Neural differentiation assay

Neural differentiation strategy

Methods

N2B27 serum free induce monolayer neural differentiation.

Reagents

2i Medium (Life technologies)

N2B27 Medium:

  DMEM/F12 (50/50) medium (Life technologies)

  N2 supplement (Life technologies, 100*) 

  Neurobasal medium (Life technologies)

  B27 supplement (Life technologies, 50*)

Procedure:

1. Day1-Day2: Mouse embryonic stem cells (mESCs) were cultured in “2i” medium for 24hrs
2. Day2-Day6: mESCs were then passaged with N2B27 medium for neural differentiation.
3. Day 6: Add mouse EGF and FGF-2 (10ng/ml each) at day6.
4. Day9: Collect cells for sequential experiments.

Protocol:

1. mESCs are plated at relative high density (1.6-2.4*10⁶ per 60mm tissue culture dish) and cultured for 24hrs in “2i” medium.
2. N2B27 medium preparation: mix 25ml DMEM/F12 (50/50) medium with 0.25ml N2 supplement, mark “1”; mix 25ml Neurobasal medium with 0.5ml B27 supplement, mark “2”; then mix “1” and “2” together.
3. Harvest mESCs by trypsin digestion at 37 degree for 5min, the trypsin is neutralized with serum containing medium (ES cells standard medium without LIF).
4. ES cells are spun down (800rpm, 5min, and room temperature) and resuspended directly in N2B27 medium. 
5. Cell number counting
6. Prepare 3*10⁵ ES cells in 3ml N2B27 medium and plate onto 60mm tissue culture dish (pre-coated with 0.1% gelatin)
7. There after medium is changed every day.
8. Add mouse EGF and FGF-2 (10 ng/ml each) at day6.
9.  Monitoring monolayer differentiation from ES cells
10. Neural precursors can be identified by fixation and immunostaining for Sox1 and for Nestin. Then RT-qPCR be introduced here to check Oct4, Nestin and Sox1 expression dynamics.

Morphology and Marker Expression of Neural Progenitor Cells

(A) Typical NPC morphology is observed in cultures (shown at day 6 of passage 1). (B-D) NPCs maintained in Neural Progenitor Medium express the CNS-type NPC markers PAX6 (B, D; red), SOX1 (C; red) and NESTIN (C; green), but not the neural crest marker SOX10 (D; green, single channel shown in inset). B-D were taken at the same magnification.

Reference:

1. Ying QL and Smith AG, 2003, Methods enzymol

2. Ying QL et al, 2003, Nature biotechnology