A single-cell platform to discover and study regulators of human development

发现和研究人类发育调节因子的单细胞平台

• 批准号: 8425506
• 负责人: Sean Curtis Bendall
• 金额: $ 9万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8425506
• 关键词: Adult; Affect; Apoptosis; Behavior; Biological Assay; Biological Models; Bone Marrow; Cell Culture Techniques; Cell Cycle; Cell Death; Cell physiology; Cells; Communities; Complement; Cytometry; Data; Data Analyses; Data Collection; Developmental Biology; Developmental Process; Doctor of Philosophy; Frequencies; Functional RNA; Gene Targeting; Genes; Genetic; Goals; Growth Factor; Heterogeneity; Human; Human Development; In Vitro; Investigation; Left; Maps; Measures; Methods; Modeling; Modification; Mus; Nature; Normal Cell; Outcome; Patients; Pharmaceutical Preparations; Physiological; Pluripotent Stem Cells; Population; Post-Translational Protein Processing; Process; Proteins; Proteomics; Regenerative Medicine; Relative (related person); Research; Research Personnel; Resolution; Resources; Role; Shadowing (Histology); Stem cells; Study models; System; Time; Tissues; Undifferentiated; Work; analytical method; analytical tool; base; cell behavior; cell type; computerized tools; cytokine; follow-up; functional gain; genetic regulatory protein; human disease; human stem cells; instrumentation; interest; meetings; next generation; novel; pluripotency; programs; protein expression; public health relevance; research study; response; single cell analysis; stem cell differentiation; stem cell fate; tool

DESCRIPTION (provided by applicant): A single-cell platform to discover and study regulators of human development Human pluripotent stem cells (PSCs), which have the potential to form virtually any cell type in the body, have been the subject of intense research focus due to their potential application to regenerative medicine and the more recent potential for patient specific stem cells via reprogramming, allowing differentiated adult cells to be turned into pluripotent ones. However, in the shadow of this excitement, the potential to exploit pluripotent stem cells for fundamental studies in human developmental biology has been overlooked. This is especially pertinent given the fundamental physiological differences between the human and mouse - the closest relative where such studies are typically carried out. The long-term goal of my future research program is to use PSCs to discover and characterize new regulators (i.e. genes, metabolites, growth factors, non-coding RNA) of the human developmental processes. In order to accomplish this it will be necessary to create a platform (set of methods and analytical tools) to allow the modeling, tracing, and comparison of the paths by which human stem cells differentiate under natural and investigator-controlled situations. To this end, I will use a next-generation single-cell analysis instrumentation (CyTOF mass cytometry), which I have previously used to measure levels of protein expression, regulatory modifications, cell cycle and cell death across most known cell types in human bone marrow in response to drugs, cytokines, and growth factors, to characterize this system. I will first create a single cell templte of pluripotent cells undergoing differentiation using standard differentiation conditions. I will ten create a set of computational tools that will not only take into consideration the dynamic nature of cellular differentiation, but also enable the comparison of differentiating cell-types in differnt investigator controlled conditions. Finally, I will use stable gene knockdown approaches to target genes of interest based on proteins previously identified as differentially expressed in human PSCs versus their differentiated counterparts. Together this pipeline of experimentation will result in the establishment of a new and unique platform (a method for data collection, a method for data analysis and interpretation, and a proof-of-concept of its implementation) for the characterizing putative regulators of cellular differentiation.

描述（由申请人提供）：用于发现和研究人类发育调节因子的单细胞平台人类多能干细胞（PSC）具有在体内形成几乎任何细胞类型的潜力，一直是研究热点的主题由于它们在再生医学方面的潜在应用以及最近通过重新编程产生患者特异性干细胞的潜力，允许分化的成体细胞转变为多能细胞。然而，在这种兴奋的阴影下，利用多能干细胞进行人类发育生物学基础研究的潜力却被忽视了。考虑到人类和小鼠（通常进行此类研究的近亲）之间的基本生理差异，这一点尤其相关。我未来研究计划的长期目标是利用 PSC 来发现和表征人类发育过程的新调节因子（即基因、代谢物、生长因子、非编码 RNA）。为了实现这一目标，有必要创建一个平台（一套方法和分析工具），以允许对人类干细胞在自然和研究者控制的情况下分化的路径进行建模、追踪和比较。 为此，我将 使用下一代单细胞分析仪器（CyTOF 质谱流式细胞术），我之前曾用它来测量人类骨髓中大多数已知细胞类型对药物的反应的蛋白质表达水平、调节修饰、细胞周期和细胞死亡，细胞因子和生长因子来表征该系统。我将首先创建一个使用标准分化条件进行分化的多能细胞的单细胞模板。我将创建一套计算工具，不仅考虑细胞分化的动态性质，而且还能够在不同的研究者控制条件下比较分化细胞类型。最后，我将使用稳定的基因敲除方法来靶向目标基因，该方法基于先前确定的在人类 PSC 中与其分化对应物中差异表达的蛋白质。这些实验将共同建立一个新的、独特的平台（一种数据收集方法、一种数据分析和解释方法以及其实施的概念验证），用于表征细胞分化的假定调节因子。