Differentiation of Neural Progenitor Cells into Neurons

Abstract

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

The video showcased a cell culture method for differentiating neural progenitor cells into neurons. By incubating cells in a nutrient-rich medium supplemented with growth factors, the progenitor cells matured into neurons with developed axons and dendrites. A stabilizing agent ensured cell viability and protected against oxidative damage throughout the differentiation process.

Protocol

1. Differentiation from NPCs to neurons (Phase II)

1. Seed about 5 x 10⁵ mouse embryonic stem cells or mESC derivatives within 2 mL of basal differentiation medium I per well onto the 0.1% gelatin-coated 6-well plates. Randomly divide the mESC derivatives into 3 groups, as Phase II protocol 1, protocol 2, and protocol 3, respectively.
2. Place the plate into the 5% CO₂ incubator at 37 °C to allow for attachment for 6 h. Wash them twice with 2 mL of PBS.
3. Add 2 mL of basal differentiation medium I, N2B27 medium I and N2B27 medium II (Table 1), respectively, to each well of the above g....

Results

Table 1: Details of the 3 protocols used in phase II differentiation.

Differentiation Phase II (10d)
	Protocols	Media
Protocol 1	Differentiation naturally: With basal differentiation medium I only	Basal differentiation medium .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-β-Tubulin III antibody produced in rabbit	Sigma Aldrich	T2200	stored at -80 °C, avoid repeated freezing and thawing
B-27 Supplement (50X), serum free	Gibco	17504044	stored at -20 °C, and protect from light
DME/F-12 1:1 (1x)	HyClone	SH30023.01B	stored at 4 °C
Fetal bovine serum	HyClone	SH30084.03	stored at -20 °C, avoid repeated freezing and thawing
Fluorescence microscopy	Olympus	CKX53
Gelatin	Gibco	CM0635B	stored at room temperature
GlutaMAX Supplement	Gibco	35050061	stored at 4 °C
Immunol Staining Primary Antibody dilution Buffer	Beyotime	P0103	stored at 4 °C
KnockOut DMEM/F-12	Gibco	12660012	stored at 4 °C
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
Normal goat serum	Jackson	005-000-121	stored at -20 °C
Neurobasal Medium	Gibco	21103049	stored at 4 °C
Nonadhesive bacterial dish	Corning	3262
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
2-Mercaptoethanol	Gibco	21985023	stored at 4 °C and protect from light
4% paraformaldehyde	Beyotime	P0098	stored at -20 °C
6 - well plate	Corning	3516
60 mm cell culture dish	Corning	430166
15 ml centrifuge tube	NUNC	339650