Af 100 31

mTSCs were obtained from the Zhou laboratory (Institute of Zoology, Chinese Academy of Sciences, Beijing, China) and isolated from the mouse blastocyst [50 (link)]. Briefly, there were four steps, prepare 4-well plates of mouse embryonic fibroblasts, collect blastocysts, disaggregate blastocyst outgrowths and isolate mTSC colonies. mTSCs were grown as previously described [51 (link)], at 37 °C in 95% air and 5% CO₂. mTSCs medium contained 20% fetal bovine serum (FBS; 04-001-1A, Biological Industries, Israel), 1 mM sodium pyruvate (11360-070, Gibco, USA), 50 μg/mL penicillin/streptomycin, 5.5 × 1 0⁻⁵ M β-mercaptoethanol (21985023, Gibco, USA), 25 ng/mL basic fibroblast growth factor (FGF4) (AF-100-31, PeproTech, USA), and 1 μg/mL heparin (07980, Stemcell, Canada) in RPMI 1640 (11875-093, Gibco, USA), with 70% of the medium preconditioned (mTSC culture medium without heparin and FGF4) by incubation with embryonic fibroblasts for 72 h.

For all spheroid culture-based assays where inhibitors or growth factors were used, cells were seeded into medium containing the vehicle (DMSO or H₂O), inhibitor, or growth factor. The following day, media were aspirated and replaced with methylcellulose-containing media that also contained the vehicle, inhibitor, or growth factor. The vehicle concentration was 0.1% for all experiments. Inhibitors used in these experiments are as follows: YKL-05-099 (MedChem Express HY-101147), apilimod (MedChem Express HY-14644), and CQ (MedChem Express HY-17589A). Growth factors used in these experiments are as follows: EGF (PeproTech AF-100-15), FGF4 (PeproTech AF-100-31), and HGF (PeproTech 100-39H).