HER1 Signaling Mediates Extravillous Trophoblast Differentiation in Humans: MATERIALS AND METHODS

HER1 Signaling Mediates Extravillous Trophoblast Differentiation in Humans: MATERIALS AND METHODSTissue Collection
First trimester placentae and decidua were obtained at the time of elective terminations of pregnancy. Informed consent was obtained from each patient, and collections were approved by the Mount Sinai Hospital’s Review Committee on the Use of Human Subjects. Tissue was collected into ice-cold PBS for villous explant or decidual cell culture.
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Placental Villous and Decidual Explant Culture and Treatment
Villous explant cultures were established from first trimester human placentae using the method of Caniggia et al.. Small fragments (15-20 mg wet wt) of placental villi from 6- to 8-wk gestation placentae were dissected from the placenta, teased apart, and placed on Millicell-CM culture dish inserts (pore size 0.4 im; Millipore Corp., MD), precoated with 0.2 ml undiluted phenol red-free Matrigel substrate, and cultured at 3% O2/5% CO2 and 37°C (Becton Dickinson, Mississauga, ON). Explant Media serum-free Dulbecco modified Eagle medium-Ham F-12 media (400 il; Invitrogen, Burlington, ON) supplemented with 100 ig/ml streptomycin (Sigma, St. Louis, MO), 100 U/ml penicillin (Sigma), pH7.4 was placed in the well surrounding the outside of the culture insert. Explants were allowed to attach to the matrigel and were covered in 200 il Explant Media. Explants were treated 48 h later once the villous outgrowth was established. Patient-matched decidua parietalis was dissected to 2-mm2 pieces and placed in a 24-well plate, with approximately 100 mg of tissue per well. Decidua explants were cultured for 48 h at 3% Q,JS% CO2 and 37°C in Explant Media. DCM was collected, centrifuged at 2000 rpm for 5 min, filtered to remove contaminating cells, and supplemented with 2 mM L-glutamine (Invitrogen) prior to treatment of established EVT outgrowths. For experimental treatments, DCM was supplemented with vehicle (0.0025% dimethyl sulfoxide [DMSO]) or HER1 antagonist AG1478 (2 iM in 0.0025% DMSO; Cedarlane, Burlington, ON). Established outgrowths were also treated with 10 ng/ml EGF (Sigma) 6 2 iM AG1478. Explants from a single placenta were cultured in triplicate for each treatment point. Each experiment was repeated with at least three placentae.

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