Isolation of endothelial-like stromal cells that express Kit ligand and support in vitro hematopoiesis

R. A. Fleischman, F. Simpson, T. Gallardo, X. L. Jin, S. Perkins

Research output: Contribution to journalArticlepeer-review

20 Scopus citations


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/Sl(d) 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.

Original languageEnglish
Pages (from-to)1407-1416
Number of pages10
JournalExperimental Hematology
Issue number13
StatePublished - 1995


  • endothelial
  • hematopoiesis
  • immunomagnetic separation
  • kit ligand
  • stromal cell

ASJC Scopus subject areas

  • Molecular Biology
  • Hematology
  • Genetics
  • Cell Biology
  • Cancer Research


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