Isolation and Culture of Human LSCs and SESCs

Human LSCs and SESCs were isolated from donors and cultured in feeder-free plates as previously described^{17 (link)}. Briefly, the limbus tissues were isolated from postmortem human eyeballs and cut into small pieces, followed by digestion with 0.2% collagenase IV (Gibco) at 37 °C for 2 h and further digestion using 0.25% trypsin–EDTA (Gibco) at 37 °C for 15 min. Then, the cells were seeded and cultured on polystyrene plates (Corning) coated with 2% Matrigel (BD Biosciences). The components of LSC medium included DMEM/F12 and DMEM (1:1) with 1% penicillin/streptomycin, 10% fetal bovine serum, 10 ng/ml EGF, 5 μg/ml insulin, 0.4 μg/ml hydrocortisone, 0.1 nM cholera toxin, and 2 nM 3,3′,5-triiodo-l-thyronine. Human epidermal tissues were obtained from eye lids, cut into small pieces, digested, and seeded on coated polystyrene plates following the above steps. SESCs were cultured using LSC medium.