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，在完整的早期人类胚胎结构融入培养物中，人类胚胎干细胞系仍得出。我们最近采用了一种新的方案，其中首先在培养开始之前将胚胎细胞彼此分离。通过这种方式，我们已经产生了正常的稳定干细胞系，我们将其称为“幼稚的层茎（HNE）细胞”。这种新方法有几个优点，因为它可以从一个人那里产生单个克隆。从发育生物学的角度来看，培养前的胚胎细胞的分离阻止了驱动细胞规范的细胞间通信，从而将它们保留在“幼稚”状态下。而且，可以从每个胚胎中产生多个克隆。我们建议在派生过程中使用这种新的机会在各个阶段进行单个克隆进行采样，同时将剩余的克隆扩展到HNES细胞系中。采样的克隆将分为单个细胞，每个细胞将仅用于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推导过程，以确定从胚胎细胞到稳定的HNES细胞系的过渡期间发生的变化（如果有）。这种方法消除了妨碍迄今为止所有相关研究的个体之间的遗传变异性。该项目的另一个目的是利用体外受精的人类胚胎的经常趋势（约60％），以表现出镶嵌性非整倍性。对于这些实验，我们建议分别从单个外植的胚胎扩展每个菌落，并检查每个细胞系的核型。然后，由此产生的HNES细胞系将在英国干细胞库中丢失，以分发给有趣的研究人员。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。具有和不带有染色体缺陷的单独细胞线条提供了一个理想的系统，可以比较各种体外衍生的组织中的非整倍性的后果，作为与唐纳德综合症相同的相同背景的各种疾病的模型，作为一种相同的综合症，将其作为一种相同的疾病，与无与伦比的组织相同。我们已经表明，与所有其他报道的线相比，我们的衍生方案产生的胚胎干细胞系与早期胚胎中的细胞最接近。因此，我们的细胞系是研究子宫内植入期间和之后发生的早期发育过程的理想模型，在体内无法访问。我们特别有兴趣确定在怀孕期间，胚胎最有流产的风险，在怀孕期间涉及生成早期胎儿的物理过程所需的生物力学变化。我们将研究改变细胞结构以监测培养物的影响的方法。我们将使用高分辨率显微镜查看每个细胞中重要的蛋白质位置。这些研究将增强我们对人类发展的理解，并提供有用的信息，这些信息可以纳入新方案的设计，以在生物医学研究和疾病建模中生产特定的组织。

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

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

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