Developing stem cell-derived macrophages to study host pathogen interactions and advance strategies to combat globally important disease in the pig

开发干细胞衍生的巨噬细胞来研究宿主病原体的相互作用并推进对抗全球重要猪疾病的策略

• 批准号: NC/V001140/1
• 负责人: Thomas Burdon
• 金额: $ 44.72万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FV001140%2F1
• 关键词: Developing; stem; cell; derived; macrophages

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.

对于世界各地的许多人来说，驯养猪是饮食动物蛋白的重要来源，确保养猪的生产力是全球当务之急。目前，商品化养猪业面临着两种高度传染性的病毒性疾病--非洲猪瘟病毒(ASFV)和猪繁殖与呼吸综合征病毒(PRRSV)的严重威胁。ASFV引起一种死亡率为90%-100%的出血热，具有高度传染性。目前还没有针对ASFV的有效疫苗，严格控制和扑杀是遏制其传播的唯一战略；这一战略具有严重的福利影响和非常高的经济成本。最近爆发的ASFV疫情导致中国屠宰了1-2亿头猪，占全球生猪产量的25%。PRRSV的毒性较小，但在商业猪群中广泛传播，导致怀孕母猪流产、仔猪高发病率和死亡率，是养猪场最重要的经济病毒病原体(&gt；10亿欧元)。抗击ASFV和PRRSV是一个优先事项，学术机构和动物保健公司的大量研究资源正被用于制定应对这些病原体的战略。ASFV和PRRSV的首选细胞宿主是白细胞，即巨噬细胞。这种直接从猪中提取的细胞类型，是研究病毒-猪感染的“黄金标准”实验培养模型。然而，这些原代细胞不会在培养中生长，会受到不同批次的影响，必须使用动物来源的样本不断补充。有其他病毒宿主细胞株可用，但要么缺乏关键的巨噬细胞特征，要么难以在培养中生长。为了提供不依赖猪的持续供应的正常巨噬细胞，我们开发了一种新的系统，可以从培养中的猪干细胞产生无限数量的巨噬细胞。干细胞可以无限期地大量生长，经过基因改造，很容易分化为巨噬细胞。巨噬细胞与从猪分离的细胞具有相同的特征，包括感染ASFV和PRRSV。我们认为，这个基于干细胞的系统可以为研究病毒和猪巨噬细胞之间的相互作用提供一个新的实验平台，并将显著减少这一研究领域对猪的依赖-以及开发战略，以减轻ASFV和PRRSV疾病对商业农场的破坏性影响。在这项建议中，我们将进一步开发这一培养系统，以最大限度地提高巨噬细胞的产量，从而减少ASFV和PRRSV研究中对猪的要求。该提案有两个目标：1)通过开发能够有效选择有效培养方案的报告细胞系来优化干细胞分化方法；2)测试如何使用永生化因子来分离和选择性地繁殖干细胞来源的巨噬细胞前体细胞。干细胞来源的巨噬细胞的无限供应，加上随意对细胞进行基因修改的能力，为ASFV和PRRSV的研究提供了一个强大而灵活的工具，包括开发和生产新的病毒疫苗。该细胞/技术可以很容易地应用于任何标准的实验室，因此我们相信干细胞来源的巨噬细胞将显著减少对猪作为巨噬细胞来源的需求，并减少用于病毒攻击实验的猪的数量。由于干细胞可以分化为许多其他类型的细胞，这种方法在研究其他感染猪的病毒或病原体方面有更广泛的应用，如冠状病毒、戊型肝炎、弓形虫等。最后，这种基于干细胞的方法可以应用于其他物种，并将对家畜研究产生更广泛的影响，减少研究中使用的动物，并改善实验室和农场的动物健康和福利。