Mapping proteomic signatures in iPSC-derived neutrophils: Modeling neutropenia

Abstract Severe congenital neutropenia (SCN) encompasses a diverse range of genetic defects that impair the differentiation and function of neutrophil granulocytes. Our study aims to characterize proteomic differences in distinct developmental stages of neutrophils with pathogenic mutations in ELANE, HAX1, SMARCD2, and VPS45.Using CRISPR-Cas9 and homology-directed repair (HDR), we modeled pathogenic variants in human iPSCs at specific loci. Control (WT) and isogenic, sequence-verified iPSC lines containing patient-specific SCN mutations were differentiated into neutrophils in vitro. To analyze proteome compositions at distinct maturation stages of neutrophil differentiation, we sorted distinct subsets (ProNeu, PreNeu, ImmatureNeu, and MatureNeu) from control and SCN-mutated iPSC-derived cells via flow cytometry. FACS analysis showed a significant increase in ImmatureNeu cells in SCN-mutated iPSC-derived neutrophils compared to the WT. In the SMARCD2-mutated cells, we observed a complete absence of mature neutrophils. To identify abnormal molecular networks involved in SCN, we performed LC-MS/MS-based proteomics on iPSC-derived neutrophils at defined differentiation stages. Proteomics analysis detected an average of 7,837 proteins per sample across different neutrophil genotypes and maturation stages. Notably, we identified stage-specific proteins, including ITGAM, MPO, and LTF. We also achieved clear separation between cycling (early myeloid progenitors) and non-cycling cells (immature and mature neutrophils), which appeared as distinct clusters in heatmaps. This finding aligns with previous data indicating two major protein clusters with opposing regulation, reflecting differences in cell-cycle states. Among 874 differentially abundant proteins seen in neutrophil precursors and circulating blood neutrophils, 683 are also observed (78%) as differentially expressed in our WT cell line, indicating a high degree of similarity between significantly enriched proteins in our WT iPS cell line and published data. Protein identification depth decreased as cells matured, with differences between stages accounting for the main variance in the dataset. Between maturation stages, 1,015 to 4,258 proteins were significantly altered. We observed genotype-specific differences in GO term enrichment across the edited iPS cell lines and WT, suggesting alterations in neutrophil protein abundance related to genotype. Proteomics analysis is ongoing to identify candidate genes and pathways underlying the molecular mechanisms of SCN. Our iPSC-based platform for two- and three-dimensional neutrophil differentiation may provide new insights into gene regulation pathways and support the development of novel therapeutic targets.