Single-cell analysis at the protein level delineates intracellular signaling dynamic during hematopoiesis

Abstract

Abstract Background Hematopoietic stem and progenitor cell (HSPC) subsets in mice have previously been studied using cell surface markers, and more recently single-cell technologies. The recent revolution of single-cell analysis is substantially transforming our understanding of hematopoiesis, confirming the substantial heterogeneity of cells composing the hematopoietic system. While dynamic molecular changes at the DNA/RNA level underlying hematopoiesis have been extensively explored, a broad understanding of single-cell heterogeneity in hematopoietic signaling programs and landscapes, studied at protein level and reflecting post-transcriptional processing, is still lacking. Here, we accurately quantified the intracellular levels of 9 phosphorylated and 2 functional proteins at the single-cell level to systemically capture the activation dynamics of 8 signaling pathways, including EGFR, Jak/Stat, NF-κB, MAPK/ERK1/2, MAPK/p38, PI3K/Akt, Wnt, and mTOR pathways, during mouse hematopoiesis using mass cytometry. Results With fine-grained analyses of 3.2 million of single hematopoietic stem and progenitor cells (HSPCs), and lineage cells in conjunction with multiparameter cellular phenotyping, we mapped trajectories of signaling programs during HSC differentiation and identified specific signaling biosignatures of cycling HSPC and multiple differentiation routes from stem cells to progenitor and lineage cells. We also investigated the recovery pattern of hematopoietic cell populations, as well as signaling regulation in these populations, during hematopoietic reconstruction. Overall, we found substantial heterogeneity of pathway activation within HSPC subsets, characterized by diverse patterns of signaling. Conclusions These comprehensive single-cell data provide a powerful insight into the intracellular signaling-regulated hematopoiesis and lay a solid foundation to dissect the nature of HSC fate decision. Future integration of transcriptomics and proteomics data, as well as functional validation, will be required to verify the heterogeneity in HSPC subsets during HSC differentiation and to identify robust markers to phenotype those HSPC subsets.

Keywords

• Mass cytometry
• Single-cell analysis
• Signaling biosignatures
• Hematopoietic stem and progenitor cells
• Hematopoiesis
• Trajectory