Tesr1

The maintenance of human ARPE‐19 cells has been described (Dunn et al., 1996 (link)). Human induced Pluripotent stem cells (CB6.2 line) were maintained in TeSR1 (STEMCELL Technologies) for 10 days, and placed in differentiation medium for 50 days, and plated onto Matrigel‐coated plates in RPE medium as described (Maruotti et al., 2013 (link)). RPE cells were grown for 3 months and exposed to Cigarette Smoke Extract (CSE; Murty Pharmaceuticals) for 24 hr.

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).

One hESC line (H9, WiCell) one neonatal iPSC line (Neo1, male 38, 39) and one adult iPSC line (AD3, from a 37‐year‐old female 40) were adapted from MEF‐supported to feeder‐free culture conditions on 6 well plates coated with growth factor‐reduced Matrigel basement membrane matrix (GFRM; Corning, New York) in TeSR1 (Stem Cell Technologies, Vancouver, Canada) defined medium. Cells were fed daily and passaged every 5 days at a ratio of up to 1:10 using 0.02% versene EDTA (Lonza, Basel, Switzerland). Cells were then differentiated by initiating EB formation, followed by long‐term culture (up to 150 days). EBs were generated either by mechanical, enzymatic, or dissociation–reaggregation approaches (Fig. 1). Each method was tested in the presence or absence of ROCK inhibitor (Y‐27632, Chemdea, NJ) for the first 48 hours of differentiation, during the early phase of EB formation. All cultures were tested in parallel under either stationary (static, “St”) or shaking (“Sh”) conditions throughout differentiation. Shaking cultures were achieved by placing cells on an orbital shaker (30 rpm, Stuart) housed inside the incubator for the entire period of differentiation, or in the case of EBs generated via the dissociation–reaggregation method, from day 12 onward. Biological replicates were performed in triplicate (n ≥ 3) for all experimental conditions tested.