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S. Singbrant et al.
hierarchy of functionally diverse splenic stress-progenitors during irradiation-induced recovery, providing 100-fold improved enrichment of stress-BFU-E compared to the current state-of-the-art.
Analysis of megakaryocytic-erythroid differentiation kinetics has been hampered by the lack of markers expressed by mature erythrocytes and platelets. By trans- planting sorted stress-progenitor populations from trans- genic mice expressing Kusabira Orange in all cells includ- ing erythrocytes and platelets,20 we determined their kinetics and full differentiation potential in vivo. Although sBFU-E gave rise to megakaryocytic colonies in vitro and CD41+ cells in vivo, only sMPP produced platelets in vivo, demonstrating the importance of in vivo experiments and the ability to trace all mature cell types for defining pro- genitor potential. In agreement with previous studies,33 this shows that CD41 is not specific for the megakary- ocytic lineage.
We further demonstrate that splenic sBFU-E provide a massive but transient erythroid wave, followed by multi- lineage reconstitution from sMPP. Harandi et al.14 previously suggested that cKit+CD71–Ter119– cells were erythroid restricted.14 However, using the same starting population of cells and in vivo reconstitution we now demonstrate that these cells contain both multi-potent progenitors (CD150+CD9+Sca1+) and more erythroid restricted BFU-E (CD150+CD9+Sca1–) and CFU-E (CD150+CD9–). The same group also proposes that stress-BFU-E are Sca1+ , whereas we show that Sca1+ stress-progenitors are multi-potent with the capacity to give rise to more restricted Sca- sBFU- E. Sca1 is known to mark multi-potent HSC, and that Sca1 as a single surface marker can differentiate between multi- lineage and BFU-E potential is well in line with cellular hier- archy mapping in steady-state hematopoiesis.34 Interestingly, CD9 has recently been reported to be expressed in murine HSC with MegE differentiation bias.34 While Pronk et al.9 have identified that CD150 marks “PreCFU-E” colony-forming potential in the BM during steady-state, we for the first time describe a FACS method for identifying progenitors with BFU-E potential, which resides in the cKit+CD71low/CD24alowCD150+CD9+ popula- tion. This demonstrates that these markers can be used to enrich for both steady-state BFU-E and stress-BFU-E.
BMP-regulation of stress-erythropoiesis has previously been described by Paulson and colleagues, whereas we have shown that steady-state erythropoiesis remains unaf- fected by disruption of canonical BMP-signaling. Investigation of the functional importance of BMP-signaling in our system revealed that transplantation of BMP-defi- cient BM resulted in an impaired stress-response with sub- stantially smaller spleens and decreased potential to form stress-progenitors, despite 50% of the transplanted BM cells being wild-type. In addition, genes upregulated in sBFU-E compared to their steady-state counterpart were associated with gene sets activated downstream of the BMP signaling pathway.33 Within the stress-erythroid progenitor popula- tions, sCFU-E expressed the same set of BMP-responsive
genes to a higher degree than sBFU-E and sMPP. Taken together, these results model stress-erythroid progenitors in general and sCFU-E in particular as BMP-responsive cells. sCFU-E also displayed the highest expression of a set of genes that is down-regulated in response to inactivation of Cbfa2t3,26 a transcriptional co-repressor that promotes degradation of hypoxia regulating protein Hif1a,35 regulates Gata1-target genes critical for erythroid differentiation,36 and maintains an erythroid-specific genetic program in pro- genitors primed for rapid activation when terminal ery- throid differentiation is induced.37 Stress-erythropoiesis is severely impaired in mice lacking Cbfa2t3.26 Collectively, this makes Cbfa2t3 an interesting target to study further in stress-erythropoiesis regulation.
Analysis of active regulatory DNA elements across the genome using ATAC-seq revealed that sBFU-E displayed a stronger prevalence for the binding motif of chromatin-loop- ing transcription factor CTCF compared to steady-state BFU-E which are less efficient in producing erythrocytes. CTCF has previously been implicated in marking active pro- moters in primary human erythroid cells,30 and LDB1-CTCF enhancer looping underlies activation of a substantial frac- tion of erythroid genes.31 In accordance, ectopic expression of CTCF in K562 cells promotes erythroid differentiation whereas CTCF knock-down significantly inhibits differenti- ation into the erythroid lineage.32 Taken together, chromatin accessibility of CTCF is the most striking epigenetic differ- ence between stress- and steady-state BFU-E, suggesting a role for CTCF-dependent mechanisms in stress-BFU-Es.
In conclusion, combining novel surface markers with the KuO tracing mouse, we have for the first time defined a cel- lular hierarchy of stress-progenitors, separating sMPP from sBFU-E and more mature sCFU-E based on their kinetics and differentiation potential in vivo. We further demonstrate that sBFU-E express gene signatures more associated with erythropoiesis and proliferation compared to steady-state BFU-E, and have enhanced and differential accessibility to CTCF binding sites. Our findings open up the field for fur- ther mechanistic and functional studies of how stress-ery- thropoiesis is regulated, and how sBFU-E contribute during recovery of erythroid disorders. Since the mechanisms reg- ulating stress-erythropoiesis may be targeted for treatment of anemia, this is an important step towards identifying and studying novel erythropoiesis stimulating agents.
Acknowledgments
The authors would like to thank Dr. Hiromitsu Nakauchi for kindly providing the Kusabira Orange mice.
Funding
This research was supported by the Ragnar Söderberg Foundation (fellowship JF), the Swedish Cancer Society (fellow- ship SS), the Swedish Research Council and the Swedish Foundation for Strategic Research (JF), the Swedish Foundation for Medical Research, the Crafoordska Foundation, the Åke Wiberg Foundation, the Clas Groschinsky's Memory Foundation and the Harald & Greta Jeansson Foundation (SS).
References
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2. Peslak SA, Wenger J, Bemis JC, et al. EPO- mediated expansion of late-stage erythroid progenitors in the bone marrow initiates recovery from sublethal radiation stress. Blood. 2012;120(12):2501-2511.
3. Harandi OF, Hedge S, Wu DC, McKeone D, Paulson RF. Murine erythroid short-term
radioprotection requires a BMP4-dependent, self-renewing population of stress erythroid progenitors. J Clin Invest. 2010; 120(12):4507-4519.
4. Perry JM, Harandi OF, Paulson RF. BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid pro-
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