Isolation and culture of endothelial cells from human bone marrow

Adhesive interactions between haemopoietic progenitor cells and bone marrow sinusoidal endothelium are potentially important in the homing of these cells back to the extravascular compartment of the marrow to re-establish haemopoiesis following stem cell transplantation. A simple method for the isolation and culture of human bone marrow endothelial cells is described using bone marrow aspirates obtained from patients undergoing bone marrow harvests for autologous or syngeneic bone marrow transplantation. The method is based on the selective binding of the lectin Ulex europaeus agglutinin-1 (UEA-1) to endothelial cells. Magnetic Dynabeads coupled with UEA-1 were incubated with single cell suspensions of bone marrow following red cell lysis, and bound cells were isolated with a magnet. The isolated cells demonstrated positive immunofluorescence staining for von Willebrand factor. Cells were plated onto tissue culture flasks coated with extracellular matrix derived from human umbilical vein endothelial cells in an endothelial serum-free medium together with 5% fetal calf serum for 24 h. Cells were then cultured in endothelial serum-free growth medium supplemented with 5% fetal calf serum, endothelial cell growth supplement and heparin. After 2-4 weeks in culture, two morphologically different cell populations can be identified. One has a polygonal spindle-shaped morphology with a rapid growth rate, the other a rounded morphology and a slow growth rate. Both populations have a vesiculated cytoplasm. Positive immunostaining of the cells was demonstrated with a number of endothelial cell markers including von Willebrand factor, and antibodies to ICAM-1, VCAM-1, E-selectin, CD31 and BMA120. Weibel-Palade bodies were observed by electron microscopy. Culture of these cells will allow detailed in vitro studies of adhesion mechanisms in the homing of haemopoietic progenitor cells.