Sodium pyruvate solution

Xylan, stearic acid (Sa), 4-(dimethylamino)-pyridine (DMAP), anhydrous dimethyl sulphoxide (DMSO), dichloromethane (CH₂Cl₂), and N, N′-dicyclohexylcarbodiimide (DCC) were all provided by J&K Scientific Ltd. (Shanghai, China). Capecitabine (Cap) was obtained from Macklin Inc. (Shanghai, China). RPMI 1640 medium, fetal bovine serum (FBS), glucose solution, sodium pyruvate solution, trypsin-EDTA solution, and 4’,6-diamidino-2-phenylindole (DAPI), fixation/permeabilization concentrate, permeabilization buffer, and fixation/perm diluent were all purchased from Thermo Fisher Scientific Inc (Waltham, USA). 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide (DiR), D-luciferin potassium, penicillin G sodium solution, streptomycin sulfate solution and dialysis bag (MWCO 3.5 kDa) were purchased from Meilun Biotech Co., Ltd, (Dalian, China). All other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) with analytical grade and used without further purification.

Murine squamous cell carcinoma (SCCVII) cells [22 (link)] (generously provided by Walter T. Lee, Duke Cancer Institute, Durham NC) were grown in 10% RPMI-1640 with L-glutamine (ThermoFisher Scientific, Waltham, MA) supplemented with 10% heat-inactivated FBS (Atlas Biologicals, Fort Collins, CO), MEM Eagle Nonessential Amino Acid Solution, Penicillin-Streptomycin, L-glutamine, HEPES Buffer (Lonza, Basel, Switzerland), and Sodium Pyruvate Solution (ThermoFisher Scientific). Antibodies for flow cytometry include anti-mouse CD8α-PerCP-Cy5.5 (clone 5H10, ThermoFisher Scientific), CD4-FITC (clone RM4-5, eBioscience, San Diego, CA), CD154/CD40L-PE (clone MR1, eBioscience), anti-mouse IFNγ-APC (clone XMG1.2, eBioscience) and TNF-PE-Cy7 (clone MP6-XT22, BD Biosciences, Franklin Lanes, NJ). Peptides for restimulation were synthesized by A&A Labs (San Diego, CA). H-2K^k MHC-tetramers incorporating the defined EEKKGNYV peptide (EGFRvIII murine epitope) were obtained from the NIH Core Facility at Emory University, (Atlanta, GA).

All DMG cell lines used in the study (except for SF7761) were cultured in ultra-low attachment flasks in culture medium with 1:1 ratio of DMEM/F-12 (Invitrogen, 11330-032) and Neurobasal A (Invitrogen, 10888-022) and ten percent each of HEPES Buffer Solution 1 M (Thermo Fisher, 15630080), Sodium Pyruvate solution 100 nM (Life Technologies, 11360070), MEM non-essential amino acids solution 10 mM (Thermo Fisher, 11140050), Glutamax-I Supplement (Thermo Fisher, 35050061), and Penicillin/Streptomycin solution (Life Technologies, 15140122). The media was supplemented with B27 Minus Vitamin A (Invitrogen, 12587-010), epidermal growth factor (EGF; StemCell Tech. Inc., 78006), fibroblast growth factor (FGF; GF003, StemCell Tech., Inc., 78003) and heparin solution, 0.2% (StemCell Tech. Inc., 07980), as well as PDGF-AA (Shenandoah Biotech, 100-16) and PDGF-BB (Shenandoah Biotech, 100-18). SF7761 cell line was cultured in medium with Neurobasal A (Invitrogen, 10888-022) and N-2 Supplement (Invitrogen, 17502), further supplemented with B27 Minus Vitamin A (Invitrogen, 12587-010), epidermal growth factor (EGF; StemCell Tech. Inc., 78006), fibroblast growth factor (FGF; GF003, StemCell Tech. Inc., 78003) and heparin solution, 0.2% (StemCell Tech. Inc., 07980). Cells were dissociated using Accutase (StemCell Tech. Inc., 07922) and passaged every 3 or 4 days.

