Generation of hematopoietic colony-forming cells from embryonic stem cells: synergy between a soluble factor from NIH-3T3 cells and hematopoietic growth factors.
Murine embryonic stem cells are able to differentiate into embryoid bodies (EBs) in vitro in the absence of leukemia-inhibitory factor with the formation of different types of hematopoietic precursors within these EBs. With the aim of determining the in vitro requirements for the continued development of hematopoietic colony-forming cells (CFCs) and their progeny from embryonic stem-derived cells, cells from EBs disrupted after 9 days of formation in the absence of leukemia-inhibitor factor were cultured under different conditions. Low numbers of day-9 EB cells (5 x 10(5) or less) cultured in the presence of several growth factors (interleukin-3 [IL-3], IL-1, c-kit ligand, basic fibroblast growth factor, insulin growth factor-1, IL-6, granulocyte colony-stimulating factor, fetal liver kinase-2 ligand) develop few or no CFCs after 1 week of culture. When these cells are plated on irradiated NIH-3T3 with IL-3 or c-kit ligand or combinations containing these and other growth factors, they are able to generate CFCs for at least 3 weeks. These cultures were found to include granulocytic, monocytic, erythrocytic, and megakaryocytic cells. Transwell cultures in which NIH-3T3 cells were separated from the EB cells and cultures in which cells were replaced by NIH-3T3 conditioned medium showed that the interaction between EB-derived cells and NIH-3T3 is via a soluble factor(s). These studies show that maximal generation of hematopoietic CFCs from precursors present in day-9 EBs is stimulated by a combination of known hematopoietic growth factors and a soluble factor(s) produced by NIH-3T3 cells.
Bigas
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Pediatric Oncology Program, Fred Hutchinson Cancer Research Center 98109, USA.
Martin
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Bernstein
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D016475:3T3 Cells / Q000378:metabolism* / Q000528:radiation effects; D000818:Animals; D001685:Biological Factors / Q000378:metabolism / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology*; D004357:Drug Synergism; D004622:Embryo, Mammalian / Q000166:cytology; D006410:Hematopoiesis / Q000187:drug effects*; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D051379:Mice; D009924:Organ Culture Techniques; D009940:Organoids; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects*
D001685:Biological Factors; D017077:Culture Media, Conditioned; D016207:Cytokines; D016298:Hematopoietic Cell Growth Factors