Directed Differentiation of hESCs to hPGCLCs

Naive H9 and naive WIBR3 OCT4-dPE-GFP hESCs were propagated in serum-free N2B27 medium (N2 & B27; Life Technologies) supplemented with 20 ng/mL hLIF (Cambridge Stem Cell Institute [SCI]), 1 μM MEK inhibitor PD0325901 (SCI), 3 μM GSK3 inhibitor CHIR99021 (SCI), and 2 μM protein kinase C inhibitor Gö6983 (Sigma-Aldrich), as described previously (Guo et al., 2016 , Takashima et al., 2014 (link)). The medium was refreshed every day and cells were passaged every 4–5 days. hEpiLC were induced by plating 2 × 10⁵ naive hESCs on a well of a 6-well plate coated with growth factor reduced Matrigel (Corning) in N2B27 medium supplemented with 1 ng/mL TGF-β1 (Peprotech), 12 ng/mL bFGF (SCI), and 1% KSR (Gibco). The medium was changed every day. hPGCLCs were induced by plating 3–4 × 10³ day-4 hEpiLCs in a well of an Ultra-Low attachment U-bottom 96-well plate (Corning) in GK15 medium (Glasgow’s minimal essential medium [Life Technologies] with 15% KSR [Life Technologies], 0.1 mM non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.1 mM β-mercaptoethanol) supplemented with 500 ng/mL hBMP4 (R&D Systems), 20 ng/mL hLIF (SCI), 100 ng/mL hSCF (R&D Systems), and 50 ng/mL hEGF (R&D Systems). Cells were cultured in 5% O₂ and 5% CO₂ in a humidified incubator at 37°C.