Dissecting the molecular basis of cell-cell variability in mouse embryonic stem cell populations using systems approaches

Stem cells are present during all phases of development, from the embryo to the adult, and are responsible for orchestrating tissue growth and repair. Embryonic stem cells (ESCs), which are derived from the very early embryo, have the ability to produce all of the body's tissues, a property that is known as pluripotency. By contrast, adult stem cells are only multipotent: they are able to produce a limited number of tissues similar to their tissue of origin. Due to their remarkable regenerative capacity, stem cells (of all different types) have become a point of focus for regenerative medicine strategies, which aim to clinically replace or restore tissue damaged due to disease or trauma. However, the molecular bases of stem cell multi- and pluripotency are still incompletely understood, hindering their clinical use.

In the body, stem cells are either found in small numbers (as is the case for ESCs during early development) or are extremely rare (as is the case for stem cells found in adult tissues) and can, therefore, be difficult to examine. Typically this problem is overcome by examining the average behaviour of (perhaps small) populations of cells. However, a number of recent studies have used advanced single cell gene expression profiling to examine stem cell populations in more detail. These reports have uncovered a surprising degree of cell-to-cell variability within apparently functionally homogeneous stem cell populations, indicating that population-based experiments may, in fact, be masking significant cell-cell variability. This is important because cell-cell variability may profoundly affect the long-term regenerative potential of the population as a whole in ways that are hard to decipher using population-based experiments. So, before new regenerative medicine strategies can be safely used in the clinic it is therefore necessary to fully understand cell-cell variability in stem cell populations.

This project aims to make use of the latest "high-throughput" single cell profiling technologies (which are able to profile hundreds of individual cells per experiment) in combination with computational and mathematical analysis to dissect the origin and functional consequences of cell-cell variability in mouse ESC populations as a model system. Mouse ESC populations have been chosen since they are easily obtained, experimentally reproducible, well studied and ethically uncontroversial. However, we anticipate that understanding of mouse ESC biology will inform our understanding of human pluripotent stem cells and, ultimately, provide wide-ranging clinical benefits.

A number of recent studies using single cell gene expression profiling have uncovered a surprising degree of cell-to-cell variability within apparently functionally homogeneous embryonic cell (ESC) populations. In particular, a number of regulators of the pluripotent stem cell identity, including key transcription factors such as Nanog, Rex1, Stella and Klf4, have been found to exhibit significant expression fluctuations at the single cell level. Such fluctuations give rise to robust functional heterogeneity within ESC cell populations, profoundly affecting their long-term regenerative potency. Collectively, these studies indicate a remarkably dynamic view of stem cell identity: individual stem cells are apparently not closely regulated, yet well-defined and robust population-level behaviour emerges from apparently stochastic dynamics at the single cell level. However, the molecular basis for these intracellular fluctuations and their functional consequences for stem cell populations remain unclear.

This project aims to take an integrated quantitative approach, employing high-throughput single cell profiling with time-lapse microscopy, image analysis and bioinformatic/mathematical analyses, to dissect further the molecular basis of cell-cell variability and its functional consequences in mouse ESC populations. This is important since a more complete understanding of these mechanisms will advance our understanding of early development. Furthermore, understanding of individual ESC identity and the relationships between single cell identity and population function will facilitate the maintenance of more defined pluripotent populations and the development of more robust differentiation protocols which will be necessary if human pluripotent stem cells are to be used safely and routinely in clinical applications.

This proposal aims to take a multidisciplinary approach, involving international collaboration, to a timely problem in stem cell biology. As such there are a number of direct beneficiaries beside the immediate scientific research community.

1) Training the next generation of multidisciplinary scientists. The proposed project will employ 2 post-doctoral research associates (PDRAs): one (primarily) experimental and one (primarily) computational. However, both PDRAs will be expected to actively engage with the other's expertise and learn how to communicate effectively within a multidisciplinary team. BDM has invested significant time in working between experimental and computational communities, and has considerable expertise in this area. A key benefit of this proposal is that BDM will provide mentoring to both PDRAs in effective multidisciplinary work. Training the next generation of scientists literate in more than one core science and engineering discipline will be essential to advance UK science in the 21st century and address the current (and future) grand challenges in science and technology. By directly contributing to the training and development of such scientists this project will directly benefit the competitiveness of UK science. Furthermore, the ability to work flexibly between disciplines is a highly transferrable skill (in science, technology and business) and it is expected that this training will therefore directly benefit to the UK economy.

2) Strengthening UK economy via international exchange of skills. This project will help build an on-going collaboration between BDM and Dr. Fumio Arai, at the Department of Cell Differentiation, Keio University, Tokyo, Japan. Thus, both UK and Japanese science will directly benefit from the shared transfer of knowledge and specialised skills between these two countries. By helping to establish a connection with world-leading international expertise this project will be of direct benefit to UK science competitiveness, with directly related economic benefits.

3) Public engagement. Stem cell research is vital to the future of biomedicine, yet commonly is misunderstood by the public. It is important, therefore, that the public are well informed and involved in dialogue by the scientific research community in ways that make the science accessible and relevant. A number of public engagement events are specifically planned as part of this project that are intended to inspire school children to consider a career in science. This will be of direct benefit to the UK public, both culturally and educationally and, ultimately, economically.

4) Health and well being. This project aims to better understand stem cell identity at the single cell level. Although this project is concerned with pluripotency in mouse cells, ultimately, this may lead to advances in human stem cell biology including protocols for the maintenance of more defined pluripotent populations and the development of more robust differentiation protocols. Such advances will be necessary if pluripotent stem cells are to be used safely and routinely in clinical applications.

5) Industry interactions. It is anticipated that advances in understanding of the molecular basis of pluripotency, and protocols for purifying stem cell populations will ultimately be of significant interest to commercial private sector investors in regenerative medicine/biotechnology. This work may therefore ultimately lead to industrial impact, although we anticipate that any such advances will be indirect and long-term.