Fig. 9. Effect of hypoxia and decidua-derived soluble factors on trophoblastic expression of CCR1. (A-D) Chorionic villi (8-weeks gestation) were cultured for 48 hours in either 20% O₂ (A,B) or 1% O₂ (C,D), followed by immunostaining with anti-CCR1 mAb. Explanted villous tips are traced with broken lines. In 20% O₂, round cells grew out from the explanted villous tips to form a cell sheet, from which spindle-shaped cells migrated (A). CCR1 expression is detected clearly on these cells (B). In 1% O₂, the individual outgrown cells are larger and the intercellular spaces are wider (C) and CCR1 expression is weak (D). (E) EVTs that outgrew in 20% O₂ were isolated and cultured for a further 24 hours in the presence of decidua-conditioned medium (either with or without heat-inactivation). These EVTs were treated with either anti-CCR1 mAb or isotype-matched control mAb and analyzed by FACScalibur. There is a distinct shift in the fluorescence intensity of the cells treated with intact decidua-conditioned medium compared to those treated with heat-inactivated decidua-conditioned medium. Scale bars: 200 µm.