Isolation and characterization of primitive hematopoietic progenitors of murine fetal liver.
We have isolated hematopoietic progenitors from mouse fetal liver using a sequential protocol of density gradient centrifugation, panning, and cell sorting. Isolated AA4.1+Ly-6A/E+CD43++ cells with density ranging from 1.0631 to 1.0770 g/cm3 were 480- to 600-fold enriched for multipotential progenitors relative to unfractionated cells and showed 40 to 60% colony-forming efficiency. We then examined the effects of various cytokines on the colony formation from enriched fetal liver cells. Steel factor (SF), interleukin-3 (IL-3), IL-4, IL-6, IL-11, and granulocyte colony-stimulating factor (G-CSF) as single agents supported formation of significant numbers of colonies, but flt3/flk-2 ligand (FL) and IL-12 did not. When the cytokines were combined, FL, SF, IL-3, and IL-4 each synergized individually with IL-6, IL-11, IL-12, or G-CSF to support formation of various types of colonies. Next we analyzed the growth factor requirements for proliferation and differentiation of lymphohematopoietic progenitors by using the two-step methylcellulose culture assay we established recently. None of the early-acting factors were effective as a single agent, but combinations of SF or FL with IL-6, IL-11, or G-CSF were effective in supporting B cell potential of the primary colonies. Of these, the combination of FL plus IL-11 appeared to be the most effective.
Fujimoto
K
K
Department of Medicine, Medical University of South Carolina, Charleston 29401-5799, USA.
Lyman
S D
SD
Hiryama
F
F
Ogawa
M
M
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003114:Colony-Forming Units Assay; D003433:Crosses, Genetic; D016207:Cytokines / Q000494:pharmacology*; D005260:Female; D005434:Flow Cytometry; D006411:Hematopoiesis, Extramedullary; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D007163:Immunosorbent Techniques; D008099:Liver / Q000166:cytology / Q000196:embryology*; D008297:Male; D008565:Membrane Proteins / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D011994:Recombinant Proteins / Q000494:pharmacology
D016207:Cytokines; D016298:Hematopoietic Cell Growth Factors; D008565:Membrane Proteins; D011994:Recombinant Proteins; C084881:flt3 ligand protein