In this study, PGC lines from two males (W4 and W19) and one female (W2), derived from WL embryos, were used. PGC lines of two males (K8 and K10) and two females (K5 and K6), derived from
Kurokashiwa embryos, were also used. PGC lines were cultured as described by Whyte et al. (2015) [18 (link)] with slight modification. PGCs
(5 × 104 cells) were seeded in 500 μl of the culture medium in a 24-well plate (Greiner Bio-one, Stonehouse, UK). The total volume of the medium in each well was changed every 2
day. The culture medium contained avian Knockout Dulbecco’s Modified Eagle Medium (DMEM) basal medium (250 mOsm/kg, 12.0 mM glucose, and calcium-chloride-free [18 (link)]), B-27 supplement, 2.0 mM GlutaMax, 1 × non-essential amino acids (NEAA), 0.1 mM β-mercaptoethanol, 1 × nucleosides, 1.2 mM sodium pyruvate, 2 mg/ml ovalbumin (Sigma
Aldrich, St Louis, MO, USA), 100 μg/ml sodium heparin (Sigma Aldrich), 20 μg/ml ovotransferrin (Sigma Aldrich), 0.2% chicken serum, 25 ng/ml Human Activin A (PeproTech, Rocky Hill, NJ, USA),
and 4 ng/ml human basic fibroblast growth factor (FGF) (R&D Biosystems, Minneapolis, MN, USA). Unless otherwise specified, all reagents were purchased from Thermo Fisher Scientific
(Waltham, MA, USA).
Kurokashiwa embryos, were also used. PGC lines were cultured as described by Whyte et al. (2015) [18 (link)] with slight modification. PGCs
(5 × 104 cells) were seeded in 500 μl of the culture medium in a 24-well plate (Greiner Bio-one, Stonehouse, UK). The total volume of the medium in each well was changed every 2
day. The culture medium contained avian Knockout Dulbecco’s Modified Eagle Medium (DMEM) basal medium (250 mOsm/kg, 12.0 mM glucose, and calcium-chloride-free [18 (link)]), B-27 supplement, 2.0 mM GlutaMax, 1 × non-essential amino acids (NEAA), 0.1 mM β-mercaptoethanol, 1 × nucleosides, 1.2 mM sodium pyruvate, 2 mg/ml ovalbumin (Sigma
Aldrich, St Louis, MO, USA), 100 μg/ml sodium heparin (Sigma Aldrich), 20 μg/ml ovotransferrin (Sigma Aldrich), 0.2% chicken serum, 25 ng/ml Human Activin A (PeproTech, Rocky Hill, NJ, USA),
and 4 ng/ml human basic fibroblast growth factor (FGF) (R&D Biosystems, Minneapolis, MN, USA). Unless otherwise specified, all reagents were purchased from Thermo Fisher Scientific
(Waltham, MA, USA).
