Basic fibroblast growth factor bfgf

Chicken embryonic fibroblasts (CEF) were isolated according to our lab’s previously reported method [11 (link)]. Chicken embryos hatched for 6–10 days were cut up after removing their head, tail, limbs and viscera; digested with trypsin; centrifuged; and cultured with Dulbecco’s Modified Eagle Medium (DMEM) (Hyclone, Logan, UT, USA) containing 10% Fetal Bovine Serum (FBS) (Gibco, Grand Island, NY, USA). The isolation and culture methods of the chicken ESCs also followed that of our previous report [12 ]. The blastoderm was aseptically separated from the freshly fertilized eggs; the yolk on it was rinsed with PBS; the blastoderm was blown off, centrifuged, and cultured with ESC medium. The chicken ESC medium was composed of 43.5 mL Knockout DMEM (Gibco, Grand Island, NY, USA), 0.1 mmol/L β-mercaptoethanol (Sigma, St. Louis, MO, USA), 0.4% non-essential amino acids (Sigma, St. Louis, MO, USA), 2% chicken serum (Gibco, Grand Island, NY, USA), 5 ng/mL Stem Cell Factor (SCF) (Sigma, St. Louis, MO, USA), 10 ng/mL Fibroblast Growth Factor-basic (bFGF) (Sigma, St. Louis, MO, USA), 1 ng/mL Leukemia Inhibitory Factor (LIF) (Merck Millipore, Burlington, MA, USA) and 0.5% penicillin (Solarbio, Beijing, China).

Five thousand cells per well were seeded in ultra-low attachment 6-well plates (Corning, Tweksburg, MA) and incubated in DMEM/F12 (1:1) supplemented with B27 (Invitrogen, Life Technologies Inc. Grand Island, NY), 25 ng/ml fibroblast growth factor-basic (bFGF, Sigma, St, Louis, MO) and 20 ng/ml epidermal growth factor (EGF, Sigma, St, Louis, MO). The number of spheres with a diameter of >50 μm was quantified by Image J software.

Non-adherent spheres of salivary mucoepidermoid carcinoma cells (salispheres), previously characterized in normal salivary cells [57 (link)], were cultured in DMEM/F-12 (Invitrogen) supplemented with 20 ng/ml EGF (Sigma-Aldrich), 20 ng/ml basic fibroblast growth factor (bFGF; Millipore), 1% penicillin/streptomycin (Invitrogen), 1% glutamax (Invitrogen), 1% N-2 supplement (Invitrogen), 1 μM dexamethasone (Sigma-Aldrich), and 10 μg/ml insulin (Sigma-Aldrich) [39 (link)]. Cells were counted, diluted to 2,000 per 1.5 ml, and added to 6-well ultra-low attachment plates (Corning; Corning, NY, USA). For in vitro passaging, salispheres were collected and exposed to 0.25% trypsin for 5-10 minutes, and then mechanically dissociated. The trypsin was neutralized using a trypsin neutralizing solution (TNS; Lonza). Colonies of 50 cells or more were considered salispheres.

PLC/PRF/5 and Hep3B were obtained from the American Type Culture Collection (Manassas, VA, USA). SMMC-7721 cells were obtained from the Cell Bank of the Institute of Biochemistry and Cell Biology, China Academy of Sciences (Shanghai, China). Huh7 cells were purchased from the Riken Cell Bank (Tsukuba, Japan). MHCC-LM3 cells were provided by the Liver Cancer Institute of Zhongshan Hospital, Fudan University (Shanghai, China). HCC-LY5 was established from primary HCC tissue in our lab. All cell lines were cultured in DMEM (Sigma-Aldrich, St. Louis, Missouri, USA) supplemented with 10% bovine serum (Hyclone, Logan, Utah, USA) and incubated in 5% CO₂ at 37 °C. For the in vitro tumor-sphere formation assay with CSCs, single HCC cells were cultured suspension in Ultra low attachment multiwell plates (Costar, St. Louis, Missouri, USA) in the conditional medium (CDM). The CDM consisted of DMEDM/F12 supplemented with 0.5 × B27 supplement, 10 ng/ml basic fibroblast growth factor (bFGF) (Millipore, Billerica, Massachusetts, USA) and 10 ng/ml epithelial growth factor (EGF) (Millipore, USA).

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), and cell culture media were purchased from Invitrogen (Carlsbad, CA, USA), unless otherwise indicated. Basic fibroblast growth factor (bFGF) and epithelial growth factor (EGF) were supplied by Millipore (Billerica, MA, USA). Hybridoma cell lines of F4/80 and Mac-2 were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). The mouse TNF-α ELISA kit (430901) and mouse IL-1β ELISA kit (432601) were from Biolegend (San Diego, CA, USA). Click-iT^® EdU Alexa Fluor^® 555 Imaging Kit (C10338) and all secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies used in the experiment are listed in Table 1.

Primary NSCs were isolated from 14.5-day-old embryos (E14.5) of C57BL/6 mice for in vitro studies and from enhanced green fluorescence protein (EGFP)-transgenic mice [C57BL/6-Tg (CAG-EGFP) 1Osb/J] for in vivo studies (all from the Institute of Model Animal, Nan Jing, China). For proliferation, dissociated single cells were cultured in the proliferation culture medium (PCM) containing Dulbecco's modified Eagle's medium/F-12 (DMEM/F-12) (Invitrogen, Carlsbad, CA, USA) supplemented with 1% B27 (Invitrogen), 20 ng/ml basic fibroblast growth factor (bFGF) (Sigma, St. Louis, MO, USA), 20 ng/ml epidermal growth factor (EGF) (Sigma) and 100 U/ml penicillin/streptomycin (Invitrogen). Primary neurospheres were formed after 5–7 days of culture. NSCs at passages 3–5 were used in the present study. For differentiation, NSCs were seeded onto poly-D-lysine (150 µg/ml, Sigma) and laminin (20 µg/ml, Invitrogen) -coated coverslips, cultured in the differentiation medium for 7 to 14 days and analyzed by immunocytochemistry. The differentiation medium contains Neurobasal medium (Invitrogen) supplemented with 1% B27 supplement, 10 ng/ml bFGF, 100 µM dibutyryl cyclic-adenosine monophosphate (dBcAMP) (Sigma) and 100 U/ml penicillin/streptomycin (Invitrogen), which is an efficient neuronal differentiation medium [17] (link), [18] (link).