Lif1010

hPGCLCs were induced from hiPSCs via iMeLCs as described previously [5 (link)]. For the induction of iMeLCs, hiPSCs were plated at a density of 4×10⁴ to 5×10⁴ cells/cm² onto a human fibronectin (Millipore)-coated 12-well plate in GK15 medium (Life Technologies) with 15% KSR, 0.1 mM NEAA, 2 mM L-glutamine, 1 mM sodium pyruvate and 0.1 mM 2-mercaptoethanol) containing 50 ng/ml of ACTA (R&D Systems), 3 μM CHIR99021 (Tocris Bioscience) and 10 μM of a ROCK inhibitor. After 31 h, iMeLCs were harvested and dissociated into single cells with TrypLE Select. For induction of hPGCLCs, iMeLCs were then plated at 3,500 cells per well into a low-cell-binding V-bottom 96-well plate (Thermo Fisher Scientific) containing GK15 medium supplemented with 200 ng/ml BMP4 (R&D Systems, 314-BP-010), 100 ng/ml SCF (R&D Systems, 255-SC-010), 50 ng/ml EGF (R&D Systems, 236-EG), 10 ng/ml LIF (Millipore, #LIF1010) and 10 μM of Y-27632 (GK15+BSELY). The plates were incubated at 37°C under a 5% CO₂ atmosphere until use in downstream assays.

Briefly, human iPSCs (hiPSCs-99-2) were generated from dermal fibroblasts (Fib-99) of a 26-year-old male by overexpression of the Yamanaka factors OCT3/4, SOX2, KLF4 and c-MYC, as described previously [30 (link)]. hiPSCs-99-2 and ESCs (H1) were maintained on feeder in standard medium contained DMEM/F12 (Gibco, 11320-033), 20% KSR (Gibco, 10828-028), 2 μM L-glutamine (Sigma, G8540), 0.1 μM NEAA (Gibco, 11140-050), 0.1 μM 2-Mercaptoethanol (Gibco, 21985-023) and 10 ng/ml human bFGF (Invitrogen, PHG0021). Mouse ESCs were isolated and cultured in mouse ESC medium containing DMEM/F12 (Gibco, 11320-033), 20% KSR (Gibco, 10828-028), 1 μM sodium pyruvate (Sigma, 10828), 2 μM L-glutamine (Sigma, G8540), 0.1 μM NEAA (Gibco, 11140-050), 0.1 μM 2-Mercaptoethanol (Gibco, 21985-023) and 10 ng/ml LIF (Millipore, LIF1010). The medium was replaced daily. Male ESCs lines were identified by PCR using Sry gene.

Cells were passaged from 2D culture using L7^TM passaging solution in order to generate small clumps. Cells were plated with L7^TM medium onto L7^TM matrix coated plates at a 1:10 to 1:20 ratio depending on the harvest cell density. After 16–20 h, the medium was switched to Lonza neural induction medium (3 mL/well of a six well plate, day 0) consisting of primary neuron basal medium (PNBM, Lonza, CC-3256), 1x B27 (ThermoFisher, 17504044), 1x Glutamax (ThermoFisher, 35050-061), 4 µm CHIR99021 (Tocris, 4423), 3 µm SB43152 (Stemgent, 04-0010), and 10 ng/mL human leukemia inhibitory factor (hLIF, Millipore, LIF1010). The media was changed every other day until day 7. The cells were passaged by exposing cells to Accutase for 8–10 min. Y27632 (5 µm/mL) was added to the neural induction medium after plating. The cells were then plated at 1.0 × 10⁶/well in a six well plate that was pre-coated with poly-L-ornithine/Laminin (NSCs-P1). On the following day, the medium was changed using neural induction medium (no Y27632). The medium was changed every other day until cells reached 95%–100% confluency (4–5 days). The cells were then passaged as described above and re-inoculated into new pre-coated (Poly-L-Ornithine/Laminin) wells of a six well plate. Further expansion of the cells was conducted to enable flow cytometry and immunostaining at P3 and cryopreserved at P4.

46 C mESCs, kindly provide by professor Qi-Long Ying (University of Southern California), were seeded on 0.1% gelatin-coated cell culture plates at 37 °C in an incubator supplemented with 5% carbon dioxide. The composition of the mESC basic medium is DMEM (2122149, Biological Industries, Israel) supplemented with 10% FBS (FND500, ExCell Bio, Australia), 1× MEM nonessential amino acids (N1250, Solarbio, China), 0.1 mM β-mercaptoethanol (M3148, Sigma), and 1000 U/ml LIF (LIF1010, Millipore, USA). i-Gadd45g mESCs were maintained in basic medium supplemented with PD0325901 (1 μM, HY-10254, MedChemExpress) and CHIR99021 (3 μM, HY-10182, MedChemExpress). Human transgenes-free induced pluripotent stem cells were kindly provided by NuwaCell.Ltd, China (ZSSY-001) and were cultured in ncTarget medium (RP01020, NuwaCell.Ltd, China). MCF7 and Hs578T cells were purchased from the National Collection of Authenticated Cell Cultures (Chinese Academy of Sciences) and were cultured in DMEM medium supplemented with 10% FBS.

The 46C mouse ES (mES) cells were a gift from Dr. John Mason (University of Edinburgh, UK). The cell line was maintained in Glasgow’s Minimum Essential Medium (GMEM) (BHK-21; Gibco™, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, USA); 1% MEM non-essential amino acids (Gibco™, USA), 1 mM sodium pyruvate (Gibco, USA), 0.1 mM 2-mercaptoethanol (Gibco, USA), 2 mM L-glutamine (Gibco ™, USA), and 10 μg/mL human recombinant leukemia inhibitory factor (LIF 1010; Millipore, USA). The cells were seeded at a cell density of 4.0 × 10⁴ cells/cm² into 25 cm² cell culture flasks coated with 0.1% (w/v) gelatin (Sigma, USA). The cells were subcultured every other day when they were 70–80% confluent.

D425, D458, and HDMB03 cell lines were kind gifts from Yoon-Jae Cho (Oregon Health and Science University, USA). The D425 and D458 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin–streptomycin and 10% FBS. The histological features of the primary tumors of HDMB03 showed large cell medulloblastoma, with its gene expression markers consistent with the expression pattern of MYC-amplified MB. We maintained the HDMB03 cells in culture media with Neurobasal medium supplemented with B27 (Gibco), FGF (GF003, Merck Millipore, Darmstadt, Germany), EGF (02653, Stem Cell Technologies, Vancouver, BC, Canada), Heparin (07980, Stem Cell Technologies, Vancouver, BC, Canada), and LIF (LIF1010, Merck Millipore, Darmstadt, Germany).