Non essential amino acids (neaa)

ESCs carrying Dox-inducible Dmrt1 allele were seeded in 6-well plates on feeder cells at 2 × 10⁴ cells/well in TSC medium (RPMI 1640 [Gibco], 100× Nonessential amino acids [Nacalai Tesque], 100× sodium pyruvate [Gibco], 100 U/mL penicillin and 100 μg/mL streptomycin [Nacalai Tesque], 20% FBS [Gibco], 0.11 mM mercaptoethanol and 1.5 μg/mL heparin [Sigma], and 37.5 ng/mL FGF4 [Sigma]) containing 2.0 μg/mL Dox. After four days, the cells were passaged into 0.2% gelatin-coated 6-cm dishes, followed by a second and third passage into 10 cm dishes. From the first passage onward, the cells were cultured under the MEF medium without Dox. Second and third passages were performed when the cells were approximately 80% confluent. For inoculation of Dmrt1-induced cells, 3 × 10⁶ cells in 25 μL of PBS were injected into the testis of 6-week-old C57BL/6 N male mice (Japan CREA). Formed tumors were excised and evaluated at four weeks after the injection.

HuH-7, Huh-7.5, HepG2, and 293FT cells were grown in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum, 100 U/ml nonessential amino acids (Nacalai Tesque, Kyoto, Japan), and Antimycotic Mixed Stock Solution Penicillin 100 units/ml, Streptomycin 100 µg/ml, Amphotericin B 0.25 µg/ml (Nacalai Tesque, Kyoto, Japan). HuS-E/2 cells were cultured as previously described [10] (link). PHH were purchased from Gibco (Grand Island, NY, USA), the hepatocyte donor was a 58-year-old male who did not show any evidences of liver abnormalities. PHH were cultured in serum-free hepatocyte maintenance medium (Gibco, NY, USA) for one week before starting each experiment.

NMR cells were cultured in RPMI1640 medium (Wako, Osaka, Japan) supplemented with l-glutamate, 10% (vol/vol) foetal bovine serum (FBS) (EQUITECH-BIO, INC., Kerrville, TX, USA or SIGMA-ALDRICH, St. Louis, MO, USA), 1% penicillin/streptomycin (Nacalai Tesque, Kyoto, Japan), 0.1 mM non-essential amino acids (Nacalai Tesque), and 0.1 mM 2-2-mercaptoethanol. NMR cells were incubated at 32 °C in a 5% O₂ and 5% CO₂ atmosphere.

The culturing conditions for the reprogramming assays are summarized in Supplementary Fig. 16. In brief, cells were cultured in maintenance media with or without EGF, bFGF, or HGF for the first several days, then in GMEM (Wako) supplemented with 10% Knockout Serum Replacement (Thermo Fisher Scientific), 1 μM adrenocorticotropic hormone, 1 mM sodium pyruvate (Nacalai Tesque), 1 × nonessential amino acids (Nacalai Tesque), 100 μM β-mercaptoethanol, and 1000 U/ml of leukemia inhibitory factor (LIF) (Millipore), and/or N2B27 supplemented with 2i inhibitor and 1000 U/ml of LIF. The medium was replaced with ESC medium (GMEM supplemented with 10% FBS (Thermo Fisher Scientific), 1 mM sodium pyruvate, 1 × non-essential amino acids, 100 μM β-mercaptoethanol, and 1000 U/ml of LIF (Millipore)). Dox was added at the concentrations indicated in the figures. Media were replaced with fresh media every 2 or 3 days. For N31, N31P, and UOKMSCN, cultured cells were stained for alkaline phosphatase expression using ALP Staining Kit (Muto Pure Chemicals). For HOKMSCN, cultured cells were analyzed for EGFP expression as an indicator of Nanog expression using FACS BD Accuri (BD Biosciences). Statistical analyses were performed by F-test and one-sided Student’s t-test to test enhancement and repression of reprogramming efficiencies unless stated otherwise.

Cells were cultured in RPMI complete medium (RPMI 1640 medium (Nacalai tesque) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 U/mL penicillin (Nacalai tesque), 100 μg/mL streptomycin (Nacalai tesque), 1 mM sodium pyruvate (Nacalai tesque), 0.1 mM nonessential amino acids (Nacalai tesque), and 5 × 10⁻⁵ M 2-mercaptoethanol (Gibco)). Bone marrow-derived basophils (BMBAs) were generated by culturing bone marrow cells in the presence of murine IL-3 (300 pg/mL; BioLegend) for 6 days^{49 (link)}. BMBAs were then sensitized with anti-TNP IgE antibody (clone: IgE-Lb4: 1 μg/mL) for 24 h, followed by the magnetic enrichment of CD49b⁺ fractions by using biotinylated CD49b antibody (clone: DX5, catalog#: 108904, dilution 1:400; BioLegend) and MojoSort Streptavidin Nanobeads (BioLegend). BMBAs were then incubated with TNP-OVA or control OVA (10 ng/mL each) for 18 h. Ly6C^hi monocytes isolated from the bone marrow of BALB/c mice or Il4ra^−/−mice were incubated for 24 h or 48 h with the culture supernatant of BMBAs. In some experiments, Ly6C^hi monocytes (1 × 10⁵ cells/well) from WT mice were incubated for 24 h or 48 h with recombinant mouse IL-4 (20 ng/mL; BioLegend), apoptotic neutrophils (1 × 10⁶ cells/well) or control PBS.