Collagen 4

Glomeruli were isolated as previously described (Kindt et al., 2017 (link)). 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) were coated with collagen IV (Corning, New York, United States) and glomeruli were grown with phenol red-free RPMI-1640 medium supplemented with 10% FBS (both Thermo Fisher Scientific, Waltham, MA, United States) at 37°C and 5% CO². To prevent glomeruli from drying but still ensure gas exchange, we used adhesive Seals (4titude Ltd., Berlin, Germany). Glomerular viability was verified by propidium iodide (Merck KGaA, Darmstadt, Germany) staining performed after manufacturer’s instructions.

Coating selection was performed using model 2. Freshly extracted keratinocytes were seeded on several coatings known for their high cell adhesion properties: collagen I (Corning, New York, NY, USA), laminin (Corning), fibronectin (Corning), collagen IV (Corning). After 10 min (selected in previous step) of adhesion, LA were removed and fresh medium was added to RA. One day later, RA were detached with trypsin-EDTA (Sigma), counted and cultured for 14 days until the observation of colonies.
Then, fresh collagen I was compared to the feeder layer using model 1. Freshly extracted keratinocytes were seeded on collagen I. After 10 min of adhesion, LA were removed, counted and seeded in 3 flasks containing feeder layers for CFU. Fresh medium was added to RA. Both RA and LA were cultured for 14 days until the observation of colonies.

RPE cells from 9–11-day-old wt or RCS rats for primary culture were isolated as previously published in detail [12 (link)]. In brief, the cornea, lens, iris, and vitreous body were removed from freshly enucleated eyes. Eyecups were incubated in 1 mg/mL hyaluronidase (#H3506, Sigma-Millipore) in Hank’s balanced saline solution without Ca²⁺ and Mg²⁺ for 45 min at 37 °C. The neural retina was removed, and eyecups were further incubated in 2 mg/mL trypsin (#27250018, Thermofisher) in Hank’s balanced saline solution with Ca²⁺ and Mg²⁺ for 30 to 45 min at 37 °C. RPE sheets were manually collected from the underlying choroid. Purified RPE patches were grown in 96-well plates with collagen IV (#354233, Corning)-coated glass coverslips in DMEM (#D6429, Sigma-Millipore) 10% FBS (#F4135, Sigma-Millipore) at 37 °C, 5% CO₂ for 5–7 days before experiments.

hTSCs were cultured as previously described^{8 (link),10} . Prior to seeding, 6-well plates were coated with 5 μg/mL Collagen IV (Corning, 354233) at 37 °C overnight. Cells were cultured in 2 mL hTSC medium [DMEM/F12 (Gibco, 11320) supplemented with 0.1 mM 2-mercaptoethanol (Millipore Sigma, 8.05740), 0.2% FBS (Millipore Sigma, ES-009-B), 0.5% Penicillin-Streptomycin (Gibco, 15140), 0.3% BSA (Gibco, 15260), 1% ITS-X (Gibco, 51500), 1.5 μg/ml L-ascorbic acid (Wako, 013-12061), 50 ng/ml EGF (Peprotech, AF-100-15), 2 μM CHIR99021 (Stemgent, 04-0004), 0.5 μM A83-01 (BioVision, 1725), 1 μM SB431542 (BioVision, 1674), 0.8 mM VPA (Sigma, P4543), and 5 μM Y-27632 (Stemgent, 04-0012)] and in 5% CO₂ and 20% O₂. Tissue culture media were filtered using a 0.22 μm filter. Media were changed every 2 days, and cells were passaged using TrypLE Express (Gibco, 12604). Unless otherwise specified, hTSCs within 30 passages were used for experiments. The cell culture is regularly tested and confirmed negative for mycoplasma contamination.

hNE (R1162X/R1162X) cells were seeded in T75 flasks coated with collagen IV (Sigma) or Purecol (Advanced Biomatrix) in 12 ml pre-warmed complete PneumaCult ALI Ex⁺ medium (StemCell kit) at 37 °C in 5% CO₂. To 500 ml medium the following supplements were added: 0.5 ml hydrocortisone (StemCell), 10 ml 50X Ex⁺ supplement (StemCell kit), and for some preparations 2 ml of amphotericin B (12.5 µg ml⁻¹; Sigma), 500 µl ceftazidime (100 mg ml⁻¹; Sigma), 500 µl vancomycin (100 mg ml⁻¹; Sigma), and 500 µl tobramycin (100 mg ml⁻¹; Sigma). Cells were expanded for 3–5 days, until they reached 70–80% confluency.
Cells were then detached by 0.05% trypsin-EDTA (Pan Biotech) or enzymatic (StemCell) treatment and seeded onto 12- or 24-ALI Transwells (0.4-µm pore polyethylene terephtalate membrane inserts, Corning) coated with collagen IV at a confluency of 1.5 × 10⁵ to 2 × 10⁵ per well, and Complete Ex⁺ medium (without antibiotics) was added as following, 0.5–0.6 ml (basolaterally) and ~0.5 ml (apically). Cells were grown for 3–4 days at 37 °C with 5% CO₂. Medium were changed every day on the apical and basolateral side. On day 4, the apical medium was removed, and basolateral bathing solution exchanged for ALI complete medium (StemCell), followed by additional exchanges three times per week for at least 21 days until reaching a fully differentiated state.

For short-term pre-exposure, hPSCs were exposed for 4 days to naïve conditions. Naïve cocktails used were: CDK8/19i cocktail [0.4 μM of CNIO-CDK8/19 inhibitor and 20 ng/mL hrLIF (Peprotech)], as reported [20 (link)], or the 2i-based cocktail PXGL [1 μM PD0325901 (Axon Medchem), 2 μM Gö6983 (Selleckchem), 2 μM XAV939 (Selleckchem) and 20 ng/mL hrLIF (Peprotech)] [8 (link)]. TSCs derivation was performed as previously reported by Kojima and colleagues [23 (link)]. Naïve and primed hPSCs were single-cell dissociated by TrypLE Express, and 0.5 × 10⁶ cells were seeded in a 6-well plated pre-coated with 5 mg/mL Collagen IV (#354233, Corning) and cultured in TS medium [DMEM/F12 (D6421, Sigma), 0.1 mM 2-mercaptoethanol (Gibco), 0.2% FBS (Gibco), 0.3% BSA (Sigma), 1% ITS-X (Gibco), 1.5 μg/mL L-ascorbic acid (Sigma), 50 ng/mL EGF (PeproTech), 2 μM CHIR99021 (Axon Medchem), 0.5 μM A83-01 (Tocris, Bristol, UK), 1 μM SB431542 (PeproTech), 0.8 mM VPA (Sigma) and 5 μM Y-27632 (Selleckchem)] [24 (link)]. Cells were passaged every 5–7 days in single cells using TrypLE Express on pre-coated Collagen IV plates.

The TSCs were a gift from P. Liu (School of Biomedical Sciences, The University of Hong Kong)^{43 (link)}. The TSCs were cultured as previously described^{52 (link)} with minor modifications. Briefly, the cells were maintained in TSC medium (DMEM/F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.2% fetal bovine serum, 0.5% penicillin–streptomycin, 0.3% BSA, 1% ITS-X supplement, 150 µM l-ascorbic acid, 50 ng ml⁻¹ EGF, 2 mM CHIR99021, 0.5 mM A83-01, 1 mM SB431542, 0.8 mM VPA and 5 mM Y27632). Tissue culture plates were coated with 5 µg ml⁻¹ collagen IV (Corning) at 37 °C for 1 h. The cells were cultured at 37 °C with 5% CO₂. TSCs at 20–30 passages were harvested for analyses.