Use of ER-MP12 as a positive marker for the isolation of murine long-term in vitro repopulating stem cells.

Monoclonal antibody ER-MP12 defines an antigen (Ag) on murine hematopoietic stem cells that is differentially expressed by the various subsets in the hematopoietic stem cell compartment. To test whether ER-MP12 could be an asset for further subfractionation of these subsets, we physically sorted our previously defined low-density ER-MP20- (i.e., Ly-6C-) Rhodamine-123dull (Rh123dull), and wheat germ agglutinindim (WGAdim) stem cell populations on the basis of ER-MP12 Ag expression. 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 sorting on the basis of ER-MP12 improves the separation of primitive and more mature stem cell subsets in the Rh123dull but not in the WGAdim subpopulation. However, the combination of sorting cells on the basis of an intermediate ER-MP12 expression and a low WGA affinity (ER-MP12mediumWGAdim) allows an 840-fold enrichment for in vitro long-term repopulating cells (day-28 CAFC) when compared with unseparated bone marrow. 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, suggesting that the level of Ag expression is not dependent on cell-cycle status. Together, the combination of ER-MP12 and WGA offers the advantage of a positive selection strategy for hematopoietic stem cells, allowing different stem cell subsets to be distinguished on the basis of their primitiveness. Since no mature bone marrow cells are found within the WGAdimER-MP12medium subpopulation, the combination of ER-MP12 and WGA enables hematopoietic stem cells to be highly enriched and thus makes the use of a cocktail of lineage-specific antibodies redundant.