Characterization of the human erythromyeloblastic islands (EMBIs) Free

The erythroblastic island (EBI) was first described in 1958, by Marcel Bessis, as a niche composed of a central macrophage (Μφ) surrounded by maturing erythroblasts. Many researchers, over the following decades, demonstrated that terminal erythropoiesis and enucleation occurs within this specialized microenvironment. Our recent work in murine models revealed that both myeloid and erythroid precursors in the bone marrow (BM) develop around the central Μφ, reclassifying this specialized niche as an erythromyeloblastic island (EMBI) (Romano and Seu, et al. Blood 2022). These findings shift the paradigm from a two-way interaction between the central Μφ and erythroblasts to a three-way interaction where the central Μφ fulfills different functions as a nurse cell for both lineages, thus balancing erythropoiesis and granulopoiesis. Single cell RNA-sequencing (scRNA-seq) analysis of EMBI-enriched fraction of mouse BM identified a small population (scRNA-seq cluster) of Μφs that specifically increased in number following administration of erythropoietin (Epo), concomitant with a similar increase in total BM erythroblasts and EMBIs, indicating that this cluster, which expressed all of the defining EMBI Μφ genes such as Mertk, Dnase2a, and Hmox1, indeed consisted of EMBI Μφs. This population also exhibited strong and restricted expression of the heme-induced transcription factor Spic. Follow-up fluorescence-activated cell sorting of GFP⁺ macrophages from the bone marrow of Spic-GFP mice achieved isolation of thousands of these cells providing a detailed transcriptomic profile of the central EMBI Μφs by scRNA-seq.

Building on this foundational work, we focused on translating these findings to human systems to better understand EMBI Μφ biology. Using discarded surgical tissue from patients who underwent hip replacement surgery or from BM biopsies, through an IRB approved protocol, we were able to isolate intact human EMBIs which were fixed for imaging flow cytometry (IFC) and were cryopreserved for coordinated evaluation by scRNA-Seq. Additionally, EMBIs in human BM in situ were evaluated by confocal immunofluorescence microscopy. Evaluation of isolated EMBIs by IFC demonstrated CD163⁺ Μφs specifically enriched in BM clusters collected by density gradient sedimentation and surrounded by CD71⁺ erythroblasts.

scRNA-Seq of over 26,000 cells captured via 10x Chromium revealed a cluster of 233 cells that had a transcriptomic profile consistent with classical EMBI macrophages. The majority of the other cells were different populations of erythroid precursors with smaller populations of granulocyte precursors and monocytes. Comparative analysis with the mouse EMBI Mφ scRNA-seq results showed conservation of key genes between mouse and human EMBI macrophages, including SPIC, C1QA, C1QB, C1QC, CD163, MRC1, and VCAM1. From the genes encoding efferocytosis receptors, TIMD4 was expressedin the majority of the EMBI macrophages, likely playing a role in apoptotic cell clearance, while MERTK and AXL appeared to characterize a subcluster, possibly those of the EMBI macrophages involved in enucleation. Additional notable genes that were robustly expressed in both species were HMOX1, SELENOP, FTH1, and SLC40A1 highlighting the central macrophage role in antioxidant defense, and iron metabolism. VCAM1 expression in human and mouse EMBI macrophage suggests a shared mechanism for erythroblast adhesion and niche formation.

We next examined the native structure of EMBIs in situ in bone sections using confocal microscopy. Previous efforts to identify EMBIs in intact bone marrow have been hindered by the high cellular density. We were able to observe a delicate CD163⁺ cytoplasmic network forming a scaffold throughout the marrow, supporting CD71⁺ erythroblasts and CD15⁺ granulocyte precursors. Notably, the central macrophages and associated cells forming an “island” span a much larger area in situ than the compact rosettes typically observed by IFC following mechanical disruption during marrow extraction.

In summary, our studies have validated the presence of EMBIs in human bone marrow and characteristics and core functions of EMBI Μφs across species. Further analysis of these findings will provide new insights into the biology of the niche for terminal erythropoiesis and granulopoiesis and may open new avenues for therapeutic targeting in anemia of inflammation and other acquired or genetic BM disorders.