Interrogation of individual cells to identify lung progenitors and their niches

询问单个细胞以识别肺祖细胞及其生态位

• 批准号: 7678335
• 负责人: MARK A KRASNOW
• 金额: $ 3.25万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7678335
• 关键词: Animal Model; Cells; Cellular biology; Classification; Color; Complex; Development; Disease; Drosophila genus; Gene Expression; Genetic; Genomics; In Situ Hybridization; In Vitro; Individual; Libraries; Lung; Lung diseases; Maintenance; Mesenchyme; Methods; Microfluidics; Mouse Strains; Mus; Pathway interactions; Pattern; Population; Principal Investigator; Resolution; Signal Pathway; Signal Transduction; Signaling Molecule; Stem cells; Structure of parenchyma of lung; Tissues; Transgenic Mice; in vivo; lung development; prevent; progenitor; programs; public health relevance; research study; response; three dimensional structure; tool

DESCRIPTION (provided by applicant): Identifying and characterizing lung progenitor cells and their signaling niches is crucial for understanding how a healthy lung is built and maintained, how alteration of development and maintenance pathways cause or contribute to lung disease, and how disease can be prevented and damaged lung tissue restored or replaced. Identifying and characterizing lung progenitor cells and their niches has been hampered by the complex three-dimensional structure of the lung and the lack of tools to mark, follow the fate, and manipulate gene expression in individual lung cells in vivo. Here we propose to develop such tools, by adapting for use in mouse lung the systematic genetic approaches and single cell resolution genetic tools ("clonal analysis") that have been used over the past decade to elucidate progenitor and stem cells and their signaling niches in the model organism Drosophila. We combine this in vivo approach with a high throughput in vitro approach that takes advantage of recent advances in genomics and microfluidics to characterize individual lung progenitor cells and their developmental responses to the signals identified in the in vivo experiments. This combined approach is general and applicable to progenitor cells throughout the lung and other mouse tissues, although we focus on the poorly characterized progenitor cells in lung mesenchyme. The specific aims are: To establish a general multi-color in vivo cell marking method to follow the fate of individual mouse lung cells, and to use this method to identify the number and types of progenitors cells in the mesenchyme of developing and mature mouse lung. To use high throughput in situ hybridization studies to localize all of the signaling and receiving centers in progenitor cell niches in the mouse lung; To determine the in vivo functions of the identified signaling and receiving centers in lung progenitor cell biology by constructing a library of transgenic mouse strains that can be used to create ectopic signaling centers and clonally inactivate each signaling pathway in the mouse lung; To establish a high throughput, microfluidic approach to isolate, propagate, profile, and assess the developmental potential of identified lung progenitor cells under defined culture conditions, and to use this to determine the number of molecularly and functionally distinct progenitor cell populations in lung mesenchyme; To establish methods for directing development of lung progenitor cells along specific lineages using the high throughput microfluidic approach to expose isolated, identified progenitor cells systematically to different concentrations, gradients, temporal patterns, and combinations of signaling molecules identified in the in vivo experiments.

描述(由申请人提供)：识别和表征肺祖细胞及其信号定位对于了解如何构建和维持健康的肺、发育和维持途径的改变如何引起或促成肺部疾病以及如何预防疾病和修复或替换受损的肺组织至关重要。由于肺的复杂三维结构，以及缺乏标记、跟踪命运和操纵体内单个肺细胞基因表达的工具，识别和表征肺前体细胞及其利基环境一直受到阻碍。在这里，我们建议开发这样的工具，通过在小鼠肺中使用系统遗传方法和单细胞分辨遗传工具(克隆分析)，这些工具在过去十年中一直被用来阐明模式生物果蝇中的祖细胞和干细胞及其信号生态位。我们将这种体内方法与高通量体外方法相结合，该方法利用基因组学和微流体学的最新进展来表征单个肺祖细胞及其对体内实验中确定的信号的发育反应。这种结合的方法是通用的，适用于整个肺和其他小鼠组织中的祖细胞，尽管我们关注的是肺间充质中特征不佳的祖细胞。具体目标是： 建立一种跟踪单个小鼠肺细胞命运的通用体内多色细胞标记方法，并用该方法鉴定发育和成熟小鼠肺间充质中祖细胞的数量和类型。 利用高通量原位杂交研究定位小鼠肺内祖细胞壁龛中的所有信号和接收中心； 通过构建转基因小鼠品系文库，确定已鉴定的信号和接收中心在肺祖细胞生物学中的体内功能，该文库可用于创建异位信号中心，并克隆灭活小鼠肺中的每一条信号通路； 建立一种高通量的微流控方法来分离、增殖、分析和评估特定培养条件下已鉴定的肺祖细胞的发育潜能，并利用该方法来确定肺间充质中分子和功能上不同的祖细胞群体的数量； 建立使用高通量微流控方法引导肺祖细胞沿特定谱系发育的方法，以系统地将分离的、经鉴定的祖细胞暴露于体内实验中确定的不同浓度、梯度、时间模式和信号分子组合中。