Human Pluripotency

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

Pluripotency is the flexible capacity of individual cells to give rise to all somatic lineages. In the human embryo the period of pluripotency extends for more than 10 days, from emergence in the late blastocyst through to lineage commitment during gastrulation. In mice two types of pluripotent stem cell line derived in vitro are thought to represent respectively the initial naïve condition and a late phase primed for differentiation. Human pluripotent stem cells exhibit attributes of priming, reflected in molecular features such as DNA methylation and, most significantly for application purposes, in variable differentiation biases. Recently, however, we have established human stem cell cultures that exhibit properties anticipated for naïve pluripotency. Here we will further validate naïve identity and enhance the stability and consistency of these cultures. We will then characterise their transition towards lineage commitment. Understanding this process and faithfully recapitulating in utero pluripotency progression may substantially improve the robustness and fidelity of directed differentiation for biomedical applications. In vivo reference material is lacking, however, because the peri- and early post-implantation period is essentially inaccessible in human embryo development. Therefore, exploitation of naïve human pluripotent stem cells offers both a unique challenge as well as a substantial opportunity.

The phase of naïve pluripotency in human is only partially conserved with rodents and key factors that support the robust self-renewal of mouse embryonic stem cells are missing. Our first task therefore is to implement both candidate gene studies and genome-wide screens to identify pivotal regulators that can enhance the stability of human naïve pluripotent cells in vitro. We will then optimise the generation of naïve stem cells both by derivation from donated embryos and by reprogramming. In parallel we will establish the conditions for naïve cells to transition to an intermediate, or formative, population that recapitulates the embryonic disk, and subsequently to progress efficiently to lineage specification. We will characterise these conversions by deep transcriptome and epigenome profiling taking advantage on next generation sequencing technology. Having defined the path to lineage specification, we will be positioned to determine the extent to which differentiation biases commonly observed among conventional human pluripotent stem cells may be muted by resetting to naïve pluripotency. Observed restoration of developmental potential will be characterised further to reveal whether "correction" is mediated by transcriptional rewiring and/or epigenome erasure.

Our laboratory possesses the experience and expertise for all the stem cell culture and manipulation aspects of this project. We also have outstanding collaborators in domains of early embryology, CRISPR screening, sequencing informatics, DNA methylome, transposable elements, media formulation, physical biology, computational modelling and genome integrity.

Technical Summary

The goals of the proposed research are: (i) to fully characterise human naïve pluripotency; (ii) to define parameters for robust self-renewal and resetting pluripotency; (iii) to delineate the pathway to multi-lineage specification and commitment. Acquiring the knowledge to harness the naïve form of human pluripotency and thence to reproduce the molecular and cellular ontogeny of pluripotency progression in the embryo should enable more consistent and efficient differentiation behaviour. The concept that differentiation biases observed among conventional human pluripotent stem cells may be muted by transition through naïve pluripotency will then be examined.

The specific research objectives and tasks are:

I. Regulatory network of the human naïve state
i. Examination of core pluripotency regulators
ii. Differential transcriptome analysis
iii. Functional genetic screens
iv. Validation and characterisation of lead candidates
v. Modelling human naïve gene regulatory circuitry

II. Culture optimisation and transgene free resetting
i. Improvement to naïve PSC culture
ii. Global characterisation of consistent identity
iii. Homogeneity of self-renewal
iv. Transgene-free resetting

III. Commitment and differentiation
i. Pathway to multilineage competence
ii. Consistent multilineage differentiation
iii. Restoration of balanced differentiation

The study is focussed on human pluripotent stem cells with associated human embryo studies.
Key methods to be deployed include: genetic engineering of reporters and inducible transgenes; CRISPR/Cas9 based genetic screens; high throughput transcriptome profiling, methylome profiling, ATAC-seq and ChIP seq with associated bioinformatics; human embryo culture and cell line derivation.

Exploitation of the results will be led by the academic research sector but new know-how and intellectual property is also anticipated to foster collaborations and licencing arrangements with service industry and Pharma.

Planned Impact

Stem cell biology is a priority area for UK science investment. The research proposed here is specifically applicable to improving the authenticity, efficiency and reproducibility of directed differentiation of pluripotent stem cells for 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 in pluripotent stem cell research, as detailed in the Academic Beneficiaries section.
Industry - new insight into culture formulations and protocols for stem cell expansion, differentiation and quality assessment will benefit 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 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 - ultimately this research is expected to feed through to improved medical care and treatment by enabling more effective exploitation of human 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. Knowledge of early human development may also benefit assisted conception by providing additional criteria for assessment of embryo quality.
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 human development. For the second, the long-term goal is to enable treatments for debilitating disease through new resources for developing personalised medicine and for implementing cell-based therapy.

