Basic fibroblast growth factor (bfgf)

Multiple doses of growth factors were used to determine the optimal dose to use for subsequent studies (data not shown). Vascular endothelial growth factor (VEGF; R&D Systems) was used at 10–20 ng/mL, hepatocyte growth factor (HGF; R&D Systems) at 100 ng/mL basic fibroblast growth factor (bFGF; Invitrogen) at 50 ng/mL, and epidermal growth factor (EGF; Invitrogen) at 20 ng/mL. To block VEGF and HGF signaling, antibodies to VEGF (Bevacizumab; 10 µg/mL) and HGF (R&D Systems; 10 µg/mL) were used. To examine whether additional growth factors were dependent on each other to induce cords, inhibitors to VEGFR2 (Ramucirumab), bFGF (bFGF antibody; Invitrogen), or EGFR (Gefitinib) were used in basal, VEGF, bFGF, and EGF driven cords. Finally, a TGF-β inhibitor (LY2157299), an inactive control (LY596144), and a multikinase inhibitor (SU11248, Sutent) were tested to examine increases in cord or tumor vessels.

Growth factors for the experimental treatment of DPSC isolates included Epidermal growth factor or EGF (catalog #PHG0311) and basic fibroblast growth factor or bFGF (catalog #13256029) were obtained from Gibco (Waltham, MA, USA). Concentrations of EGF at 20 ng/mL and bFGF at 20 ng/mL were used in the experimental assays, which were consistent with concentrations utilized in other studies of DPSC neuronal differentiation [31 (link),32 (link)]. Negative controls for each experimental assay involved the use of 1X PBS in place of the addition of growth factors into the cell culture media.

Human embryonic kidney (HEK) 293FT cells (Life Technologies) were maintained in DMEM (Dulbecco's Modified Eagle's medium) medium supplemented with 10% FBS (fetal bovine serum), 0.1 mM Minimum Essential Medium non-essential amino acids (MEM NEAA), and 1 mM sodium pyruvate. Cells were passaged every two days using TrypLE.
K562 cells were maintained in RPMI-1640 medium supplemented with 15% FBS at a seeding density of 2 × 10⁻⁵ and passaged every two days.
Pluripotent stem cells were either maintained on mouse embryonic fibroblast (MEF) feeder cells in KSR-hESC culture medium (DMEM Ham's F-12 medium, 20% Knockout Serum Replacement (KSR), 1% non-essential amino acids, 2 mM L-glutamine, 0.1 mM beta-mercaptoethanol, and 100 ng/ml basic fibroblast growth factor (bFGF), all from Gibco, except for bFGF, which was from Invitrogen) or on extracellular matrix (ECM) Gel (from Sigma) in MEF-conditioned KSR-hESC medium, at 37 °C with 5% CO₂. Subculture was performed on day 7 using manual passaging of clones. Cells were regularly tested for mycoplasma.

A human NPC line, established from human-induced pluripotent stem cells (hiPSCs), was obtained from Royan Institute, Tehran, Iran [23 (link)], and used. NPCs were passaged in a 1:3 ratio for expansion on poly-d-lysine (PDL)-coated plates and cultured in neurobasal medium (Gibco) supplemented with 1× penicillin/streptomycin, 25 ng/ml bFGF, 20 ng/ml epidermal growth factor (EGF), and 2 mM L-glutamine (all from Invitrogen).
At about 70% confluency, OPC differentiation was induced according to a previously published protocol with minor modifications [24 ]. Briefly, NPCs were grown for 3 weeks in the oligo medium containing serum-free DMEM/HAMS F12 medium (Gibco) supplemented with 1% bovine serum albumin, 2 mM L-glutamine, 50 μg/ml gentamicin, 1× N2 supplement, 3 nM T3 (SIGMA), 2 ng/mL Shh (SIGMA), 2 ng/mL NT-3 (SIGMA), 20 ng/mL bFGF, and 10 ng/mL PDGF-AA (SIGMA). Differentiation of OPCs to OLs was initiated by growth factors withdrawn for 2 days.
Human embryonic kidney cells (HEK293T) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Hyclone, USA) and 1% antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin sulfate). Cells were grown in a humidified atmosphere containing 5% CO² at 37 °C.