ES cell culture and monolayer neural differentiation procudures
Sox1-GFP mESCs should be maintained in the stem cell culture medium to keep the pluripotent state (Figure 1).
For monoculture neural differentiation, dissociate and plate the undifferentiated Sox1-GFP mESCs on to 0.1% gelatin-coated tissue culture plastic at a density of 1-3x104/cm2 in serum-free N2B27 medium. Renew N2B27 medium every 2 days. GFP positive cells can be observed on Day 5 (Figure 2).
Figure 2 | Monoculture neuronal differentiation of Sox1-GFP mESCs on Day 5
Growth Medium
Embryonic stem cell medium
DMEM: 415ml
FBS: 75ml (HYCLONE LABORATORIES INC, Cat# SH30070.03EH)
L-Glutamine: 5ml
MEM NEAA: 5ml
1-Thioglycerol: 4.3ul
LIF: 50ul LIF: 50ul (10ng/ml final concentration) (PrimCells, Cat# PFHSA01)
Total Volume: 500ml
N2B27 medium for neural differentiation
N2B27 is a 1:1 mixture of DMEM/F12 (Gibco, Paisley, UK) supplemented with modified N2 (25 μg/ml insulin, 100 μg/ml apo-transferrin, 6 ng/ml progesterone, 16 μg/ml putrescine, 30 nM sodium selenite and 50 μg/ml bovine serum albumin fraction V (Gibco)) and Neurobasal medium supplemented with B27 (both from Gibco).
Alternatively, Ndiff227 (StemCells INC, Cat# SCS-SF-NB-02) is ready-to-use medium for the neural differentiation of mESCs in adherent monoculture conditions as described in Ying QL et al (2003)2.
References
1. Aubert, J. et al. Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice. Proc Natl Acad Sci U S A 100 Suppl 1, 11836-11841 (2003).
2. Ying, Q.L., Stavridis, M., Griffiths, D., Li, M. & Smith, A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 21, 183-186 (2003).
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