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IO impairs normal HSPCs and survival in MDS mice
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Figure 1. RUNX1S291fs induced mice model can phenotypically recapitulate human myelodysplastic syndrome. (A) Experimental scheme of our model mouse using RUNX1S291fs mutant. (B) Chimerism of GFP+ cells post transplantation. (C) Successful expression of RUNX1S291fs protein detected by western blotting. (D) White blood cell (WBC) count. (E) Hemoglobin (HG) count. (F) Platelet (PLT) count. (G) Mean corpuscular volume (MCV). (H) The morphological abnormality observed in the bone marrow and spleen size. (I) Pathological changes in the femur, liver and spleen. *P<0.05, **P<0.01, ***P<0.001.
of white pulp and narrow red pulp, whereas such patho- logical changes were not found in Empty control mice (Figure 1I). Collectively, our RUNX1S291fs-induced mice model showed neutropenia, anemia, thrombocytopenia and multilineage dysplasia, together with less than 20% blasts, matching the criteria of MDS in the Bethesda pro- posal and phenotypically recapitulating human MDS.
Next, we examined whether iron overload model can be established in RUNX1S291fs induced MDS mice. We and others previously reported that iron overload can cause liver and spleen enlargement.12,13 There was a clear increase in liver and spleen weight in both control mice and in MDS mice administered iron dextran treatment, implying that iron can deposit in the liver and spleen in RUNX1S291fs-induced MDS mice (Figures 1H, and 2A and B). We further performed Perl’s iron staining to verify iron deposition in mice tissues and organs. Significant iron deposition could be observed in the liver, spleen and BM in RX291/FE group compared to RX291/NS mice (Figure 2C), supporting iron overload in RX291/FE mice. In addi- tion, our data showed the level of ferritin was almost undetectable in Empty/NS, while it was significantly high- er in RX291/NS mice (Figure 2D), which can be clearly explained by ineffective erythropoiesis in MDS. However, the ferritin in Empty/FE and RX291/FE group was compa- rable, and was more significant than in Empty/NS and RX291/NS mice, respectively (Figure 2D), indicating that iron overload can also be established in MDS mice. Taken
together, administration of iron dextran by intraperitoneal injection confers iron deposition in organs, liver and spleen enlargement, and can be used to facilitate the estab- lishment of an iron overload model in RUNX1S291fs- induced MDS mice.
Iron overload impairs the frequency of normal HSPCs and affects erythroid maturation in MDS mice
To understand how iron overload influences the hematopoietic system, we first evaluated WBC, PLT and HG counts. However, our experiment showed no statisti- cal difference but a downward trend in the PB between the group treated with iron and that treated with normal saline in both control mice and MDS mice (Figure 1D-F). We further analyzed the frequency of HSCs (Lin–c-Kit+Sca1+/LSK) and HPCs (Lin–c-Kit+Sca1–/LK) in BM (Figure 3A) and we found that there were significantly more normal (GFP–) HSCs in MDS mice than in control mice, which may due to extremely active myeloid hyper- plasia in MDS. In addition, iron overload can significantly decrease the number of GFP– HSCs in both control and MDS mice. However, we did not observe any significance about mutant (GFP+) HSCs in RX291/NS and RX291/FE mice (Figure 3B). A similar result can be seen regarding the change in normal and mutant HPCs in these groups (Figure 3C). Next, we analyzed erythroid differentiation through CD71 and Ter119 gating (Figure 3D). We referred to proerythroblasts, basophilic erythroblasts, polychro-
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