title: Isolation of endothelial-like stromal cells that express Kit ligand and support in vitro hematopoiesis.
abstract: Although macrophages account for 70-90% of the adherent cells in mouse long-term bone marrow cultures (LTBMC) and CFU-F colonies, the predominant nonhematopoietic stromal cell is endothelial-like (EL), expressing cytoplasmic collagen IV, laminin, and an antigen recognized by the monoclonal antibody MECA-10. We report the isolation of this stromal cell lineage from primary LTBMC by immunomagnetic cell selection using MECA-10. More than 95% of the cells in the MECA-10-positive fraction are EL cells as judged by morphology, surface staining for MECA-10, cytoplasmic staining for collagen IV, and electrophoretic analysis of MECA-10-positive cells isolated from radiation chimeras. When plated under LTBMC conditions, EL cell monolayers recharged with either wild-type or Sl/Sld marrow support an increased density and number of clonogenic and mature hematopoietic cells in short-term cultures. In accord with this finding, Northern blots of mRNA from unstimulated EL cells demonstrate constitutive expression of Kit ligand (KL). Moreover, in situ two-color immunofluorescence staining for cytoplasmic collagen IV and surface KL suggests that EL cells are the exclusive source of membrane-bound KL in mouse cultures. The ability to isolate EL cells from primary cultures without the need for repeated cell passage or immortalization provides a novel approach to dissecting the molecular basis of stem cell-stromal cell interactions.
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001854:Bone Marrow Cells; D019070:Cell Lineage; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D003114:Colony-Forming Units Assay; D003239:Connective Tissue Cells; D004727:Endothelium / Q000166:cytology* / Q000276:immunology; D005260:Female; D015870:Gene Expression; D006410:Hematopoiesis; D018189:Immunomagnetic Separation; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D011828:Radiation Chimera; D019089:Stem Cell Factor / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D019089:Stem Cell Factor; D016173:Macrophage Colony-Stimulating Factor; D003094:Collagen