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

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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.