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

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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.