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

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移植到培养中后获得的。我们最近应用了一种新的方案，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“人类幼稚上皮干细胞(HNES细胞)”。这种新方法有几个优点，因为它可以从单个个体生产单个克隆。从发育生物学的观点来看，胚胎细胞在培养前的分离阻止了驱动细胞规范的细胞间通信，从而使它们保持在一种天真的状态。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的不同阶段对单个克隆进行采样，同时将剩余的克隆扩展到HNES细胞系中。取样的克隆将被分成单个细胞，每个细胞将同时进行基因表达和表观遗传修饰。这将使我们能够在分子水平上跟踪衍生过程，以确定从胚胎细胞到稳定的HNES细胞系的转变过程中发生了什么变化。这种方法消除了个体之间的遗传变异性，阻碍了迄今为止进行的所有相关研究。该项目的另一个目标是利用体外受精人类胚胎呈现嵌合体非整倍体的频繁趋势(约60%)。对于这些实验，我们建议分别从单个移植胚胎中扩增每个克隆，并检查每个细胞系的核型。由此产生的具有特征性的HNES细胞系将被保存在英国干细胞库中，以分发给感兴趣的研究人员。在已报道的非整倍体中，最常见的是小染色体21或22的三体。具有和不具有染色体缺陷的不同细胞系提供了一个理想的系统，可以用来比较各种体外衍生组织中非整倍体的后果，作为唐氏综合症等同源疾病的模型，并直接与相同遗传背景下的未受影响的组织进行比较。我们已经证明，我们的衍生方案产生的胚胎干细胞株与所有其他报道的株系相比，与早期胚胎内的细胞具有最接近的相似之处。因此，我们的细胞系是研究子宫内植入期间和之后的早期发育过程的理想模型，这些过程在体内是无法获得的。我们尤其感兴趣的是，在胚胎最有可能流产的怀孕期间，确定协调早期胚胎产生所涉及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的方法，以监测培养过程中对发育的影响。我们将使用高分辨率显微镜来观察每个细胞中的重要蛋白质是如何定位的。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，可用于设计新的方案，以在培养中产生特定组织，用于生物医学研究和疾病建模。

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.