Human stem cell derived neurones and astrocytes as an in vitro model of human prion infection and replication

朊病毒疾病对动物和人类健康产生深远影响。在英国，牛海绵状脑病（BSE）和变型克雅氏病（vCJD）的影响仍然是一个活生生的公共卫生问题，在北美，鹿的慢性消耗性疾病被认为是对人类健康的潜在威胁，但在世界范围内也是如此，每年有百万分之一的人死于散发或家族形式的疾病。朊病毒疾病与阿尔茨海默病等更常见的痴呆症病因类似，因为它们的特点是大脑中异常折叠的蛋白质沉积，但与阿尔茨海默病不同的是，朊病毒疾病明显可以从一个人或一只动物传播到另一只动物。这种相对容易在个体之间传播的情况意味着，几十年来，动物实验一直是朊病毒疾病研究的主要手段，首先使用啮齿动物和猴子，然后使用越来越复杂的转基因小鼠来模拟动物和人类的朊病毒疾病。人类朊病毒疾病的转基因小鼠模型是目前可用的人类神经退行性疾病的转基因小鼠模型中最有用的模型之一，然而它们同时具有经济和伦理成本。用人类朊病毒感染培养的人类细胞的能力将是一项重大进步，并将加速我们在分子和细胞水平上对朊病毒疾病的理解，从而提高我们开发治疗方法的能力。以前用人类朊病毒感染人类细胞的尝试基本上是不成功的，迄今为止还没有广泛使用的任何人类朊病毒疾病的细胞培养模型。我们假设之前的失败是由于在培养中生长和繁殖良好的人类细胞种类与人类大脑中朊病毒的自然目标（称为星形胶质细胞和神经元的特殊脑细胞）之间的差异造成的。从人类胚胎和诱导多能干细胞中制造多种功能性人类细胞（包括星形胶质细胞和神经元）的能力为生物学和医学（包括细胞移植疗法）提供了很多帮助，但它也可能为朊病毒疾病研究提供一些极其有用的东西。我们已经鉴定了一系列人类干细胞系的序列和正常朊病毒蛋白的表达，并根据我们从患者和实验动物研究中了解到的情况，选择了我们预测的易受vCJD朊病毒感染的干细胞系。然后，我们将这些干细胞制成星形胶质细胞，并将它们暴露于死于vCJD的患者的少量人脑组织中。我们的研究结果表明，人类朊病毒确实可以在培养的人类细胞中复制，只要注意使用与患者大脑中受影响的细胞非常相似的细胞。我们试图利用这一观察结果，找出(i)神经元（除了星形胶质细胞）是否可以被vCJD朊病毒感染，（ii）与其他形式的克雅氏病相关的朊病毒是否可以感染干细胞衍生的星形胶质细胞和神经元，（iii）细胞培养系统是否复制已知的感染遗传屏障，（iv）朊病毒感染是否损害神经元和胶质细胞；(v)是否影响星形胶质细胞和神经元之间的相互支持关系。最后，我们将开发一种确定的方法来测量细胞中的朊病毒复制。这些实验结果将为提倡在特定种类的人类朊病毒基础和应用研究中使用干细胞衍生的神经元和星形胶质细胞替代实验动物提供良好的基础。

Prion diseases have a profound impact on animal and human health. This is especially true in the UK where the effects of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) remain a live public health issue, and in North America where chronic wasting disease in deer is considered a potential threat to human health, but it is also true worldwide where one in a million people every year die from sporadic or familial forms of the disease. Prion diseases resemble more common causes of dementia, such as Alzheimer's disease, in that they are characterised by the deposition of abnormally folded protein in the brain, but unlike Alzheimer's disease, prion diseases are demonstratively transmissible from one person or one animal to another. This relative ease of transmission between individuals has meant that animal experimentation has been the main stay of prion disease research for decades, first using rodents and monkeys and then using increasingly sophisticated genetically modified mice to model animal and human prion diseases. Transgenic mouse models of human prion disease are among the most useful of any of the currently available transgenic mouse model of human neurodegenerative disease, however they carry both an economic and ethical cost.The ability to infect human cells grown in culture with human prions would be a major advance and would lead to an acceleration in our understanding of prion diseases at the molecular and cellular level and consequently our ability to develop treatments for them. Previous attempts to infect human cells with human prion agent have been largely unsuccessful and to date there is no widely used cell culture model of any human prion disease. We have hypothesised that previous failures result from differences between the kinds of human cells that grow and multiply well in culture and the natural targets of prion in human brains, specialised brain cells called astrocytes and neurones. The ability to make functional human cells of many types (including astrocytes and neurones) from human embryonic and induced pluripotent stem cells offers much to biology and medicine, including cellular transplantation therapies, but it may also offer something extremely useful to prion disease research.We have characterised a series of human stem cell lines for the sequence and expression of the normal prion protein and selected the ones that we predict would be susceptible to vCJD prion infection based on what we learned from patients and from experimental animal studies. We then made these stem cells into astrocytes and exposed them to small quantities of human brain tissues from patients who died from vCJD. Our results show that human prions can indeed replicate in human cells in culture, provided that care is taken to use cells that closely resemble the cells affected in the brains of patients.We seek to capitalise on this observation by finding out (i) whether the neurones (in addition to astrocytes) can be infected with the vCJD prions, (ii) whether the prions associated with other forms of Creutzfeldt-Jakob disease can infect stem cell derived astrocytes and neurones, (iii) whether the cell culture system reproduces known genetic barriers to infection, (iv) if prion infection harms neurones and glia and (v) whether it affects the mutually supportive relationship between astrocytes and neurones. Lastly, we will develop a definitive method for measuring prion replication in cells. The results of these experiments will provide a sound basis on which to advocate the use of stem cell derived neurones and astrocytes as a replacement for experimental animals in specific kinds of basic and applied human prion research.