The study of adhesive interactions between haemopoietic progenitor cells and bone marrow sinusoidal endothelial cells
The study of adhesive interactions between haemopoietic progenitor cells and bone marrow sinusoidal endothelial cells
A method was developed to isolate EC from aspirates of human bone marrow using a technique based on the selective binding of the lectin, Ulex europaeus to the EC surface via fucose residues. Endothelial cells were positively selected from red cell lysed suspensions of BM aspirates using UEA-1 coated magnetic Dynabeads. Bone marrow endothelial cells (HBMECs) were established in culture on either extracellular matrix derived from umbilical vein (HUVECs), human plasma fibronectin or 0.1% gelatin, in endothelial serum-free medium supplemented with 5% FBS, EC growth supplement and heparin. Two EC populations were identified within the same cultures, a faster growing cell with a polygonal, more spindle shaped morphology and one with a slower growing, more rounded morphology. Both had a vesiculated cytoplasm. Cultured cells demonstrated positive immunostaining with vWF, CD32 and also expressed a number of adhesion molecules. Ultrastructural examination by electron microscopy revealed the presence of Weibel-Palade bodies which are unique to human endothelium.
Two new, in vitro static adhesion assays were developed to examine the potential adhesive homing interactions between EC and PC. These assays used either chromium-51 or PKH2 fluorescently labelled haemopoietic cell lines (KG1a, HL60, K562, CEM, NALM-6), which are representative of different stages and lineages of haematopoietic development, and non-activated and rh TNFα activated HUVECs and HBMECs. KG1a and HL60 adhesion to HUVECs and HBMECs was further studied under various conditions, including: assay medium free of divalent cations; in the presence of synthetic tetrapeptides (RGDS/RGES); blocking monoclonal antibodies to adhesion molecules/ligands; and carbohydrates (synthetic neoglycoproteins), to identify molecules which may be of functional importance in PC:EC interactions. A possible role of adhesion molecules of the integrin family (VLA-4) and carbohydrates (fucose) in the binding of haemopoietic cells to marrow endothelium was indicated.
University of Southampton
1997
Masek, Lisa Christina
(1997)
The study of adhesive interactions between haemopoietic progenitor cells and bone marrow sinusoidal endothelial cells.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
A method was developed to isolate EC from aspirates of human bone marrow using a technique based on the selective binding of the lectin, Ulex europaeus to the EC surface via fucose residues. Endothelial cells were positively selected from red cell lysed suspensions of BM aspirates using UEA-1 coated magnetic Dynabeads. Bone marrow endothelial cells (HBMECs) were established in culture on either extracellular matrix derived from umbilical vein (HUVECs), human plasma fibronectin or 0.1% gelatin, in endothelial serum-free medium supplemented with 5% FBS, EC growth supplement and heparin. Two EC populations were identified within the same cultures, a faster growing cell with a polygonal, more spindle shaped morphology and one with a slower growing, more rounded morphology. Both had a vesiculated cytoplasm. Cultured cells demonstrated positive immunostaining with vWF, CD32 and also expressed a number of adhesion molecules. Ultrastructural examination by electron microscopy revealed the presence of Weibel-Palade bodies which are unique to human endothelium.
Two new, in vitro static adhesion assays were developed to examine the potential adhesive homing interactions between EC and PC. These assays used either chromium-51 or PKH2 fluorescently labelled haemopoietic cell lines (KG1a, HL60, K562, CEM, NALM-6), which are representative of different stages and lineages of haematopoietic development, and non-activated and rh TNFα activated HUVECs and HBMECs. KG1a and HL60 adhesion to HUVECs and HBMECs was further studied under various conditions, including: assay medium free of divalent cations; in the presence of synthetic tetrapeptides (RGDS/RGES); blocking monoclonal antibodies to adhesion molecules/ligands; and carbohydrates (synthetic neoglycoproteins), to identify molecules which may be of functional importance in PC:EC interactions. A possible role of adhesion molecules of the integrin family (VLA-4) and carbohydrates (fucose) in the binding of haemopoietic cells to marrow endothelium was indicated.
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Published date: 1997
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Local EPrints ID: 463137
URI: http://eprints.soton.ac.uk/id/eprint/463137
PURE UUID: 2b0c2e5e-b10d-431e-866c-1fb629581fb0
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Date deposited: 04 Jul 2022 20:45
Last modified: 04 Jul 2022 20:45
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Author:
Lisa Christina Masek
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