Biomechanical prerequisites for pluripotency

多能干细胞系是生物医学和临床研究的宝贵资源，具有分化成身体任何组织的能力。胚胎干细胞来源于植入前小鼠胚胎的外胚层。外胚层（胎儿的创始者）在其从胚泡内细胞团中的原始内胚层（其将产生胚外卵黄囊）分离期间获得多能性。这种分离是通过物理分选从“盐和胡椒”分布到两个不同的组织中来实现的。这一过程已经通过活体成像观察到，并在转录水平上进行了表征，但对上胚层和原始内胚层组织分离的生物力学机制知之甚少，从而建立了胚胎的多能区室。为了阐明这一重要问题，我们从基因表达数据（RNA测序）和蛋白质表达（质谱）中生成了细胞细胞骨架结构所需的候选者列表。我们将根据外胚层和原始内胚层中的代表性以及影响培养中细胞生物力学的可能性来形成我们的候选名单。有了最初的候选名单，我们将通过在一个经过验证的胚胎干细胞系中人工改变它们的表达来操纵这个名单上的~50个基因的表达。这条线是独特的，因为它允许在体外建模的原始内胚层分离外胚层的异位表达的原始内胚层特异性基因，从而提供了一个很好的模型的早期小鼠胚胎的内细胞团。通过改变候选人在我们细胞系中的表达，我们将使用生物力学代理，如集落形成测定和细胞形状的测量来缩小我们的名单。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，这是细胞分选的主要驱动力。为了巩固我们的实验结果，我们将选择最多10个候选蛋白质的短名单。我们将使用野生型植入前胚胎体外探索该生物力学短名单的影响。为此，我们将使用两种不同类型的实验，使用我们的细胞系，对候选基因进行扰动。首先，我们将在2或4细胞阶段的一个或多个卵裂球（第一胚胎细胞）中强制表达候选名单的成员。我们期望看到使用该测定对所有三种早期胚胎谱系的分离的影响，包括滋养外胚层（将形成胎盘）。第二，为了更集中于在外胚层/原始内胚层分选期间建立多能性，我们将使用我们的细胞系，其具有使用嵌合体的相关候选扰动，通过将供体细胞注射到8细胞阶段的宿主野生型胚胎中。显示最显著表型的嵌合体将被转移到野生型寄养小鼠中，以允许发育进行，直到谱系变得更加明显（最多一周，在妊娠的前三个月内）。这项工作的结果将更清楚地了解胚胎中获得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育的了解，并为自我更新或定向分化的培养方案的改进提供信息。

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.