Capturing formative pluripotency

Lead Research Organisation: University of Cambridge
Department Name: Wellcome Trust - MRC Cam Stem Cell Inst


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

Technical Summary

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.

Planned Impact

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, 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.


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Description We have developed conditions based on minimal growth factor stimulation for propagating stem cells representing a distinct stage of early embryo development. These formative pluripotent stem cells are at a more advanced stage than classical mouse embryonic stem cells. They are poised to respond to inductive signals and produce lineages of the embryo but have not acquired any lineage. Using the same culture conditions we established new pluripotent stem cells from human embryos. We also investigated livestock embryos and with a modified culture condition derived pluripotent stem cells from pig, sheep and cattle. This is the first demonstration that a common and fully defined culture condition can be applied to establish pluripotent stem cells from these economically important species. Finally, in a side project we investigated why loss of DNA methylation capacity leads to compromised developmental potential in pluripotent stem cells. We found that de novo methylation is necessary for efficient cell fate decisions by silencing transcription factor genes that induce aberrant fates.
Exploitation Route The concept of formative pluripotency has become widely accepted and it is likely that there will be considerable interest in the culture conditions we have developed for formative stem cells. They provide a new resource for fundamental research and for directed differentiation applications in stem cell medicine. The progress we have made in establishing pluripotent stem cells from livestock species is already being licensed commercially for applications in genetic enhancement and for production of cell-based meat.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

Description The new livestock pluripotent stem cells derived as part of this project in collaboration with Prof Alberio are being commercially licensed by the University of Nottingham (in partnership with University of Cambridge) to companies in the emerging cultured meat industry.
First Year Of Impact 2021
Sector Agriculture, Food and Drink
Impact Types Economic

Description Plasticity of the Pluripotency Network
Amount € 2,499,970 (EUR)
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 01/2020 
End 12/2024
Title Human formative pluripotent stem cells. 
Description Formative pluripotent stem cell lines derived from human embryos. Registering with the UK Stem Cell Steering Committee for deposition in the UK Stem Cell Bank. 
Type Of Material Cell line 
Year Produced 2020 
Provided To Others? Yes  
Impact To be determined 
Title Mouse formative pluripotent stem cells 
Description Formative pluripotent stem cell lines derived from mouse E5.5-6.5 epiblast as described in Kinoshita et al, 2020. 
Type Of Material Cell line 
Year Produced 2020 
Provided To Others? Yes  
Impact To be determined 
Description Austin Smith - Ramiro Alberio 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Pluripotency in sheep and pig
Collaborator Contribution Pluripotency in sheep and pig
Impact None yet
Start Year 2018
Description PluriMes Consortium 
Organisation Technical University of Dresden
Country Germany 
Sector Academic/University 
PI Contribution Coordinator: A consortium of 12 European partners awarded €6million by the European Commission for a research and development project focused on directing stem cells to become bone and muscle.
Collaborator Contribution The project combines the expertise of ten academic and two industrial partners to bring together stem cell experts, genetic engineers, developmental biologists, cell therapy pioneers, bioengineers and specialist SMEs in a cross-disciplinary collaborative effort. PluriMes is supported through the European Commission's Framework 7 HEALTH research programme and Coordinated by Professor Austin Smith from the Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute at the University of Cambridge.
Impact PMID: 28765214 PMID 25215486
Start Year 2014
Description Potential of pluripotent stem cells for use in regenerative and transplantation medicine 
Organisation University of Tokyo
Country Japan 
Sector Academic/University 
PI Contribution Potential of pluripotent stem cells for use in regenerative and transplantation medicine
Collaborator Contribution Potential of pluripotent stem cells for use in regenerative and transplantation medicine
Impact Publication: Development 2021 Vol. 148 Issue 23 DOI: 10.1242/dev.199901
Start Year 2019
Title Livestock pluripotent stem cells 
Description Development of pluripotent stem cell lines from pigs, sheep and cattle in collaboration with Prof Ramiro Alberio, University of Nottingham. Cell lines are being licensed jointly by University of Cambridge and University of Nottingham to commercial companies interested in manufacturing cell-derived meat. 
IP Reference  
Protection Trade Mark
Year Protection Granted 2021
Licensed Yes
Impact Cell lines have been commercially licensed to companies as a renewable source for biomanufacturing meat..
Description BBC Science & Technology 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Commentary for BBC Science & Technology, Jan 2018
Year(s) Of Engagement Activity 2018
Description PER Awards 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact University of Cambridge, Vice-Chancellor's Public Engagement with Research Awards - member of the judge panel.
Year(s) Of Engagement Activity 2017