Developing stem cell-derived macrophages to study host pathogen interactions and advance strategies to combat globally important disease in the pig
Lead Research Organisation:
University of Edinburgh
Department Name: The Roslin Institute
Abstract
Domesticated pigs are an important source of dietary animal protein for many people across the world, and securing the productivity of pig farming is a global imperative. Currently, commercial pig farming faces severe threats from two highly transmissible viral diseases, African Swine Fever Virus (ASFV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). ASFV causes a haemorrhagic fever that has a mortality rate of 90-100% and is highly contagious. There is no effective vaccine against ASFV, and strict containment and culling are the only strategies available for combating its spread; a strategy that has severe welfare implications and very high economic costs. A recent ASFV outbreak has led to the slaughter of 100-200 million pigs in China, which represents 25% of global pig production. PRRSV is less virulent, but widespread in commercial herds causing abortions in pregnant sows, high morbidity and mortality in piglets, and is the most economically important virus pathogen in pig farming (> 1 billion Euros). Combating ASFV and PRRSV is a priority, and considerable research resources in academic institutions and animal health companies are being directed to developing strategies to deal with these pathogens.
The preferred cellular host for ASFV and PRRSV is a white blood cell, the macrophage. This cell type, prepared directly from pigs, is the "Gold Standard" experimental culture model for studying virus-pig infection. However, these primary cells do not grow in culture, are subject to batch-to-batch variation and must be continuously replenished using animal-derived samples. Alternative virus host cell lines are available, but, either lack key macrophage characteristics, or are difficult to grow in culture. To provide normal macrophages that do not rely on a constant supply of pigs, we have developed a new system to generate unlimited numbers of macrophages from pig stem cells in culture. The stem cells can be grown indefinitely in large numbers, genetically modified, and be readily differentiated into macrophages. The macrophages have the same characteristics as cells isolated from pigs, including infection by ASFV and PRRSV. We propose that this stem cell based system can provide a new experimental platform for studying the interaction between the viruses and pig macrophage, and will significantly reduce reliance upon pigs in this area of research - as well as developing strategies to mitigate the devastating effects of ASFV and PRRSV disease on commercial farms.
In this proposal we will further develop this culture system to maximise output of macrophages, thus reducing a requirement for pigs in ASFV and PRRSV research. The proposal has two objectives: 1) To optimise the stem cell differentiation methodology by developing reporter cell lines that allow efficient selection of effective culture protocols; and 2) To test how immortalisation factors can be used to isolate and selectively propagate stem cell derived macrophage progenitor cells.
An unlimited supply of stem cell derived macrophages, combined with the ability to genetically modify the cells at will, provides a powerful and flexible tool in ASFV and PRRSV research, including the development of, and production of, new viral vaccines. The cells/technology can be readily applied in any standard laboratory and therefore we believe that stem cell-derived macrophages will significantly reduce the requirement for pigs as a source of macrophages and reduce the number of pigs used in viral challenge experiments. Since stem cells differentiate into many other cell types, this approach has wider applications in studying other viruses or pathogens that infect pigs, e.g. Coronavirus, Hepatitis E, Toxoplasma etc. Finally, this stem cell based approach can be applied to other species, and will have a broader impact on livestock research, reducing animal use in research, and improving animal health and welfare in the laboratory and on the farm.
The preferred cellular host for ASFV and PRRSV is a white blood cell, the macrophage. This cell type, prepared directly from pigs, is the "Gold Standard" experimental culture model for studying virus-pig infection. However, these primary cells do not grow in culture, are subject to batch-to-batch variation and must be continuously replenished using animal-derived samples. Alternative virus host cell lines are available, but, either lack key macrophage characteristics, or are difficult to grow in culture. To provide normal macrophages that do not rely on a constant supply of pigs, we have developed a new system to generate unlimited numbers of macrophages from pig stem cells in culture. The stem cells can be grown indefinitely in large numbers, genetically modified, and be readily differentiated into macrophages. The macrophages have the same characteristics as cells isolated from pigs, including infection by ASFV and PRRSV. We propose that this stem cell based system can provide a new experimental platform for studying the interaction between the viruses and pig macrophage, and will significantly reduce reliance upon pigs in this area of research - as well as developing strategies to mitigate the devastating effects of ASFV and PRRSV disease on commercial farms.
In this proposal we will further develop this culture system to maximise output of macrophages, thus reducing a requirement for pigs in ASFV and PRRSV research. The proposal has two objectives: 1) To optimise the stem cell differentiation methodology by developing reporter cell lines that allow efficient selection of effective culture protocols; and 2) To test how immortalisation factors can be used to isolate and selectively propagate stem cell derived macrophage progenitor cells.
An unlimited supply of stem cell derived macrophages, combined with the ability to genetically modify the cells at will, provides a powerful and flexible tool in ASFV and PRRSV research, including the development of, and production of, new viral vaccines. The cells/technology can be readily applied in any standard laboratory and therefore we believe that stem cell-derived macrophages will significantly reduce the requirement for pigs as a source of macrophages and reduce the number of pigs used in viral challenge experiments. Since stem cells differentiate into many other cell types, this approach has wider applications in studying other viruses or pathogens that infect pigs, e.g. Coronavirus, Hepatitis E, Toxoplasma etc. Finally, this stem cell based approach can be applied to other species, and will have a broader impact on livestock research, reducing animal use in research, and improving animal health and welfare in the laboratory and on the farm.
Technical Summary
African Swine Fever Virus (ASFV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) are currently responsible for unparalleled financial losses and welfare concerns in commercial pig farming across the world. ASFV causes a lethal haemorrhagic fever that, in the absence of an effective vaccine, necessitates strict containment and mass culling to eradicate disease. PRRSV is less virulent but is widespread in pig herds and causes infertility and respiratory problems that make PRRS the most costly viral disease in commercial pig farming. Both viruses preferentially infect pig macrophages and studying these viruses until now has relied on primary macrophage cultures or transformed cell lines: both of which have major limitations. To overcome this limitation we have developed a stem cell-based culture system for producing normal pig macrophages that are readily infected with ASFV and PRRSV. We propose that macrophages generated from pig stem cell lines provide a biologically relevant and scalable system for ASFV/PRRSV research and vaccine development, significantly reducing the requirement for pigs as either a source of ex vivo macrophages, or as subjects in viral challenge experiments.
The aim of this programme is to maximise the efficiency of generating pig macrophages in culture by 1) Optimising macrophage differentiation using haematopoietic/macrophage reporter cell lines to characterise, monitor and refine the differentiation process, and 2) Deriving conditionally immortalised macrophage progenitor cell lines. Reporter activity and transcriptional profiling of key stages in stem cell-macrophage differentiation will inform rational development of optimised protocols and facilitate the establishment of new pig macrophages progenitor cell lines. This novel stem cell based platform will reduce the number of pigs required in ASFV/PRRSV research and will illustrate the potential for applying stem cell-based technology more generally in livestock research.
The aim of this programme is to maximise the efficiency of generating pig macrophages in culture by 1) Optimising macrophage differentiation using haematopoietic/macrophage reporter cell lines to characterise, monitor and refine the differentiation process, and 2) Deriving conditionally immortalised macrophage progenitor cell lines. Reporter activity and transcriptional profiling of key stages in stem cell-macrophage differentiation will inform rational development of optimised protocols and facilitate the establishment of new pig macrophages progenitor cell lines. This novel stem cell based platform will reduce the number of pigs required in ASFV/PRRSV research and will illustrate the potential for applying stem cell-based technology more generally in livestock research.
