The transcriptomic landscape of normal and ineffective erythropoiesis at single-cell resolution.

Document Type

Article

Publication Date

6-23-2023

Publication Title

Blood Adv

Keywords

washington; isb

Abstract

The anemias of myelodysplastic syndrome (MDS) and Diamond Blackfan anemia (DBA) are generally macrocytic, always reflect ineffective erythropoiesis, yet result from diverse genetic mutations. To delineate shared mechanisms that lead to cell death, we studied the fate of single erythroid marrow cells from individuals with DBA and MDS-5q. We defined an unhealthy (vs. healthy) differentiation trajectory using transcriptional pseudotime and cell surface proteins. The pseudotime trajectories diverge immediately after cells upregulate transferrin receptor (CD71), import iron, and initiate heme synthesis, although cell death occurs much later. Cells destined to die highly express heme responsive genes, including ribosomal protein and globin genes, while surviving cells downregulate heme synthesis and upregulate DNA damage response, hypoxia and HIF1 pathways. Surprisingly, 24±12% of cells from control subjects follow the unhealthy trajectory, implying that heme might serve as a rheostat directing cells to live or die. When heme synthesis was inhibited with succinylacetone, more DBA cells followed the healthy trajectory and survived. We also noted high numbers of messages with retained introns that increased as erythroid cells matured, confirmed the rapid cycling of CFU-E, and demonstrated that cell cycle timing is an invariant property of differentiation stage. Including unspliced RNA in pseudotime determinations allowed us to reliably align independent datasets and accurately query stage-specific transcriptomic changes. MDS-5q (unlike DBA) results from somatic mutation, so many normal (unmutated) erythroid cells persist. By independently tracking erythroid differentiation of cells with and without chromosome 5q deletions, we gained insight into why 5q+ cells cannot expand to prevent anemia.

Department

Institute for Systems Biology

Department

Hematology

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