Mitochondrial Dynamics in the Control of the Pluripotent States

At the beginning of development of an embryo, cells start off with the potential to give rise to all the cell types and tissues that will form the newborn organism. This potential is called pluripotency, and those cells that are pluripotent hold great promise for regenerative medicine. This promise is not only due to their future clinical applications in cellular therapies, but also because now patient specific pluripotent cells can be generated for "disease-in-a-dish" approaches to understand human diseases. Stem cell therapies are being developed for diseases ranging from diabetes to neurodegenerative disorders and a stem cell based mechanism is thought to underlie different forms of cancer.

Over the last few years considerable efforts have been invested into identifying the signalling and gene regulatory networks that regulate the pluripotent state and control the first steps of differentiation, when cells start to acquire a lineage specific identity. These studies have identified the key molecular features that provide pluripotent cell identity. In contrast to this, we know relatively little about the cell biology changes that accompany exit from the pluripotent state, for example how the different organelles of the cell adapt to the different environments they encounter during the first stages of differentiation. Of particular interest are mitochondria, that are not only the powerhouse of the cell, but also regulate a diverse range of cellular processes, including the ability of the cell to respond to cell death signals, their potential to differentiate, as well as how they respond to the different signalling pathways that regulate cell identity and function.

We have identified that during the first stages of differentiation, cells undergo a dramatic change in the morphology of their mitochondria, suggesting that mitochondrial dynamics change during exit of pluripotency. Additionally we have found that the first stages of differentiation, cells alter their metabolism and their response to cell death stimuli, becoming hypersensitive to death signals. In this proposal we will investigate how mitochondrial dynamics impact of these changes. We will do this by using a combination of experiments performed in pluripotent mouse stem lines and mouse embryos, that allow us to readily determine the in vivo relevance of our data, as well as human embryonic stem cell models, that provide us with insight of the conservation in human of our findings. Specifically we will do three things.

In the first place we will study in detail, at the ultrastructural level, the precise changes that occur in mitochondrial morphology as cells exit the pluripotent state. We will do this by analysing the distribution of mitochondrial proteins in pluripotent and differentiating cells, as well as by imaging mitochondrial dynamics upon exit of pluripotency. We will then manipulate mitochondrial dynamics and study how this affects cell identity. For this we will study three cellular processes that are likely to be affected by mitochondrial changes, the cells metabolism, it's ability to respond to cell death stimuli, and its potential to differentiate into the different lineages that will form the embryo. Finally, we will exploit the significant knowledge of the signalling pathways that maintain pluripotency and that drive differentiation, to identify novel signalling inputs that regulate mitochondrial dynamics and function in stem cells. Together, these experiments will provide a unique insight into an essential, but understudied aspect of early embryonic development, the dynamics of the mitochondria, as well as addressing how they impact on the process of differentiation.

Understanding how pluripotent stem cells initiate differentiation is a key question not only for developmental and stem cell biology, but is also essential for the use of stem cells in regenerative medicine. Over the last few years, significant research efforts have been focussed on analysing the signalling, transcriptional and epigenetic landscape of pluripotent stem cells, as well as unravelling the molecular changes that underpin the initiation of differentiation. In contrast to this, very little is understood about how pluripotent cells adapt their organelle activity to the new environments they face during the onset of differentiation. Of specific interest, given their fundamental role in determining the cells apoptotic response and metabolic output are mitochondria. We have found that at the onset of differentiation, pluripotent stem cells dramatically change their mitochondrial morphology and dynamics, and that accompanying these changes they modify their response to cell death stimuli and metabolism. Here we will investigate in both mouse, and humans, using a combination of stem cell and embryological approaches, the links between these events. In the first place we will study in detail the precise changes that occur in mitochondrial morphology and dynamics as cells exit the pluripotent state. We will then manipulate mitochondrial dynamics and study how this affects the metabolism of pluripotent stem cells, their ability to respond to cell death stimuli, and their developmental potential. Finally, we will exploit the significant knowledge of the signalling pathways that maintain pluripotency and that drive differentiation, to identify novel signalling inputs that regulate mitochondrial dynamics and function in stem cells. Together, these experiments will provide a unique insight into an essential, but understudied aspect of early embryonic development, the dynamics of the mitochondria, as well as addressing how they impact on the process of differentiation.

Our studies will uncover the mechanisms that ensure the fitness of pluripotent stem cells. The large promise that pluripotent stem cells hold for regenerative medicine, where stem cell are induced to differentiate towards specialised cells that are then transplanted into patients, has carried with it a large degree of interest both from the academic and medical communities. Furthermore, with the advances in reprogramming there is an enormous scope for applying in vitro differentiation of patient-derived induced pluripotent stem (iPS) cells to study diseases. However, increased knowledge about how specific cell types are induced in an irreversible way as well as how to differentiate cells down specific lineages is vital to control that the correct cell types are generated before these stem cell therapy becomes meaningful clinically. Therefore, the results obtained in this study will be of direct relevance to translational research. Given that understanding how cellular fitness is regulated is a key question in cancer, our work will also be of interest to researchers working in this field.

The primary beneficiaries and users of this research are members of the academic sector - research workers, teachers and students. Other beneficiaries are the general public and commercial sector. In particular, the data obtained in this project will benefit the companies developing stem cell-based assays for drug screening (e.g. AstraZeneca) as well as companies developing media for pluripotent stem cell maintenance and differentiation (e.g. STEMCELL Technologies, Millipore), and pharmaceutical and biotech companies developing hESC-based cell replacement therapies for treatment of degenerative diseases (e.g. Advanced Cell Technologies, Viacyte).

A further beneficiary of our work will be the lay public. Given the medical relevance and ethical implications of understanding developmental processes, and its impact on stem cell biology, our work will provide factual input to and therefore benefit the public discussion about the advantages and risks of stem cell therapy.

The results of this research will be conveyed to other researchers through the publication of findings in peer-reviewed journals, by reporting unpublished work at conferences and through personal communication with other scientists. Though the results will primarily be disseminated through scientific journals, we will liaise with dedicated Media Teams at Imperial College and the University of Sheffield to issue a press release when appropriate. We take seriously the responsibility of scientists to engage with the lay public, to raise awareness among them of the results of publicly funded research, to openly debate ethical issues relating to our research so that public opinion may be formed in an informed manner and to take the excitement of our research to the children of today, who will be the scientists of tomorrow. For these reasons we engage through the University but also as individuals in activities aimed at the public dissemination of science.

This project will also train early-career researchers in emerging methodologies, contributing to their career development, as well as producing individuals capable of carrying out future research in the biomedical sciences. At a time when industry is moving in the direction of interdisciplinary research, such individuals will be highly sought-after not only in academia but also in the commercial sector.