Differential regulation by CD47 and thrombospondin-1 of extramedullary erythropoiesis in mouse spleen (Bulk RNA-Seq)

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from cd47-/- mice. Elevated expression of several genes associated with early stages of erythropoiesis was observed in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors including erythropoietin receptor, aquaporin-1, glycophorin A, and erythrocyte membrane-associated protein were more abundant in cd47-/- spleens but significantly depleted in thbs1-/- spleens. Single cell transcriptome analysis indicated that loss of CD47 is associated with accumulation and increased proliferation of CD34- committed erythroid progenitors in spleen, consistent with the known function of CD47 to limit turnover of aging erythrocytes. Conversely, loss of thrombospondin-1 delays turnover, which suppresses erythropoiesis in thbs1-/- spleens relative to the basal level in wild type mice. Overall design: CD8+ T Cells were Isolated CD8a+ T Cell Isolation Kit mouseCD8a+ T Cell Isolation Kit (Order no. 130-104-075) from Miltenyi biotec according to manufactures instructions. The freshly isolated CD8+ T cells were washed with filtered buffer solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 Mm EDTA. The clumps of isolated cells were removed using Pre-Separation Filters (30 µm) and subjected to negative selection of CD8+ T cells. The cells were immediately placed at -80 °C until the RNA was extracted.