Basic fibroblastic growth factor bfgf

Bone marrow was collected aseptically from the sternebrae of horses being euthanized for unrelated reasons immediately following euthanasia. Using an 11-gauge Jamshidi bone marrow biopsy needle (VWR Scientific, Bridgeport, NJ) and 60 mL syringe containing 30,000 U of heparin, 30 mL of bone marrow was aspirated. Bone marrow samples were processed via density centrifugation with Ficoll-Paque Plus (GE Healthcare, Chicago, IL, USA) prior to seeding into flasks containing medium consisting of Dulbecco's Modified Eagle Medium (DMEM) with 1 g/L of D-glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate (ThermoFisher Scientific, Hampton, NH), penicillin (100 U/mL)-streptomycin (100 μg/mL) solution (Invitrogen, Carlsbad, CA), 10% fetal bovine serum (FBS) (VWR Life Science Seradigm, VWR, Radnor, PA), and basic fibroblastic growth factor (bFGF, 1 ng/mL) (Invitrogen, Carlsbad, CA). Medium was changed every 48 h. Cells were passaged when they reached ~80% confluency using Trypsin-EDTA Cell Dissociation Reagent (ThermoFisher Scientific, Waltham, MA). Passage 2 (P2) cells were used for differentiation assays. Cell number and viability was determined using the Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience, Lawrence, MA) and ViaStain™ AOPI staining solution (Nexcelom Bioscience LLC, Lawrence, MA).

The neural differentiation from dental pulp stem cells was obtained as reported in our previous study^{5 (link)}. Briefly, cells were cultured for 6 days in vitro (DIV) in the basic medium (DMEM), subsequently the medium was added with the following neural induction cocktail: 0.5 mM Isobutyl Methyl Xanthine (IBMX), 20 ng/ml human Epidermal Growth Factor (hEGF) 1 mM dibutyrylcAMP (dbcAMP), 10 ng/ml Nerve Growth Factor (NGF) and 10 ng/ml Brain-Derived Neurotrophic Factor (BDNF), 40 ng/ml basic Fibroblastic Growth Factor (bFGF), (all reagents were purchased from Invitrogen, Milan, Italy). Obtained neuronal differentiation, cells were grown in culture for 15 days in DMEM/FBS added with 10 µM retinoic acid²¹ .

H460 cells were suspended in serum-free DMEM/F12 medium (Gibco) containing B27 (Invitrogen), human recombinant epidermal growth factor EGF 20 ng/ml (PeproTech), basic fibroblastic growth factor bFGF 20 ng/ml (PeproTech) and plated at 500 to 30,000 cell/ml in ultralow-attachment 24- or 6-well plates (Corning, USA). The medium was replaced twice a week. Self-renewal capacity of the CSCs was examined by re-populating tumorspheres. Briefly, tumorspheres were collected by centrifugation, trypsinized, passed through a 45 mm strainer (BD, Biosciences), counted and replated in tumorsphere culture medium. Tumorspheres were defined as spheres with a diameter > 100 μm size. The numbers of tumorspheres were quantitated using Image J software (NIH, USA).