Differential regulation by CD47 and thrombospondin-1 of extramedullary erythropoiesis in mouse spleen (scRNA-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: Single cell suspensions from WT, thbs1-/- and cd47-/- mice spleens were depleted of all mature hematopoietic cell lineages including erythroblasts and mature RBC by passing through CD8a (Ly-2) microbeads and CD8+ T cell untouched negative selection microbeads (Miltenyi Biotec). Single cell suspensions were incubated with the supplied antibody cocktail of the CD8+ T cell isolation kit for 15 min. on ice and then passed through the column as per manufacturer's instructions. After 3 washes, the flow through was centrifuged and incubated with the CD8a (Ly-2) microbeads and then passed through the magnetic column to obtain the lineage-depleted cell populations.