Tryple express enzyme 1

Cells in the supernatant were dissociated into single cell suspension with TrypLE Express Enzyme (1×) (ThermoFisher) and resuspended in 2% FBS in PBS solution with the appropriate concentrations of the antibodies (Table S2) and DAPI (CyStain® DNA, PARTEC). Samples were analysed on BD™ LSR II flow cytometer (BD Biosciences). The data was analysed on BD FACSDiva software (BD Biosciences).

CRCOs were cultured using previously described methods (10 (link), 11 (link), 14 (link)). The culture medium of CRCOs was refreshed every 3 days. CRCOs were subcultured every 3–14 days depending on the growth rate of organoids. CRCOs were passaged by mechanical dissociation into small fragments through shearing with 1% Bovine Serum Albumin (BSA)-coated glass pipette tip. For those dense organoids, they were resuspended in prewarmed TrypLE™ Express enzyme (1×) (GIBCO, 12605-010) before mechanical dissociation. After dissociation, CRCOs were washed with cold PBS several times to clear out the Matrigel. Finally, CRCO fragments were resuspended in fresh Matrigel, seeded into a prewarmed 24-multiwell plate, and cultured as described above.
For CRCO cryopreservation, organoids were harvested and mechanically dissociated into small fragments as described above. Then, organoid fragments were mixed with freezing medium (CELLBANKER™ 2, ZENOAQ, 170905) and frozen following standard procedures. As required, the frozen CRCOs were thawed according to standard procedures and cultured as mentioned before. The culture medium was supplemented with 10 μM RHOK inhibitor Y-27632 for the first 3 days of culture after thawing.

Human limbal epithelial cells at passage two were harvested with 0.02% EDTA in PBS. After washing with PBS + Ca/Mg, a batch of cells was cultured as HLE non-sorted cells control. The remaining cells were spun down at 500 g for 5 min and resuspended in HRG (Ringer’s lactate solution containing 2.5% BSA and 0.4% glucose.); then incubated with magnetic beads coated with mouse anti-ABCB5 monoclonal antibody (mAb) (Clone 3C2–1D12) (TICEBA) for 25 min at RT under constant motion. The beads-bound cells (considered HLE ABCB5-positive cells) were magnetically separated from unbound cells (considered HLE ABCB5-negative cells). To detach the ABCB5-positive cells from beads, they were incubated with TrypLE™ Express Enzyme (1×) (Gibco™,Grand islan, NY, USA) for 3 min at 37 °C, 5%CO_2, then washed 3 times with HRG solution. HLE ABCB5-positive and negative fractions were cultured until 60 to 90% confluence before using them for the next experiment.

Flow cytometry analyses cells were detached by TrypLE™ Express Enzyme (1×) (Gibco™, Grand islan, NY, USA), counted, and blocked in PBS supplied with 0.5%BSA and 2 mM EDTA for 30 min at 37 °C, 5%CO₂. After 2 washes the cell suspensions were incubated with 10 μg/mL mouse anti-ABCB5 monoclonal antibody (mAb) (Clone 3C2–1D12) conjugated with FITC or isotype-matched control mAb Mouse IgG1 κ (Isotype Control (P3.6.2.8.1), FITC, eBioscience™) for 1 h at 37 °C, 5%CO₂; then washed twice with PBS supplemented with 2% FCS and incubated with 7-AAD (Biolegend)1:100 for 5 min at RT before analyzing. Subsequently, the cells were analyzed using a BD FACSCanto™ II (BD Biosciences, Qume Dr San Jose, CA, USA. Forward scatter (FSC) and side scatter (SSC) were used to remove cell debris and doublets, and 7AAD-negative cells were defined as live limbal epithelial cells. The ABCB5+ live population was defined as the fraction of live limbal epithelial cells with the highest contrast against the isotype control. The data were analyzed using the FlowJo (version 10, BD Biosciences, Qume Dr San Jose, CA, USA) software program.

JAR cells (5 × 10⁵ cells) were seeded into 60 mm disks and cultured for 24 h. The culture medium was then replaced with a fresh complete growth medium plus a final concentration of 5 μM cyclopamine or 0.5 μg/ml recombinant human Shh and cultured for the next 24 h. Complete growth medium plus an equal volume of alcohol was used as a negative control. At the endpoint of treatment, cells were gently detached by TrypLE™ Express Enzyme (1×) (Gibco by Life Technologies, USA). Cells were washed and resuspended in 100 μl binding buffer, and 5 μl PE-conjugated Annexin V and 5 μl 7-AAD were added immediately thereafter and incubated for 15 min in the dark at room temperature. After incubation, 400 μl of binding buffer was added to all samples, and apoptosis was immediately quantified by using a FACSCanto II flow cytometer (BD Biosciences, USA) according to the manufacturer’s instructions.