Transcriptional and functional architecture of the whole neutrophil compartment [RNA-seq IPSCs]

In this study, we present NeuMAP, a comprehensive single-cell analysis of neutrophils spanning over 40 anatomical, physiological, and pathological contexts in mice. NeuMAP confirms and expands previous models of neutrophil diversity, revealing the organization of neutrophils into distinct functional hubs under both normal and pathological conditions. Furthermore, we delineate prototypical trajectories as key organizers of granulopoiesis and examine neutrophil dynamics along these trajectories during acute inflammation and cancer. Integrating insights from fate mapping, mutant mouse models, and in vitro experiments, we identified immunological signals guiding neutrophil through these typical trajectories of granulopoiesis, that balance immune protection, tissue homeostasis and repair. Specifically, we identified IFNB, GMCSF, and TGFB as drivers of pro-inflammatory, cancer-associated, and mature neutrophil states, respectively and found that the transcription factor JUNB drives the angiogenic and immunosuppressive function of neutrophils. Additionally, we uncover conserved transcriptional signatures for human neutrophil states, validate their prognostic significance in cancer patients, and introduce a proof-of-concept strategy for exploring the diagnostic potential of neutrophil functional diversity across various diseases using single-cell transcriptomics of blood neutrophils. Our study provides a model that delineates the global architecture of the neutrophil compartment in mammals, and stablishes a robust framework for further exploration of neutrophil biology. Human iPS cells (SCTi003-A; STEMCELL Technologies) were maintained in 6-well sterile tissue culture plates (ThermoFisher) coated with Matrigel (Corning), using mTeSR™ Plus serum-free medium (STEMCELL Technologies). The medium was replaced daily. iPS cell colonies were cultured at a density of fewer than 5 colonies per well in a 6-well tissue culture plate coated with Matrigel (Corning). When individual colonies reached approximately 500 um of diameter, mTeSR™ Plus medium was replace with Stemline II hematopoietic stem cell expansion medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with insulin-transferrin-selenium (ITS, Invitrogen) and cytokines as follows: bone morphogenetic protein (BMP) 4 (20 ng/mL, R&D Systems) was added for four days and then replaced with vascular endothelial growth factor (VEGF) 165 (40 ng/mL, R&D Systems) on Day 4. On Day 6, VEGF 165 was replaced with a combination of stem cell factors (SCF, 50 ng/mL, R&D Systems), interleukin-3 (IL-3, 50 ng/mL, R&D Systems), thrombopoietin (TPO, 5 ng/mL, Merck), and granulocyte colony-stimulating factor (G-CSF, 50 ng/mL; Merck). Thereafter, the medium was replaced every five days. Neutrophils were collected every five days starting on day 11 of differentiation. Neutrophils were collected after 24h of treatment with different cytokines: TGFβ, IFNβ and GM-CSF. Total RNA was prepared with the RNA Extraction RNeasy Plus Mini-kit (QIAGEN)