An immunophenotype-coupled transcriptomic atlas of mouse hematopoietic progenitors

Decoding the bone marrow (BM) hematopoietic progenitor compartment is central to understanding the development of mature immune cells in health and disease. Single-cell multimodal approaches are transforming the analysis of this heterogeneous compartment by simultaneously capturing transcriptomic and proteomic information. Still, integrated studies covering the full spectrum of murine hematopoietic stem and progenitor cells (HSPCs) remain scarce. Here, we report a Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) based atlas of 9,281 low-frequency mouse BM HSPCs, profiled using a 123 antibody-derived tag (ADT) panel coupled with genome-wide single-cell transcriptomes. Multimodal analysis resolved 28 progenitor stages across eight blood lineages, each defined by distinctive surface marker and transcription factor (TF) profiles, and provided novel marker combinations to dissect transcriptionally closely related progenitor stages. Classical mouse progenitor surface markers, such as CD117, CD34, and CD115, were detected in accordance with well-established lineage relationships, whereas CD43 and CD48 showed, unexpectedly, broader expression patterns. Neglected surface molecules such as CD27, CD36, CD106, and PIR-A/B are nominated as novel stage-specific markers. ADT versus transcriptome comparison revealed transcription-translation inconsistencies for markers, including CD54 and CD68. A CITE-seq-guided gating strategy translates these population definitions into FACS-compatible panels for prospective isolation and functional interrogation of previously unresolved progenitor stages. Comprehensive TF network analysis delineated lineage- and stage-specific transcriptional programs. This contributes to clarifying unresolved ontogenies and functions, including the lympho-myeloid dual origin of pDCs or rapid neutrophil turnover. This new and comprehensive resource enables exploration of mouse hematopoietic progenitor states driving discoveries in hematology and immunology.