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 clinical and industry researchers while 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 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 design and host two similar types of specific activity, reaching out to different communities.
 
Description European Molecular Biology Laboratory policy on human embryonic stem cell and human embryo research
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
 
Description International Society for Stem Cell Research Standards Taskforce
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
 
Description FP7 Project Pluripotent Stem Cell resources for mesodermal medicine (PluriMes) 
Organisation European Research Centre for Biology and Medicine (Centre Européen de Recherche en Biologie et en Médecine)
Country France 
Sector Academic/University 
PI Contribution Austin Smith is responsible for overall scientific coordination of the project and leads work package 1, translating current advances and understanding of rodent pluripotency to develop and refine conditions that promote homogeneous naïve state for human pluripotent stem cells.
Collaborator Contribution By interlinking complementary biological and computational expertise, the partners aim to drive the generation of new knowledge on the characteristics of normal and abnormal stem cells. By harnessing the capacity of pluripotent stem cells to produce functional cell types with precision and at scale, researchers hope to enable new treatment modalities for degenerative diseases. The PluriMes project is specifically targeted at therapies for muscle, bone and cartilage.
Impact DOI: http://dx.doi.org/10.1016/j.cell.2014.08.029 DOI:10.1038/nprot.2014.102
Start Year 2014
 
Description FP7 Project Pluripotent Stem Cell resources for mesodermal medicine (PluriMes) 
Organisation Leiden University Medical Center
Country Netherlands 
Sector Academic/University 
PI Contribution Austin Smith is responsible for overall scientific coordination of the project and leads work package 1, translating current advances and understanding of rodent pluripotency to develop and refine conditions that promote homogeneous naïve state for human pluripotent stem cells.
Collaborator Contribution By interlinking complementary biological and computational expertise, the partners aim to drive the generation of new knowledge on the characteristics of normal and abnormal stem cells. By harnessing the capacity of pluripotent stem cells to produce functional cell types with precision and at scale, researchers hope to enable new treatment modalities for degenerative diseases. The PluriMes project is specifically targeted at therapies for muscle, bone and cartilage.
Impact DOI: http://dx.doi.org/10.1016/j.cell.2014.08.029 DOI:10.1038/nprot.2014.102
Start Year 2014
 
Description FP7 Project Pluripotent Stem Cell resources for mesodermal medicine (PluriMes) 
Organisation Pluriomics BV
Country Netherlands 
Sector Private 
PI Contribution Austin Smith is responsible for overall scientific coordination of the project and leads work package 1, translating current advances and understanding of rodent pluripotency to develop and refine conditions that promote homogeneous naïve state for human pluripotent stem cells.
Collaborator Contribution By interlinking complementary biological and computational expertise, the partners aim to drive the generation of new knowledge on the characteristics of normal and abnormal stem cells. By harnessing the capacity of pluripotent stem cells to produce functional cell types with precision and at scale, researchers hope to enable new treatment modalities for degenerative diseases. The PluriMes project is specifically targeted at therapies for muscle, bone and cartilage.
Impact DOI: http://dx.doi.org/10.1016/j.cell.2014.08.029 DOI:10.1038/nprot.2014.102
Start Year 2014
 
Description FP7 Project Pluripotent Stem Cell resources for mesodermal medicine (PluriMes) 
Organisation Sapienza University of Rome
Country Italy 
Sector Academic/University 
PI Contribution Austin Smith is responsible for overall scientific coordination of the project and leads work package 1, translating current advances and understanding of rodent pluripotency to develop and refine conditions that promote homogeneous naïve state for human pluripotent stem cells.
Collaborator Contribution By interlinking complementary biological and computational expertise, the partners aim to drive the generation of new knowledge on the characteristics of normal and abnormal stem cells. By harnessing the capacity of pluripotent stem cells to produce functional cell types with precision and at scale, researchers hope to enable new treatment modalities for degenerative diseases. The PluriMes project is specifically targeted at therapies for muscle, bone and cartilage.
Impact DOI: http://dx.doi.org/10.1016/j.cell.2014.08.029 DOI:10.1038/nprot.2014.102
Start Year 2014
 
