Defining the gene regulatory mechanisms controlling the entry of human cells into naïve pluripotency

Human pluripotent stem cells (hPSCs) are unspecialised cells that can form any tissues of the body. There is much hope that hPSCs will provide cell-based therapies for studying and treating diseases, for replacing worn out tissues, and for improving our understanding of human development. One of the recent, exiting advances in this research area has been the capture of hPSCs in different states of development. The two main states have been termed naïve and primed hPSCs to reflect their different identities. Naïve hPSCs have properties that recapitulate the cells of the human pre-implantation embryo, whereas primed hPSCs resemble cells from the embryo soon after it implants. These differences in developmental identity are important because they alter how hPSCs are controlled and respond, and also the range of cell types that the hPSCs are capable of specialising into. For example, only naïve hPSCs can efficiently turn into early placental and amnion cell types, and this is an important distinction because these specialised cell types are naturally anti-inflammatory and anti-immunogenic which mark them out as potentially useful sources for cell-based therapies. We currently know little about how naïve hPSCs are controlled and stabilised, but it is important that we find out so that we can exploit the full potential of these cells.

In our research so far, we have made exciting progress towards understanding how human cells can be converted into a naïve state during a process called reprogramming. My group has completed a large screen to identify the genes that are needed for naïve hPSC reprogramming, and also the genes that act normally to impede reprogramming. Interestingly, the top 'hits' in our completed screen identified many or all of the components within the same small number of complexes, strongly implicating these complexes as having important roles in this process. These complexes have not been studied before in naïve hPSCs or in reprogramming human cells. This work has led us to form the specific hypothesis that two complexes, called PRC1.3 and SAGA, are required to activate a set of critical genes during the initiation of reprogramming, and this activation is counteracted by an inhibitory pathway called HDAC2. The overall aim in this research proposal, therefore, is to discover how these newly identified complexes control naïve hPSC reprogramming, and to use this knowledge to develop methods to improve naïve hPSC production. We have carefully planned three main objectives to test our hypothesis:

The first and second objectives are to determine why the PRC1.3 and SAGA complexes are essentially required for naïve hPSC reprogramming. We will achieve this by identifying the genes that the two complexes need to activate during reprogramming, whether they function by transferring 'activating marks' to the genome, and by asking what happens precisely to cells that lack either of the complexes.

The third objective is to investigate a pathway that acts normally to impede the reprogramming process and we will focus on HDAC2. We predict that HDAC2 normally restricts naïve hPSC generation by removing the 'activation marks' that are required to promote transcriptional activation and effective reprogramming. We will use chemical inhibitors of HDAC2 to ask what happens when we prevent HDAC2 from working, and we will follow up on existing leads to identify the other factors required for HDAC2 to do this.

The successful completion of this research will develop strategies to understand and overcome critical barriers of naïve hPSC reprogramming, leading to improved conditions for naïve hPSC reprogramming and proliferation. More generally, the identified processes are likely to be co-opted in other situations to activate stem cell pathways in disease or are involved in the onset of human developmental disorders, and so the new mechanisms we uncover could be investigated in these other contexts.

Naïve and primed human pluripotent stem cells (hPSCs) differ substantially in molecular and functional properties that reflect their discrete developmental identities. Investigating naïve and primed hPSCs has enormous potential for uncovering the pathways that control different stages of pluripotency and for producing early developmental cell types that have sought after properties for biomedical applications. Although the reprogramming to an induced primed state has been studied in detail, much less is known about how naïve human pluripotency is acquired and stabilised. In response to this fundamental gap, we will determine the gene regulatory mechanisms that control the entry of human cells into naïve pluripotency.

We have completed a genome-wide, loss-of-function screen (presented as preliminary data in the grant proposal) that identified unanticipated regulators of naïve hPSC reprogramming. Remarkably, 10 of the top 15 ranked genes encode the components of two complexes: PRC1.3 and SAGA. Our hypothesis is that PRC1.3 and SAGA recruit two distinct histone acetyltransferases (HATs) to activate transcription, and that these events are jointly required for driving effective cell state change. Our screen also identified Histone Deacetylase 2 (HDAC2) as a significant impediment to reprogramming, which suggests that HDAC2 counteracts HAT-mediated transcriptional activation during the initial phase of reprogramming.

The central aim of this proposal is to investigate the newly identified barriers and facilitators of human naïve cell reprogramming and to develop a mechanistic understanding of how they act. This work will lead to important advances in understanding how human pluripotency is controlled, beyond that inferred from other species. Modulating the identified pathways will improve naïve cell production and open up new ways to deliver cell types with useful translational properties.

Our work will have important impact on a wide range of stakeholders, as outlined below.

The academic research community. The primary impact of our work will come from the gained understanding of the gene regulatory mechanisms that control human naïve pluripotent cell reprogramming. These results will inform stem cell and regenerative medicine, as well as provide insights into the basic principles of transcriptional control and chromatin complexes, with direct benefits to the scientific community (see Academic Beneficiaries).

The MRC. We will contribute to the successful delivery of MRC's mission. Our discovery research in stem cell biology falls centrally within Research Priority Theme 1 'Resilience, repair and replacement'. Specifically: "We [MRC] aim to harness the potential of stem cell biology and cell reprogramming technologies to provide renewable supplies of defined cell and tissue products for transplantation", which we address directly in this grant by establishing the molecular facilitators and barriers to human naïve pluripotent cell reprogramming. The identification and modulation of the pathways will lead to major and much needed improvements in the efficiency and stability of generating naïve cells, thereby providing a greater source of material for potential therapies. Our findings will likely also apply to gene regulatory mechanisms in other cell types, in which chromatin modifying complexes such as PRC1.3 or HDAC2 might control a switch in transcriptional responses and in cell state. Our work will therefore also deliver on the 'Molecular datasets and disease' area by addressing "We [MRC] will build on the UK's strengths in epigenetics to better understand what triggers epigenetic changes and how they influence disease."

Industry. Knowledge gained through this research could generate important intellectual property with the potential to be commercialised and exploited leading in the longer term to wealth creation. In particular, designing strategies to overcome barriers to human naïve cell reprogramming might lead to improvements in stem cell production that can be either commercialised directly or taken advantage of by the biotechnology sector in their R&D activities. Where relevant, we will pursue commercial opportunities generated from this research by developing projects with industrial collaborators (see Pathways to Impact for details). We will engage in industrial collaborations with the assistance of Babraham Institute Enterprise, which are the trading arm of the Babraham Institute and manages, develops and commercialises the Institute's intellectual property portfolio.

The health-care sector, charities and patients. This research has the potential to generate safer and potentially an expanded range of cell types that are available for regenerative medicine. In addition, our research will shed new light on the function of gene regulatory complexes and their target regions, which is information that could be exploited by clinical geneticists to associate genetic variants with developmental disorders and degenerative diseases. More broadly, our research will advance our understanding of the mechanisms that might control stem cell regeneration in adult tissues.

Staff development. This project will provide professional development for the PI, and train the PDRA and RA in modern molecular, cellular and bioinformatic methods. These research skills are relevant for both a career in academic or industrial science, and will therefore contribute to the competitiveness of UK for bioscience research.

The general public. There is significant public interest in the understanding of human pluripotent stem cells and research in human developmental biology. We will contribute to STEM understanding through our public engagement activities, which are important for facilitating two-way discussions about the benefits and challenges of stem cell research and regenerative medicine.