Evi1 Is a Stem Cell-Specific Regulator of Self-Renewal Capacity In the Definitive Hematopoietic System

Abstract 838

Self-renewal is a defining property of stem cells. Although a number of molecules have been implicated in the regulation of hematopoietic stem cell (HSC) self-renewal, loss of these genes is accompanied with other hematological abnormalities. Thus, it is unclear what will happen with a specific loss of self-renewal capacity of HSCs. Evi1 is an oncogenic transcription factor in myeloid malignancies. Evi1 expression is limited to hematopoietic stem/progenitor fraction, and Evi1 is essential for the maintenance of HSCs, but is dispensable for blood cell lineage commitment. Thus, we hypothesized that Evi1 expression could distinguish hematopoietic stem and progenitor cells, and reduction of Evi1 gene dosage might cause a specific loss of self-renewal activity. First, to elucidate Evi1 expression within the hematopoietic system, we have generated Evi1-IRES-green fluorescent protein (GFP) knock-in mice, in which GFP was expressed under the endogenous transcriptional regulatory elements of Evi1 gene. We found that Evi1 was predominantly expressed in the hematopoietic stem/progenitor fraction (Lin- Sca-1+ c-kit+ (LSK)), but its expression was rapidly extinguished during early stages of lineage commitment. Among the LSK compartment, Evi1 was expressed at the highest level in long-term HSCs (LT-HSCs; Flk2- CD34-, CD48- CD150+, or SP-tip fractions in LSK cells). Next, we hypothesized that Evi1 would have the potential to mark LT-HSCs effectively. To test this, we compared GFP+ and GFP- cells in the LSK fraction, and revealed that GFP+ LSK cells were more immature and quiescent with a higher colony-forming capacity than GFP- LSK cells. In addition, in vivo long-term multilineage repopulating cells were exclusively enriched in the GFP+ LSK fraction. In the embryo, Evi1 was highly expressed in the hematopoietic stem/progenitor fraction; that is, CD34+ c-kit+ cells in embryonic day 10.5 (E10.5) aorta-gonad-mesonephros, CD34+ c-kit+ CD48- cells in E12.5 placenta, and Mac-1+ Sca-1+ Lin- (MSL) CD48- cells in E14.5 fetal liver (FL). In vivo competitive repopulation assay showed that, in the MSL fraction of FL, GFP+ MSL cells exclusively had a long-term multilineage repopulating capacity. These results implied that Evi1 plays a more specific role in HSCs than in other hematopoietic cells. To clarify this, we analyzed heterozygous Evi1 knockout mice (Evi1 +/− mice), as it seems difficult to elucidate the function of a small population of HSCs in Evi1 conditional knockout mice due to the leaky expression of Cre recombinase. We have previously showed that haploinsufficiency of Evi1 leads to decreased numbers of LSK and CD34- LSK cells, and impaired long-term repopulating activity. Here we demonstrated the number of each fraction in Evi1 +/− LSK cells was reduced in proportion to their expression level of Evi1. But, there were no significant differences in the numbers of lymphoid and myeloid progenitors between Evi1 +/+ and Evi1 +/− mice. Evi1 +/− CD34+ LSK cells had an equivalent in vitro colony-forming capacity and day 11 colony-forming unit-spleen activity to Evi1 +/+ CD34+ LSK cells. However, in vivo short-term repopulation assay using CD34+ LSK cells showed that the percentage of donor-derived cells from Evi1 +/− mice was significantly declined at 4 weeks after transplantation. Moreover, Evi1 +/− CD34- LSK cells had a pronouncedly impaired in vivo repopulating capacity. These data suggested that the differentiation capacity of Evi1 +/− HSCs was maintained, but their self-renewal capacity was specifically reduced. Although flow cytometric analysis of cell-cycle status and apoptosis showed no differences in CD34- LSK cells between Evi1+/+ and Evi1 +/− mice, the G0 fraction of Evi1 +/− CD34+ LSK cells was significantly reduced, indicating that these cells might proliferate more rapidly to compensate for the impaired self-renewal capacity of HSCs. In conclusion, we showed that Evi1 is predominantly expressed in HSCs and its expression can mark long-term repopulating HSCs in the fetal and adult hematopoietic system. Moreover, functional loss caused by haploinsufficiency of Evi1 is limited to a defect of self-renewal capacity of HSCs, and the increased cell-cycle progression of CD34+ LSK cells in Evi1 +/− mice seems to be the consequence of the impaired self-renewal capacity. Our data may help to understand the unrevealed effects of loss of self-renewal activity of HSCs and compensative mechanism of their defects.