ISOLATION AND CULTURE OF HUMAN BONE-MARROW ENDOTHELIAL-CELLS

Bone marrow endothelial cells are likely to play an important role in the homing of hematopoietic progenitor cells. In view of analyzing the interactions between endothelial cells and hematopoietic progenitor c ells, we studied several methods of isolating endothelial cells from h uman bone marrow, including fluorescence activated cell sorting (FAGS) and separation by immunomagnetic beads. FAGS sorting gave the best re sults as contamination with other cells did not occur. After density-g radient centrifugation of bone marrow aspirates, the mononuclear cell (MNC) fraction was depleted for T cells, B cells, and myeloid cells by immunomagnetic separation. Further enrichment of endothelial cells wa s achieved by FAGS sorting using BNH9 or S-Endol monoclonal antibodies (MAbs). These MAbs, in contrast to several other endothelial-cell rea ctive MAbs, were found to react highly specifically with sinus endothe lial cells as tested by immunohistochemistry on bone marrow tissue sec tions and cell culture preparations and by double-colored FAGS analysi s on bone marrow MNCs (BMMNC). Sorted cells, which formed 0.05% of the MNC fraction, showed strong intracytoplasmic von Willebrand factor po sitivity. Ultrastructural analysis revealed cells with endothelial cha racteristics. Cells were cultured in fibronectin-coated, 24-well cultu re plates in endothelial-cell culture medium or long-term bone marrow culture medium. After 1 to 3 weeks of culture, a monolayer of spindle- shaped cells developed expressing endothelial cell antigens. Cells cou ld be kept in culture for 4 to 6 weeks. In conclusion, the method desc ribed provides highly purified preparations of human bone marrow endot helium that may permit in vitro adhesion experiments with normal and l eukemic hematopoietic progenitor cells.