Characterisation of Stromal Cells Which Support Hematopoietic Niches in Murine Spleen
Authors: C O’Neill Helen ,   Clem Jones Centre for Regenerative Medicine, Bond University, Gold Coast, QLD, Australia., Lim Hong Kiat ,   Research School of Biology, Australian National University, Canberra ACT, Australia.
Published: July 2024

The role of spleen in hematopoiesis is not well understood. Recent studies now identify the presence of hematopoietic stem cells (HSC) in spleen although their numbers are far lower than HSC in bone marrow. Neonatal and pregnant mice contain higher numbers of HSC than do normal adults and have been used here as a model to investigate the localisation of HSC within the spleen. Immunocytochemical staining of spleen sections has been used to identify stromal cell types in the microenvironment of spleen. These studies have demonstrated the stromal structure and splenic architecture of pregnant adult mice and neonatal mice compared with normal adult mouse spleen. Six-day (D6) neonatal spleen is characterised by small T and B cell areas in poorly formed white pulp areas, and a surfeit of red pulp marked by the presence of numerous sinusoids and stromal cells, and notably the presence of gp38-staining cells. These are absent from adult red pulp region, being confined to the white pulp region as fibroblastic reticular cells. Another model tested was extramedullary hematopoiesis induced in adult mouse given Flt3L. Quantification of hematopoietic stem and progenitor cells (HSPC) in the various models revealed that pregnant mice contained significantly more long-term (LT)-HSC than did neonatal or adult normal mice, and that the spleen of Flt3L treated mice was populated more by multipotential progenitors (MPP) than LT-HSC. The pregnant and neonatal mice were subsequently analysed for localisation of LT-HSC in relation to various stromal cell types including endothelial cells (CD31), angiogenic vasculature (CD105), mesenchymal cells (CD29), fibroblastic reticular cells (gp38), myeloid cells (F4/80) and perivascular reticular cells (CD140b, CD51, Thy1.2/CD90). Multi-colour staining was used to colocalise LT-HSC with different region-specific stromal subsets. In both neonatal and pregnant mice, LT-HSC were located in red pulp. While many HSC colocalised with CD140b-exppressing stroma, some were found in association with myeloid cells, Thy1.2+ cells and in the case of neonates, with gp38+ cells. CD140-expressing cells in spleen were investigated more fully through gene expression analysis using the Fluidigm platform. Clustering analysis of expressed genes identified 3 distinct cell types, one reflecting follicular dendritic cells that do not support HSC niches. These were in the white pulp of adult mice. Two further clusters reflected perivascular reticular cells, one subset expressing type-specific genes as well as mesenchymal lineage genes at a higher level. It was concluded that a subset of mature and immature perivascular reticular cells cells may exist in spleen. These data confirm the presence of HSC and HSC niches in spleen and identify mesenchymal CD140b-expressing perivascular reticular cells as important niche elements in line with similar findings on HSC niches in bone marrow.