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

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.

Pluripotent stem cells give rise to multiple lineages of differentiated mature cell types. We currently think that stem cells change their chromatin accessibility patterns in a different manner in each differentiation lineage. The regulation of this process is of fundamental importance for regenerative medicine and reprogramming and its deregulation causes human disease and cancer. However, how stem cells orchestrate these changes to activate multiple differentiation programs into a myriad of cell types is still unknown. Planarians are an ideal model organism to investigate this question as their stem cells constantly differentiate into all cell types to turn over their tissues. The advent of single cell technologies, together with the properties of the planarian model, will enable this research now. We have already applied single cell transcriptomics to planarian stem cell differentiation, revealing for the first time their full differentiation lineage tree. Here I propose to measure chromatin interactions and accessibility at the single cell level in the bulk cell population of the planarian Schmidtea mediterranea. By using and combining Chromatin Conformation Capture techniques to monitor chromatin interactions and single cell ATAC-seq to measure chromatin accessibility we will elucidate the changes in chromatin accessibility that take place during stem cell differentiation into each differentiated cell lineages and their order. By measuring chromatin accessibility after knocking down candidate conserved regulators, we will be able to detect the chromatin changes they induce, the genomic regions where they happen and the differentiation lineages where they are needed. Since many of the genes involved are conserved from humans to planarians, this knowledge will inform human stem cell therapies and regenerative medicine. This will lay a cornerstone into our understanding of the epigenetic bases that regulate stem cell differentiation to multiple cell types.

Worldwide academic advancement:

We will be the first to investigate with novel single cell techniques how a pluripotent stem cell differentiates into multiple cell types at the epigenetic level. We will measure the changes in chromatin accessibility and chromatin contacts and study their regulation. This will mark a significant milestone in our efforts to advance our understanding of stem cells, and will hence be of interest to all research communities working on the stem cell, developmental biology, single-cell, regenerative medicine, systems biology community and planarians. Understanding the fundamental principles that govern stem cell differentiation is a key question to biomedical sciences. To understand how to reprogram and differentiate human cells in vitro to cure patients and treat diseases we need to understand how these processes work in vivo. The planarian Schmidtea mediterranea is a key model organism for this research. We will disseminate our findings in major journals and international meetings, but also make our data available through web repositories, web interactive interfaces and we will actively engage with the major public with outreach activities.

Training highly skilled researchers:

This program will influence the careers of the three main researchers involved. The technician will be trained in the experimental techniques of the planarian model organism, as well as very novel single cell analysis techniques. As a result, this technician will be a highly skilled researcher upon completion of this program, with very high chances of being hired by me or another research group. The computational postdoctoral researcher will receive training in single cell analysis methods by me and by a projected visit to the David Garfield laboratory in Berlin. After the completion of this program this researcher will be and expert in the analysis of single cell sequencing data, a very novel and demanded skill. This will allow her or him to start another postdoc position or even start a new lab in a very active emerging field of biology. Finally, as the first funding instrument of my lab, and taking advantage of Oxford Brookes leadership courses this program will allow me to establish my position in the field of single cell technologies applied to planarian stem cells.

Innovative methodologies and cross-disciplinary approaches:

Single cell technologies are quickly emerging as a powerful way to disentwine the complexity of tissues, organs and even entire organisms as we have previously shown. These technologies are highly novel and being intensely developed. We will contribute to this development by implementing these approaches in the UK, sharing our experience of their use and developing new analysis tools that will can benefit the community. Our cross-disciplinary approach, blending physics, mathematical modelling and wet lab biology experiments is an example of the new directions the biological sciences have taken in recent years, and this program of research will contribute to this paradigm shift.