scRNA-seq analysis of EBI constituent cells reveals terminal granulopoiesis alongside terminal erythropoiesis. (A) Uniform Manifold Approximation and Projection (UMAP) plot generated by ICGS2 (Iterative Clustering and Guide-gene Selection version 2) depicting the different populations of cells composing the EBI fraction. The naming of cell populations was marker gene–driven, with an emphasis on prior well-defined hematopoietic lineage notations.²⁰ (B) Visualization in the UMAP plot of the sorted F4/80⁺ (green), CD11b⁺ (yellow), and CD71⁺ (purple) populations, representing the main component populations within the EBIs, showing their alignment to the unsorted cells (pink, blue) of the EBI fraction from C57BL/6 WT BM. (C) Individual contribution of each capture to the UMAP plot pictured in A. Cell numbers for each sample were as follows: WT1 unsorted, n = 4963; WT2 unsorted, n = 7224; CD11b, n = 2668; F4/80 n = 1335; and CD71, n = 1264. (D) Comb plot showing the relative expression level of genes characteristic of each population (erythroblasts, granulocytic precursors, macrophages). ERP1 to ERP4 are marked by high expression of erythroid commitment and differentiation genes such as Gata1, Epor, Klf1, and Gfi1b, whereas transcripts of membrane and cytoskeletal proteins such as transferrin receptor CD71 (Tfrc), glycoprotein A (Gypa), band 3 (Slc4a1), and α-spectrin (Spta1) are most significantly expressed in ERP2 to ERP4. ERP5 was marked by genes known to be expressed in orthochromatic erythroblasts and reticulocytes such as Bpgm and Xpo7^21-23 and was the most frequent erythroblast population in unsorted and in CD71⁺ population sorted from the EBIs, as expected for the most mature erythroblasts. CD11b⁺ sorted cells segregate into 3 transcriptionally distinct clusters that represent granulocytic precursors associated with previously defined neutrophil specification (proNeu) and commitment cell states (preNeu to immNeu).²⁴ Downregulation of cell cycle–related genes Mcm2, Aurkb, and Cdkn2c, especially in ERP5 and immNeu, confirms that these populations consist of terminally differentiated erythroid and granulocytic cells.