N1250

46C mESCs, kindly provided by Qi-Long Ying (University of Southern California, USA), were cultured in 0.1% gelatin-coated dishes at 37°C in 5% carbon dioxide. The medium used for routine maintenance was DMEM (Biological Industries, Israel) supplemented with 15% FBS (FND500, ExCell Bio, Australia), 1× MEM nonessential amino acids (N1250, Solarbio, China), 0.1 mM β-mercaptoethanol (M3148, Sigma) and 1000 U/ml LIF (LIF1010, Millipore, USA). Cells were digested by 0.05% Trypsin-EDTA solution every 2–3 days.

The 46C mouse ESCs were kindly provided by Qi-Long Ying (The University of Southern California) and were cultured in 0.1% gelatin-coated dishes at 37 °C in 5% CO₂. The conventional cell culture conditions were Dulbecco's modified Eagle's medium (Biological Industries) supplemented with 10% fetal bovine serum (FND500, ExCell Bio), 1× MEM nonessential amino acids (N1250, Solarbio), 1× penicillin/streptomycin (P1400, Solarbio), 0.1 mM β-mercaptoethanol (M3148, Sigma), and LIF (made in house). Human transgene-free iPSCs were kindly provided by Nuwacell Ltd (ZSSY-001) and were cultured in ncTarget (RP01020, Nuwacell Ltd). Cells were dissociated using EDTA solution (RP01007, Nuwacell Ltd) every 3 to 5 days.

We mainly referred to two previous studies for the induction protocols (14 (link), 18 (link)). Briefly, for mouse PGCLC formation, 1 × 10⁵ 46C ESCs were seeded in fibronectin bovine plasma (16.7 μl/ml, F1141-5MG, Sigma) coated plate and were cultured in activin A (20 ng/ml, C678, Novoprotein) and basic fibroblast growth factor (12 ng/ml, C044, Novoprotein) containing medium to induce EpiLCs. The medium was changed every day. After 2 days, 3 × 10⁵ EpiLCs were incubated in GK15 medium, consisting of Glasgow's minimal essential medium (11710035, Gibco), 15% KSR (10828028, Invitrogen), 1× penicillin/streptomycin (P1400, Solarbio), 1× MEM nonessential amino acids, 0.1 mM β-mercaptoethanol, and 1 mM sodium pyruvate (N1250, Solarbio), to generate PGCLCs in the presence of BMP4 (500 ng/ml, 315-27-10, PeproTech), LIF (1000 U/ml, Millipore), SCF (100 ng/ml, AF-250-03, PeproTech), and EGF (50 ng/ml, AF-100-15, PeproTech).
For human PGCLC induction, 1 × 10⁵ human iPSCs were differentiated into iMeLCs with 50 ng/ml activin A, 3 mM CHIR99021, and 10 mM ROCK inhibitor (Y27632). iMeLCs (3 × 10⁵) were then incubated in GK15 medium supplemented with BMP2 (200 ng/ml, C012, Novoprotein) or BMP4 (500 ng/ml, 120-05-5, PeproTech), LIF (1000 U/ml, Millipore), SCF (100 ng/ml, C034, Novoprotein), EGF (50 ng/ml, AF-100-15, PeproTech), and 10 mM ROCK inhibitor for 4 days.