Differentiation of Neural Progenitor Cells into Neurons

Protocollo

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 groups.
4. Place the plates into the incubator and allow them to differentiate for another 10 days. Change the corresponding medium every 2 days.
5. Check the differentiation status and record the morphological changes as mentioned in step 3.
6. On Day 18, evaluate the generation of neurons (β-Tubulin III positive) and determine the differentiation efficiency of the 3 protocols used in step 4.

Risultati

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 ...

Materiali

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