Immunological phenotype of freshly isolated human microvascular endothelial cells from human term placenta

Abstract

Objectives: It is known that endothelial cells may play an important role in the pathogenesis in angiogenetic events as well as in disturbances of pregnancy like preeclampsia, intrauterine growth retardation. In order to examine pathophysiological mechanisms, particularly, in the endothelium of placenta, we isolated human placental microvascular endothelial cells . Methods: Fine minced specimens from fresh placenta cotyledons were washed three times in phosphate buffered saline, then incubated overnight in 1.2 units dispase II per ml Puck's solution at 4°C. Afterwards specimens were gently pressed by using syringes. Supernatant cells were plated in tissue culture flasks that had been coated in 1% gelatine and cultured in endothelial cell basal medium supplemented with 20% normal human serum, 5 ng epidermal growth factor per ml, 2 mM L-glutamine, 2mM L-arginine, 100µg streptomycin per ml,100 U penicillin per ml, 250 ng amphotericin B per ml.Primary cultures, if necessary, were purified using immunomagnetic beads recognizing CD 31 according to the manufacturer's protocol. Immunohistochemical procedures as well as flow cytometer analysis were done in order to characterize the phenotype and to assess the purity of the obtained placental microvascular endothelial cells. Results: Confluent monolayers of placental endothelial cells showed the typical cobblestone morphology. Cytospin preparation and flow cytometer analysis revealed positive immunostaining with Ulex europaeus 1 lectin, von Willebrand factor, PECAM-1 (CD31), VE-cadherin, CD34, thrombomodulin. Using anti-fibroblast antibodies did not show any staining. Glut -1 staining was dependent on endothelial cell origin (maternal versus fetal) Stimulation of placental microvascular endothelial cells with TNF-alpha 200 U per ml for 4 hours resulted in expression of E-Selectin-1 as a further evidence of endothelial cells. Conclusions: Our method is suitable to isolate purified microvascular placental (maternal and fetal) endothelial cells as assessed morphologically, by immunohistochemical phenotype and functionally (cytokine-induced E-Selectin-expression). These microvascular endothelial cells are different from endothelial cells deriving large vessels such as umbilical veins and therefore are important for further investigation of endothelial cell related disturbances in pregnancy.