Comparative transcriptional control of establishment, maintenance and collapse of naive pluripotency in rodents and primates in vivo

The value of stem cell lines that can multiply in culture and retain the capacity to become any tissue type in the body is immense, both from the point of view of generating animal models for research, and to produce patient-specific human tissue for drug screening or cell replacement therapy. Stem cells derived from the founder tissue of the foetus in the early mouse embryo, known as pluripotent embryonic stem (ES) cells, have been proven to generate any type of adult tissue, including the germ cells, by observing their contribution to animals produced after integrating ES cells into a host embryo. Human ES cells, however, have characteristics distinct from mouse ES cells; this is evident in their molecular attributes, morphology and culture requirements. In fact, human ES cells resemble much more closely the cells found in more mature mouse embryos that have already implanted in the uterus and are considered to be 'primed' for differentiation. These cells are harder to grow and are more restricted in their differentiation repertoire compared with mouse ES cells. In the interests of therapeutic relevance, it would be highly desirable to understand how to capture cells from human embryos that are more akin to mouse ES cells. Although attempts have been made to produce such cells, either by manipulating the culture conditions in which they are grown, or by means of 'reprogramming' strategies that can revert adult cell types to a more embryonic condition, it has not been possible to capture human cells that can thrive in such a state. We believe that the only way to overcome this obstacle is to understand how these founder embryonic cells are formed in mouse embryos, from which true ES cell lines can be readily obtained. The ultimate goal is to use this knowledge as a blueprint to probe the development of early human embryos (or non-human primate embryos as a model for humans) and exploit alternative pathways to capture similar pluripotent cells from primates. In addition to discovering how to derive the ideal type of stem cells from human embryos, this knowledge will be used to generate useful cells from adult tissues and patient samples. In this project we plan to expand on our previous work to produce a detailed molecular portrait of mouse embryos before, during and after the stage in animal development when pluripotent cells are naturally produced. We will make use of genetic modification strategies to explore the importance of master control genes in this process. In addition, we can add highly specific chemicals to the culture medium that activate or suppress different cellular behaviours to reveal how these control genes are turned on or off. We have already begun to identify molecular differences between rodent (mouse) and primate (marmoset) embryos. We will extend this concept and then test how marmoset embryos respond to external signals that we predict may be beneficial to development. We will use this information to target specific pathways that could be enhanced or inhibited to allow pluripotent cells to be captured, thereby providing the essential starting point to create useful tissues for drug screening and, ultimately, cell replacement therapy.

To determine the molecular requirements for naïve pluripotency, we will produce a quantitative profile of transcription factor expression patterns and interactions during the establishment of epiblast in the murine embryo using single cell RNA-seq and targeted qRT-PCR. To determine the essential transcriptional network for establishing and maintaining naïve pluripotency, we will probe the phenotypic effects of deletion of key pluripotency regulators in uninterrupted development and during embryonic diapause. The induction of gene expression by extrinsic signals will also be interrogated by application of agonists and antagonists to candidate signalling pathways to cultured embryos. The state of naïve pluripotency during normal development is transient, and the epiblast rapidly becomes primed after implantation incurring a loss of naïve pluripotency. We recently showed that the bHLH transcription factor Tfe3 is exported from the nucleus in epiblast cells soon after implantation, coincident with this state change. We will explore its subcellular localisation by immunohistochemistry during embryonic diapause, when the state of naïve pluripotency is extended. To ascertain whether this interesting intracellular protein shuttling activity is necessary for exit from naïve pluripotency in vivo, we will make use of a novel transgenic mouse line in which Tfe3 protein can be ectopically directed to the nucleus, and characterise the subsequent developmental potential of the epiblast. The functional and molecular blueprint we generate from these combined experiments will be compared with transcriptional profiles and immunohistochemistry of marmoset embryos before and after explant culture, with or without agonists or antagonists of potentially relevant pathways. Ultimately, we aim to attempt to direct the development of primate embryos or isolated cells/tissues to explore the possibility of capturing authentic pluripotent cell lines.

Who will benefit from this research?
The primary beneficiaries of this project will be scientists engaged in basic developmental biology and stem cell research, especially members of the group and our collaborators. Members of the clinical sector and companies engaged in developing and manufacturing regenerative medicine applications of human ES and iPS cells (e.g. Neusentis, GSK, ACT, Viacyte) will also be interested, as will companies providing tools to support these developments (e.g. LifeTechnologies, TAP Biosystems, Stem Cell Technology), and the Cell Therapy Catapult. Project outputs will also benefit companies developing in vitro assay systems for drug discovery, disease modelling and toxicology (e.g. GSK, AstraZeneca, Cellartis, SC4SM). The ultimate beneficiaries of our work will be clinicians and patients.

How will they benefit from this research?
Preliminary data will be discussed informally in lab meetings and internal seminars and symposia; as the output develops into cohesive results they will be discussed in the wider scientific community. Members of the research group will benefit from the skills developed from this very specialist programme. They will be stretched intellectually because of the nature of the research question and the need to develop novel approaches to processing the material and data. They will be encouraged to present their work informally, at international conferences and in the form of outreach activities. We are confident that this research will be of widespread interest, and therefore publishable in high impact journals. This will assist both post docs to achieve independent positions by the end of the project.
Within the short term, biomedical companies may benefit considerably from the availability of non-human primate models of pluripotent stem cell lines, which we expect to develop within 3 years, that have the potential to be rigorously validated before the protocols we use to develop them have been translated to the human system.
The inevitable expansion of knowledge pertaining to the process and molecular control of early primate development achieved during this project may enable the improvement of culture media for IVF programmes (human and non-human primate) and inform diagnosis and treatment of early developmental defects.
The potential to improve the differentiation capacity of human pluripotent stem cell lines resulting from our molecular characterisation and experimental manipulation of culture regimes will enhance understanding of the signalling requirements for directed exit from pluripotency and is likely to enrich the repertoire of tissues available for drug screening and ultimately, transplantation to patients.