title: Antibacterial capacity of differentiated murine embryonic stem cells during defined in vitro inflammatory conditions.
abstract: Embryonic stem (ES) cells are a powerful model for the development of cells responsible for the cellular immune response. Therefore, we analyzed the defense and phagocytic capacity of embryoid bodies (EBs) derived from ES cells using in the vitro inflammatory conditions caused by Escherichia coli. Further, we used this phagocytic activity to purify activated immune cells. Our data show that spontaneously differentiated 18-day-old EBs of the cell line CGR8 contained immune cells, which were positive for CD45, CD68, CD11b, F4/80, and CD19. Exposure of these EBs to E. coli with defined infection doses of bacterial colony-forming units (CFUs) led to a significant time-dependent reduction of CFUs, indicating the immune responses exerted by EBs. This was paralleled by an upregulation of inflammatory cytokines, that is, IL-1β and TNF-α. Western blot analysis of infected EBs indicated an upregulation of CD14 and cytochrome b-245 heavy chain (NOX2). Silencing of NOX2 significantly reduced the antibacterial capacity of EBs, which was partially explained by reduction of F4/80-positive cells. To identify, isolate, and further cultivate phagocytic active cells from differentiated EBs, a cocultivation assay of differentiated ES cells with green fluorescent protein (GFP)-labeled E. coli was established. Colocalization of GFP-labeled E. coli with cells positive for CD45, CD68, and F4/80 revealed time-dependent phagocytotic uptake, which was underlined by colocalization with the LysoTracker-Red(®) dye as well as preincubation with cytochalasin D. In conclusion, a primitive immune response with efficient phagocytosis was responsible for the antibacterial capacity of differentiated EBs.
authors: Department of Cardiology, University Heart Center, Clinic of Internal Medicine I, Jena University Hospital, Jena, Germany. andreas_finkensieper@gmx.net
authors: Andreas
authors: A
authors: Finkensieper
authors: Mohamed M
authors: MM
authors: Bekhite
authors: Heike
authors: H
authors: Fischer
authors: Saskia
authors: S
authors: Nitza
authors: Hans-R
authors: HR
authors: Figulla
authors: Joerg P
authors: JP
authors: Müller
authors: Heinrich
authors: H
authors: Sauer
authors: Maria
authors: M
authors: Wartenberg
mesh_terms: D000588:Amines; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology; D000943:Antigens, Differentiation / Q000235:genetics / Q000276:immunology; D002454:Cell Differentiation; D002460:Cell Line; D018920:Coculture Techniques; D015169:Colony Count, Microbial; D015638:Cytochalasin D; D058732:Embryoid Bodies / Q000166:cytology / Q000276:immunology*; D004926:Escherichia coli / Q000254:growth & development* / Q000276:immunology; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D053583:Interleukin-1beta / Q000235:genetics / Q000276:immunology; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D051379:Mice; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D010587:Phagocytosis / Q000276:immunology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000276:immunology
chemical_list: D000588:Amines; D015703:Antigens, CD; D000943:Antigens, Differentiation; D053583:Interleukin-1beta; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; C493330:Red DND-99; C036763:monocyte-macrophage differentiation antigen; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases