Stem cell Differentiation media iPSCs or hESCs differentiation into cerebellar neuroepithelium (NE)

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Upstream tips
hESCs were dissociated to single cells and quickly reaggregated using low-cell-adhesion 96-well culture plate with V-bottomed wells (PrimeSurface MS-9096V; Sumitomo Bakelite)
Protocol tips
cerebellar differentiation medium [Iscove’s modified Dulbecco’s medium/Ham’s F-12 1:1, 1 chemically defined lipid concentrate, penicillin/streptomycin, monothioglycerol (450 M) and purified BSA (99% purified by crystallization; Sigma) ](6,000 cells/well) containing growth-factor-free chemically defined medium (gfCDM), insulin (7 μg/ml), 10 μM Y-27632, and 10 μM SB431542 (Tocris Bioscience)
Upstream tips
NMM Contains: DMEM/F12, Glutamax (500 ml, Life Technologies), Neurobasal medium (500 ml, Life Technologies), N2 supplement (1×, Life Technologies), B27 supplement (1×, Life Technologies), insulin (5 μg ml−1, Imgen), L-glutamine (1.5 mM, Life Technologies), non-essential amino acids (NEAA, 100 µM, Life Technologies), penicillin/streptomycin (100 U l−1, Life Technologies), beta-mercaptoethanol (10 µM, Life Technologies). Medium was stored at 4°C and used within 3 weeks.
Protocol tips
Neural Maintenance Medium (NMM)+FGF2 (4 ng ml−1, Peprotech)+RI (10 µM), and distributed to anti-adhesive (Poly-HEMA, Sigma, P3932-10G) coated 6WPs in a 2 or 3:1 ratio (with 2.5 ml medium per well)
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