Fibroblast growth factor 2 (fgf2)

HPS cells were differentiated to the hematopoietic lineage as previously described (Ng et al., 2008 (link); Ng et al., 2005 (link)). The day prior to differentiation, iPS cells were passaged with TrypLE Express at a high density in mTeSR with Rock inhibitor. On Day 0 of the differentiation, iPS cells were dissociated into a single cell suspension and plated in a 96-well U-bottom plate (Genesee Scientific) at 3000 cells per well in APEL2 (StemCell Technologies) with 40 ng/mL BMP4 (R and D Systems), 40 ng/mL SCF (R and D Systems), 20 ng/mL VEGF (R and D System), 10 ng/mL FGF-2 (StemCell Technology), and 5 nM Rock inhibitor. On Day 2 of the differentiation, the embryoid bodies (EB) were treated with F7L6 (5 nM), Wnt3a (5 nM), CHIR (250 nM) or an equivalent volume of Wnt storage buffer. On Day 4 of the differentiation, the media was removed from the EBs and replaced with fresh APEL2 with BMP4, SCF, VEGF, and FGF-2. On Day 7 of the differentiation, EBs were transferred to gelatin coated plates. On Day 8 of the differentiation one volume of APEL with BMP4, SCF, and VEGF was added. EBs were dissociated on Day 14 of the differentiation for analysis by flow cytometry.

pd‐GBSCs were cultivated under stem cell conditions following the procedure described in [18 (link)] in order to retain as many of the original tumour features as possible. Cell expansion and subsequent experiments were conducted in Dulbecco's Modified Eagle Medium (DMEM/F12, Cat. No: 1331020, Gibco, Waltham, MA, USA) supplemented with N2 (100×: 25 μg·mL⁻¹ insulin (Cat. No: I6634, Sigma, Darmstadt, Germany)), 100 μg·mL⁻¹ apo‐transferrin (Cat. No: 11108016, Gibco), 50 μg·mL⁻¹ bovine serum albumin fraction V 7.5% (Cat. No: 15260037, Gibco), 0.02 μg·mL⁻¹ progesterone (Cat. No: P8783, Sigma), 16 μg·mL⁻¹ putrescine (Cat. No: P5780, Sigma) and 5.18 ng·mL⁻¹ sodium selenite (Cat. No: S5261, Sigma), 1× B‐27 (Cat. No: 17504044, Gibco), 1 mm sodium pyruvate (Cat. No: S8636, Sigma), 500 units·mL⁻¹ penicillin/streptomycin (Cat. No: P4333, Sigma) and supplemented daily with 10 ng·mL⁻¹ EGF (Cat. No: 78006.1, Stem Cell Tech., Vancouver, BC, Canada), and 10 ng·mL⁻¹ FGF‐2 (Cat. No: 78003, Stem Cell Tech). Cells were cultivated in flasks and plates previously coated with 10 μg·mL⁻¹ laminin (Cat No: L2020, Sigma) for 4 h at 37 °C. Cell cultures were incubated at 37 °C in a humidified atmosphere with 5% CO₂ and were routinely checked to ensure they were mycoplasma‐free (Mycoplasma Gel Detection Kit, Cat. No: 90.022‐4544, Biotools, Madrid, Spain).

TS603, TS667, TS543 were obtained from Memorial Sloan Kettering Cancer Center (MSKCC). SU-AO3, NCH612 glioma lines were described previously [23 (link)]. The patient-derived IDH1-mutant glioma lines were routinely subjected to panel sequencing analysis [26 (link)] and immunoblotting to confirm the expression of mutant IDH1 (R132H). Glioma lines were maintained as neurospheres in NeuroCult (StemCell Technologies) with 2 µg/ml heparin sulfate (Sigma), 20 ng/ml of EGF (StemCell Technologies), and FGF2 (StemCell Technologies). PC12 cells (provided by Dr. Agarwal, Heidelberg University; Dr. Chang, Gachon University) were maintained in DMEM (D5796, Sigma) supplemented with 10% horse serum (H1138, Sigma) and 1% penicillin–streptomycin. Human neural progenitor cells (SCC008, Sigma) were maintained in DMEM (D5796, Sigma) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin. All cells were grown in a 5% CO₂ humidified incubator at 37 °C.

To determine the osteogenic stimulatory effect of BMP-2 and FGF-2 on BM-MSCs, the cells were treated with 100 ng/mL of BMP-2 (Stem Cell Technologies, Grenoble, France, cat. no. 78004.1) and/or 20 ng/mL FGF-2 (Merck, Saint Louis, MO, USA, cat. no. F0291). BM-MSCs cultured in the αMEM complete medium were used as a control. The supplemented and control media were changed every three days. The experiments on osteogenic induced BM-MSCs were performed after 7, 14, and 21 days of incubation.