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.
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.
Rebel
V I
VI
Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada.
Dragowska
W
W
Eaves
C J
CJ
Humphries
R K
RK
Lansdorp
P M
PM
D000818:Animals; D000950:Antigens, Ly / Q000032:analysis*; D001854:Bone Marrow Cells; D002455:Cell Division; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004921:Erythropoietin / Q000494:pharmacology*; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology*; D015850:Interleukin-6 / Q000494:pharmacology*; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D008810:Mice, Inbred C57BL; D019089:Stem Cell Factor; D014909:Wheat Germ Agglutinins / Q000378:metabolism*
D000950:Antigens, Ly; D016895:Culture Media, Serum-Free; D016298:Hematopoietic Cell Growth Factors; D015850:Interleukin-6; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D019089:Stem Cell Factor; D014909:Wheat Germ Agglutinins; D004921:Erythropoietin