Evaluating diverse technologies for detecting and validating senescent cells in vivo

项目摘要 单细胞分析最近已经成为主流生物学。单细胞分析已经被采用 在生物学的无数应用中，包括多种疾病、胚胎发育、比较 进化研究和衰老。最近，单细胞分析已经告知了细胞的现象， 衰老该提案有两个阶段，UG 3（模型系统）和UH 3（人类验证）。 细胞衰老是一个多方面的细胞命运，基本上不可逆地阻止细胞增殖， 激活促炎细胞因子、趋化因子、生长因子、蛋白酶的产生和分泌 和脂质，称为衰老相关分泌表型（SASP）。SASP可以影响组织 微环境，因此衰老细胞可以强烈影响组织功能，并可能影响全身环境。 衰老细胞随着年龄的增长而增加，并且可以驱动越来越多的与年龄相关的病理学， 神经退行性变到癌症，部分通过SASP。 越来越多的证据表明，衰老细胞没有通用的标志物。相反， 细胞，虽然共享某些特征和生物标志物，是显着异质性，不同的， 基因型、细胞和组织类型、衰老诱导物、组织（和细胞培养物） 微环境和时间顺序（初始衰老诱导后的时间）。 虽然一些更常用的衰老标记物在表面上鉴定衰老过程中具有实用性， 衰老细胞从头开始，责任仍然在研究人员，以证明为什么一个细胞应该考虑 衰老，而不是依赖于历史标记，如p16 INK 4a或p21 Cip 1。因此，新技术 设计来鉴定新的衰老细胞和表型是必要的，这将需要验证 无论是在培养中还是在组织中。 这项提案的最终目标是开发新技术，将衰老的签名映射回 完整的人体组织这一目标将使我们能够识别和空间表征衰老细胞在每个 组织，揭示取决于组织和细胞类型的独特标记。 试验1：识别小鼠血管系统中的衰老细胞，并确定它们是否可以通过 试验2：开发微生理学离体组织芯片， 卵巢衰老和通过SASP的人类组织-组织相互作用。 这两个试点将使用我们最初分析中确定的细胞表面标志物的组合， 空间分析（DSP，Nanostring）或Visium（10倍），将衰老特征定位到形态结构 和组织切片中的细胞。我们的提案将在多种组织中开发有效的衰老标记物， 细胞类型以前没有被表征，以将这些技术部署到更广泛的社区。

PROJECT SUMMARY Single cell profiling has recently exploded into mainstream biology. Single cell profiling has been employed in myriad applications in biology, including multiple diseases, embryonic development, comparative evolutionary studies and aging. Most recently, single cell profiling has informed the phenomena of cellular senescence. This proposal has two phases, UG3 (model systems), and UH3 (human validation). Cellular senescence is a multi-faceted cell fate that arrests cell proliferation, essentially irreversibly, and activates the production and secretion of pro-inflammatory cytokines, chemokines, growth factors, proteases and lipids, termed the Senescence Associated Secretory Phenotype (SASP). The SASP can influence tissue microenvironments, and thus senescent cells can strongly affect tissue function and likely the systemic milieu. Senescent cells increase with age and can drive a growing list of age-related pathologies, ranging from neurodegeneration to cancer, in part through the SASP. There is increasing evidence that there are no universal markers for senescent cells. Instead, senescent cells, while sharing certain characteristics and biomarkers, are remarkably heterogeneous, varying in characteristics with genotype, cell and tissue type, senescence inducer, tissue (and cell culture) microenvironment, and chronology (time after initial senescence induction). While some of the more commonly employed senescence markers have utility in superficially identifying senescent cells de novo, the onus remains on the investigator to demonstrate why a cell should be considered senescent, rather than relying on historical markers such as p16INK4a or p21Cip1. Thus, new technologies designed to identify novel senescent cells and phenotypes are necessary that will require validation both in culture and in tissue. The ultimate goal of this proposal is to develop new technologies to map senescent signatures back to intact human tissue. This goal will enable us to identify and spatially characterize senescent cells in each tissue, uncovering unique markers depending on tissue and cell type. Pilot 1: Identify senescent cells in the mouse vasculature, and determine if they can be detected by ultrasound, and verified in human tissue; Pilot 2: Develop a microphysiologic ex vivo tissue-on-a-chip to model ovarian senescence, and human tissue-tissue interactions via the SASP. These two pilots will use a combination of cell surface markers identified in our initial profiling, and Digital Spatial Profiling (DSP, Nanostring) or Visium (10x) to localize senescent signatures to morphological structures and cells in tissue sections. Our proposal will develop validated markers of senescence in multiple tissues and cell types not previously characterized to deploy these technologies to the broader community.