Hyaluronan

The cells were washed with phosphate-buffered saline (PBS), resuspended (1.0 × 10⁴ cells/ml) in DMEM/F-12 (Gibco) supplemented with 50 ng/ml EGF (R&D), 10 ng/ml bFGF (R&D), 25 μg/ml insulin (Gibco), and 2% B27 supplement (Gibco), and cultured for 4 days in ultra-low attachment surface plates (Corning) [21 (link)]. Images of spheroids were acquired with a BZ-700×0 microscope (Keyence), and the Heywood diameter of the spheroids was determined. The number of spheroids (>30 or >100 μm in Heywood diameter) in 5 low-power fields (×100) was counted [35 (link), 37 (link)]. Frequency of spheroids (>100 μm) (%) was calculated as follows: [the number of spheroid (>100 μm)/the total number of spheroids (>30 μm)] × 100. In some experiments, 4-methylumbelliferone (4MU) (100 μΜ) (Sigma), hyaluronan (100 μg/ml) (Seikagaku Corp.), SB431542 (10 μm) (R&D), anti-CD44 monoclonal antibody (mAb) (clone: BRIC235, 10 μg/ml) (IBGRL), or anti-activin A (betaA subunit) mAb (Clone: #69403, 10 μg/ml) (R&D) was added to the culture medium.

d-luciferin (Summit Pharmaceuticals International Corporation) was dissolved in saline or 1% hyaluronan (Seikagaku Corporation) to a final concentration of 15 mg/ml and then filtered.