Pet transwell membranes

Kidneys were dissected from E12.5 embryos and cultured at 37°C with 5% CO₂ on 0.4 µm PET Transwell membranes (Corning, New York, NY). The culture medium consisted of MEM (SIGMA, UK M5650), 10% FCS, and 1% Pen/Strep. Isolated mesenchyme was induced with spinal cord as previously described (Davies, 1994 (link); Davies and Garrod, 1995 (link)). Media was changed to contain IWR1 or CHIR after 48 hr and cultures kept for an additional 48 hr. Isolated Six2⁺ cells were pelleted for 5 min at 800×g and induced with spinal cord for 24 hr. The cultures were treated with CHIR or IWR1. CHIR99021 (University of Dundee, UK), ICG001 (Enzo LifeSciences, UK), IWR1 (TOCRIS), salinomycin (SIGMA), BIO (TOCRIS, UK), Ly294002 (TOCRIS), LDN-193189 (STEMGENT, Cambridge, MA), methotrexate (SIGMA), were used as specified in the text.

TER assay was conducted using the EVOM2 voltohmmeter (World Precision Instruments, Sarasota, Florida, USA), following the manufacturer’s instructions. RPE cells and RPE spheroids were seeded onto PET Transwell membranes (3 μm pore size, Corning) coated with Matrigel at a same density and cultured for 2 weeks. TER values were measured from three replicates on day 14. Background resistance was determined from a blank culture insert. The TER value (Ωcm²) was calculated using the following formula. $$\mathrm{TER}(\mathrm{\Omega }/{\text{cm}}^2)=({\text{R}}_{\text{total}}-{\text{R}}_{\text{insert}})/\text{A}$$
R_total represents the total resistance measured (Ω), R_insert represents the resistance of the blank insert, and A represents the membrane area (cm²) of the insert.

hESC-RPE cells were seeded at 1.5 × 10⁵ cells/cm² onto PET Transwell® membranes (Corning) coated with GFR-MG and cultured for 6 weeks in XFD-RMM (Table S1). Weekly TEER values (Ω•cm²) were obtained using an STX2 electrode (World Precision Instruments, Sarasota, FL, USA) from 3 replicate cultures each measured in triplicate. Background resistance was measured in triplicate equivalent wells containing no cells. Final values were obtained by subtracting background resistance from cell culture readings.