Defining the prerequisites of naive pluripotent human embryo cells for self-renewal in culture

定义幼稚多能人类胚胎细胞在培养物中自我更新的先决条件

• 批准号: MR/P010423/1
• 负责人: Jennifer Nichols
• 金额: $ 87.13万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FP010423%2F1
• 关键词: Defining; prerequisites; naive; pluripotent; human

Until recently human embryonic stem cell lines were derived following explantation of intact early human embryonic structures into culture. We have recently applied a novel protocol in which the embryonic cells are first separated from one another before culture is begun. By this means, we have generated normal stable stem cell lines that we named 'human naïve epiblast stem (HNES) cells'. There are several advantages with this new approach, since it enables production of individual clones from a single individual. From a developmental biology point of view, separation of the embryonic cells prior to culture prevents the inter-cell communication that drives cell specification, thus retaining them in a 'naïve' state. Also, multiple clones can be produced from each embryo. We propose to utilise this novel opportunity to sample individual clones at various stages during the derivation process, whilst expanding the remaining clones into HNES cell lines. The sampled clones will be separated into single cells, each of which will be profiled simultaneously for gene expression and epigenetic modifiers. This will allow us to track the process of derivation at a molecular level to determine what changes, if any, occur during the transition from embryonic cell to stable HNES cell line. This approach removes the genetic variability between individuals hampering all related studies performed so far. Another objective of this project is to take advantage of the frequent tendency (around 60%) of in vitro fertilised human embryos to exhibit mosaic aneuploidy. For these experiments, we propose to expand each of the colonies from a single explanted embryo separately and check the karyotype of each cell line. The resulting characterised HNES cell lines will then be lodged in the UK Stem Cell Bank for distribution to interested researchers. Most frequent amongst the reported aneuploidies is trisomy of the small chromosomes 21 or 22. Having separate cell lines with and without the chromosomal defect provides an ideal system with which to compare the consequences of aneuploidy in various in vitro-derived tissues as a model for such congenic disorders as Down's syndrome, in direct comparison to unaffected tissues on the same genetic background. We have already shown that our derivation protocol produces embryonic stem cell lines that bear the closest resemblance to cells within the early embryo compared with all other reported lines. Our cell lines are therefore ideal models with which to investigate the early developmental process occurring during and after implantation in the uterus that are inaccessible in vivo. We are particularly interested in identifying the biomechanical changes required to orchestrate the physical processes involved in generation of the early foetus during the period of pregnancy during which the embryo is most at risk of miscarriage. We will investigate ways of altering the structure of the cells to monitor the effects on development in culture. We will use high resolution microscopy to see how important proteins are localised within each cell. These studies will enhance our understanding of human development and provide useful information that may be incorporated in the design of new protocols to produce specific tissues in culture for biomedical research and disease modelling.

直到最近，人类胚胎干细胞系是在将完整的早期人类胚胎结构培养后衍生的。我们最近采用了一种新的方案，即在开始培养之前首先将胚胎细胞彼此分离。通过这种方法，我们已经产生了正常稳定的干细胞系，我们将其命名为“人类幼稚外胚层干细胞（HNES）细胞”。这种新方法有几个优点，因为它能够从单个个体产生单个克隆。从发育生物学的角度来看，在培养之前分离胚胎细胞会阻止细胞间的通讯，从而使细胞保持在“幼稚”状态。此外，每个胚胎可以产生多个克隆。我们建议利用这一新的机会，在衍生过程中的各个阶段的样品个别克隆，同时扩大到HNES细胞系的其余克隆。将采样的克隆分离成单细胞，同时分析每个细胞的基因表达和表观遗传修饰剂。这将使我们能够在分子水平上跟踪衍生过程，以确定在从胚胎细胞到稳定的HNES细胞系的过渡期间发生了什么变化（如果有的话）。这种方法消除了个体之间的遗传变异性，这阻碍了迄今为止进行的所有相关研究。该项目的另一个目的是利用体外受精的人类胚胎经常（约60%）表现出嵌合非整倍体的趋势。对于这些实验，我们建议分别从单个胚胎中扩增每个集落，并检查每个细胞系的核型。由此产生的HNES细胞系将被存放在英国干细胞库中，以分发给感兴趣的研究人员。在报道的非整倍性中最常见的是小染色体21或22的三体性。具有单独的具有和不具有染色体缺陷的细胞系提供了一种理想的系统，通过该系统，可以直接与相同遗传背景下未受影响的组织进行比较，来比较作为唐氏综合征等同类疾病模型的各种体外衍生组织中非整倍性的后果。我们已经表明，我们的衍生方案产生的胚胎干细胞系与所有其他报道的细胞系相比，与早期胚胎中的细胞最相似。因此，我们的细胞系是研究子宫植入期间和植入后发生的早期发育过程的理想模型，而这些过程在体内是无法接近的。我们特别感兴趣的是确定所需的生物力学变化，以协调在怀孕期间胚胎最有流产风险的早期胎儿生成所涉及的物理过程。我们将研究改变细胞结构的方法，以监测对培养物中发育的影响。我们将使用高分辨率显微镜来观察每个细胞内重要的蛋白质是如何定位的。这些研究将提高我们对人类发展的理解，并提供有用的信息，这些信息可能被纳入新协议的设计中，以在文化中产生用于生物医学研究和疾病建模的特定组织。

项目成果

Single cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development.

• DOI: 10.1242/dev.167833
• 发表时间: 2018-11-09
• 期刊: Development (Cambridge, England)
• 作者: Boroviak T;Stirparo GG;Dietmann S;Hernando-Herraez I;Mohammed H;Reik W;Smith A;Sasaki E;Nichols J;Bertone P
• 通讯作者: Bertone P

Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency.

• DOI: 10.1016/j.stem.2020.11.005
• 发表时间: 2021-03-04
• 期刊: Cell stem cell
• 影响因子: 23.9
• 作者: Kinoshita M;Barber M;Mansfield W;Cui Y;Spindlow D;Stirparo GG;Dietmann S;Nichols J;Smith A
• 通讯作者: Smith A

Entropy sorting of single cell RNA sequencing data reveals the inner cell mass in the human pre-implantation embryo

单细胞RNA测序数据的熵排序揭示了人类植入前胚胎的内细胞团

• DOI: 10.1101/2022.04.08.487653
• 发表时间: 2022
• 作者: Radley A

Entropy sorting of single-cell RNA sequencing data reveals the inner cell mass in the human pre-implantation embryo.

• DOI: 10.1016/j.stemcr.2022.09.007
• 发表时间: 2023-01-10
• 期刊: STEM CELL REPORTS
• 影响因子: 5.9
• 作者: Radley, Arthur;Corujo-Simon, Elena;Nichols, Jennifer;Smith, Austin;Dunn, Sara-Jane
• 通讯作者: Dunn, Sara-Jane

The blueprint of primate preimplantation development

灵长类动物植入前发育蓝图

• DOI: 10.1016/j.mod.2017.04.107
• 发表时间: 2017
• 期刊: Mechanisms of Development
• 影响因子: 2.6
• 作者: Boroviak T