In Situ Optoguided Microsampling Single-cell Mass Spectrometry for Elucidating Cell Heterogeneity

 产品说明：细胞异质性群体的形成对胚胎发生和正常发育至关重要;细胞异质性产生不同类型的组织，并且还涉及疾病的发生和进展。了解细胞异质性对人类健康具有重要意义，但需要专门的方法，如质谱法（MS），提供高检测选择性和灵敏度。然而，包括单细胞MS在内的相当大一部分生物分析方法都是离体工作或依赖于长期的细胞培养。这些条件潜在地改变蛋白质组学，尤其是细胞的代谢组学组成和功能，并使细胞间差异结果的解释复杂化。在此，我们建议引入一个基于MS的原位单细胞分析平台，并揭示在南非爪蟾（Xenopus laevis）和斑马鱼（Danio rerio）的实际的、活的、自由发育的胚胎中形成的单细胞之间的代谢组学和蛋白质组学差异，这两种动物都是细胞和发育生物学以及人类疾病研究中成熟的脊椎动物模型。该平台的关键方面是原位和高通量操作，以直接在样本中识别和测量任何给定的感兴趣细胞，重复测量细胞形态和生化组成的能力，对不同类型的代谢物和肽的无标记识别，而不必在实验前知道它们的存在，以及可扩展到不同的细胞尺寸;因此避免了单细胞的培养和分离。该平台使用16-和32-细胞非洲爪蟾胚胎中的单细胞进行验证，所述单细胞具有高度可再现的组织命运，并且在水平和垂直身体平面中都表现出已知的转录组细胞异质性。在沿着背腹轴和动物-植物轴的策略性选择的鉴定细胞中测量代谢物和肽，并对所得的复杂化学信息进行数学评估，以揭示不同胚胎中具有相同身份的细胞之间的相似性。此外，我们建议将单细胞研究扩展到1至16细胞的斑马鱼胚胎，其中细胞本身较小，并且在早期发育过程中对细胞异质性了解较少。除了开发新技术外，预期结果还提供了有关实际发育胚胎中细胞异质性时空动态的新信息，为细胞和发育生物学以及人类健康提供了重要的生化数据。这些结果与RFA-RM-13-021“单细胞分析的特殊创新工具和技术”的目标非常匹配。“所提出的方法适用于不同的物理和时间分辨率，广泛类型的生物分子和不同的模型系统，以帮助健康研究和下一代药物的开发。

 DESCRIPTION: Formation of a heterogeneous population of cells is critical to embryogenesis and normal development; cell heterogeneity gives rise to different types of tissues and is also implicated in the onset and progression of diseases. Understanding cell heterogeneity holds important implications in human health, but requires specialized approaches, such as mass spectrometry (MS), that deliver high detection selectivity and sensitivity. A substantial portion o bioanalytical methodologies including single-cell MS, however, work ex vivo or rely on long-term cell cultures. These conditions potentially change the proteomic and, especially, the metabolomic composition and function of cells and complicate the interpretation of results on cell-to-cell differences. Herein we propose to introduce an in situ single-cell analysis platform based on MS and uncover metabolomic and proteomic differences among single cell that form in the actual, live, freely developing embryo of the South African clawed frog (Xenopus laevis) and the zebrafish (Danio rerio), both of which are well-established vertebrate models in cell and developmental biology and human disease research. Key aspects of this platform are in situ and high-throughput operation to identify and measure any given cell of interest directly in the specimen, a capability for repeated measurement of cell morphology and biochemical composition, label-free identification of diverse types of metabolites and peptides without having to know their presence before experiments, and scalability to different cell dimensions; thus, culturing and isolation of single cells are avoided. The platform is validated using single cells i the 16- and 32-cell Xenopus embryo that have highly reproducible tissue fates and exhibit known transcriptomic cell heterogeneity both in the horizontal and vertical body plan. Metabolites and peptides are measured in strategically selected identified cells along the dorso- ventral and animal-vegetal axes, and the resulting complex chemical information is mathematically evaluated to uncover similarity between cells that have the same identity in different embryos. Furthermore, we propose to extend single-cell investigations to the 1- to 16-cell zebrafish embryo, where cells are inherently smaller and cell heterogeneity is less understood during early development. Besides developing a new technology, the anticipated results provide new information on the spatiotemporal dynamics of cell heterogeneity in the actual developing embryo, providing important biochemical data for cell and developmental biology and human health. These outcomes are matched well with the goals of RFA-RM-13-021, "Exceptionally Innovative Tools and Technologies for Single Cell Analysis." The proposed approach is adaptable to different physical and temporal resolutions, broad types of biomolecules, and different model systems to aid health research and the development of next-generation pharmaceuticals.