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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
VI Rebel, W Dragowska, CJ Eaves, RK Humphries and PM Lansdorp
Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada.
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 > or = to 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.
Volume 83,
Issue 1,
pp. 128-136,
01/01/1994
Copyright © 1994 by The American Society of Hematology
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