Isolation of Primary Human Trophoblasts

Primary human first trimester trophoblasts were isolated as previously described [33 (link),41 (link)]. Briefly, first trimester placental tissue was digested with Dispase/DNAse and Trypsin (Gibco, Invitrogen, Carlsbad, CA, USA). After Percoll (Gibco) gradient centrifugation, fibroblasts and common leukocyte-antigen expressing cells were removed by negative immunoselection with anti-CD90 (Dako, Glostrup, Denmark) and anti-CD45 conjugated magnetic beads (Thermo Fisher Scientific, Rockford, IL, USA), respectively. Purity of the trophoblast preparations was determined by human chorionic gonadotropin secretion as well as positive cytokeratin 7 (Dako, 1:750) and negative vimentin (Dako, 1:250) immunostaining. Only isolations with a purity ≥95% were used.