Knockout serum replacement (ksr)

DE cells that were FOXA2 and SOX17 dual positive were differentiated to generate hepatocyte progenitor cells by combining methods as published before [9 (link), 17 (link)]. Briefly, DE cells were maintained for 7 days in KnockOut DMEM (Thermo Fisher Scientific, Cat. no. 10829018) containing 20% knockout serum replacement (STEMCELL Technologies, Cat. no. 10828028), 1% nonessential amino acids MEM (HyClone, Cat. no. SH40003.01), 0.1 mM beta-mercaptoethanol (Thermo Fisher Scientific, Cat. no. 31350010), 1% L-glutamine (Thermo Fisher Scientific, Cat. no. 24030081), and 1% DMSO (Sigma, Cat. no. D8418). Media were changed every alternate day.
In the second method, as published by Carpentier et al., DE cells were maintained in hepatocyte progenitor media as described above along with 100 ng/ml HGF for 7 days [6 (link), 10 (link)] and media were replaced every alternate day.

Forebrain organoids were generated as previously described^{60 (link)}. On Day 0, iPSC colonies were detached by ReLeSR and aggregated to form EBs by Aggrewell (STEMCELL Technologies, #34811). The following day, EBs were resuspended and transferred to 6-well plate (Corning Costar) rotating at 110 rpm, containing DMEM/F12, 20% KnockOut Serum Replacement, 1X Non-essential Amino Acids, 1X GlutaMax, 1 μM LDN193189, 5 μM SB-431542 and 2 μg/mL heparin (STEMCELL Technologies). On Day 6, half of the medium was replaced with induction medium consisting of DMEM/F12, 1X N2 Supplement, 1X Non-essential Amino Acids, 1X GlutaMax, 1 μM CHIR99021 and 1 μM SB-431542. On Day 7, organoids were embedded in Matrigel and cultured stationarily in Low-attachment plate (Corning Costar) in the induction medium. On Day 14, organoids were mechanically dissociated from Matrigel by manual pipetting in a 10 mL pipette tip. Organoids were transferred to 6-well plate rotating at 110 rpm, containing differentiation medium consisting of DMEM:F12/Neurobasal, 1X N2 and 1X B27 Supplements, 1 × 2-Mercaptoenthanol, 1X Non-essential Amino Acids, and 2.5 mg/ml human Insulin. From Day 35 to Day 60, 1% Matrigel was supplemented in differentiation medium.