Homo sapiens
Protocols: Placental samples were obtained from healthy women undergoing elective terminations in the first trimester of pregnancy. Written and informed consent was obtained in accordance with the guidelines in The Declaration of Helsinki 2000. Ethical approval for the use of these tissues was obtained from the Cambridge Local Research Ethics Committee (REC 04/Q0108/23). This now forms part of the Centre for Trophoblast Research biobank for the 'Biology of the Human Uterus in Pregnancy and Disease Tissue Bank' at the University of Cambridge. Overall biobank ethical approval is from the East of England–Cambridge Central Research Ethics Committee (17/EE/0151). Trophoblast organoids were obtained by sequential digestion of placental villi in 0.2% trypsin-250 (PAN-Biotech P10-025100P), 0.02% EDTA (Sigma E9884) in PBS, followed by collagenase (1.0 mg ml−1 collagenase V, Sigma C9263) in Ham's F12/10% FBS. Pooled digests were washed in Advanced DMEM/F12 medium, then resuspended in Matrigel on ice (Corning, 356231) and plated in 25 μl droplets into 48 well culture plates and overlaid with 250 μl trophoblast organoid medium (TOM). Cultures were maintained in 5% CO2 in a humidified incubator at 37°C and medium was replaced every 2–3 days. After passaging into 35mm dishes, organoids are grown in TOM for 3-4 days then switched into EVT differentiation medium (EVTM) with NRG1. After ~4-7 days, when outgrowths of cells are observed from the organoids, the medium is switched to EVTM without NRG1 (EVTM-NRG1) for a further 4 days. To study the effects of uNK cytokines on EVT differentiation, cytokines are added to the EVTM-NRG1 medium as follows: CSF2 (10 ng/mL), XCL1 (100 ng/mL), CSF1 (20 ng/mL), and CCL5 (50 ng/mL). Cells were collected after (3h, 24h, 48h and 96h) addition of cytokines. Nucleic acid was extracted based on the Chromium Single Cell 3′ Kit v3 from 10x Genomics. Single-cell library preparation was performed based on the Chromium Single Cell 3′ Kit v3 from 10x Genomics to obtain between 2,000 and 10,000 cells per reaction.