A previously reported protocol was used for neural differentiation of hiPSCs (Nori et al., 2011 (link)). Briefly, hiPSCs were dissociated and cultured in suspension as EBs, which were subsequently dissociated and cultured in MHM supplemented with B27 (Life Technologies), 20 ng/ml fibroblast growth factor 2 (FGF-2, Peprotech), and 10 ng/ml hLIF (Millipore).
Neural rosette differentiation was performed as previously described (Koch et al., 2009 (link)). Four-day-old EBs were transferred to poly-L-ornithine (PO)-coated tissue culture dishes and propagated in ITSfn medium (DMEM/F-12 [Wako] containing 25 μg/ml insulin [Wako], 100 μg/ml transferrin [Nakalai-tesque], 5 ng/ml selenite [Sigma-Aldrich], and 2.5 μg/ml fibronectin [Sigma-Aldrich]). Within 10 days, neural tube-like structures developed in the EB outgrowth.
For neural induction from single hiPSCs, hiPSCs were incubated with TrypLE Select (Life Technologies) for 5 min and dissociated into single cells by pipetting. Cells were plated into a T75 flask (Nunclon), then 103 cells were plated for the neurosphere formation assay, and cultured in MHM supplemented with B27, 20 ng/ml FGF-2, 10 μM Y-27632 (Wako), and 10 ng/ml hLIF in 4% oxygen for 14 days. Neurospheres were repeatedly passaged by dissociation into single cells, and then cultured in the same manner. Neurospheres at passages 3–7 were typically used for analysis. For terminal differentiation, dissociated neurospheres were allowed to adhere to PO- (Sigma-Aldrich) and fibronectin-coated coverslips and cultured in MHM containing B27, 10 ng/ml brain-derived neurotrophic factor (BDNF; R&D systems), 10 ng/ml glial cell-derived neurotrophic factor (GDNF; R&D systems), 200 μM ascorbic acid (Sigma-Aldrich), and 1 mM dibutyryl-cAMP (Sigma-Aldrich) for 10–70 days.
Neural rosette differentiation was performed as previously described (Koch et al., 2009 (link)). Four-day-old EBs were transferred to poly-L-ornithine (PO)-coated tissue culture dishes and propagated in ITSfn medium (DMEM/F-12 [Wako] containing 25 μg/ml insulin [Wako], 100 μg/ml transferrin [Nakalai-tesque], 5 ng/ml selenite [Sigma-Aldrich], and 2.5 μg/ml fibronectin [Sigma-Aldrich]). Within 10 days, neural tube-like structures developed in the EB outgrowth.
For neural induction from single hiPSCs, hiPSCs were incubated with TrypLE Select (Life Technologies) for 5 min and dissociated into single cells by pipetting. Cells were plated into a T75 flask (Nunclon), then 103 cells were plated for the neurosphere formation assay, and cultured in MHM supplemented with B27, 20 ng/ml FGF-2, 10 μM Y-27632 (Wako), and 10 ng/ml hLIF in 4% oxygen for 14 days. Neurospheres were repeatedly passaged by dissociation into single cells, and then cultured in the same manner. Neurospheres at passages 3–7 were typically used for analysis. For terminal differentiation, dissociated neurospheres were allowed to adhere to PO- (Sigma-Aldrich) and fibronectin-coated coverslips and cultured in MHM containing B27, 10 ng/ml brain-derived neurotrophic factor (BDNF; R&D systems), 10 ng/ml glial cell-derived neurotrophic factor (GDNF; R&D systems), 200 μM ascorbic acid (Sigma-Aldrich), and 1 mM dibutyryl-cAMP (Sigma-Aldrich) for 10–70 days.
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