Prospective isolation of radiation induced erythroid stress progenitors reveals unique transcriptomic and epigenetic signatures enabling increased erythroid output

Abstract

Massive expansion of erythroid progenitor cells is essential for surviving anemic stress. Research towards understanding this critical process, referred to as stress-erythropoiesis, has been hampered due to lack of specific marker-combinations enabling analysis of the distinct stress-progenitor cells capable of providing radioprotection and enhanced red blood cell production. Here we present a method for precise identification and in vivo validation of progenitor cells contributing to both steady-state and stress-erythropoiesis, enabling for the first time in-depth molecular characterization of these cells. Differential expression of surface markers CD150, CD9 and Sca1 defines a hierarchy of splenic stress-progenitors during irradiation-induced stress recovery in mice, and provides high-purity isolation of the functional stress-BFU-Es with a 100-fold improved enrichment compared to state-of-the-art. By transplanting purified stress-progenitors expressing the fluorescent protein Kusabira Orange, we determined their kinetics in vivo and demonstrated that CD150+CD9+Sca1- stress-BFU-Es provide a massive but transient radioprotective erythroid wave, followed by multi-lineage reconstitution from CD150+CD9+Sca1+ multi-potent stem/progenitor cells. Whole genome transcriptional analysis revealed that stress-BFU-Es express gene signatures more associated with erythropoiesis and proliferation compared to steady-state BFU-Es, and are BMP-responsive. Evaluation of chromatin accessibility through ATAC sequencing reveals enhanced and differential accessibility to binding sites of the chromatin-looping transcription factor CTCF in stress-BFU-Es compared to steady-state BFU-Es. Our findings offer molecular insight to the unique capacity of stress-BFU-Es to rapidly form erythroid cells in response to anemia and constitute an important step towards identifying novel erythropoiesis stimulating agents.

Enhancing mitochondrial function in vivo rescues MDS-like anemia induced by pRb deficiency

“…Steady-state erythropoiesis occurs at a constant rate in the BM, while anemia induces an extramedullary stress response in the spleen to rapidly produce large numbers of new erythrocytes [16,17]. The spleen weight of pRbdeficient mice was increased 2-fold compared with wild type ( Figure 1H,I).…”

Finding erythroid stress progenitors: cell surface markers revealed

“…The findings by Singbrant et al 3 provide the field with an important tool to isolate stress-BFU-E in mice. This allows researchers to investigate stress erythropoiesis in a variety of mouse model systems that mimic disease con-ditions with acute anemia.…”

“…Interestingly, the authors also found CD150 + CD9 + BFU-E progenitors in steady-state bone marrow but not the spleen, demonstrating that these markers are useful to identify BFU-E progenitors in both steady-state and stress conditions. Previous studies indicated that steady BFU-E migrate from the bone marrow to the spleen during stress erythropoiesis to become stress BFU-E, 12 while more recent findings suggest that endogenous splenic stress BFU-E during stress erythropoiesis are distinctive from steady BFU-E. 7 The CD150 + CD9 + BFU-E progenitors Singbrant et al 3 discovered in both steady state and stress conditions would be helpful to resolve this contradiction in future studies.…”

“…2 However, finding the stress erythroid progenitor cells for detailed mechanistic investigation remains a demanding task. In this issue of Haematologica, Singbrant et al 3 discovered cell surface markers that could aid the identification of these progenitors.…”

“…To identify and enrich the stress erythroid progenitors, Singbrant et al 3 used an irradiation-induced anemia mouse model to mimic stress erythropoiesis. The authors previously showed that fetal erythroid BFU-E can be identified with high purity as lineage -cKit + and CD71/CD24a low Sca1 -CD34in mouse fetal liver.…”