Anti ck7

Immunofluorescence staining of organoids was performed essentially as described previously^{25 (link)}. Briefly, organoids were fixed with 4% PFA for 2 hours at RT and washed twice with PBS. Organoids were blocked and permeabilized with 5% BSA-PBS including 0.2% Triton-X for 1 hour at RT. Organoids were washed once with PBS and incubated with primary antibody diluted in 2.5% BSA-PBS O/N at 4 ºC. Primary antibodies included anti-AFP (Thermo Fisher, PA5-16658, 1:250); anti-ALB (Bethyl, A80-229A, 1:500); anti-Ki-67 (Thermo Fisher, 14-5698-82, 1:1000); anti-CK7 (Thermo Fisher, MA5-11986, 1:400); anti-CK19 (Cell Signaling Technology, 13092 S, 1:500); anti-MRP2 (Abcam, ab3373, 1:500); anti-BCAT (Santa Cruz, sc-7199, 1:500); anti-A1AT (Bethyl, A80-122A, 1:250); anti-HNF4A (Abcam, ab201460, 1:250); anti-CYP3A4 (Thermo Fisher, MA5-17064, 1:250); anti-ZO1 (Thermo Fisher, PA5-19090, 1:250). Organoids were washed with PBS three times and subsequently incubated with the appropriate secondary Alexa Fluor antibodies diluted in 2.5% BSA-PBS for 3 hours at RT. After one wash with PBS, organoids were incubated with 1 μg/ml DAPI (Thermo Fisher, 62248), and cell membranes were optionally stained with Phalloidin-Atto 647N (Sigma–Aldrich, 65906, 1:1000), both in PBS for 20 min at RT. After two washes with PBS, organoids were transferred to a 96-well Sensoplate and imaged on a Leica Sp8 microscope.

Healthy and PE‐complicated term placentas obtained soon after C‐section were used for the isolation of primary human trophoblasts (PHTs) as previously described.¹⁹ Briefly, 50 g of placental tissue was cut into small pieces and washed with PBS at room temperature. The tissue was enzymatically digested with sequential trypsin‐DNase digestions at 0.25%. Then, a discontinuous gradient of Percoll (5%–60%, in four steps; Sigma–Aldrich, Germany) was applied to remove red blood cells and cell debris. The middle‐layer cells were collected and washed with DMEM/F12 (Gibco). Then, the cells were resuspended in DMEM/F12 supplemented with 5 μg/ml insulin (Sigma–Aldrich, Germany), 10 μg/ml transferrin (Sigma–Aldrich, Germany), 20 nM sodium selenite (Sigma–Aldrich, Germany), 5 ng/ml EGF (Sigma–Aldrich, Germany), 400 U/L hCG (Abcam, UK), 10% FBS (Gibco), 1% penicillin/streptomycin (Gibco), 50 μg/ml gentamycin (Gibco) and 0.25 μg/ml amphotericin (Gibco). The PHTs were further verified by flow cytometry with anti‐CD31 (Thermo Fisher) and anti‐CK7 (Thermo Fisher) antibodies (Figure S1A).