USE OF ER-MP12 AS A POSITIVE MARKER FOR THE ISOLATION OF MARINE LONG-TERM IN-VITRO REPOPULATING STEM-CELLS

Monoclonal antibody ER-MP12 defines an antigen (Ag) on murine hematopo ietic stem cells that is differentially expressed by the various subse ts in the hematopoietic stem cell compartment. To test whether ER-MP12 could be an asset for further subfractionation of these subsets, we p hysically sorted our previously defined low-density ER-MP20(-) (i.e., Ly-6C(-)) Rhodamine-123(dull) (Rh123(dull)), and wheat germ agglutinin (dim) (WGA(dim)) Stem cell populations on the basis of ER-MP12 Ag expr ession. In addition, we determined the distribution of the ER-MP12 Ag on bone marrow 6 days after 5-FU treatment. Long-term and transiently repopulating stem cell subsets were both identified in vitro using the cobblestone area-forming cell (CAFC) assay. The data show that sortin g on the basis of ER-MP12 improves the separation of primitive and mor e mature stem cell subsets in the Rh123(dull) but not in the WGA(dim) subpopulation However, the combination of sorting cells on the basis o f an intermediate ER-MP12 expression and a low WGA affinity (ER-MP12(m edium) WGA(dim)) allows an 840-fold enrichment for in vitro long-term repopulating cells (day-28 CAFC) when compared with unseparated bone m arrow. The distribution of the ER-MP12 Ag on 5-FU-treated bone marrow stem cells was similar to that in normal bone marrow stem cells, sugge sting that the level of Ag expression is not dependent on cell-cycle s tatus. Together, the combination of ER-MP12 and WGA offers the advanta ge of a positive selection strategy for hematopoietic stem cells, allo wing different stem cell subsets to be distinguished on the basis of t heir primitiveness. Since no mature bone marrow cells are found within the WGA(dim)ER-MP12(medium) subpopulation, the combination of ER-MP12 and WGA enables hematopoietic stem cells to be highly enriched and th us makes the use of a cocktail of lineage-specific ic antibodies redun dant.