ICAM4 Mediates Human Erythroid Centered Island Formation
Authors: Han Xu , Ren Kehan , Wang Pan , Bi Honghao , Aydemir Inci , Li Ermin , Ji Peng 
Published: December 2024

Terminal erythropoiesis occurs in specific niches termed erythroblastic islands (EBI), which comprise a central macrophage surrounded by developing erythroblasts. Our knowledge of EBI mainly comes from in vitro assays, which involve the reconstitution of mixed cell populations that do not recapitulate in vivo niches. Moreover, these understandings come from mice, and the erythroid niche in humans is still unknown. Here, we utilized multiple spatial mapping technologies, including spatial transcriptomics and multiplexed imaging studies, to characterize erythropoiesis in human hematopoietic tissues during development and under stress.

We first utilized the Visium platform from 10X Genomics to study the spatial transcriptomic profiles of human fetal liver. The Visium platform contains barcoded spots with a diameter of 55 µm, which is suboptimal for single-cell analyses but ideal for the investigation of cell-cell interactions within their microenvironment. Previously we found that C1q+ macrophages served as classical EBI central macrophage in mice. However, in human fetal liver, after data processing, deconvolution of the clusters and co-analysis with corresponding single-cell RNA sequencing data revealed that there was no positive correlation between erythroid cells and all kinds of macrophages including C1q+ macrophages and other macrophages. Interestingly, there was a strong positive correlation between erythroid cells and erythroid progenitor cells, indicating that erythroid centered islands instead of classical macrophage centered EBIs were present in humans. Xenium subcellular spatial transcriptomic assay in situ confirmed the erythroid centered island existence. We then applied similar strategy in human adult bone marrow using bone marrow clot sections. In the bone marrow samples from 4 normal cases without hematologic diseases and 7 myelodysplastic syndromes (MDS) patients, there was positive correlation between erythroid cells and erythroid progenitor cells, but not between erythroid lineage cells and macrophages, indicating that the erythroid centered island was conserved in human during development and under stress conditions. Interestingly, there is an increased correlation coefficient between erythroid cells and erythroid progenitor cells in post-therapy bone marrow compared to their pre-therapy counterparts, demonstrating that reduced physical connection among erythroid cells could be a dysplastic feature in MDS, which could be improved with therapy. We also performed multiplexed immunofluorescence (IF) stains and further proved the existence of erythroid centered island in bone marrow core biopsy samples and an induced pluripotent stem cell (iPSC) derived-bone marrow organoid model.

To explore the underlying mechanisms mediated the formation of erythroid centered island, we analyzed the ligand-receptor pairs among erythroid lineages using human bone marrow single cell RNA-seq dataset. A list of ligand-receptor pairs was identified and the interactions between ICAM4 and RHAG or ITGA2B appear to be prominent among erythroid cells and erythroid progenitor cells. To test this, we used specific antibodies to block their interactions in iPSC-bone marrow organoid model and colony-forming unit assay with a medium containing only erythropoietin, and we found that blocking ICAM4 significantly reduced the number and size of the erythroid centered island and inhibited erythropoiesis. These findings support that ICAM4 plays a critical role in the formation of erythroid-centered EBIs in humans.

Taken together, our findings identify a novel kind of erythroid island which is erythroid centered, and its formation is mediated by ICAM4, which provides a comprehensive understanding of human erythropoiesis under normal and disordered conditions.