Additionally, we differentiated iPSCs toward NSCs by treatment with a chemically defined medium comprising of hLIF, SB431542, CHIR99021, Compound E, and dorsomorphin (Liu et al., 2012).
Following 1.5 h incubation in a shaking water bath at 37°C, the cell suspension was filtered through a 40 µm mesh (Fisher Scientific, Loughborough, UK) to remove undigested material and the filtrate was resuspended in washing medium, comprising of HBSS with 10% fetal bovine serum (FBS, Sigma).
For experiments, 90% confluent cells 2 to 4 days after collection were disassociated using Accutase (Sigma), resuspended in complete medium, comprising of DMEM (without phenol red)/Hams F-12 nutrient mixed 1∶1 (Sigma), supplemented with 10% FBS, 20 mM Hepes, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mM l-glutamine (all Sigma).
The growth medium was then aspirated off and replaced with 100 μl of maintenance medium comprising of 100 ml DMEM, 2 ml Fetal Bovine Serum (FBS), 1 ml Penstrep, 1 ml Amphotericin B, 1 ml L-Glutamate and 0.1 ml Gentamycin.
Well-fed larvae were reared on apple juice plates with standard medium comprising of 80 g/l maize, 18 g/l dried yeast, 10 g/l soya flour 80 g/l malt extract, 40 g/l molasses, 8 g/l agar, 6.6 ml acid mix (comprising 50% propionic acid and 3.2% phosphoric acid)/l throughout larval life.
After incubation, 1,000-fold dilutions were prepared and 100 μL of the diluted samples was plated on a solid medium comprised of TSB.
The medium comprised of (g/l) soluble starch 47.0, Yeast extract 3.0, peanut meal 22.0, (NaCl2.7O4 2.7, NaCaCo37, CaCo3, pH 6.8 was sterilized at 121 °C, 15 lbs for 15 min.
The cells were cultured in a specialized growth medium comprised of a triple-sugar minimal essential medium α L-15 base supplemented with 10% fetal bovine serum, designated as GTSF-2 [85], and incubated at 37°C at 5% CO2 environment.
The medium comprises of DMEM/F12 with GlutaMax, BSA 1.8%, β-Mercaptoethanol 0.1 mM, 1× StemPro protein cocktail (All from Invitrogen) and 8 ng/ml of basic fibroblast growth factor (FGF2, Sigma).
Media was replaced with hESC differentiation medium, comprised of Glasgow Minimum Essential Medium(GMEM) supplemented with 10% knockout serum replacement, 0.1 mM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Sigma-Aldrich, St . Louis U. S. A ., and 0.1 mM β-mercaptoethanol (Millipore).
The loosely adherent RPE cell layer was gently peeled off each eyecup using fine forceps, transferred to a 15 ml tube containing culture medium comprised of Medium199, 15% FBS (Gibco, Carlsbad, CA) and 1x MEM with non-essential amino acids (Sigma, St . Louis, and then triturated with a transfer pipette to form a dissociated cell suspension.
