Here we provide evidence that the Ikaros family of DNA binding factors is critical for the activity of hemopoietic stem cells (HSCs) in the mouse. Mice homozygous for an Ikaros null mutation display a >30-fold reduction in long-term repopulation units, whereas mice homozygous for an Ikaros dominant negative mutation have no measurable activity. The defect in HSC activity is also illustrated by the ability of wild-type marrow to repopulate unconditioned Ikaros mutants. A progressive reduction in multipotent CFU-S14 (colony-forming unit-spleen) progenitors and the earliest erythroid-restricted precursors (BFU-E [burst-forming unit-erythroid]) is also detected in the Ikaros mutant strains consistent with the reduction in HSCs. Nonetheless, the more mature clonogenic erythroid and myeloid precursors are less affected, indicating either the action of a compensatory mechanism to provide more progeny or a negative role of Ikaros at later stages of erythromyeloid differentiation. In Ikaros mutant mice, a decrease in expression of the tyrosine kinase receptors flk-2 and c-kit is observed in the lineage-depleted c-kit+Sca-1+ population that is normally enriched for HSCs and may in part contribute to the early hemopoietic phenotypes manifested in the absence of Ikaros.

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.

Although hematopoiesis is known to proceed from stem cells through a graded series of multipotent, oligopotent, and unipotent precursor cells, it has been difficult to resolve these cells physically one from another. There is, therefore, corresponding uncertainty about the exact distribution and timing of the expression of genes known to be important in hematopoietic differentiation. In earlier work, the generation of a set of amplified complementary DNAs (cDNAs) from single precursor cells was described, whose