Amplification of Sca-1+ Lin- WGA+ cells in serum-free cultures containing Steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential

V. I. Rebel, W. Dragowska, C. J. Eaves, R. K. Humphries, P. M. Lansdorp

Research output: Contribution to journalArticle

102 Scopus citations

Abstract

Normal 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 repopulating 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.

Original languageEnglish (US)
Pages (from-to)128-136
Number of pages9
JournalBlood
Volume83
Issue number1
Publication statusPublished - Jan 1 1994

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ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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