Advanced Human Pluripotent Stem Cell Kidney Organoid Model for Investigating Development and Disease

肾功能对生命至关重要，治疗肾衰竭每年花费NHS 20亿英镑。大多数研究肾脏发育和疾病衰竭的科学家使用动物体内和体外模型，特别是小鼠。然而，小鼠肾脏与人类肾脏不同，无论是在总体组织还是在发育过程中的精确分子组成。这可以解释为什么具有特定基因缺陷的小鼠通常不会表现出与具有这些变化的人类相同的症状。该提案的目的是使用非常早期的干细胞（人类多能干细胞：hPSC）以非常小的规模（微型类器官）建立发育中肾脏的人类3D模型。目前，我们和其他人制造的标准肾脏类器官需要大量的细胞/类器官（约100，000+），并且缺乏某些细胞类型，包括关键的免疫系统细胞，称为巨噬细胞。这些巨噬细胞在胎儿肾脏的发育中发挥作用，其作用不同于它们在出生后的炎症中的作用。它们促进血管发育和肾脏流出区的发育，这两者在目前的hPSC-肾脏模型中发育不良。我们假设，通过引入巨噬细胞和改善营养物/废物扩散，可以使肾脏类器官成为显着更好的肾脏模型。我们将根据已发表的方法，在我们经验丰富的合作者的帮助下，从我们的hPSC中产生巨噬细胞，并测试不同比例的巨噬细胞在改善我们的干细胞-肾类器官中的作用，首先是在传统的“大”类器官中，然后是在细胞数量为1/10或更少的微类器官中。我们将利用这个平台来研究免疫巨噬细胞是否可以促进肾类器官的发育。在我们的研究中，类器官将以一种补偿缺乏血液流过的方式生长，通过在专门设计的专用腔室中培养它们来在微小的肾脏微型类器官周围产生流动。该模型将允许增加培养时间，以便肾组织保持健康并进一步发育。微型类器官将比目前的干细胞衍生的大型类器官更复杂，并且在组成上与发育中的肾脏更相似。这个人类系统将更好地理解人类肾脏发育和影响人类肾脏发育的疾病，特别是那些由基因的有害变化引起的疾病。它将取代将类似的基因改变引入小鼠体内并观察其肾脏发育的需要，而肾脏发育可能会受到不同的影响。我们可以将该模型适用于其他组织，如肝、肠或肺hPSC或组织类器官模型。因此，该平台将适用于更好地了解人类发育和疾病，并通过产生许多微型类器官（按比例放大），用于测试可能缓解肾脏疾病的药物，或测试它们是否对肾脏有害。从长远来看，它可以提供一种产生补充肾脏组织的途径，以帮助生病的肾脏。

Kidney function is essential for life and treatment for kidney failure costs the NHS £2 billion each year. Most scientists investigating kidney development and failure in disease use animal in vivo and ex-vivo models particularly mice. However, mouse kidneys are not the same as human kidneys, either in gross organisation or in the precise molecular composition during development. This may explain why mice with particular gene defects often do not show the same symptoms as a human with these changes. The aim of this proposal is to use very early stem cells (human pluripotent stem cells: hPSCs) to establish a human 3D model of the developing kidney at very small scale (micro-organoids). At the moment the standard kidney organoids we and others make, require a large number of cells/organoid (about 100,000+) and lack certain cell types including critically, cells of the immune system called macrophages. These macrophages play roles in development of the fetal kidney, roles that are different from their role in inflammation after birth. They promote blood vessel development and that of the outflow regions of the kidney, both of which develop poorly in the current hPSC-kidney models. We hypothesise that kidney organoids can be made dramatically better kidney models by introducing macrophages and improving nutrient/waste diffusion. We will generate macrophages from our hPSCs according to published methods, aided by our experienced collaborators, and test the effect of different proportions of these in improving our stem cell-kidney organoids, first in the conventional 'macro'-organoids, then in micro-organoids with 1/10th the number of cells or fewer. We will use this platform to ask whether immune macrophages can enhance kidney organoid developmental. In our study, the organoids will be grown in a way that compensates for lack of blood flowing through, by generating flow around the tiny kidney micro-organoids through culturing them in specially designed purpose built chambers. This model will allow the time in culture to be increased so that the kidney tissue remains healthy and develops further. The micro-organoids will be more complex and more similar in composition to developing kidneys than current stem cell derived macro-organoids. This human system will be better for understanding human kidney development and diseases affecting development of the human kidney, particularly those caused by detrimental changes in genes. It will replace the need to introduce similar gene changes into mice and look at their kidney development-which may anyway be affected differently. We can adapt the model for other tissues like liver gut or lung hPSC- or tissue-organoid models. Thus, this platform will be suitable for use in better understanding of human development and disease and by generating many micro organoids (scale up), for use in testing drug, which may alleviate kidney disease, or testing if they are harmful to the kidney. In the long term, it could provide a route for generating supplementary kidney tissue to aid ailing kidneys.