Collagen 1

First trimester placenta collections were approved by the AMC Biobank review committee, and were obtained at the time of elective terminations of pregnancy. Informed consent was obtained from each patient. Small fragments of placental villi from 5 to 12 weeks gestation were dissected from the placenta and placed in culture plate wells coated with collagen I (R&D systems). Placental villous explants were cultured for a maximum of 5 days at 5% O₂ and 37 °C in serum free DMEM/F12 media supplemented with pen-strep. Images of the explants were taken daily to monitor changes in outgrowth. At day 4 or 5 explants were fixed in 4% PFA and processed for immunohistochemistry.

The heart samples were fixed by 4% paraformaldehyde followed by embeddedness in paraffin. Sections were stained with primary antibody against IL-17 (1:200) (Cell Signaling Technology, MA, United States), collagen I and collagen III (1:200) (R&D Systems, Minneapolis, MN, United States) overnight at 4°C, respectively. After washing three times with phosphate buffered saline (PBS), the sections were incubated with corresponding secondary antibody at room temperature for 1 h. Diaminobenzidine and neutral gum were used to stain sections. Images were captured by fluorescence microscopy (Nikon 80i, Otawara, Tochigi, Japan). The resulting figures were then processed by Image-Pro Plus 6.0 to quantify the data.

Human first trimester placenta specimens (n = 12) were obtained from the HIS Mouse Facility of the Amsterdam UMC, location AMC, Amsterdam. All material has been collected from donors at the time of elective terminations of pregnancy from whom a written informed consent for the use of the material for research purposes had been obtained by the Bloemenhove clinic. Small fragments (15–20 mg wet weight) of placental villi from 5 to 12 weeks gestation were dissected from the placenta and placed in 48 well culture plates coated with 3 mg/ml collagen I (R&D systems). Transfection was done 24 h later with 80 pmol PCBP1, PCBP2, or YBX1 siRNAs (Qiagen, Germany, Flexitube Genesolution, a package of four preselected siRNAs targeting the different genes) or non-targeting siRNAs (Qiagen) using 2.5 μl XtremeGENE HP transfection reagent (Sigma) in a total volume of 250 μl. Placental villous explants were cultured for a maximum of 5 days at 5% O₂ and 37 °C in serum free DMEM/F12 media supplemented with pen/strep. Images of the explants were taken on a Leica microscope daily to monitor changes in outgrowth. At day 4 or 5 explants were fixed in 4% PFA and embedded for immunohistochemistry.

We coated 96-well plates overnight at 4 °C with recombinant Npnt (5 μg/ml, R&D Systems), collagen I (10 μg/ml, Nippi), collagen IV (10 μg/ml, Nippi), laminin I (10 μg/ml, Nippi), and fibronectin (10 μg/ml, Nippi), then they were washed with PBS and blocked with 3% bovine serum albumin for 1 hour at 37 °C. M3H1 cells were then plated at a concentration of 2 × 10⁴ cells/well and cultured for 48 hours. Sox2+ cells were determined by immunohistochemistry using an anti-Sox2 antibody. Total cells were counted using DAPI, then the ratio of Sox2+ cells was determined under a fluorescence microscope.