Advance dmem f12

Human cell line BG02 and H9 embryonic stem cells maintained on MEFs as previously described54 (link). To induce cortical neuroepthelial stem cells, the ESCs were digested into small clumps for suspension culture on low-cell-adhesion dishes in the medium containing Advance DMEM/F12 (Gibco): Neurobasal media (Gibco) (1:1), 1×B27 (Gibco), and 1% Glutmax (Sigma), 10 ng/mL bFGF (Gibco), 3 μM CHIR99021 (StemRD), 5 μM SB431542 (Cellagen technology), 0.2 μM Compound E (Calbiochem), 0.1 μM LDN193189 (Selleck), and 50 μg/ml Vitamin C (Vc, Sigma). After 6 days, EBs were transferred to 5 μg/ml laminin (Gibco) and poly-ornithine (Sigma, 10 μl/well)-coated plates for attachment culture and the media was switched to CHbFSB + LIF (NESC) culture medium35 (link)36 (link). The NESC culture medium is composed of Neurobasal media surplus with 1×B27, 1×N2, 1XNEAA (Sigma), 1% Glutmax (Sigma), 3 μM CHIR99021, 5 μM SB431542, 10 ng/ml bFGF, and 1000 U/ml hLIF (Millipore).

hESCs were digested into small clumps for suspension culture on ultra-low attachment plates (Corning, 3471) in the NESC-derived medium [26 (link)], which is composed of Advance DMEM/F12 (Gibco, 10565-018): Neurobasal media (Gibco, 21103-049) (1:1) supplemented with 1% N2 (Gibco, 17502-048), 2% B27 (Gibco, 17504-044), 1% Glutmax (Gibco, 35050-061), 10 ng/mL OsrbFGF (Oryza sativa recombinant human basic fibroblast growth factor, Wuhan Healthgen, China, HYC005M01), 3 μM CHIR99021 (Selleck, S2924), 5 μM SB431542 (Cellagen technology, C7243), 0.2 μM Compound E (Calbiochem, 565790), 0.1 μM LDN193189 (Selleck, S2618), and 0.1 mM β-mercaptoethanol (Sigma, M3148). After suspension culture for 6 days, neuron bodies (NBs) were digested into single cells and inoculated into ultra-low attachment plates with CHbFSB+LIF culture medium. The CHbFSB+LIF culture medium [26 (link), 28 (link), 31 (link)] is composed of Neurobasal medium, 1% N2, 2% B27, 1% NEAA (Gibco, 11140-050), 1% Glutmax, 3 μM CHIR99021, 5 μM SB431542, and 10 ng/ml OsrbFGF surplus with 1000 U/ml hLIF (Millipore, LIF1050).

The proliferating HepLPCs were dissociated with TrypLE Express (GIBCO), which was neutralized with pre-warmed medium, and then the cells were washed in phosphate-buffered saline (PBS). Cells were seeded at a density of 2 × 10⁵ proliferative cells per well in ultra-low attachment 6-well-plates (Corning). The culture media was based on Advance DMEM/F12 (GIBCO) supplemented with GlutaMAX-I (GIBCO), HEPES (Basal Media), Primocin (InvivoGen), 1 × B27 (GIBCO), 1.56 mM N-Acetylcysteine (Sigma, Germany), 0.5 μm A83-01 (Tocris, United State), 10 μm Y27632 (Selleck), 3 µM CHIR-99021 (SELLECK), 50 ng/ml EGF (PeproTech, United State) and 25 ng/ml HGF (PeproTech). Primary spheroids had formed by the next day. The medium was replaced every two or 3 days.