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On the second day, the medium was switched to neural induction medium (50% of each DMEM/F12/GlutaMax and Neurobasal A, 1 GlutaMax, 50 ug/ml BSA, 1 N2 supplement, 1 B27 supplement without Vitamin A, 10 ng/ml human LIF, 4 M CHIR99021, 3 M SB431542, 0.1 M Compound E and 10 M Y-27632)