Heterogeneity and cellular population dynamics in the placenta

与胎盘相关的妊娠并发症是对妇女健康的重大挑战。死产是围产儿死亡的主要原因，每年在英国造成3000多人死亡，全球约260万人死亡。胎儿生长受限会导致短期发病率，教育效果不佳，并容易在以后的生活中患糖尿病和心血管疾病。目前缺乏对这些并发症的机制认识和有效的治疗方法。这里提出的工作将提供胎盘中所有不同细胞类型的详细图谱--目前我们不知道有多少不同的细胞类型和亚型。我们还将确定这些细胞中存在哪些RNA，这与细胞功能直接相关。了解不同类型的细胞是如何形成的是了解正常胎盘发育的关键。单细胞内信使核糖核酸的分析方法是最近发展起来的。然而，由于母血和胎儿组织之间的屏障是多核的合胞体，目前的单细胞RNA测序方法是不合适的，需要新的方法。本提案描述了这些新方法及其在胎盘中的应用。目的本工作的主要目的是使用单细胞RNA测序来表征小鼠和人胎盘细胞的异质性，并确定滋养层细胞分化的调控步骤。为了解决这一目标，我们将回答以下3个具体问题：1)胎盘中有多少种细胞类型，它们的特征是什么？这将需要开发新的方法，特别是允许分析单个核和数千个单核中的非编码RNA。2)胎盘细胞群体如何随着时间的推移而变化？我们将通过从妊娠早期和足月收集的5个人胎盘组织的单核测序来评估不同的细胞群。我们将把这些数据与从怀孕期间5个不同时间收集的小鼠胎盘样本进行比较。3)调节这种异质性的机制是什么？小鼠滋养层干细胞可以在诱导分化的条件下生长。我们将对分化过程中多个时间点的单个细胞进行测序。在单细胞水平上对分化细胞群体进行采样，可以识别分化过程中的中间群体。我们将确定这一过程中分支点的调控因素，并测试过度表达或敲除这些基因是否会改变分化。潜在的应用和好处即使是预防一小部分死产和其他妊娠并发症也将是一个重大的好处。良好的产科护理意味着许多妊娠合并胎盘发育不良导致活产。然而，宫内生活受到损害的婴儿有患长期慢性疾病的风险，如心血管疾病和糖尿病。更好地了解胎盘的生长和功能有可能改善妊娠结局，这将有短期和长期的健康和经济利益。这些都是遥远的好处，但更直接的好处将是在胎盘、发育和基因调控生物学领域。建议的方法是创新的，需要跨学科的工作(微流体学、化学、分子生物学和生物信息学)，因此将使所有这些学科受益。这里建议的工作将与一个合作的工业合作伙伴-球流体有限公司进行。剑桥大学、巴拉罕研究所和球形流体公司之间的密切合作将进一步加强剑桥周围生物技术/制药公司和学术界之间的互动。这将对这三方有利，并可能吸引研发投资并增加经济活动，使英国经济受益。

ContextPlacenta-related complications of pregnancy are a major challenge for women's health. Stillbirth is a major cause of perinatal death, causing more than 3,000 deaths each year in the UK and ~2.6million worldwide. Fetal growth restriction results in short term morbidity, poor educational outcomes and predisposes to diabetes and cardiovascular disease in later life. Mechanistic understanding and effective therapies for these complications are lacking. The work proposed here will provide a detailed atlas of all the different cell types in the placenta -currently we don't know how many different cell types and sub-types there are. We will also determine which RNAs are present in each of these cells and this is directly relevant to cell function. Knowledge of how the different cell types arise is key to understanding normal placental development. Methods for the analysis of the mRNA within single cells have recently been developed. However, as the barrier between maternal blood and fetal tissue is a multi-nucleated syncytium current single "cell" RNA-sequencing methods are inappropriate and new approaches are needed. This proposal describes such new methods and their application to the placenta.AimsThe overarching aim of this work is to use single cell RNA sequencing to characterise placental cellular heterogeneity in mouse and man and to define regulatory steps in trophoblast differentiation. To address this aim we will answer the following 3 specific questions:1) How many cell types are there in the placenta and what are their distinguishing features? This will necessitate the development of new methods, specifically to allow the analysis of individual nuclei and the analysis of non-coding RNA in many thousands of single nuclei.2) How does the placental cell population change over time? We will evaluate the different cell populations by single nucleus sequencing from tissue collected from 5 human placentas in the first trimester and also at term. We will compare these with data from samples of mouse placenta collected at 5 different times in pregnancy.3) What are the mechanisms that regulate this heterogeneity?Murine trophoblast stems cells can be grown under conditions that induce differentiation. We will sequence single cells from multiple time points during differentiation. Sampling a population of differentiating cells at the single cell level allows identification of the intermediate populations during differentiation. We will identify regulatory factors at the branch points in this process and test whether over expression or knocking out these genes alters the differentiation.Potential applications and benefitsPreventing even a small proportion of still births and other complications of pregnancy would be a significant benefit. Good obstetric care means that many pregnancies complicated by poor placental development end in live births. However, babies whose intrauterine life was compromised are at risk for long-term chronic diseases, such as cardiovascular disease and diabetes. Better understanding of placental growth and function has the potential to improve pregnancy outcome and this will have both short and long term health and economic benefits. These are distant benefits but more immediate gains will be in the fields of placental, developmental, gene regulatory biology. The methods proposed are innovative and require cross-disciplinary work (microfluidics, chemistry, molecular biology and bioinformatics) and so will benefit all these disciplines.The work proposed here is to be carried out with a collaborative industrial partner - Sphere Fluidics Limited. Close collaboration between the University, the Babraham Institute and Sphere Fluidics will further enhance interactions among biotech/pharma and academia around Cambridge. This will be a benefit to these three parties and may attract R&D investment and increase economic activity to the benefit of the UK economy.