Evaluating diverse technologies for detecting and validating senescent cells in vivo

项目总结 单细胞图谱最近已迅速进入主流生物学领域。已经采用了单细胞分析 在生物学中的无数应用，包括多种疾病，胚胎发育，比较 进化论研究和老龄化。最近，单细胞图谱已经揭示了细胞的现象 衰老。该提案分为两个阶段，UG3(模型系统)和UH3(人工验证)。 细胞衰老是一种多方面的细胞命运，它基本上不可逆转地阻止细胞增殖，并且 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 和脂类，称为衰老相关分泌表型(SASP)。SASP可影响组织 微环境，因此衰老的细胞可以强烈地影响组织功能，并可能影响全身环境。 衰老细胞随着年龄的增长而增加，并可能导致越来越多的与年龄相关的病理变化，范围从 神经退行性变为癌症，部分通过SASP。 越来越多的证据表明，衰老细胞没有通用的标记物。相反，它正在衰老 细胞，虽然共享某些特征和生物标记物，但非常不同，在 基因、细胞和组织类型、衰老诱导剂、组织(和细胞培养)的特征 微环境和年表(初始衰老诱导后的时间)。 虽然一些更常用的衰老标记在表面上识别 衰老细胞从头开始，研究人员仍有责任证明为什么细胞应该被考虑 老化，而不是依赖历史标记，如p16INK4a或p21Cip1。因此，新技术 用于识别新的衰老细胞和表型的设计是必要的，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的技术，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并在空间上表征每个 组织，根据组织和细胞类型揭示独特的标记。 实验1：识别小鼠血管系统中的衰老细胞，并确定它们是否可以被 超声波，并在人体组织中得到验证；试验2：开发微生理学体外芯片组织模型 卵巢衰老，以及通过SASP的人类组织-组织相互作用。 这两个试点将使用在我们的初始分析中确定的细胞表面标记的组合，以及数字 空间分析(数字信号处理器、纳米串)或维西姆(10x)，用于将衰老特征定位为形态结构 和组织切片中的细胞。我们的计划将在多个组织和组织中开发有效的衰老标记 以前没有被描述为将这些技术部署到更广泛的社区的细胞类型。

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.