Neural differentiation of AAVS1-TRE3G-NGN2 pluripotent stem cells

Abstract

This protocol is about neural differentiation of AAVS1-TRE3G-NGN2 pluripotent stem cells.

Before start

Prepare ND1 Medium and ND2 Medium:

ND1 Medium

DMEM/F12

N2 (100x) 1x

BDNF 10 ng/ml

NT3 10 ng/ml

NEAA (100X) 1x

Laminin 0.2 μg/ml

Doxycycline 2 μg/ml

ND2 Medium

Neurobasal medium

B27 (50x) 1x

Glutamax (100x) 1x

BDNF 10 ng/ml

NT3 10 ng/ml

Doxycycline 2 μg/ml

Note

* Doxycycline can be withdrawn on Day 10 (or even earlier-not tested yet).* Cells can be split from Day 5 to Day 7 (or even earlier-not tested yet).

Attachments

Neural_differentiation_of_AAVS1-TRE3G-NGN2-pluripotent_stem_cells.pdf

Steps

Day 0:

Treat H9 AAVS1-TRE3G-NGN2 cells with Accutase and plate the dissociated cells in matrigel-coated 6-well plates (2x10⁵ cells/well) in ND1 Medium supplemented with Y27632 (10micromolar (µM)).

Day 1:

Replace the medium with ND1 Medium .

Day 2:

Replace the medium with ND2 Medium .

Day 4:

Exchange 50% of the medium from each well.

Day 6:

Treat the cells with Accutase and replate the dissociated cells in matrigel-coated 6-well plates (2-4x10⁵ cells/well) in ND2 Medium .

Day 8 and thereafter:

Exchange 50% of the medium from each well every other day.