Abstract
AbstractNormal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties, Sca-1 expression (Sca-1+), lack of staining with a cocktail of mature hematopoietic lineage markers (Lin†), and binding of wheat germ agglutinin (WGA + ). This approach allowed the reproducible isolation of a very small subpopulation (0.037% ± 0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+ Lin+WGA+ cells resulted in ≥20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients, respectively. When Sca-1 + Lin† WGA+ cells were cultured in serum-free medium supplemented with Steel factor, interleukin-6 (IL-6), and erythropoietin (with or without IL-3), a large increase in total cell number, including cells with an Sca-1+ Lin†WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly, this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulat ing potential in vivo, as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1 + Lin†WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum- and stroma cell-free culture, providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1 + Lin† WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype. © 1994 by The American Society of Hematology.
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