Type: Oral
Session: 101. Red Cells and Erythropoiesis, Excluding Iron: Effects of Inflammation on Erythropoiesis and the Niche
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Hematopoiesis, Biological Processes
To further explore the utility of Spic as a specific marker for EMBI Μφs, we examined EMBIs from Spicigfp/igfp mice which express GFP under the control of the Spic promoter and found that nearly 90% of the EMBI central Μφs were GFP+ indicating their expression of Spic. Moreover, evaluation of single BM cells by flow cytometry demonstrated that Spic-GFP+ Μφs are specifically enriched in the EMBI fraction and nearly 100% of the Spic-GFP expression in total BM cells belongs to the population of F4/80hi Μφs consistent with EMBI Μφs. Utilizing Spic-GFP as an endogenous marker for the EMBI Mφs, we were able to trypsinize whole BM from Spic-GFP mice stimulated with Epo or vehicle control (three biological replicates each) and the spleen of the Epo-treated mice (two biological replicates), and flow-sort the GFP+ cells for scRNAseq, avoiding Mφ fragmentation and reducing contamination from non-Mφ cells surrounded by Mφ membrane. This yielded more than 45,000 cells with the transcriptomic characteristics of classically-defined EMBI Mφs from eight 10x Chromium captures. Despite their many common EMBI Μφ genes, multiple distinct clusters could be identified in the murine BM at baseline and under stress erythropoiesis conditions, demonstrating the transcriptomic and surface marker heterogeneity that we and others have previously reported. Moreover, Epo stimulation led to differential gene expression within clusters, increases in certain Mφ populations, and the appearance of novel EMBI Μφ populations in both the BM and spleen during stress-erythropoiesis, indicating plasticity of existing macrophages and/or expansion of the EMBI Μφ pool via monocyte recruitment.
We next examined the native structure of EMBIs in situ in Spicigfp/igfp mouse BM by confocal microscopy. Previous attempts to identify EMBIs in intact BM were typically confounded by the high density of cells in close proximity in space. Leveraging the expression of Spic-GFP to label the cell bodies, and powerful image analysis software (Imaris), we were able to visualize the thin lacy network of F4/80+ cytoplasmic extensions expanding as a scaffold throughout the marrow and cradling the Ter119+ erythroblasts and CD11b+ granulocyte precursor cells. The central Μφs and associated cells constituting an “island” span a much more extended area in situ than the small rosette of cells typically recognized as an erythro(myelo)blastic island in cytospins or by imaging flow cytometry, after typical mechanical fragmentation during BM extraction and preparation for studies. In order to examine these large extended EMBIs without disruption, we enzymatically dissociated spic-GFP+ BM with collagenase and DNAse and deposited the intact cell-clusters on clean glass coverslips; the Spic-GFP+ EMBI Mφs adhered, and each appeared to have at least 60-100 CD71+ and CD11b+ cells attached to their projections. These transcriptomic, imaging, and functional assay data implicate specialization of various EMBI Mφ populations to nurture erythropoiesis and granulopoiesis at different stages in order to have optimized production of blood cells at steady-state or stress conditions.
Disclosures: Kalfa: Novo Nordisk: Research Funding; Agios Pharmaceuticals: Research Funding.
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