Ascorbic acid

DN differentiation was induced as previously described with minor modifications (brain derived neurotrophic factor [BDNF] was excluded in the second step) [16 ]. In the first step, 1.5 × 10⁵ SHED were plated onto a 6-well culture plate or glass coverslips coated with 0.01% poly-L-lysine (Sigma-Aldrich), in the same culture medium as described above. They were incubated overnight at 37 °C in the presence of 5% CO₂, and were then cultured in serum-free Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 20 ng/mL epidermal growth factor (Sigma-Aldrich), 20 ng/mL basic fibroblast growth factor (Peprotech, NJ, USA), and 1% N2 supplement (Life Technologies) for 2 days at 37 °C, in the presence of 5% CO₂. In the second step, DMEM was replaced with neurobasal medium (Life Technologies) supplemented with 2% B27 supplement (Life Technologies), 1 mM dibutyryladenosine 3,5-cyclic monophosphate (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), and 200 μM ascorbic acid (Nacalai Tesque, Kyoto, Japan), and cells were incubated for 5 days, at 37 °C, in the presence of 5% CO₂.

For the induction of 3D osteogenic induction from MC3T3-E1 and MLO-Y4, 1.5 X 10⁶ or 6 X 10⁵ hiPSCs were seeded in a 3.5-cm glass bottom dish (for confocal imaging) or a 12-well plate (for RNA extraction and tissue sectioning) coated by collagen gel, and cultured with osteogenic induction medium, which was composed of α-MEM (Gibco) supplemented with 10% FBS (Hyclone), 0.1 mM dexamethasone (Sigma), 50 mg/mL ascorbic acid (Nacalai Tesque), and 10 mM β-glycerophosphate (Sigma)) and the medium was refreshed at day 2, 4, 7, 9, and 11.

Cells were seeded at 2 × 10⁴ cells per well in 24-well plates (Becton Dickinson Labware, Lincoln Park, NJ) in 10% FBS/α-MEM as control medium (CM) or CM containing 2 mM β-glycerophosphate (Sigma, St. Louis, MO), 50 mg/ml ascorbic acid (Nacalai Tesque, Kyoto, Japan), and 1 × 10⁻⁷ M dexamethasone (Merck Millipore, Darmstadt, Germany) as osteoblastic differentiation medium (DM). Half of the medium was exchanged every 2 or 3 days. After 3 weeks of culture, the cells were fixed with 4% paraformaldehyde (PFA; Merck Millipore) and then washed with distilled water and stained with Alizarin red S as described previously [9 (link)]. The area of each Alizarin red S-positive region was imaged under a Biozero digital microscope (Keyence, Osaka, Japan). Total RNA was isolated from each culture after 3 days.

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.