Description FP7 Project Pluripotent Stem Cell resources for mesodermal medicine (PluriMes) 
Organisation Stem Cell Technologies
Country Canada 
Sector Private 
PI Contribution Austin Smith is responsible for overall scientific coordination of the project and leads work package 1, translating current advances and understanding of rodent pluripotency to develop and refine conditions that promote homogeneous naïve state for human pluripotent stem cells.
Collaborator Contribution By interlinking complementary biological and computational expertise, the partners aim to drive the generation of new knowledge on the characteristics of normal and abnormal stem cells. By harnessing the capacity of pluripotent stem cells to produce functional cell types with precision and at scale, researchers hope to enable new treatment modalities for degenerative diseases. The PluriMes project is specifically targeted at therapies for muscle, bone and cartilage.
Impact DOI: http://dx.doi.org/10.1016/j.cell.2014.08.029 DOI:10.1038/nprot.2014.102
Start Year 2014
 
Description FP7 Project Pluripotent Stem Cell resources for mesodermal medicine (PluriMes) 
Organisation Swiss Federal Institute of Technology in Lausanne (EPFL)
Country Switzerland 
Sector Public 
PI Contribution Austin Smith is responsible for overall scientific coordination of the project and leads work package 1, translating current advances and understanding of rodent pluripotency to develop and refine conditions that promote homogeneous naïve state for human pluripotent stem cells.
Collaborator Contribution By interlinking complementary biological and computational expertise, the partners aim to drive the generation of new knowledge on the characteristics of normal and abnormal stem cells. By harnessing the capacity of pluripotent stem cells to produce functional cell types with precision and at scale, researchers hope to enable new treatment modalities for degenerative diseases. The PluriMes project is specifically targeted at therapies for muscle, bone and cartilage.
Impact DOI: http://dx.doi.org/10.1016/j.cell.2014.08.029 DOI:10.1038/nprot.2014.102
Start Year 2014
 
Description FP7 Project Pluripotent Stem Cell resources for mesodermal medicine (PluriMes) 
Organisation Technical University of Dresden
Country Germany 
Sector Academic/University 
PI Contribution Austin Smith is responsible for overall scientific coordination of the project and leads work package 1, translating current advances and understanding of rodent pluripotency to develop and refine conditions that promote homogeneous naïve state for human pluripotent stem cells.
Collaborator Contribution By interlinking complementary biological and computational expertise, the partners aim to drive the generation of new knowledge on the characteristics of normal and abnormal stem cells. By harnessing the capacity of pluripotent stem cells to produce functional cell types with precision and at scale, researchers hope to enable new treatment modalities for degenerative diseases. The PluriMes project is specifically targeted at therapies for muscle, bone and cartilage.
Impact DOI: http://dx.doi.org/10.1016/j.cell.2014.08.029 DOI:10.1038/nprot.2014.102
Start Year 2014
 
Description FP7 Project Pluripotent Stem Cell resources for mesodermal medicine (PluriMes) 
Organisation University College London
Department Division of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Austin Smith is responsible for overall scientific coordination of the project and leads work package 1, translating current advances and understanding of rodent pluripotency to develop and refine conditions that promote homogeneous naïve state for human pluripotent stem cells.
Collaborator Contribution By interlinking complementary biological and computational expertise, the partners aim to drive the generation of new knowledge on the characteristics of normal and abnormal stem cells. By harnessing the capacity of pluripotent stem cells to produce functional cell types with precision and at scale, researchers hope to enable new treatment modalities for degenerative diseases. The PluriMes project is specifically targeted at therapies for muscle, bone and cartilage.
Impact DOI: http://dx.doi.org/10.1016/j.cell.2014.08.029 DOI:10.1038/nprot.2014.102
Start Year 2014
 
Description FP7 Project Pluripotent Stem Cell resources for mesodermal medicine (PluriMes) 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution Austin Smith is responsible for overall scientific coordination of the project and leads work package 1, translating current advances and understanding of rodent pluripotency to develop and refine conditions that promote homogeneous naïve state for human pluripotent stem cells.
Collaborator Contribution By interlinking complementary biological and computational expertise, the partners aim to drive the generation of new knowledge on the characteristics of normal and abnormal stem cells. By harnessing the capacity of pluripotent stem cells to produce functional cell types with precision and at scale, researchers hope to enable new treatment modalities for degenerative diseases. The PluriMes project is specifically targeted at therapies for muscle, bone and cartilage.
Impact DOI: http://dx.doi.org/10.1016/j.cell.2014.08.029 DOI:10.1038/nprot.2014.102
Start Year 2014