Gibco™ DMEM/F-12, GlutaMAX™ supplement

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	All medium was changed once every 3 days after day 18

Protocol tips
All medium was changed once every 3 days after day 18

Publication protocol

The differentiation culture of hESCs was performed using the SFEBq (serum-free floating culture of embryoid body-like aggregates with quick reaggregation) method as previously described (Sakaguchi et al., 2019). In brief, hESCs were dissociated to single cells in TrypLE Express (Gibco) containing 1.25 U/ml DNase I (Takara Bio) and 10 μM Y-27632 (Wako), and quickly reaggregated using low-cell-adhesion-coated V-bottomed 96-well plates (PrimeSurface MS-9096V; Sumitomo Bakelite) in differentiation medium (9,000 cells per well, 100 μl) under 5% CO2. The differentiation medium was Glasgow’s MEM (Gibco) supplemented with 20% (vol/vol) KSR, 0.1 mM NEAA, 1 mM sodium pyruvate (Sigma-Aldrich), 0.1 mM 2-ME, 100 U/ml penicillin, and 100 μg/ml streptomycin. Defining the day on which the SFEBq culture was started as day 0, 5 μM SB431542 (transforming growth factor β inhibitor; TOCRIS) and 3 μM IWR1e (Wnt inhibitor; Calbiochem) were added to the culture from day 0 to day 18. 50 μM Y-27632 was added from day 0 to day 3. Half the medium was changed once every 3 days from day 3 to day 15. At day 18, the floating aggregates were transferred to 10-mm non- adhesive dishes (EZSPHERE; Iwaki) and further cultured in suspension using DMEM/F- 12 GlutaMAX (Gibco) supplemented with 1% (vol/vol) N-2 Supplement (Gibco), 1% (vol/vol) Chemically Defined Lipid Concentrate (CDLC; Gibco), 0.25 μg/ml Amphotericin B (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin under 40% O2/5% CO2 conditions. The floating aggregates were cut into halves or thirds with micro scissors (Bio Research Center, #16324319) under a stereo microscope at day 35, and the aggregates were transferred to gas-permeable dishes (Lumox dish; Sarastedt) at day 50. All medium was changed once every 3 days after day 18. When 5-ethynyl-2'- deoxyuridine (EdU) labeling of 6w- or 10w-organoids was performed, 10 μM EdU (Sigma-Aldrich) was added to the medium for 24 h.

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