Cnt 07

iPSCs were maintained feeder-free on Geltrex-coated (ThermoFisher Scientific) tissue culture plastic in TesR1 (STEMCELL Technologies, Vancouver, BC, Canada). For keratinocyte differentiation, mid-passage iPSCs at ~50% confluency in six-well plates had media changed to defined keratinocyte-SFM media supplemented with 25 ng/ml BMP-4 (Bio-Techne, Minneapolis, MN, USA) and 1 μM RA (STEMCELL Technologies) for the first 96 h, at which point the BMP4 and RA were removed, followed by media changes every 72 h thereafter until epithelial cell morphology became apparent (~10 days). At this timepoint, the media was switched to Cnt-07 (CELLnTEC, Bern, Switzerland) and the cells cultured until confluency. At this point they were pre-treated with ROCK inhibitor (Y-27632, VWR) for at least 1 h and passaged using Accutase (STEMCELL Technologies) onto 10 cm² tissue culture plates coated with collagen I/collagen IV, where rapid attachment-mediated enrichment of Krt14+ cells was performed as previously described. Resultant iPSC-keratinocyte cultures were cultured in Cnt-07 media containing 10 μM ROCK inhibitor until first media change after plating (CELLnTEC).

Cell line: human OKF6/TERT2 OECs (BWH Cell Culture and Microscopy Core, USA) were amplified in defined keratinocyte-SFM basal medium (K-SFM) with growth supplements (Thermo Fischer Scientific, USA).
Primary cells: human OECs and gingival fibroblasts (GFs) were isolated from gingival samples of healthy controls using the direct explant technique as previously described for the skin⁴³ . Briefly, cells were characterized by their respective morphology and by RT-qPCR amplification of markers. OECs were tetrahedral and positive for keratin 14 (KRT14), whereas GFs were fusiform and positive for CD90. OECs were cultured in a serum-free keratinocyte growth medium CnT-07 (CELLnTEC, Switzerland) with 0.1% penicillin-streptomycin. GFs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) glutaMAX® (Thermo Fischer Scientific, USA) supplemented with 10% FCS, 1% penicillin-streptomycin. OECs were used at passage 0 for all experiments except for challenging with TLR ligands (passage 1). GFs were used at passage 3.

We isolated and cultured the primary mouse SMG cells. Briefly, harvested SMG tissues from 8 weeks old female C57/BL6 mice were digested with 5 mL DMEM containing 10% FBS, 1% penicillin-streptomycin and collagenase (8 mg/mL). Then cells were filtered through a 100 μm nylon mesh and digested with 0.05% Trypsin-0.02% EDTA. The digested cells were re-suspended with epidermal keratinocyte medium, CnT-07 (CELLnTEC, Bern, Switzerland). Primary mSMG cells were used between passages 5 to 11. The primary mSMG cells were seeded at a density of 2 × 10⁵ cells per well in 24-well culture plates in the presence or absence of recombinant murine IL-22 (20 ng/mL) for 3 days.