2015 – A HIGHLY MULTIPLEXED SINGLE CELL PROTEOMIC SCREEN REVEALS THE PHENOTYPIC AND FUNCTIONAL LANDSCAPE OF THE HUMAN LYMPHO-MYELOID DIFFERENTIATION AXIS

While single-cell sequencing techniques have elucidated transcriptomic and epigenetic heterogeneities among human hematopoietic stem and progenitor cell (HSPC) populations, the corresponding proteomic level information, where the action of these regulatory networks manifests, is still missing. As cell sorting relies on surface markers, the functional capabilities and lineage specificities of HSPCs can only be evaluated after interrogation of the proteome at a single cell resolution. To that end, based on a highly-multiplexed single-cell screening framework in our lab (Glass and Tsai et al. Immunity in press) we quantified the simultaneous expression of 353 surface molecules and 79 functional intracellular molecules (TFs, chromatin regulators, metabolic enzymes) with mass cytometry. In doing this, we created a core panel with probes against intracellular markers associated with lymphoid potential to better illuminate the lympho-myeloid axis. In total, we analyzed 556,226 CD34+ bone marrow HSPCs across 3 individuals and identified 81 molecules expressed by HSPCs, with heterogeneous expression among conventionally-defined HSPC cell types. Using unsupervised clustering, we identified 11 populations and defined their unique proteomic composition. At the same time, we were able to infer the identity of HSPC cell types based on prior knowledge and impute the functional proteomic data onto canonical cell types. Most interestingly, a population mostly consists of SATB1-high CMPs exhibits a distinct epigenetic proteomic profile, such as high CTCF and H3k4me3 levels, indicating a possible decision making point in lympho-myeloid differentiation. Overall, we supply a quantified summary of the proteomes of human HSPCs and create a framework to identify and characterize progenitor populations with unique functional states along the lympho-myeloid developmental process.