RPMI-1640 was from Sigma-Aldrich (St. Louis, MO, USA) and Nakarai Tesque (Kyoto, Japan). Phosphate buffered saline (PBS), citric acid and 0.5M EDTA solution were from Wako Pure Chemical Corp. (Osaka, Japan). TRIsure™ reagent was from Nippon Genetics (Tokyo, Japan). Fetal bovine serum (FBS) was from HyClone (Logan, UT, USA). Normal rat IgG was from MP Biomedicals (Santa Ana, CA, USA) and neutralizing antibody against mouse GM-CSF (clone MP122E9) was from BioLegend (San Diego, CA, USA). Thioglycollate (TG) was from Difco Laboratories (Detroit, MI, USA). L-Glutamine, penicillin/streptomycin, trypsin-EDTA, sodium pyruvate solution and RNAlater were from Life Technologies (Gaithersburg, MD, USA). Proteinase K and the High Pure RNA Isolation Kit were from Roche (Mannheim, Germany). Anti-Ly6G rat monoclonal IgG (clone 1A8) and anti-FoxP3 rat monoclonal IgG (clone MF-14) were from BioLegend. Anti-F4/80 rat monoclonal IgG (clone BM8) was from eBioscience (San Diego, CA, USA).

For teratoma-derived cell and keratinocyte cultures, 3.0 × 10⁶ (teratoma-derived cells) and 1.5–1.8 × 10⁷ cells (keratinocytes) were placed into the system and cultured using the media described above. The medium exchanges were performed twice a week.
For EB-explant outgrowth culture, iPSCs were dissociated into single cells with accutase (Thermo Scientific, MA, USA) after exposure to the rock inhibitor (Y-27632: A11105-01, Wako, Japan) and then passaged into the 90 mm dishes coated with 0.1% gelatin solution (Sigma-Aldrich, St. Louis, MO, USA) at a density of 10,000 cells/dish in the EB medium containing 76% Knockout DMEM, 20% Knockout Serum Replacement (Life Technologies, CA, USA), 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, and 50 μg/mL L-ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, MO, USA). Thereafter, the EBs collected from two dishes were placed into the system and cultured using DMEM/Nutrient Mixture F-12 (Life Technologies) supplemented with 20% FBS, 1% Pen-Strep solution, 1% NEAA (Life Technologies), 1% sodium pyruvate solution (Life Technologies), and GlutaMAX supplement (Life Technologies). The medium exchanges were performed twice a week. When sufficient cell outgrowth from EBs was observed, the cells were passaged (on day 7), and the cultivation was continued further.

RPMI-1640 medium was purchased from Sigma-Aldrich (St. Louis, MO, USA) and Nakarai Tesque, Kyoto, Japan. Dulbecco’s modified Eagle’s medium (DMEM) with high glucose was from Nakarai Tesque. Phosphate buffered saline (PBS) and citric acid were from Wako Pure Chemical Corp, Osaka, Japan. TRIsure^® reagent was from Nippon Genetics, Tokyo, Japan. Fetal bovine serum (FBS) was from HyClone, Logan, UT, USA and Gibco, Grand Island, NY, USA. L-glutamine, penicillin/streptomycin, trypsin-EDTA, sodium pyruvate solution and RNAlater were from Life Technologies, Gaithersburg, MD, USA. High Pure RNA Isolation Kit was from Roche, Mannheim, Germany. BCA protein assay kit was from Takara, Tokyo, Japan. A mouse monoclonal Ab against human PDGF-A (clone E-10) was from Santa Cruz, Dallas, TX, USA. Anti-Ly6G rat monoclonal IgG (clone 1A8), recombinant mouse PDGF-AA and BB were from BioLegend, San Diego, CA, USA. Anti-F4/80 rat monoclonal IgG (clone BM8) was from eBioscience, San Diego, CA, USA. A rabbit polyclonal Ab against αSMA was from Abcam, Cambridge, MA, USA. Trapidil and crenolanib were from Selleck Chemicals, Osaka, Japan.

A panel of 30 human cell lines including MLL-r leukemias, MLL-wt leukemias, solid tumour cell lines and control, non-malignant cells was used for in vitro experiments (Table 2). The PER-485, PER-490, PER-703A, PER-785A, PER-826A cell lines were established from infant patients with pre-B cell acute lymphoblastic leukemia [75 (link), 76 (link)]. The MOLM-13 cell line was kindly provided by R. D'Andrea, IMVS, South Australia, Australia. THP-1 cells were a kind gift from W. Jessup, Centre for Vascular Research, New South Wales, Australia. Hal-01 and UOC-B1 cells were kindly provided by A. Thomas, Dana-Farber Cancer Institute, Massachusetts, USA. BE(2)-C cells were a kind gift from J. Biedler, Memorial Sloan-Kettering Cancer Centre, NY, USA. All other cells were purchased from ATCC (Manassas, Virginia, USA) or DSMZ (Braunschweig, Germany). Cells were maintained in DMEM, IMDM or RPMI 1640 media containing 20% or 10% Foetal Calf Serum (FCS), 1% Non-essential Amino Acid Mix (100x) and 1% 100mM Sodium Pyruvate Solution (100x) (Life Technologies, Scoresby, Victoria, Australia).