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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=763289
• 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，生殖生物学研究较少。尽管胎盘具有维持胎儿存活直到出生的基本功能，但它是人体内研究最少的器官。搜索有关“胎盘”的研究文章发现90,909篇论文，而肾脏有80多万篇，大脑有100多万篇。这种缺乏胎盘研究的原因有很多。首先，胎盘的生长或功能障碍往往会导致死产或流产，这意味着没有病人需要治疗(除了悲痛的父母)。第二，传统上对女性特有的生物学研究不足。我们有严重的医疗问题，我们无法解决。最常见的妊娠疾病，如先兆子痫和早产(合并影响所有妊娠的近10%)，除了分娩和转入重症监护外，没有其他治疗方法。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繁殖和发育最关键的器官。推进滋养层细胞的基础生物学研究。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，以表征细胞类型的多样性及其在发育过程中的变化。为了做到这一点，我们将使用单细胞基因表达方法(RNA测序)和统计聚类。此外，利用剪接的RNA与非剪接的RNA的比率的新的生物信息学方法被用来识别这些细胞簇之间的发育连接。将使用小鼠和人类滋养层细胞的干细胞模型来表征细胞信号和表达滋养层细胞发育所必需的基因。信号是使用激活剂和抑制物来引导细胞进入特定的成熟滋养层细胞类型。用CRISPR-Cas9方法通过基因缺失来研究基因功能。我将特别关注印记基因。印记和滋养细胞。印迹是一个过程，基因的表达受到不同来源的亲本的不同调控。基因印迹的进化是随着滋养细胞和胎盘的进化而发生的。印迹通常由不同的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是在与一个或多个长的非编码RNA相关的集合中发现的。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来识别印记基因，即只有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，我将识别新的和人类特有的印记基因缺失，以评估它们在滋养细胞发育中的功能。