Objective: To develop a reliable method for the isolation and longterm culture of microvessel endothelial cells from human endometrium and to evaluate their response to angiogenic growth factors and steroid hormones in comparison to endothelial cells derived from other organs.

Methods: Endometrial tissue from hysterectomy specimens were digested sequentially with collagenase and trypsin, cultured for 24 h, then selected by adhesion to anti‐CD‐34 coated magnetic beads. Alternatively, anti‐CD‐34‐coated beads could also be substituted by Ulex europaeus agglutinin‐1, anti‐PECAM, or anti‐E‐selectin‐coated beads. Characterization of the isolated cultures included expression of endothelial cell markers, regulation of E‐selectin in response to TNF‐α, proliferative response to angiogenic growth factors, and expression of progesterone and estrogen receptors. We also analyzed the relative binding affinity of VEGF on endometrial endothelial cells in comparison to other endothelial cell types.

Results: Selection on anti‐CD‐34‐coated beads eliminated contaminating cells and resulted in a homogeneous population of human endometrial endothelial cells (HEEC), as assessed by expression of PECAM, von Willebrand's factor, and uptake of acetylated‐LDL. HEEC also upregulated E‐selectin in response to TNF‐α in a manner similar to that seen for other endothelial cell types. Expression of progesterone and estrogen receptor was revealed by immunocytochemistry and RT‐PCR consistently until passage 5. Endometrial endothelial cells were more responsive to growth stimulation by VEGF than were dermal endothelial cells isolated under similar conditions. Further characterization indicated that VEGF bound more avidly to HEEC than to other endothelial cell types.

Conclusions: Human endometrial endothelial cells were isolated to homogeneity by a two‐part protocol and successfully passaged under culture conditions similar to those used for other endothelial cell types. The HEEC were very responsive to VEGF growth‐stimulation likely due to elevated affinity, or increased levels of, KDR and FLT‐1 on the cell surface. These results indicate that HEEC are capable of maintaining a mature phenotype in culture and might provide a model for understanding the response of these cells to the recurrent cycles of proliferation imposed on the endometrium during menstruation.