Capturing formative pluripotency

All mammals, including humans, develop from a special group of cells that form in the very early embryo. These cells are described as pluripotent because they have the flexibility and potency to produce all cell types of the developing body. Pluripotent cells are present for only a few days in the embryo but scientists have devised conditions to maintain them in the laboratory. The study proposed here is aimed at understanding how pluripotent cells begin the process of specialisation into the different cell lineages that will make up the developing embryo and eventual newborn. We will investigate this first using mouse stem cells because these are better understood than human embryonic stem cells and are easier to manipulate. Furthermore, it is not possible to examine human embryos that have implanted into the uterus. However, the fundamental principles of early development should be similar across mammals and therefore our results should be translatable to human pluripotency. Much current effort is focussed on developing methods to use pluripotent stem cells as a source of mature cells for applications either in drug screening or in cell transplantation therapies. The research we propose may further these goals by improving our ability to direct differentiation along particular pathways.

The idea that motivates this project is that pluripotency is a developmental continuum. The first-formed pluripotent cells have been erased of all identity, creating a blank slate for formation of the new organism. This primitive condition is termed naïve pluripotency. Crucially, the erasure process disables the capacity to develop a specialised identity directly. Therefore naïve pluripotent cells have to acquire the competence to execute coordinated heritable programs of gene expression that define cell fates. We term this a formative transition. It has been difficult to pinpoint and study because pluripotent cells undergo continuous and seamless progression towards differentiation in the embryo. Recently, however, our laboratory has succeeded in arresting pluripotency midway by blocking signals that drive lineage specification. Cells then continue to proliferate but are suspended in a transitional stage. They show distinct properties from other pluripotent stem cells which represent either the initial naïve state or the final primed state that has already begun specialisation. We therefore consider them as formative pluripotent stem cells. Their availability as a stable and expandable resource opens the door to determining how competence is constituted at the molecular level.

In the proposed research we intend to characterise formative pluripotent stem cells in great detail. We will using mouse stem cells to reproduce events in the embryo and develop a cell culture system for seamless conversion from naïve to formative pluripotency. We will then determine if we can obtain human formative pluripotent stem cells by conversion of human naïve embryonic stem cells. We will study how formative pluripotent cells become specified for differentiation into separate lineages. The instructive signals are mostly known from studies of the embryo but we aim to shed light on the key regulatory changes between naïve and formative pluripotency that confer responsiveness to these differentiation instructions. To that end we will use a variety of techniques, in particular high throughput genome sequencing technologies which provide comprehensive information on gene activity and regulation. We will also examine whether formative pluripotent cells are a uniform population or may be heterogeneous, with individual cells already displaying potentiation or bias for a particular differentiation fate.

Overall, study of this new type of pluripotent stem cell will deepen our understanding of how formation of an embryo becomes possible and enhance our capacity to control differentiation in the laboratory for research and biomedical applications

Pluripotency is the plasticity at the single cell level to give rise to all somatic lineages in response to extrinsic cues. Two forms have been defined, naïve and primed, that correspond to the initial and final phases of pluripotency in the early embryo. These are represented by distinct types of mouse stem cell in vitro; embryonic stem (ES) cells and post-implantation epiblast-derived stem cells (EpiSC). We hypothesize that between these two stages lies an essential formative phase wherein competence is acquired for multi-lineage specification and commitment.
Here we propose to trap and characterise cells in this formative stage. We will apply selective activation and inhibition of growth factor pathways together with matrix engineering methodology to identify microenvironmental conditions that stabilise formative pluripotency. We will then establish counterpart human formative pluripotent stem cells. We will assess potency for germline and somatic differentiation in vitro and, for mouse cells, also in chimaeras. We will measure consistency, stability, and homogeneity of cultures. Signalling, transcription factor and epigenetic composition will be related to unpatterned epiblast cells in the early post-implantation mouse embryo. Single cell analyses will be deployed to measure heterogeneity of the formative stem cell compartment. Finally, we will synthesise logical models of the gene regulatory network using automated reasoning and will test model predictions by genetic perturbation.
Overall, access to an intermediate "staging post" will facilitate elucidation of requirements for seamless conversion from the tabula rasa of naive pluripotency through to discrete lineage specification. The insights gained into cellular progression through pluripotency may provide a new paradigm for stem cell transitions. Our findings should also lead to improved command over, and reproducibility of, in vitro lineage specification for bioindustry and biomedical applications.

Stem cell biology is a priority area for UK science investment. The research proposed here is ultimately applicable to improving the authenticity, efficiency and reproducibility of directed differentiation of pluripotent stem cells that can be applied to achieve biomedical goals in drug discovery and regenerative medicine.

Beneficiaries and stakeholders will include:
Academic researchers - this research will contribute to maintaining a world-leading position of the UK for innovation and discovery in pluripotent stem cell research and cognate areas, as detailed in the Academic Beneficiaries section.

Industry -insight into culture formulations and protocols for expansion and differentiation of a new pluripotent stem cell type will be of considerable interest to commercial activities in research tool provision, drug discovery and regenerative medicine. Relevant industry includes reagent companies in the stem cell sector, biotechnology service companies, and Pharma, all of whom are represented in the Cambridge cluster. The project is expected to generate new Intellectual Property, for which we will seek patent protection through the University technology transfer organisation, Cambridge Enterprise. Specialist know-how will be a further basis for collaborative engagement with industry and commercial translation, potentially involving the UK Regenerative Medicine Platform and Cell and Gene Therapy Catapult as intermediaries. Industry can also profit from the highly skilled workforce that will be developed over the course of the project. Overall the project will support retention and growth of the Life Sciences industry around Cambridge and in the UK and thereby contribute to economic activity and competitiveness.

Clinicians and patients - in the long term this research is expected to feed through to improved medical care and treatment by enabling more effective exploitation of pluripotent stem cells. This will include both applications in regenerative medicine and use of reprogrammed cells derived from patients for applications in disease modelling and drug discovery.

General public - the project aims to meet expectations for publicly funded research; (i) to increase understanding of the natural world, and (ii) to lead to improved quality of life. In the first domain the research addresses fundamental issues in the biology of early development. For the second, a long-term perspective is that new stem cell resources and increased understanding can provide a route to treatments for debilitating disease.

Outcomes of the project will be disseminated through a range of communication routes. Seminars, workshops, conference presentations and open access publications will reach relevant academics. The Cambridge Stem Cell Club provides frequent opportunity for informal dialogue and networking with the local scientific community including clinical and industry researchers. Cambridge Enterprise and the University Office for Translation provide more formal avenues for identifying and engaging with commercial partners. Austin Smith has personal contacts within management at companies such as StemCell Technologies UK (based in Cambridge), Plasticell and AstraZeneca, and is a member of the Cell and Gene Therapy Catapult Scientific Advisory Board.

The Smith laboratory has a track record in public engagement, speaking at schools and science festivals, meeting patient groups, contributing to EuroStemCell, (Europe's Stem Cell Hub http://www.eurostemcell.org/), and hosting work experience projects for sixth form pupils. In 2015 we worked with the Institute Public Engagement Officer to organise a competition for computer game developers on the theme of stem cell fate. The winning game is being taken forward for development into an outreach tool. For the present proposal we aim to host two equivalent activities, reaching out to different communities.