Placental Villous Tissue Explant Culture

The placental villous tissues were dissected into explants of 2–5 mm in diameter and explanted as previously described (Chen et al., 2018 (link); Zhang et al., 2012 (link)). A 24‐well plate was precoated with 50 μl of a 1 mg/ml Matrigel matrix solution (CA# 354234, BD Biosciences, San Jose, CA, USA) and incubated at 37°C for 4 h for solidification. Serum‐free DMEM/F12 (Gibco) medium containing 10% FBS (PAN) with 20 mM NAM (#HY‐B0150, MedChemExpress, USA), 3 μM SRT1720 (#HY‐10532, MedChemExpress, USA), 500 nmol/l lentiviral vector‐based shRNA targeting SIRT1 or an equal concentration of scrambled shRNA (GeneChem, Shanghai, China) was added to the wells; the explants were cultured in 3% oxygen and 5% CO₂ for 48 h. The explants with good attachment and outgrowth on the gel were assessed after 24 and 48 h, respectively. The evaluation of villus outgrowth was performed as previously described (Li et al., 2014 (link)).