Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Abstract

Source: Hanafiah, A., et al. Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells. J. Vis. Exp. (2020).

The video demonstrates the differentiation of mouse embryonic stem cells (mESCs) to embryoid bodies (EBs) by the hanging drop method. Upon suspending the mESCs in hanging drops and incubating, the cells aggregate to form EBs, which are further differentiated into neuronal progenitor cells (NPCs) using a differentiation medium supplemented with retinoic acid (RA).

Protocol

1. mESC culture Coat a 10 cm tissue-culture-treated plate with 0.1% gelatin and allow the gelatin to set for at least 15–30 min before aspirating it out. Seed γ-irradiated mouse embryonic fibroblasts (MEFs) one day before culturing the mESCs in the pre-warmed mESC medium (Dulbecco's modified Eagle medium (DMEM) with 15% fetal bovine serum (FBS), non-essential amino acids, β-mercaptoethanol, L-glutamine, penicillin/streptomycin, sodium pyruvate, Leukemia inhibitor…

Materials

0.05% Trypsin + 0.53mM EDTA 1X	Corning	25-052-CV
0.1% Gelatin	Sigma	G1890-100G	Prepared in de-ionized water
CHIR99021	Cayman Chemicals	13122
DMEM	Corning	10-017-CM
DMEM/F12 medium	Thermo Fisher Scientific	11320033
EB buffer	Qiagen	19086
Ethanol	111000200	Pharmco	Diluted in de-ionized water
Fetal bovine serum	Atlanta Biologicals	S10250
L-glutamine	Thermo Fisher Scientific	25030024
LIF	N/A	N/A	Collected from MEF supernatant
MEM Non-essential amino acids	Corning	25-025-Cl
PD0325901	Cayman Chemicals	13034
Penicillin/streptomycin	Corning	30-002-Cl
Phosphate-buffered saline (PBS)	N/A	N/A	Prepared in de-ionized water
Potassium chloride	P217-500G	VWR
Potassium phosphate monobasic anhydrous	0781-500G	VWR
Sodium chloride	BP358-10	Fisher Bioreagents
Sodium phosphate, dibasic, heptahydrate	SX0715-1	Milipore
Retinoic acid	Sigma	R2625	Prepared in DMSO
Sodium pyruvate	Corning	25-000-Cl
β-mercaptoethanol	Fisher BioReagents	BP176-100