New approaches in single cell biology - linking stem cell function with molecular profiles in heterogeneous populations

这一建议的重点是通过连接单细胞功能与单细胞分子谱来理解正常和突变干细胞的复杂生物学。它将结合单细胞功能和分子分析，流式细胞术指数分类和数学建模，以迭代的方式跨越正常和突变干细胞群。该项目需要在单细胞功能生物学（Kent组）、单细胞分子生物学（Gottgens组）和计算建模（GSK系统建模和转化生物学组）方面具有不同专业知识的合作伙伴。工业合作伙伴（GSK）将开发数学模型，以了解体内克隆扩增的规模和速度，并将其与体外收集的单细胞增殖和分化数据进行比较。这些数据将与本项目产生的干细胞/祖细胞的单细胞rna测序数据以及Kent/Gottgens实验室的其他项目相结合，以提供对单个干细胞不同命运选择的全面分子理解。总体而言，该项目有以下三个目标：目标1：了解体外造血干细胞异质性的程度，以及它与单个干细胞移植中观察到的不同亚型之间的关系。目的2：比较目的1中观察到的细胞动力学与自我更新活性增加（TET2功能丧失）或增加（JAK2功能获得）的突变干细胞，以确定哪些特性与持久的自我更新有关。目的3：通过指数分选和串联流式细胞术/质谱法（CyToF）在蛋白质水平上确定干细胞自我更新和功能异质性的调节因子。

This proposal focuses on understanding the complex biology of normal and mutant stem cells by linking single cell function with single cell molecular profiles. It will combine single cell functional and molecular assays with flow cytometric index-sorting and mathematical modelling in an iterative manner across both normal and mutant stem cell populations. This project requires partners with diverse expertise in single cell functional biology (Kent group), single cell molecular biology (Gottgens group) and computational modelling (GSK Systems Modeling and Translational Biology Group). Mathematicalmodels will be developed by the industrial partner (GSK) to understand the scale and speed of clonal expansion in vivo and this will be compared to indexsorted single cell proliferation and differentiation data collected in vitro. These data will be integrated with single cell RNA-sequencing data of stem/progenitor cells generated for this project and also other projects in the Kent/Gottgens labs to provide a comprehensive molecular understanding of distinct fate choices in single stem cells. Overall this project has the following three objectives: Objective 1:To understand the extent of blood stem cell heterogeneity in vitro and how it relates to the distinct subtypes observed in single stem cell transplantations. Objective 2: To compare cellular dynamics observed in Objective 1 to mutant stem cells with increased (TET2 loss-of-function) or ecreased (JAK2 gain-of-function) self-renewal activity to determine which properties are associated with durable self-renewal. Objective 3:To determine regulators of stem cell self-renewal and functional heterogeneity at the protein level by index-sorting and tandem flow cytometry / mass spectrometry (CyToF).