Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion

Abstract

To examine potential mechanisms by which hematopoiesis may be regulated by endothelial cells within the bone marrow (BM) microenvironment, we have devised a technique for the in vitro study of the interaction of human BM microvascular endothelial cells (BMEC) with hematopoietic cells. Microvessels isolated by collagenase digestion of spicules obtained from filtered BM aspirate were plated on gelatin-coated plastic dishes, and colonies of endothelial cells grown from microvessel explants were further purified by Ulex europaeus lectin affinity separation. BMEC monolayers isolated by this technique grew in typical cobblestone fashion, stained positively with antibody to factor VIII/von Willebrand factor, and incorporated acetylated LDL. Immunohistochemical studies showed that BM microvessels and BMEC monolayers express CD34, PECAM, and thrombospondin. Incubation of resting BMEC with BM mononuclear hematopoietic cells resulted in the selective adhesion of relatively large numbers of CD34+ progenitor cells and megakaryocytes. The binding of purified BM-derived CD34+ progenitor cells to BMEC was dependent on divalent cations and was partially blocked by antibodies to CD34. IL-1 beta treatment of BMEC monolayers resulted in an increase of CD34+ progenitor cell adhesion by mechanisms independent of CD34 or divalent cations. BMEC exhibit specific affinity for CD34+ progenitor cells and megakaryocytes, suggesting that the BM microvasculature may play a role in regulating the trafficking, proliferation, and differentiation of lineage specific hematopoietic elements, and possibly of pluripotent stem cells within the CD34+ population.

Adhesion Molecules Involved in Transendothelial Migration of Human Hematopoietic Progenitor Cells

“…A role for CD34 and E‐selectin (CD62E) has been described in adhesion of HPC to BMEC [28‐30]. Therefore, the contribution of these proteins in migration of HPC was investigated.…”

Thrombopoietin and Its Receptor, c-mpl, Are Constitutively Expressed by Mouse Liver Endothelial Cells: Evidence of Thrombopoietin as a Growth Factor for Liver Endothelial Cells

“…3 The endothelial nature of LEC-1 clones was assessed by flow cytometry and RT-PCR by the expression of the following specific endothelial cell markers: CD34, VCAM, ICAM-1, VIII/vWF, fms-like tyrosine kinase (flt), and fetal liver kinase-1 (flk-1). 22 LEC-1 clones were also characterized by their high WGA binding affinity. 21,23 LEC-1 clones were maintained in IMDM-20% FCS and used in the experiments described below, after these cells reached confluence within the wells.…”

“…PCR amplification of the cDNA was then performed using specific oligonucleotides, which were previously used for the detection of TPO and ␤-actin transcripts. 13,22 The primers for murine TPO were 5Ј-TCTGTCCAGCCCCGTAG-GTC-3Ј (sense) and 5Ј-GTTCCATCCACAGGTCCGTG-3Ј (antisense). Primers for ␤-actin were 5Ј-CATGATCTGGGTCATCTTTTCACGGT-TTGGC-3Ј (sense) and 5Ј-ATGGATGATGATATCGCTGCGCTGGTC-GTC-3Ј (antisense).…”

Characterization of the glycosylation profile of the human breast cancer cell line, MDA-231, and a bone colonizing variant

“…Immortalized human bone marrow endothelial cells (TrHBMEC) were kindly provided by Drs D. Paulin (University of Paris VI), K. Schweitzer, and B.B. Weksler (both of Weill College of Medicine of Cornell University) (20)(21)(22). TrHBMEC were derived by immortalizing human bone marrow endothelial cells with a construct encoding the large T antigen of SV40 under the control of a truncated human vimentin promoter (22).…”