Stem cell models of the trophoblast

滋养层干细胞模型

• 批准号: RGPIN-2019-04363
• 负责人: Cox, Brian
• 金额: $ 2.4万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=685039
• 关键词: Stem; cell; models; trophoblast

How do we sexually reproduce? Compared to other areas of human and animal biology the biology of reproduction is less investigated. Despite its essential function of keeping the fetus alive until birth, the placenta is the least studied organ in the body. Searching for research articles about the “placenta” finds 90,909 publications, while the kidney has over 800,000 and brain over 1,000,000. There are many reasons for this lack of placental research. First, failures of the placenta's growth or function often lead to stillbirth or miscarriage, meaning there is no patient to treat (aside from the grieving parents). Second is that female-specific biology has been traditionally understudied. ***We have serious medical issues that we cannot solve. The most common pathologies of pregnancy, such as the hypertensive disorder of preeclampsia and preterm birth (combined affecting nearly 10% of all pregnancies) have no treatments, other than delivery of the baby and transfer to intensive care. This failure is due to the first problem; we have not conducted sufficient basic science research to understand this most critical organ to mammalian reproduction and development. ***Advancing the basic biology of the trophoblast. Within my proposed research, my group will assess human and mouse placenta tissues to characterise the diversity of cell types and their changes during development. To accomplish this, we will use single-cell gene expression methods (RNA-sequencing) and statistical clustering. Additionally, new bioinformatics methods that utilise the ratio of spliced to non-spliced RNA are used to identify developmental connectivity between these clusters of cells. Stem cell models of mouse and human trophoblast will be used to characterise cell signalling and expressed genes essential to trophoblast development. Signalling is addressed using activators and inhibitors to guide cells into specific mature trophoblast cell types. Gene function is investigated using gene deletion by the CRISPR-CAS9 method. In particular, I will focus on imprinted genes. ***Imprinting and the trophoblast. Imprinting is a process where a gene's expression is differentially regulated depending on its parent of origin. The evolution of gene imprinting occurred with the evolution of the trophoblast and the placenta. Imprinting is typically controlled by differential DNA methylation, which we will observe this by DNA sequencing. Many known imprinted genes are found in sets associated with one or more long non-coding RNAs. RNA sequencing can identify imprinted genes as genes that are expressed by only one allele based on the presence or absence of known sequence polymorphisms between a genes alleles. Through the integrated assessment of DNA methylation, RNA-sequencing and genomic sequences I will identify novel and human-specific imprinted genes for deletion to assess their function on trophoblast cell development. **

我们如何进行有性繁殖？与人类和动物生物学的其他领域相比，生殖生物学的研究较少。尽管胎盘的基本功能是维持胎儿的生命，但它是人体中研究最少的器官。搜索关于“胎盘”的研究文章，发现有90，909篇出版物，而肾脏有80多万篇，大脑有100多万篇。缺乏胎盘研究的原因有很多。首先，胎盘生长或功能的失败通常会导致死胎或流产，这意味着没有病人可以治疗（除了悲伤的父母）。第二，女性特有的生物学传统上研究不足。** 我们有严重的医疗问题，我们无法解决。最常见的妊娠病理，如先兆子痫和早产的高血压疾病（合并影响近10%的妊娠），除了分娩和转移到重症监护室外，没有治疗方法。这一失败是由于第一个问题;我们没有进行足够的基础科学研究来了解这个对哺乳动物生殖和发育最关键的器官。* 推进滋养层的基础生物学。在我提议的研究中，我的团队将评估人类和小鼠的胎盘组织，以确定细胞类型的多样性及其在发育过程中的变化。为了实现这一点，我们将使用单细胞基因表达方法（RNA测序）和统计聚类。此外，新的生物信息学方法利用剪接与非剪接RNA的比例来识别这些细胞簇之间的发育连接。小鼠和人类滋养层的干细胞模型将用于研究滋养层发育所必需的细胞信号传导和表达基因。信号是解决使用激活剂和抑制剂，以引导细胞进入特定的成熟滋养层细胞类型。通过CRISPR-CAS9方法使用基因缺失研究基因功能。特别是，我将集中在印记基因。*** 印记和滋养层。印记是一个基因表达根据其起源亲本而受到差异调节的过程。基因印迹的进化是伴随着滋养层和胎盘的进化而发生的。印记通常由差异DNA甲基化控制，我们将通过DNA测序观察到这一点。许多已知的印记基因被发现与一个或多个长的非编码RNA相关。RNA测序可以根据基因等位基因之间是否存在已知的序列多态性来鉴定印迹基因，即仅由一个等位基因表达的基因。通过DNA甲基化，RNA测序和基因组序列的综合评估，我将确定新的和人类特异性的印迹基因缺失，以评估其对滋养层细胞发育的功能。**