Blood-brain barrier-specific properties of a human adult brain endothelial cell line

Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. Normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase and/or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution of the transduced cells, one was selected for its expression of normal endothelial markers such as CD31, VE Cadherin and von Willebrand Factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, upregulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics including tight junctional proteins and capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely utilizable research tool