A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

Abstract

Source: Mao, X., et al. Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro. J. Vis. Exp. (2020).

This video demonstrates a method for the in vitro generation of neural progenitor cells (NPCs) from mouse embryonic stem cells (mESCs). The mESCs are cultured in suspension to form embryoid bodies (EBs), which are then guided to differentiate into NPCs via retinoic acid treatment in an adherent culture.

Protocol

1. Mouse embryonic stem cell culture

1. Prepare 0.1% gelatin coated cell culture dishes or plates.
   1. Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in water) to 60 mm cell culture dishes. Rock gently to ensure even coating of the cell culture dishes.
   2. Put the dishes into a 5% CO₂ incubator at 37 °C and allow coating for 1 h.
   3. Remove the 0.1% gelatin solution before seeding the cells.
      NOTE: After removing the gelatin, there is no need to dry or wash the coated dishes.
2. Mouse embryonic stem cells (A2lox and 129) culture
   1. Incubate mESCs (A2lox....

Results

Figure 1: The morphology of the embryoid bodies. (A) Embryoid bodies cultured for 4 days. (B) Embryoid bodies cultured for 2 days.

....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
B-27 Supplement (50X), serum free	Gibco	17504044	stored at -20 °C, and protect from light
CHIR-99021 (CT99021)	Selleck	S1263	stored at -20 °C
Coverslips	NEST	801007
Fetal bovine serum	HyClone	SH30084.03	stored at -20 °C, avoid repeated freezing and thawing
Gelatin	Gibco	CM0635B	stored at room temperature
GlutaMAX Supplement	Gibco	35050061	stored at 4 °C
KnockOut DMEM/F-12	Gibco	12660012	stored at 4 °C
KnockOut Serum Replacement	Gibco	10828028	stored at -20 °C, avoid repeated freezing and thawing
MEM Non-essential amino acids solution	Gibco	11140076	stored at 4 °C
N-2 Supplement (100X)	Gibco	17502048	stored at -20 °C and protect from light
Neurobasal Medium	Gibco	21103049	stored at 4 °C
Phosphate Buffered Saline (1X)	HyClone	SH30256.01B	stored at 4 °C
Penicillin/ Streptomycin Solution	HyClone	SV30010	stored at 4 °C
PD0325901(Mirdametinib)	Selleck	S1036	stored at -20 °C
Retinoic acid	Sigma	R2625	stored at -80 °C and protect from light
Strain 129 Mouse Embryonic Stem Cells	Cyagen	MUAES-01001	Maintained in feeder-free culture system
Trypsin 0.25% (1X) Solution	HyClone	SH30042.01	stored at 4 °C
2-Mercaptoethanol	Gibco	21985023	stored at 4 °C and protect from light
60 mm cell culture dish	Corning	430166
15 ml centrifuge tube	NUNC	339650