Identification of Hematopoietic Stem Cell Subsets on the Basis of Their Primitiveness Using Antibody ER-MP12

• Published in: Blood 1995-02, Vol.85 (4), p.952-962
• Main Authors: van der Loo, Johannes C.M., Slieker, Walentina A.T., Kieboom, Dorinde, Ploemacher, Rob E.
• Format: Article
• Language: English
• Subjects: alpha-Thalassemia - therapy; Animals; Antibodies, Monoclonal; Antigens, Surface - analysis; Biological and medical sciences; Bone Marrow Cells; Cell differentiation, maturation, development, hematopoiesis; Cell physiology; Colony-Forming Units Assay; Crosses, Genetic; Female; Flow Cytometry; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - immunology; Male; Mice; Mice, Inbred BALB C; Molecular and cellular biology; Rats - immunology; Spleen
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V85.4.952.bloodjournal854952

Monoclonal antibody ER-MP12 defines a novel antigen on murine hematopoietic stem cells. The antigen is differentially expressed by different subsets in the hematopoietic stem cell compartment and enables a physical separation of primitive long-term repopulating stem cells from more mature multilineage progenitors. When used in two-color immunofluorescence with ER-MP20 (anti-Ly-6C), six sub-populations of bone marrow (BM) cells could be identified. These subsets were isolated using magnetic and fluorescence-activated cell sorting, phenotypically analyzed, and tested in vitro for cobblestone area-forming cells (CAFC) and colony-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 developed α-thalassemic chimeric mouse model. Cells with long-term repopulating ability (LTRA) and day-12 spleen colony-forming ability appeared to be exclusively present in the two subpopulations that expressed the ER-MP12 cell surface antigen at either an intermediate or high level, but lacked the expression of Ly-6C. The ER-MP12med20- subpopulation (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-MP12hi20- population (comprising 1% to 2% of the BM cells, containing no mature cells) included 80% of the early and less primitive CAFC (CAFC-5), 25% of the CFU-S-12, and only 10% of the LTRA cells and immature CAFC-28. The ER-MP12hi cells, irrespective of the ER-MP20 antigen expression, included 80% to 90% of the CFU-C (day 4 through day 14), of which 70% were ER-MP20 and 10% to 20% ER-MP20med/hi. In addition, erythroblasts, granulocytes, lymphocytes, and monocytes could almost be fully separated on the basis of ER-MP12 and ER-MP20 antigen expression. Functionally, the presence of ER-MP12 in a long-term BM culture did not affect hematopoiesis, as was measured in the CAFC assay. Our data demonstrate that the ER-MP12 antigen is intermediately expressed on the long-term repopulating hematopoietic stem cell. Its level of expression increases on maturation towards CFU-C, to disappear from mature hematopoietic cells, except from B and Tlymphocytes.