In order to identify cell surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was applied to mouse bone marrow (BM) CD34 c-Kit+ sca-1+ lineage (CD34KSL) cells which is highly enriched for HSCs. Among the identified genes, mRNA expression of Endomucin, an endothelium-specific gene encoding a CD34-like sialomucin, appeared highly specific to CD34-KSL HSCs.

To further investigate the expression of Endomucin, we generated two rat anti-mouse Endomucin monoclonal antibodies that recognize different epitopes (AE2D4, AE7F2). Taking advantage of these and another monoclonal antibody, V7c7 (1999, Blood, 93; 1; 165), detailed expression analysis was performed. Endomucin expression was largely confined to lineage markers-negative (Lin) cells. Approximately 7 % of Lin cells were Endomucin-positive. Cells strongly expressing Endomucin represented 30% of c-kit+ sca-1+ cells. Gating out CD34+ cells from Lin Endomucin+ population resulted in high yield of KSL cells. High correlation between Lin Endomucin+CD34 cells and KSL cells was confirmed by in vivo bone marrow transplantation. When Lin cells were fractionated by their expression of CD34 and Endomucin, only Lin Endomucin+CD34 cells contributed to long-term repopulation (LTR), and as few as 100 cells were enough to obtain engraftment. Furthermore, the majority of CD34KSL cells were Endomucin+, and again, only CD34KSL-Endomucin+ cells had LTR activity. These data indicate two facts: 1) A single positive marker, Endomucin can substitute for c-kit+ sca-1+, 2) All LTR -HSCs express Endomucin.

We then analyzed the expression of Endomucin during embryonic development of the hematopoietic system. Definitive HSCs arise from the hemogenic endothelium lining the wall of the dorsal aorta in embryonic aorta-gonads-mesonephros (AGM) region, then seed to the fetal liver. E10.5 AGM CD45 cells were segregated into subpopulations by their expression of Endomucin and CD41, an early marker of embryonic hematopoiesis. In vitro coculture system with a stromal cell line, OP9, was applied to detect the ability of hematopoietic potential. Hematopoietic activity was exclusively found in the CD41+Endomucin+ population, that represents 24% of CD41+ cells. Taken together, these data indicate that Endomucin marks both embryonic and adult HSCs, providing a novel useful cell surface marker for definitive HSCs throughout development.

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