Biomechanical prerequisites for pluripotency

多能性的生物力学先决条件

• 批准号: BB/P003575/1
• 负责人: Jennifer Nichols
• 金额: $ 52.73万
• 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: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP003575%2F1
• 关键词: Biomechanical; prerequisites; pluripotency

Pluripotent stem cell lines are a valuable resource for biomedical and clinical research, having the ability to differentiate into any tissue of the body. Embryonic stem cells are derived from the epiblast of preimplantation mouse embryos. The epiblast (founder of the foetus) acquires pluripotency during its segregation from the primitive endoderm (which will produce the extra-embryonic yolk sac) in the inner cell mass of the blastocyst. This segregation is achieved by means of physical sorting from a 'salt and pepper' distribution into the two distinct tissues. This process has been observed by live imaging and characterised at the transcription level, but little is known about the biomechanical mechanisms by which epiblast and primitive endoderm tissues segregate, thereby establishing the pluripotent compartment of the embryo. To shed light on this important issue, we generated a list of candidates required for the structure of the cell's cytoskeleton from gene expression data (RNA sequencing) and protein expression (mass spectrometry). We will form our candidate list based upon representation in the epiblast and primitive endoderm and likelihood to influence cell biomechanics in culture. Armed with the initial candidate list, we will manipulate expression of the ~50 genes on this list by changing their expression artificially in a validated embryonic stem cell line. This line is unique in that it allows in vitro modelling of primitive endoderm segregation from epiblast by ectopic expression of a primitive endoderm-specific gene, thereby providing an excellent model of the inner cell mass of the early mouse embryo. By changing the expression of the candidates in our cell line, we will use biomechanical proxies such as colony forming assays and measurements of cell shape to narrow down our list. Then we will further elucidate the biomechanics of the cell line by measuring stiffness and tension of the cells, which are the main drivers of cell sorting. Consolidating the results of our experiments, we will select a shortlist of up to 10 candidate proteins. We will probe the effects of this biomechanical shortlist ex vivo using wild type preimplantation embryos. To do this, we will use two different types of experiments using our cell line with perturbations on candidate genes. First, we will force expression of the members of the candidate shortlist in one or more of the blastomeres (first embryonic cells) at the 2 or 4 cell stage. We expect to see effects on segregation to all three early embryonic lineages, including trophectoderm (that will form the placenta) using this assay. Second, to focus more on the establishment of plurioptency during epiblast/primitive endoderm sorting, we will use our cell line with relevant candidate perturbations using chimaeras by injection of donor cells into host wild type embryos at the 8 cell stage. Chimaeras that show the most significant phenotype will be transferred to wild type foster mice to allow development to progress until the lineages become more distinct (up to one week, within the first trimester of pregnancy). The output from this work will be a clearer understanding of the biomechanical mechanisms governing acquisition of true naïve pluripotency in the embryo that will enhance knowledge of early mammalian development and inform refinement of culture protocols for self-renewal or directed differentiation.

多能干细胞系是生物医学和临床研究的宝贵资源，具有分化为身体任何组织的能力。胚胎干细胞源自植入前小鼠胚胎的附覆细胞。在胚泡的内细胞质量中，epiblast（胎儿的创始人）从原始内胚层（将产生胚外卵黄囊）中获取多脂蛋白。这种隔离是通过从“盐和胡椒”分布到两个不同组织的物理分类来实现的。通过实时成像观察到了这一过程，并在转录水平上表征了这一过程，但是对层表和原始内胚层组织的生物力学机制知之甚少，从而建立了胚胎的多能腔。为了阐明这一重要问题，我们生成了从基因表达数据（RNA测序）和蛋白质表达（质谱法）中获得细胞细胞骨架结构所需的候选列表。我们将根据培养细胞和原始内胚层的代表形式形成我们的候选列表，并可能影响培养细胞生物力学的可能性。在初始候选列表中，我们将通过在经过验证的胚胎干细胞系中人为地更改其表达方式在此列表中操纵50个基因的表达。这条线是独一无二的，因为它允许通过原始内胚层特异性基因的依托表达对原始内胚层分离进行体外模型，从而提供了早期小鼠胚胎的内部细胞质量的出色​​模型。通过更改候选物在我们的细胞系中的表达，我们将使用生物力学代理，例如菌落形成测定法和测量细胞形状，以缩小我们的列表。然后，我们将通过测量细胞的刚度和张力来进一步阐明细胞系的生物力学，这是细胞分类的主要驱动因素。合并实验结果，我们将选择最多10种候选蛋白的入围名单。我们将使用野生型植入前胚胎探测该生物力学候选名单的效果。为此，我们将使用我们的细胞系使用两种不同类型的实验，并在候选基因上进行扰动。首先，我们将在2或4个细胞阶段的一个或多个胚泡（第一个胚胎细胞）中强制候选候选名单的表达。我们希望看到对所有三个早期胚胎谱系的隔离的影响，包括使用此测定法（将形成plapeta）。其次，要更多地关注层次/原始内胚层分类期间的多晶型，我们将通过将供体细胞注入8个细胞阶段，将供体细胞注入宿主野生型胚胎中。显示最重要的表型的嵌合体将转移到野生型寄养小鼠中，以使发育能够发展，直到谱系变得更加鲜明（在妊娠的头三个月内长达一周）。这项工作的输出将更清楚地理解有关胚胎中真正幼稚多能性的生物力学机制，这将增强对早期哺乳动物发展的知识，并为自我更新或定向分化的文化方案提供了细化。

项目成果

Cell surface fluctuations regulate early embryonic lineage sorting.

• DOI: 10.1016/j.cell.2022.01.022
• 发表时间: 2022-03-03
• 期刊: Cell
• 影响因子: 64.5
• 作者: Yanagida A;Corujo-Simon E;Revell CK;Sahu P;Stirparo GG;Aspalter IM;Winkel AK;Peters R;De Belly H;Cassani DAD;Achouri S;Blumenfeld R;Franze K;Hannezo E;Paluch EK;Nichols J;Chalut KJ
• 通讯作者: Chalut KJ

Distinct phospho-variants of STAT3 regulate naïve pluripotency and developmental pace in vivo

STAT3 的独特磷酸化变体调节体内幼稚多能性和发育速度

• DOI: 10.1101/2022.03.08.483469
• 发表时间: 2022
• 作者: Azami T

OCT4 induces embryonic pluripotency via STAT3 signaling and metabolic mechanisms.

• DOI: 10.1073/pnas.2008890118
• 发表时间: 2021-01-19
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Stirparo GG;Kurowski A;Yanagida A;Bates LE;Strawbridge SE;Hladkou S;Stuart HT;Boroviak TE;Silva JCR;Nichols J
• 通讯作者: Nichols J