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

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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).