Phenotypic analysis of murine long-term hemopoietic reconstituting cells quantitated competitively in vivo and comparison with more advanced colony-forming progeny.

Abstract

Early hemopoietic precursors have been extensively studied using short-term assays based on colony formation or in vivo reconstitution that do not run beyond a few weeks. However, little information is available on the phenotype of the stem cells that are detectable in 6-12-mo transplantation assays, and their relationship to cells detected in short-term assays is not known. In this study, we investigated the phenotype and separability by cell sorting of a spectrum of hemopoietic precursor cells in normal adult mouse marrow, including cells quantitated in a 1 yr competitive transplantation assay in vivo as well as in short-term colony assays in vitro and in vivo. Two principal findings emerged. The first was that cells detected in a variety of short-term assays--CFU-S12 (spleen colony-forming cells), CFCmulti (multilineage colony-forming cells), pre-CFCmulti (precursors of CFCmulti), CFC-E/Mg (erythroid/megakaryocyte CFC) and CFC-G/M (granulocyte/macrophage CFC)--were phenotypically similar and could not be separated from one another using a panel of markers useful in segregating them from more differentiated cells, including buoyant density, sedimentation velocity, adhesiveness to plastic, light scatter, high rhodamine-123 retention, and expression of surface wheat-germ agglutinin (WGA)-binding carbohydrate, H-2K, CD45, AA4.1, heat stable antigen (HSA), CD71, and Ly6A/Sca-1 antigens. Long-term reconstituting (LTR) cells quantitated in vivo differed little from the other precursors in expression of many of the above markers. However, they differed somewhat in lower sedimentation velocity and lower expression of WGA-binding surface carbohydrate, and most strikingly in their conditional adhesiveness to plastic, very low retention of Rh123 and high level expression of Ly6A/Sca-1, to a degree that would permit the quantitative separation of the two precursor classes from each other. The results provide a comprehensive characterization of LTR cells measured to 12 mo in vivo and a direct and quantitative analysis of their separation from cells detected in colony assays.