Conditioned medium preparation and experiments

hPDMSCs at passages 3 to 5 and isolated primary CTBs cultured for 1–2 days were used to collect the conditioned medium. To harvest the conditioned medium, the cells were cultured with 10% FBS in DMEM until the cells were greater than 90% confluent and washed with PBS to remove detached cells. Then, the medium was replaced with serum-free DMEM. At different time points (6, 12, 24, 48, and 72 h), the cell culture supernatant was collected and centrifuged at 3500 rpm for 20 min to remove detached cells and cellular debris. Then, the placental cell CM was filtered (0.22 μm), adjusted with serum-free medium to 10 ml/5 × 10⁷ cells, and frozen at − 80 °C for future experiments.

The nonvascular placental stromal tissue between the maternal surface and fetal surface was selected for the collection of sub-cultured placental tissue conditioned medium. These placental samples were mechanically minced into small pieces and washed with PBS supplemented with 5% penicillin/streptomycin. Then, the wet placental villi tissue (1 cm³/dish) was laid uniformly in a 10-cm² Petri dish until the tissue was semidry, and serum-free DMEM was added. The next day, the supernatant was discarded, the tissue was washed, and the medium was replaced with 10 ml of serum-free DMEM. At different time points (1, 3, 5, 7, 10, and 14 days), the supernatant of the cultured placental tissue was collected, centrifuged, filtered, and then adjusted with serum-free medium to 10 ml/dish. Finally, the sub-cultured placental tissue-derived CM was stored at − 80 °C for further experiments.