title: The in vitro differentiation of mouse embryonic stem cells into neutrophils.
abstract: A reliable and effective in vitro differentiation system is described for the production of functional neutrophils from mouse embryonic stem (ES) cells. A three-step culture method was developed that enables abundant production and effective harvesting of mature neutrophils at high purity without sorting. Utilization of the OP9 stromal cell line, which does not produce macrophage colony stimulating factor (M-CSF) was found to enhance the number, percentage and duration of neutrophils produced. Based on a number of criteria, morphologically and functionally mature neutrophils can be produced using this method in approximately 16 days. This differentiation system provides a useful model system for studying neutrophil development and maturation in vitro and the many factors that regulate this process. Morphologically mature ES-derived neutrophils can be grown in culture that produce superoxide, flux calcium and directionally respond to the chemoattractant MIP-2. In addition, they express the granulocyte markers Gr-1 and the neutrophil specific antigen, as well as specific chloroacetate esterase. Interestingly, during their development in culture, regional areas of apparent neutrophil production can be identified that recapitulate certain aspects of the marrow environment. As ES cells can be genetically modified, this system enables evaluation of the effects of specific genetic alterations on neutrophil differentiation and function.
authors.affiliation: Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206, USA.
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Lieber
authors.forename: Gordon M
authors.initials: GM
authors.lastname: Keller
authors.forename: G Scott
authors.initials: GS
authors.lastname: Worthen
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D018920:Coculture Techniques; D003470:Culture Media / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology*; D051379:Mice; D009504:Neutrophils / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D017154:Stromal Cells / Q000166:cytology
chemical_list: D003470:Culture Media; D017077:Culture Media, Conditioned; D016207:Cytokines