IDENTIFICATION OF HEMATOPOIETIC STEM-CELL SUBSETS ON THE BASIS OF THEIR PRIMITIVENESS USING ANTIBODY ER-MP12

Monoclonal antibody ER-MP12 defines a novel antigen on murine hematopo ietic stem cells. The antigen is differentially expressed by different subsets in the hematopoietic stem cell compartment and enables a phys ical separation of primitive long-term repopulating stem cells from mo re mature multilineage progenitors. When used in two-color immunofluor escence with ER-MP20 (anti-Ly-6C), six subpopulations of bone marrow ( BM) cells could be identified. These subsets were isolated using magne tic and fluorescence-activated cell sorting, phenotypically analyzed, and tested in vitro for cobblestone area-forming cells (CAFC) and colo ny-forming units in culture (CFU-C; M/G/E/Meg/Mast). In addition, they were tested in vivo for day-12 spleen colony-forming units (CFU-S-12) , and for cells with long-term repopulating ability using a recently d eveloped alpha-thalassemic chimeric mouse model. Cells with long-term repopulating ability (LTRA) and day-12 spleen colony-forming ability a ppeared to be exclusively present in the two subpopulations that expre ssed the ER-MP12 cell surface antigen at either an intermediate or hig h level, but lacked the expression of Ly-6C. The ER-MP12(med)20(-) sub population (comprising 30% of the BM cells, including all lymphocytes) contained 90% to 95% of the LTRA cells and immature day-28 CAFC (CAFC -28), 75% of the CFU-S-12, and very low numbers of CFU-C. In contrast, the ER-MP12(hl)20(-) population (comprising 1% to 2% of the BM cells, containing no mature cells) included 80% of the early and less primit ive CAFC (CAFC-5), 25% of the CFU-S-12, and only 1001'o of the LTRA ce lls and immature CAFC-28. The ER-MP12(hl) cells, irrespective of the E R-MP20 antigen expression, included 80% to 90% of the CFU-C (day 4 thr ough day 14), of which 70% were ER-MP20(-) and 10% to 20% ER-MP20(med/ hl). In addition, erythroblasts, granulocytes, lymphocytes, and monocy tes could almost be fully separated on the basis of ER-MP12 and ER-MP2 0 antigen expression. Functionally, the presence of ER-MP12 in a long- term BM culture did not affect hematopoiesis, as was measured in the C AFC assay. Our data demonstrate that the ER-MP12 antigen is intermedia tely expressed on the longterm repopulating hematopoietic stem cell. I ts level of expression increases on maturation towards CFU-C, to disap pear from mature hematopoietic cells, except from B and T lymphocytes. (C) 1995 by The American Society of Hematology.