Conference UNAM University Mexico City 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact > 100 people attended from various international countries. I made two state of the art presentations on stem cell biology and set cells in disease.
Year(s) Of Engagement Activity 2016
 
Description Invited Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited lecture on "One medicine" at University College Dublin. Key Note Lecture
Year(s) Of Engagement Activity 2019
 
Description Invited Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact NHS Christmas Lecture and around 60 people attended which included postgraduate students and researchers and doctors.
Year(s) Of Engagement Activity 2017
 
Description Invited Talk, University of Hokkaido, Japan 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Sparked discussion afterwards

Supported development of MOU to provide exchange for postgraduate and undergraduate students
Year(s) Of Engagement Activity 2014