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

朊病毒病对动物和人类的健康都有深远的影响。这在英国尤其如此，在英国，牛海绵状脑病(BSE)和变种克雅氏病(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，每年每百万人中就有一人死于这种疾病的零星或家族性形式。Prion病类似于阿尔茨海默病等更常见的痴呆症病因，因为它们的特征是异常折叠的蛋白质在大脑中沉积，但与阿尔茨海默病不同的是，Prion疾病可以从一个人或一种动物传染给另一种人或动物。这种在个体之间传播的相对容易意味着，几十年来，动物实验一直是普恩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普恩病毒疾病。在目前可用的人类神经退行性疾病转基因小鼠模型中，人类Pron疾病的转基因小鼠模型是最有用的之一，但它们同时也带来了经济和伦理成本。用人类Prion感染在培养中生长的人类细胞的能力将是一个重大的进步，并将加速我们在分子和细胞水平上对Prion疾病的理解，从而促进我们开发治疗这些疾病的能力。以前用人类Pron试剂感染人类细胞的尝试大多没有成功，到目前为止还没有广泛使用的任何人类Pron疾病的细胞培养模型。我们曾假设，之前的失败是由于在培养中生长和繁殖良好的人类细胞类型与人脑中Pron的天然靶点之间的差异，人类大脑中的特定脑细胞称为星形胶质细胞和神经元。从人类胚胎和诱导的多能干细胞中制造多种类型的功能性人类细胞(包括星形胶质细胞和神经元)的能力为生物学和医学提供了很多东西，包括细胞移植疗法，但它也可能对普恩病毒疾病的研究提供非常有用的东西。我们已经为一系列人类干细胞系的正常普恩蛋白的序列和表达进行了特征描述，并根据我们从患者和实验动物研究中了解到的情况，选择了我们预测会受到vCJD普恩病毒感染的细胞。然后，我们将这些干细胞制成星形胶质细胞，并将它们暴露在死于vCJD的患者的少量人脑组织中。我们的结果表明，人类在培养中确实可以在人类细胞中复制，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，通过以下方式利用这一观察：(I)神经元(除星形胶质细胞外)是否可以感染vCJD普恩病毒，(Ii)与其他形式的克雅病相关的普恩病毒是否可以感染干细胞来源的星形胶质细胞和神经细胞，(Iii)细胞培养系统是否复制已知的感染遗传屏障，(4)Pron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普恩复制。这些实验的结果将为倡导使用干细胞来源的神经元和星形胶质细胞作为实验动物在特定类型的基础和应用人类普里安研究中的替代品提供可靠的基础。

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.