Immortalisation of human bone marrow endothelial cells: characterisation of new cell lines.

Abstract

Adhesion of haematopoietic progenitor cells (HPC) to human bone marrow endothelial cells (HBMEC) plays a key role in homing of HPC to bone marrow. Here we describe four new HBMEC cell lines that can be used to study the (specific) adhesion of HPC to HBMEC. HBMEC were immortalised with a retroviral construct containing the human papilloma virus 16 E6/E7 genes. Four cell lines were characterised. The cell lines showed their endothelial nature by the expression of von Willebrand Factor and VE-cadherin (CD144). Electron microscopic analysis revealed normal endothelial-cell characteristics, including the presence of Weibel-Palade bodies and intercellular junction structures. An extensive phenotypic analysis of the cell-lines was performed, they were found to resemble primary HBMEC. The only difference found was the absence of expression of E-selectin (CD62e) and VCAM-1 (CD106) on resting HBMEC cell lines. Upon stimulation with IL-1beta the expression of E-selectin, VCAM-1 and ICAM-1 (CD54) was upregulated. All resting cell lines bound CD34+ HPC. Adhesion was increased by addition of the phorbol ester PMA. Two cell lines showed increased binding upon IL-1beta prestimulation. Highest adhesion was observed after the combination of IL-1beta prestimulation of the endothelial cells and addition of PMA. Binding of CD34+ HPC to HBMEC was compared with the binding to human umbilical vein endothelial cell lines and to a human dermal microvascular endothelial cell line (HMEC-1). So far, we have only found relatively less binding of HPC to IL-1beta prestimulated HMEC-1 cells, which could be explained by a reduced induction of E-selectin and VCAM-1 upon IL-1beta stimulation of these cells. The immortalised HBMEC cell lines have maintained their normal phenotype for the majority of characteristics examined. The expression of E-selectin and VCAM-1, which are not constitutively expressed on the cell lines, can be induced by stimulation of the endothelial cells with IL-1beta. The cell lines have furthermore maintained their capability to bind HPC. They will therefore be useful to investigate the interactions between HPC and HBMEC involved in homing of HPC.