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

人类多能干细胞（hPSCs）是一种非特化的细胞，可以形成身体的任何组织。人类造血干细胞很有希望为研究和治疗疾病、替换受损组织以及提高我们对人类发育的理解提供基于细胞的疗法。在这一研究领域的最新进展之一是捕获处于不同发育状态的人造血干细胞。这两种主要状态被命名为naïve和引物的hPSCs，以反映它们不同的身份。Naïve人乳头状细胞具有再现人类植入前胚胎细胞的特性，而引物的人乳头状细胞类似于胚胎植入后不久的细胞。这些发育特性的差异很重要，因为它们改变了人乳头状细胞的控制和反应方式，也改变了人乳头状细胞能够特化的细胞类型范围。例如，只有naïve人造血干细胞可以有效地转化为早期胎盘和羊膜细胞类型，这是一个重要的区别，因为这些特殊的细胞类型具有天然的抗炎和抗免疫原性，这标志着它们是基于细胞的治疗的潜在有用来源。目前我们对naïve人造血干细胞是如何控制和稳定的知之甚少，但重要的是，我们发现了这一点，这样我们就可以充分利用这些细胞的潜力。到目前为止，在我们的研究中，我们在理解人类细胞如何在一个被称为重编程的过程中转化为naïve状态方面取得了令人兴奋的进展。我的小组已经完成了一个大屏幕，以确定naïve hPSC重编程所需的基因，以及正常阻止重编程的基因。有趣的是，在我们完成的筛选中，顶部的“命中”确定了相同数量的复合物中的许多或所有成分，强烈暗示这些复合物在该过程中具有重要作用。这些复合物尚未在naïve人造血干细胞或重编程人类细胞中进行过研究。这项工作使我们形成了一个特定的假设，即在重编程启动过程中，需要两个复合物PRC1.3和SAGA来激活一组关键基因，而这种激活被称为HDAC2的抑制途径抵消。因此，本研究计划的总体目标是发现这些新发现的复合物如何控制naïve hPSC重编程，并利用这些知识开发改进naïve hPSC生产的方法。我们仔细规划了三个主要目标来检验我们的假设：第一个和第二个目标是确定为什么PRC1.3和SAGA复合体是naïve hPSC重编程所必需的。我们将通过识别这两种复合物在重编程过程中需要激活的基因来实现这一目标，它们是否通过将“激活标记”转移到基因组来起作用，以及通过询问缺乏这两种复合物中的任何一种的细胞会发生什么。第三个目标是研究正常情况下阻碍重编程过程的途径，我们将重点研究HDAC2。我们预测HDAC2通常通过去除促进转录激活和有效重编程所需的“激活标记”来限制naïve hPSC的生成。我们将使用HDAC2的化学抑制剂来了解当我们阻止HDAC2起作用时会发生什么，我们将跟踪现有的线索，以确定HDAC2起作用所需的其他因素。这项研究的成功完成将制定策略来理解和克服naïve hPSC重编程的关键障碍，从而改善naïve hPSC重编程和增殖的条件。更一般地说，已确定的过程可能在其他情况下被选择来激活疾病中的干细胞途径或参与人类发育障碍的发病，因此我们发现的新机制可以在这些其他情况下进行研究。

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.