Epigenetic regulation of lineage competence in human pluripotent stem cells

人体由250多种不同类型的细胞组成。在胚胎发育过程中，这种多样性起源于一小群大约10-20个被称为“外胚层”的细胞，它们最初是相同的，但随后会继续发育并形成整个身体。这种产生身体所有细胞的能力被称为“多能性”。多能细胞将通过不同的途径最终变成不同的细胞类型（这一过程被称为“分化”）：大脑神经元、心肌细胞等。所有特化细胞（除了极少数例外）都有相同的基因，但每种细胞类型中只有特定的基因子集是活跃的——这就是细胞的独特之处。有一些特殊的机制可以开关基因的“开启”和“关闭”。其中许多都与这样一个事实有关：我们的基因不是简单的裸露的DNA分子，而是代表着DNA与许多不同蛋白质的复合物。特定的分子标签可以放置在这些蛋白质或DNA上。这些所谓的“表观遗传”修饰不会改变我们的DNA序列，它们是可逆的，可以用来“开启”和“关闭”基因。发育生物学中最令人着迷的问题之一是胚胎的多能细胞是如何做出第一个决定的，即什么是特化的细胞。值得注意的是，当外胚层在胚胎中首次出现时，这些细胞对诱导它们特化的信号并不敏感（我们称之为“naïve”）。只有在胚胎植入子宫的8-10天后，外胚层细胞才会获得对这些信号作出反应的能力（“启动”阶段），并继续发育。我们的项目旨在了解多能细胞如何成为有能力分化的机制。由于伦理方面的考虑，直接研究人类胚胎的发育存在很大的局限性。在许多情况下，解决方案是使用所谓的“多能干细胞”（PSC），它来自胚胎。使用特殊的生长因子和化学物质可以捕获PSC，使它们保持与外胚层细胞非常相似。在这些条件下，PSC几乎可以在培养皿中无限生长，并且保持不特化。如果PSC暴露在刺激发育的因素中，它们可以像胚胎一样产生成熟细胞。除了用于基础研究之外，PSC作为成熟细胞的移植来源，也是再生医学的一个很有前途的工具。先前，我们开发了人类PSC从naïve到启动阶段复制外胚层发育的条件。这个过程大约需要10天，这与实际胚胎中这个过程的长度非常相似。我们计划用这个独特的模型来了解人类胚胎发育的最初阶段。我们的假设是，表观遗传机制的作用不仅是让基因“开启”和“关闭”，而且在此之前，还为基因的激活做好了准备。我们预测，在从幼稚多能性到引物多能性的转变过程中，一些分化基因会通过表观遗传修饰“预激活”。这使得细胞对分化信号敏感。一旦这些信号出现，这些基因就会启动，多能细胞就会产生许多不同的特化细胞。在这个项目中，我们将识别“预激活”的基因和“预激活”它们的表观遗传修饰。然后，我们将应用现代基因工程工具来了解哪些因素首先“预激活”基因，以及这些基因在分化过程中如何被激活。总之，我们的结果将揭示一小群10-20个外胚层细胞如何做出第一个决定，以产生像我们这样的复杂生物体。此外，这一见解将提高我们将PSC应用于生物医学科学和潜在临床应用的能力。

The human body consists of more than 250 specialised cell types. During embryo development, this diversity originates from a small group of about 10-20 cells named "epiblast", which are initially equivalent, but will then continue development and form the whole body. This ability to produce all cells of the body is called "pluripotency". Pluripotent cells will take diverse pathways in order to eventually become different cell types (a process referred to as "differentiation"): neurons of the brain, heart muscle cells, among many others.All specialised cells (with very few exceptions) have the same genes, but only a specific subset of genes is active in each cell type - this is what makes the cells unique. There are special mechanisms to switch genes "on" and "off". Many of them are related to the fact that our genes are not simply naked DNA molecules, but rather represent DNA in complex with many different proteins. Specific molecular tags can be placed onto these proteins or onto DNA itself. These so-called "epigenetic" modifications do not alter our DNA sequence, they are reversible and employed to turn genes "on" and "off".One of the most fascinating questions in developmental biology is how pluripotent cells of an embryo make their first decisions what specialised cells to become. Remarkably, when the epiblast first emerges in the embryo, these cells are not sensitive to the signals that would induce them to specialise (we call this stage "naïve"). Only after 8-10 days, during which the embryo implants in the uterus, the epiblast cells will gain the capacity to respond to these signals ("primed" stage) and go on to develop further. Our project aims to understand the mechanism of how pluripotent cells become competent to differentiate.There are major limitations to study development of human embryos directly, due to ethical considerations. The solution in many cases is to use so called "pluripotent stem cells" (PSC), which are derived from embryos. Using special growth factors and chemicals allows for trapping PSC so that they remain very similar to epiblast cells. In these conditions, PSC can be grown in a dish practically indefinitely and remain unspecialised. If PSC are exposed to factors that stimulate development, they can produce mature cells as if they were in the embryo. Besides their use for fundamental research, PSC is a promising tool for regenerative medicine, as a source of mature cells for transplantation.Previously, we developed conditions in which human PSC reproduce development of the epiblast from the naïve to primed stage. This takes about 10 days, which is very similar to the length of this process in the actual embryo. We plan to use this unique model to understand the very beginnings of human embryo development. Our hypothesis is that epigenetic mechanisms operate not only simply to turn genes "on" and "off" but also before that, in order to prepare genes for activation. We predict that during transition from naive to primed pluripotency, some genes for differentiation become "pre-activated" by epigenetic modifications. This makes cells sensitive to differentiation signals. Once these signals appear, these genes will turn on and pluripotent cells will produce many different specialised cells.In this project, we will identify "pre-activated" genes and the epigenetic modifications that "pre-activate" them. Then, we will apply modern genetic engineering tools to understand which factors "pre-activate" the genes in the first place, and how these genes become activated later during differentiation. Altogether, our results will reveal how a small group of 10-20 epiblast cells make their first decisions in order to generate complex organisms like ourselves. Furthermore, this insight will advance our abilities to employ PSC for biomedical science and potential clinical application.