Shaping a stem cell into dozens of cell types: A single-cell epigenetic roadmap of planarian stem cell differentiation

多能干细胞可以分化成所有的成体细胞类型。了解多能干细胞如何分化成许多不同的成熟细胞类型是生物医学和再生科学的一个关键问题。基因活性的调控是干细胞及其分化的关键。在细胞核中，组蛋白和其他分子将遗传物质包裹在一种叫做染色质的物质中。染色质内DNA的可及性至关重要：染色质“打开”允许基因活动，“关闭”关闭基因活动。显微镜研究很早就表明，干细胞具有相对开放的染色质，而完全成熟的细胞具有较大的封闭区域。我们目前认为，多能干细胞通过打开和关闭每种类型的基因组的不同区域来分化成许多不同的细胞类型。在过去的十年中，我们在理解这些开启和关闭事件方面取得了很大的进展，这在一定程度上要归功于测量整个基因组中染色质可及性和相互作用的技术的发展，并在体外阐明了几种细胞类型分化的特征事件。我们还发现，这些事件的改变往往会导致癌症和疾病。然而，我们仍然不了解多能干细胞如何协调它们的染色质打开和关闭事件，以展开无数成熟细胞类型的分化程序，从而形成一个复杂的成年生物体。为了解决这个问题，我建议在地中海涡虫（Schmidtea mediterranea）的单细胞水平上测量染色质相互作用和可及性。地中海涡虫是一种理想的模式生物，我们可以通过它来研究体内多能干细胞的分化。淡水涡虫是一种无脊椎动物，与人类不同，它们成年后拥有多能干细胞。它们不断地分化成各种类型的细胞，以取代受损的细胞，并使涡虫具有非凡的再生特性：涡虫的每一块都能在几天内再生出一个完整的成虫。单细胞分析的最新技术进步以及涡虫作为模式生物的特性使这项研究成为可能。我们已经将单细胞方法应用到涡虫中，从而阐明了涡虫干细胞的完整分化树。在这里，我建议使用新的单细胞技术来测量涡虫细胞的染色质结构和可及性。这将告诉我们涡虫DNA和染色质的哪个区域在每种主要涡虫成熟细胞类型分化的每个阶段打开或关闭。我们还可以关闭几个可能调节这一过程的基因，并测量这些动物的染色质可及性。这些基因中的大多数存在于人类和涡虫中，我们知道两者的干细胞都需要它们来发挥作用，但我们仍然忽略了它们的确切作用机制。通过测量关闭它们后染色质可及性的变化，我们将了解哪些是它们调节的开启和关闭事件，以及它们在哪些细胞类型中是重要的。这一信息将使针对这些相同基因的人类干细胞分化方法和再生医学的新策略成为可能。总之，这项研究将使我们了解干细胞如何重塑其染色质以分化为多种不同的成熟细胞类型。

Pluripotent stem cells can differentiate into all adult cell types. Understanding how pluripotent stem cells differentiate into many different mature cell types is a key question for the biomedical and regenerative sciences. The regulation of gene activity is key for stem cells and differentiation. In the nucleus, histones and other molecules wrap the genetic material in a substance called chromatin. The accessibility of DNA within chromatin is of fundamental importance: chromatin "opens" to allow gene activity and "closes" to shut it down. Microscopy studies very early on showed that stem cells have relatively open chromatin while fully mature cells have large closed regions instead. We currently think that pluripotent stem cells achieve differentiation into many different cell types by opening and closing of different regions of their genomes in each type. In the last decade, we have made great advances to understand these opening and closing events in part thanks to the development of techniques that measure chromatin accessibility and interactions across the entire genome, and have elucidated the events that characterize differentiation to a few cell types in vitro. We have also discovered that alterations in these events often lead to cancer and disease. However, we still do not understand how pluripotent stem cells orchestrate their chromatin opening and closing events to unfold the differentiation programs of the myriad of mature cell types that make a complex adult organism.To tackle this question, I propose to measure chromatin interactions and accessibility at the single cell level in the planarian Schmidtea mediterranea, an ideal model organism through which we can investigate pluripotent stem cell differentiation in vivo. Freshwater planarians are invertebrates that, unlike us, have pluripotent stem cells as adults. They constantly differentiate into all cell types to replace damaged cells and to enable the remarkable planarian regeneration properties: each piece from a planarian can regenerate an entire adult in a matter of days. Recent technological advances of single-cell analysis together with the properties of planarians as a model organism enable this research now. We have already implemented single-cell approaches into planarians, resulting in the elucidation of the complete differentiation tree of planarian stem cells. Here I propose to use novel single-cell techniques to measure chromatin structure and accessibility in planarian cells. This will tell us which regions of the planarian DNA and chromatin open or close in every stage of differentiation to each of the major planarian mature cell types. We can also turn off several genes that are likely to be regulating this process and measure chromatin accessibility in these animals. Most of these genes are present in both humans and planarians and we know that stem cells from both need them to function both but we still ignore their precise mechanisms of action. By measuring how chromatin accessibility changes after turning them off we will understand which are the opening and closing events that they regulate and in which cell types they are important. This information will enable new strategies for human stem cell differentiation approaches and regenerative medicine by targeting those same genes. Altogether, this research will allow us to understand how stem cells reshape their chromatin to differentiate into multiple and different mature cell types.