Induction of PGCLC from Dissociated Cells

For PGCLC induction, cells were dissociated by incubation with Accutase and then transferred into a low-cell-binding U-bottom 96-well plate (Greiner, #650970) in GK15 medium (GMEM (Thermo, #11017035) containing 15% KSR, 0.1 mM NEAA, 1 mM sodium pyruvate (Thermo, #11360070), 2 mM GlutaMax, 0.1 mM 2-mercaptoethanol, and 1 × penicillin/streptomycin) supplemented with 200 ng/ml human BMP4 (R&D Systems, #314-BP-01 M), 100 ng/ml mouse SCF (R&D Systems, #455-MC-500), 1000 U/ml human LIF (Wako, #125-06661), 50 ng/ml human EGF (R&D Systems, #236-EG) and 10 μM ROCK inhibitor with or without 2.5 μM IWR1 (TOCRIS, #3532) and 1.0 µM BMS493 (R&D Systems, #3509/10). In each well, 5 × 10³ cells were used. In the experiments requiring enforced expression of the transgenes, 100 μM dexamethasone, 0.5 μg/ml doxycycline, and 0.5 μM Shield-1, which induce SOX17, BLIMP1, and TFAP2C expression, respectively, were added to the medium^{11 (link)}. To enforce the expression of SOX17 alone, only 100 μM dexamethasone was added.