Mapping single cell protein & biosynthesis dynamics across human embryonic & adult stem cell specification

Human embryonic lineage specification represents a major cellular transition from pluripotent identity towards lineage restriction. Cellular differentiation requires the timely expression and activity of developmental regulator proteins which control and enforce identity by organizing gene expression. Human embryonic stem cells which are pluripotent in their developmental potential maintain cell identity in part by the combined co-expression and cumulative activity of a group of protein regulators which individually can guide lineage-specific gene expression programs. While previous investigations of many of these developmental regulators shed light on their molecular characteristics individually during human pluripotent maintenance and lineages specification, there remains an incomplete model of developmental regulator protein expression dynamics as single-cells transition from pluripotent to lineage specified states. Moreover, in both embryonic and adult cellular differentiation settings, production of the macromolecules representing the central dogma of molecular biology (i.e., DNA, RNA, and protein) must be dynamically produced and eliminated to maintain cellular pools and facilitate changes in cell identity. With the work described in this Dissertation, I endeavored to uncover the molecular relationships between protein regulators during the continuous process of human lineage specification and to map single-cell biosynthesis activities during cellular differentiation. A central molecular approach used in my PhD work is multiplex single cell protein analysis with mass-cytometry, which directly facilitated the parallel assessment of single cell developmental regulator expression, biosynthesis activities, and cell cycle progression. To this end I developed two new methodological approaches and applied them to map cellular biosynthesis activities and the organization of protein regulators across the continuous process of cell differentiation in two major biological settings. Using a metabolic pulse-labeling approach to label live cells with three precursor molecules which specifically incorporate into nascent molecules of DNA, RNA, and protein, I tracked the parallel de novo activity of these three biosynthesis activities across several transitions in cell-state, including cell cycle and developmental progression, all at a single-cell resolution. I used this new approach to characterize biosynthesis activities across continuous cellular development spanning the healthy human hematopoietic continuum, and identified cellular transitions during B-cell development where cellular biosynthesis processes are highly organized. In addition, I created new reagents and molecular approaches to track multiplex developmental regulator protein expression at the single-cell level during models of human embryonic lineage specification. In order to reveal the specific relationships between protein regulators during human lineage specification, I applied single-cell computational analysis of multiplex developmental regulator protein expression data collected from time course differentiated human embryonic stem cell lines. I also leveraged in silico identification of cell-cycle dynamics and cellular differentiation trajectory in order to reveal the regulatory network architecture during the asynchronous process of embryonic lineage specification. Importantly, results obtained from this work suggest quantitative relationships of protein developmental regulators underlie transitions in cell identity during embryonic lineage specification, and are particularly modulated during progression through mitosis and cell division

Keywords: Developmental Biology.
Advisor: Bendall Sean
Fuller Margaret T.
Jarosz Daniel
Wernig Marius