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

原位光导微量采样单细胞质谱分析阐明细胞异质性

• 批准号: 8828889
• 负责人: Peter Nemes
• 金额: $ 21.66万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8828889
• 关键词: Address; African; Animal Model; Animals; Area; Aspirate substance; Biochemical; Biological Models; Blood capillaries; Caliber; Cell Culture System; Cell Culture Techniques; Cell Size; Cell physiology; Cells; Cellular Morphology; Chemicals; Collection; Complex; Coupled; Custom; Data; Detection; Development; Developmental Cell Biology; Disease Progression; Electrophoresis; Embryo; Embryonic Development; Exhibits; Fishes; Fluorescence; Goals; Health; Heterogeneity; Histocompatibility Testing; Home environment; Human; In Situ; Individual; Investigation; Label; Laboratories; Life; Mass Spectrum Analysis; Measurement; Measures; Metabolic; Methodology; Microscopy; Modeling; Monitor; Morphology; Mus; Nature; Oils; Optics; Organism; Outcome; Peptides; Pharmacologic Substance; Population Heterogeneity; Proteome; Proteomics; Protocols documentation; Rana; Recruitment Activity; Regulation; Reproducibility; Research; Resolution; Sampling; Specimen; Staging; System; Technology; Temperature; Testing; Tissues; Vertebrates; Work; Xenopus; Xenopus laevis; Xenopus sp.; Zebrafish; base; capillary; cell dimension; cell type; design; embryo cell; embryo stage 2; human disease; innovation; innovative technologies; insight; instrument; interest; ionization; metabolomics; millisecond; nano-electrospray; new technology; next generation; operation; public health relevance; research and development; research study; sample fixation; single cell analysis; spatiotemporal; tandem mass spectrometry; three dimensional structure; tool; transcriptomics; zebrafish development

 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.

 描述：异质细胞群体的形成对胚胎发育和正常发育至关重要；细胞异质性导致不同类型的组织，也与疾病的发生和发展有关。了解细胞异质性对人类健康具有重要影响，但需要提供高检测选择性和灵敏度的专门方法，如质谱仪(MS)。然而，包括单细胞MS在内的很大一部分生物分析方法在体外工作或依赖于长期细胞培养。这些条件可能会改变蛋白质组，特别是细胞的代谢组成分和功能，并使对细胞间差异的结果的解释复杂化。在这里，我们建议引入一个基于MS的原位单细胞分析平台，并揭示在南非爪蛙(Xenopus Laevis)和斑马鱼(Danio Rerio)的实际、活的、自由发育的胚胎中形成的单细胞之间的代谢和蛋白质组学差异，这两种动物都是细胞和发育生物学以及人类疾病研究中公认的脊椎动物模型。该平台的关键方面是原位和高通量操作，以直接在标本中识别和测量任何感兴趣的给定细胞，能够重复测量细胞形态和生化组成，无需标记即可鉴定不同类型的代谢物和多肽，而不必在实验前知道它们的存在，以及可扩展到不同细胞尺寸；因此，避免了培养和分离单个细胞。该平台使用16细胞和32细胞非洲爪哇胚胎的单细胞进行了验证，这些胚胎具有高度可重复性的组织命运，并在水平和垂直身体平面上都表现出已知的转录细胞异质性。在沿着背腹和动物-植物轴的战略选择的识别细胞中测量代谢物和多肽，并对产生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到16细胞的斑马鱼胚胎，在这些胚胎中，细胞天生较小，在早期发育过程中对细胞异质性的了解较少。除了开发新的技术外，预期的结果还提供了关于实际发育胚胎中细胞异质性的时空动力学的新信息，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“用于单细胞分析的特别创新工具和技术”的目标很好地匹配。提出的方法适用于不同的物理和时间分辨率、广泛的生物分子类型和不同的模型系统，以帮助健康研究和下一代药物的开发。