Fibroblast growth factor (fgf)

Cells at subconfluence in chamber slides or in 12- or 24-well plates were stimulated by one of the two induction protocols. i) Neurogenic induction medium (NIM-1) consisted of Neurobasal A (Gibco-Invitrogen) with B27 supplement (GIBCO-BRL), 20 ng/mL epidermal growth factor (EGF) (BD Biosciences), and 40 ng/mL fibroblast growth factor (FGF) (BD Biosciences) was added to the cells and cultures incubated for 4 weeks with the medium refreshed every 3 days. Cultures were then analyzed by immunocytofluorescence for the expression of the neural cell marker, βIII-tubulin. Isotype-matched antibodies were used as negative controls. ii) NIM-2 consisted of α-MEM with 10 ng/ml bFGF, 10 μM forskolin (Sigma), 25 mM KCl, 2 mM valproic acid and 5 μg/ml insulin. The cells received pre-neural induction in α-MEM medium containing 10% FBS and 10 ng/ml bFGF (Roche) for 24 h [26 ]. Subsequently, the medium was removed, cells washed with PBS and the NIM#2 added for up to 35 days incubation period with the medium refreshed every 3 days. Cells were monitored continually after neural induction for morphological changes and were lysed for RNA extraction or fixed for immunostaining. The control group received regular medium and harvested at the same time points as the neurogenic group.

Pregnant wild type CD1+ mice were used for obtaining embryos at E12 and E16. Cerebral cortices were dissected and the dissociated cells passed through a sterile nylon sieve (82 µm pores) and then pooled and seeded in culture dishes to generate cultures of mouse fetal-derived neural stem/precursor cells (NSPCs).
Cultures of NSPCs were grown floating in suspension as neurospheres in Dulbecco’s modified Eagle’s medium with B27, N2, 1 mM sodium pyruvate, 2 mM glutamine, 1 mM N-acetylcysteine (Sigma-Aldrich, St. Louis, USA), 20 ng/ml epidermal growth factor, (EGF), (BD Biosciences, Bedford, MA, USA), 10 ng/ml fibroblast growth factor, (FGF) (BD Biosciences, Bedford, MA, USA), and 2µg/ml heparin (Sigma-Aldrich St. Louis, IN, USA).
All chemical reagents were from Gibco (Invitrogen, Grand Island, NY, USA) unless otherwise specified and of the highest purity available. Neurospheres are a heterogeneous collection of cells that includes a small number of stem cells, a greater number of more specified progenitor cells, and a few differentiated cells such as astrocytes and neurons. Neurospheres, were dissociated using NeuroCult medium kit (Stem Cell Technologies, Vancouver, Canada) and the resulting individual cells were plated on laminin-coated glass bottom dishes and adhered for 24 hours prior to FLIM analysis.