Generation of an In Vivo Senescent Cell Atlas: Across the life-course and in pathology

衰老是由生理和病理应激触发的稳定的细胞周期停滞状态。虽然衰老细胞不能增殖，但它们是生物活性的，并且已经显示在各种环境中具有功能活性，包括（但不限于）胚胎发生、衰老、伤口愈合、心血管疾病和肿瘤发生。在这些不同的背景下，衰老可以是一个积极的或有害的有机体健康的组成部分。例如，衰老细胞促进伤口的迅速闭合，而它们的缺失延迟了这一过程。虽然在衰老过程中，衰老细胞在多个组织中的积累会导致组织功能障碍，但在这种情况下，它们的消融（药理学或遗传学）已被证明可以延长小鼠的健康寿命。许多这些效应被认为是由获得性分泌表型（衰老相关的分泌表型; SASP）介导的。那么衰老是如何影响这些不同的生物学过程的呢？当然，在纤维化过程中，衰老的肝细胞不可能与癌前皮肤细胞进行完全相同的程序，因为功能后果是截然不同的。为了回答这个问题，我们的工作假设是，衰老不能被描述为一个程序，不同的组织和衰老诱导的背景下，与独特的和共同的功能状态。此外，我们还证实，在一种病理生理学环境（即纤维化）中的衰老不是由一个衰老群体组成的，而是由许多亚群组成的，这些亚群的复合物共同协调了表型结果。因此，这些亚群比例的改变可能导致病理性疾病。然而，目前大多数衰老数据是在体外开发的，并且是在少数细胞系（通常是原代成纤维细胞）中开发的。因此，我们缺乏对体内衰老的基本了解，在这些不同的状态和组织背景下，重要的是在一个决议，使我们能够确定哪些亚群存在。这项建议的目的是产生一个高质量的体内图谱，这些不同的衰老状态，作为研究和生物技术产业的催化剂。我们的建议围绕使用新的工具来分离衰老细胞（p16谱系追踪小鼠，以及不需要固定和pH修改的衰老相关β半乳糖苷酶的荧光染料），创新的测序方法（使转录组，染色质可及性和甲基化数据能够从同一个单细胞中分离出来），以及开创性的计算分析（多组学数据集的询问）。结合这些数据将使我们能够识别不同的衰老亚群，并了解这些不同的功能状态是如何实现的。该提案的一个组成部分是我们注重最大限度地分配这一资源，因此，我们打算建立一个用户友好的基于网络的平台，用于快速传播处理后的数据，此外还与社区分享所有技术和原始数据。因此，这种资源有可能被学术研究界广泛使用，以根据自己的原始数据快速测试假设。此外，由于靶向衰老抑制剂已被证明可以改善小鼠的健康和寿命，因此了解功能性衰老状态对于开发未来的靶向药物至关重要，同时减少脱靶效应。在这方面，该数据集对于新兴的以健康老龄化为重点的生物技术行业也至关重要。

Senescence is a state of stable cell cycle arrest, triggered by physiological and pathological stressors. Whilst senescent cells cannot proliferate, they are biologically active, and have been shown to be functionally active in a variety of settings including (but not limited to) embryogenesis, ageing, wound healing, cardiovascular disease, and tumourigenesis. In these diverse contexts, senescence can be either a positive or detrimental component to organismal health. For example, senescent cells promote the prompt closure of wounds, and their absence delays this process. Whilst in ageing the accumulation of senescent cells across multiple tissues results in tissue dysfunction, in this context their ablation (pharmacological or genetic) has been shown to extend healthy life-span in mice. Many of these effects are believed to be mediated by the acquired secretory phenotype (Senescence-Associated Secretory Phenotype; SASP).So how do senescent impinge on these diverse biological processes, across a variety tissues? Surely a senescent liver cell during fibrosis cannot engage the exact same programme as precancerous skin cell, as the functional consequences are widely divergent. To answer this question, our working hypothesis is that senescence cannot be described as one programme, and that different tissue and senescence-inducing contexts are associated with both unique and common functional states. Furthermore, we also posit that senescence in one pathophysiological setting (i.e. fibrosis) is not composed of one senescent population, but a number of sub-populations whose composite together orchestrates the phenotypic outcome. Thus, alterations to the proportions of these sub-populations may result in a pathological disease. However, currently the majority of senescence data has been developed in vitro, and in a small number of lines (often primary fibroblasts). As such we lack a basic understanding of senescence in vivo, across these diverse states and tissue contexts, and importantly at a resolution that enables us to determine what sub-populations exist.The aim of this proposal is to generate a high-quality in vivo atlas of these diverse senescent states to act as a catalyst for the research and biotech industry. Our proposal centres around the use of novel tools to isolate senescent cells (a p16-lineage tracing mouse, and a fluorescence dye for Senescence-Associated Beta Galactosidase that requires no fixation and pH modification), innovative sequencing approaches (enabling the transcriptome, chromatin accessibility, and methylation data to be isolated from the same single cell), and pioneering computational analyses (interrogation of multi-omic datasets). Combined these data will enable us to characterise the different senescent sub-populations, as well as understand how these diverse functional states are achieved. Integral to the proposal is our focus on maximal distribution of this resource, as such we intend to generate a user-friendly web-based platform for the rapid dissemination of processed data, in addition to sharing all technical and primary data with the community. As such this resource has the potential to be used widely by the academic research community to quickly test hypotheses based on their own primary data. Furthermore, as targeting senescence pharmacologically has been shown to improve health and life-span in mice, understanding the functional senescence states is essential to develop future targeted agents, with reduced off-target effects. In this regard, this dataset will also be essential for the burgeoning healthy-ageing focused biotech industry.