2022 – CHARACTERIZING HETEROGENEITY IN HEALTHY AND MALIGNANT HEMATOPOIETIC HIERARCHIES USING SINGLE CELL PROTEOMICS

Abstract

In recent years, single-cell studies demonstrated a high degree of molecular and functional heterogeneity within complex cellular systems. Single-cell transcriptome profiling has provided the basis for our current knowledge about stem cell heterogeneity. Although single-cell transcriptomics has given tremendous insight into heterogeneity, transcript levels are not always a good proxy for protein abundance. In this study, we demonstrate for the first time that single-cell mass spectrometry (scMS)-based proteomics enables the exploration of the global protein landscape of human hematopoietic stem & progenitor cells (HSPCs) in healthy aging and hematologic malignancies. By combining fluorescence-activated cell sorting (FACS) with our current experimental and computational scMS workflow, we are able to capture heterogeneity within these complex cellular hierarchies. Our analyses encompass scMS measurement of over 6000 single-cells, quantifying ∼1000 proteins per cell, from young and elderly individuals as well as patients with myelodysplastic syndromes. Our approach is able to recapitulate the differentiation trajectories of early human hematopoiesis. Besides, our scMS data highlights that the current FACS marker combinations appear to fall short of precise delineation of early human progenitors. We further show that Endomucin, a recently published marker of human HSCs, marks the stem cells at the top of the hierarchy. Finally, by integrating scMS and CITE-seq data, we aim to provide a better resolution of disease states based on the combined analysis of proteome and transcriptome. Overall, the results presented here support the power of global single-cell proteomics as a tool to characterize normal and perturbed developmental hierarchies. In recent years, single-cell studies demonstrated a high degree of molecular and functional heterogeneity within complex cellular systems. Single-cell transcriptome profiling has provided the basis for our current knowledge about stem cell heterogeneity. Although single-cell transcriptomics has given tremendous insight into heterogeneity, transcript levels are not always a good proxy for protein abundance. In this study, we demonstrate for the first time that single-cell mass spectrometry (scMS)-based proteomics enables the exploration of the global protein landscape of human hematopoietic stem & progenitor cells (HSPCs) in healthy aging and hematologic malignancies. By combining fluorescence-activated cell sorting (FACS) with our current experimental and computational scMS workflow, we are able to capture heterogeneity within these complex cellular hierarchies. Our analyses encompass scMS measurement of over 6000 single-cells, quantifying ∼1000 proteins per cell, from young and elderly individuals as well as patients with myelodysplastic syndromes. Our approach is able to recapitulate the differentiation trajectories of early human hematopoiesis. Besides, our scMS data highlights that the current FACS marker combinations appear to fall short of precise delineation of early human progenitors. We further show that Endomucin, a recently published marker of human HSCs, marks the stem cells at the top of the hierarchy. Finally, by integrating scMS and CITE-seq data, we aim to provide a better resolution of disease states based on the combined analysis of proteome and transcriptome. Overall, the results presented here support the power of global single-cell proteomics as a tool to characterize normal and perturbed developmental hierarchies.