Aim: Quantify the permissiveness of placental cells to SARS-CoV-2 infection and ability to support viral release

Data Collection: For tissue processing, decidua was removed and the tissue was washed in PBS for 10min. Villi were scraped and digested in 0.2% Trypsin/0.02% EDTA at 37°C for 7/15min. The digest was filtered through a gauze and quenched with FBS. Undigested material was further digested with 1mg/ml Collagenase V supplemented with 10mg/ml DNase for 30/40min at 37°C. After filtering and washing, layer the cell suspension on to a Pancoll gradient ad centrifuge without break for 20min at 3000rpm. For more details and gating strategies, refer to Appios et al., 2021 (Bio Protoc) and Thomas et al., 2021 (JEM. For tissue sections, slides were fixed and permeabilised in ice-cold acetone (5min), washed in PBS, blocked in 2% serum (20min), incubated overnight at 4°C with primary antibodies - ACE2 (rabbit, ab15348), cytokeratin 7 (mouse, M7018). Slides were washed twice in PBS, and incubated with secondary/conjugated antibodies (anti-rabbit AF488, (A-11008), anti-mouse AF555 (A-31570) and CD31-AF700 (303133)) for 1hr in the dark. Slides were washed twice before staining with DAPI (D9542) and mounted ready for imaging. For in vitro assays, cells were infected with BetaCoV (B.1.1.7) strain at MOI 1 for 1hr. Inoculum was removed and cells washed with PBS before replacing with media containing 2% serum. Cells were fixed at 24h in 4% PFA. Cells were permeabilised (0.1% saponin, 10min), and blocked (1% BSA and 0.01% saponin, 20min) before incubating with the primary antibody SARS-CoV-2 spike protein (mouse, GTX632604) for 1hr. After 3 washes (5min each) in blocking solution, cells were incubated with the secondary antibody, anti-mouse AF488 (A-32766) for 45min in the dark. After washing 3 times, nuclei were stained with Hoechst 33342 (62249) for 5min, washed with PBS and maintained in PBS ready for imaging.

Data generation: Human primary Hofbauer cells were sorted and data recorded on the BD FACS Aria III using the BD FACSDiva™ software. Tissue sections and cells from infection assays were imaged using the Zeiss LSM780 AxioObserver. Brightness and contrast of images were adjusted using the Brightness/Contrast tool in the FiJi (Just ImageJ) software.