Nucleoside

mESC lines (EB5 (ref. 38 (link)); passages 35–45, LMX1A::GFP KI ESCs; passages 11-21, G4-2 (ref. 38 (link)); passages 20–30) and iPSC line (440A-3, a kind gift from Dr Okita, Kyoto University Center for iPS Cell Research and Application, Kyoto, Japan; passages 15–25) were maintained on mitotically inactivated mouse embryo fibroblast feeder layer in knockout DMEM medium supplemented with 1% penicillin/streptomycin (P/S; Gibco), 20% fetal bovine serum (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (2-ME; Wako), 2 mM L-glutamine (L-Glu; Sigma-Aldrich), 2,000 U ml⁻¹ LIF (Merck Millipore) and 1 × Nucleosides (Merck Millipore). We changed the medium every day.
For neural induction, mESCs and miPSCs were replated in low cell adhesion 96-well plates (Lipidure-Coat Plate A-96U; NOF Corporation) at a density of 9,000 cells per well in a differentiation medium containing Glasgow minimum essential medium (GMEM) (Gibco) supplemented with 5% KSR, 0.1 mM MEM non-essential amino acids solution (Gibco), 2-ME, 1 mM sodium Pyruvate solution (Pyruvate; Sigma-Aldrich) and 2 mM L-Glu. Moreover, we added both 100 ng ml⁻¹ FGF8b (R&D) and SHH (R&D) to induce midbrain and FP cells, respectively, from day 1 to day 6. On day 7, we added 200 nM Ascorbic acid (AA; Nacalai), 20 ng ml⁻¹ brain-derived neurotrophic factor (BDNF) (R&D), 1 × N-2 supplement (Gibco) and removed 5% KSR. We changed the medium every 2 days.

Mouse AG- and PG-haESCs were cultured on feeder cells with ES medium or 2i culture medium^{54 (link)}. ES medium consisted of DMEM with 15% fetal bovine serum (Thermo Fisher Scientific), 1% nucleosides (EMD Millipore), 1% glutamax (Thermo Fisher Scientific), 1% non-essential amino acid (Thermo Fisher Scientific), 1% β-mercaptoethanol (EMD Millipore), 1000 U/ml leukemia inhibitory factor (LIF; EMD Millipore), 100 U/ml penicillin and 100 μg/ml streptomycin. 2i culture medium contained the above ES medium supplemented with 3 μM CHIR99021 (Tocris) and 1 μM PD0325901 (Tocris). In general, haESCs were cultured in ES medium, unless noted otherwise. Feeder cells were not used in most of our experiments except in haploid percentage analyses, morphology analyses, and alkaline phosphatase staining. For single colony assay, 16 single colonies were segregated from PG-haESCs (the 319 line), and sequentially passaged in ES medium supplemented with 2i, PD166285/2i, or RDF/PD166285/2i.

R1 ES cells were purchased from American Type Culture Collection (ATCC) and cultured on mitomycin C-treated MEFs in ES medium containing DMEM (Merck Millipore) supplemented with 15% (v/v) fetal bovine serum (HyClone), 1-mM l-glutamine (Merck Millipore), 0.1 mM mercaptoethanol (Merck Millipore), 1% nonessential amino acid stock (Merck Millipore), penicillin/streptomycin (100×, Merck Millipore), nucleosides (100×, Merck Millipore) and 1000 U ml⁻¹ LIF (Merck Millipore). These cells tested negative for mycoplasma contamination.

mESCs (EB5; passages 35–45) and CTIP2:GFP KI mESCs (passages 11–21) were maintained on a mitotically inactivated mouse embryonic fibroblast feeder layer in KnockOut DMEM (Thermo Fisher Scientific) supplemented with 20% (vol/vol) Fetal Bovine Serum (FBS; Merck), 1% PS, 0.1 mM 2-ME, 2 mM L-Gln, 2,000 U ml⁻¹ Leukocyte Inhibitory Factor (LIF; Merck) and 1% (vol/vol) Nucleosides (Merck). The culture medium was replaced with fresh medium every day. To induce cortical neurons, the mESCs were replated in prime surface 96-well plates (Sumitomo Bakelite) at a density of 9,000 cells per well in differentiation medium containing GMEM (Thermo Fisher Scientific) supplemented with 10% (vol/vol) KnockOut Serum Replacement (KSR; Thermo Fisher Scientific), 0.1 mM MEM non-essential amino acids solution (NEAA; Thermo Fisher Scientific), 0.1 mM 2-ME, 1 mM sodium Pyruvate solution (Pyruvate; Merck) and 2 mM L-Gln. 10 μM SB-431542 (Merck), which is a TGF-β receptor inhibitor, and 20 nM WNT-C59 (Collagen Technology, San Diego, CA, USA), which is a WNT inhibitor, were added by day 6. On day 7, we switched the medium from GMEM to DMEM/F12 (Fujifilm) supplemented with 0.1 mM 2-ME, 2 mM L-Gln, 1% N2 supplement and 2% B-27 supplement. Half of the medium was replaced with fresh medium every 3 days.