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, these genes are widely expressed in the hematopoietic system, and their mutations in genetic models are exclusively accompanied with other hematological abnormalities. Thus, a bona fide stem-cell specific regulator of their function has not been identified and the functional identification of HSCs based on their ability to self-renew remains difficult. Ecotropic viral integration site 1 (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. 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.

First, I found 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 the long-term HSC (LT-HSC) fraction (Flk-2- CD34- LSK or CD48- CD150+ LSK). Next, I hypothesized that Evi1 would have the potential to mark long-term repopulating HSCs effectively. To test this issue, I compared GFP+ and GFP- cells in the LSK fraction, and revealed that LSK GFP+ cells were more immature and quiescent with a higher colony-forming capacity than LSK GFP- cells. In addition, in vivo long-term multilineage repopulating cells were exclusively enriched in the LSK GFP+ fraction. Furthermore, even within the highly subfractionated LT-HSC fraction (Flk-2- CD34- LSK or CD48- CD150+ LSK), long-term repopulating HSCs predominantly reside in the GFP+ fraction, suggesting Evi1 expression can further augment the conventional HSC purification strategy.

In the embryo, Evi1 was highly expressed in the hematopoietic stem/progenitor fraction; that is, CD45+ 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. In vivo competitive repopulation assay showed that, in the CD34+ c-kit+ CD48- fraction of E12.5 placenta or the MSL fraction of E14.5 fetal liver, GFP+ cells exclusively had a long-term multilineage repopulating capacity, suggesting a specific relationship between Evi1 expression and HSC self-renewal capacity throughout ontogeny.

These results implied Evi1 plays a more specific role in LT-HSCs than in other hematopoietic cells. To clarify this, I analyzed heterozygous Evi1 knockout mice (Evi1+/- mice). Here I demonstrated LT-HSCs (Flk-2- CD34- LSK or CD48- CD150+ LSK cells) from Evi1+/- mice exhibited a marked reduction in frequency as compared to Evi1+/+ controls. However, 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, Evi1+/- CD34- LSK cells had a pronouncedly impaired in vivo repopulating capacity. These data suggested the differentiation capacity of Evi1+/- HSCs was maintained, but their self-renewal capacity was specifically reduced. In contrast, Evi1 overexpression in HSPCs suppresses differentiation and boosts self-renewal activity in vitro. Re-introduction of Evi1, but not Mds1-Evi1, rescues the HSC defects caused by Evi1 heterozygosity.

Thus, in addition to documenting a specific relationship between Evi1 expression and HSC self-renewal activity, these findings highlight the utility of Evi1-IRES-GFP reporter mice for the identification and sorting of functional HSCs.

本研究は、造血系における重要な転写因子であるEvi1の発現・機能を明らかにするために、新規に作成されたEvi1-GFPレポーターマウスを用いてin vivoにおけるEvi1の発現の詳細な解析を試み、さらにEvi1ノックアウトマウスおよびEvi1過剰発現系を用いてEvi1の機能解析を行ったものであり、下記の結果を得ている。

1.Evi1-GFPレポーターマウス由来骨髄のフローサイトメトリー解析により、成体においてEvi1の発現が長期造血幹細胞に限局していることを明らかにされた。さらにLSK分画内でGFP+とGFP-の細胞に分離して機能解析を行い、LSK GFP+細胞はより静止期にあり、コロニー形成能が高く、未分化な細胞集団であることが示された。

2.LSK分画内でGFP+とGFP-の細胞に分離して骨髄再構築実験を行うことにより、LSK分画内において、Evi1の発現が骨髄再構築可能な細胞をマークすることが示された。さらに長期造血幹細胞分画内(SLAM-LSKおよびFlk2- CD34- LSK)において同様の実験を行い、これらの分画内においてもEvi1を発現する分画内に骨髄再構築能が限局していることが明らかにされた。

3.AGM領域、胎盤、胎児肝の各段階においてEvi1の発現が造血幹前駆細胞領域に限局していることが示された。さらにコロニー形成実験と骨髄再構築実験により機能的にも胎生期の造血幹細胞がEvi1を発現する細胞に限局していることが明らかにされた。

4.Evi1ヘテロ欠損マウスの解析により、Evi1ヘテロ欠損は分化細胞・造血前駆細胞数には影響がないが、長期造血幹細胞数の著明な減少を認めることが示された。また相互的移植実験により、このEvi1ヘテロ欠損の影響が細胞自律的であることを明らかにされた。

5.Evi1ヘテロ欠損の短期造血幹細胞と長期造血幹細胞を分けてソートして機能を比較したところ、Evi1ヘテロ欠損の短期造血幹細胞はin vitroにおけるコロニー形成能、in vivoにおけるCFU-spleen形成能ともに保たれていたのに対し、長期造血幹細胞はin vitroにおける増殖能、in vivoにおける骨髄再構築能ともに著明な低下を認めることが示された。

6.Evi1ヘテロ欠損の長期造血幹細胞では野生型と比較して細胞周期、アポトーシス、活性酸素レベルに変化を認めないことが示された。

7.Evi1を造血幹・前駆細胞に過剰発現させることにより、Evi1が造血幹・前駆細胞の分化を抑制し、自己複製を亢進させることが示された。さらにMds1-Evi1でなくEvi1の遺伝子導入により、Evi1ヘテロ欠損の造血幹細胞の機能が回復することが示された。

以上、本論文は、Evi1-GFPレポーターマウスおよびEvi1ヘテロマウスの解析から、造血発生の全ての過程においてEvi1の発現と造血幹細胞機能に密接な関係があること、機能期にはEvi1は長期造血幹細胞の自己複製の維持に重要であること、Evi1の発現を既存の幹細胞マーカーと組みわせることにより造血幹細胞の純化能を高めることができること、を明らかにした。本研究は、既知の遺伝子よりEvi1が発現・機能の両面において造血幹細胞に特異的であることを示すものであり、造血幹細胞の幹細胞性維持の分子メカニズムの解明に重要な貢献をなすと考えられ、学位の授与に値するものと考えられる。