Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Summary

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Abstract

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Introduction

Embryonic stem cells (ESCs) are pluripotent and can differentiate into neural precursor cells (NPCs) and subsequently into neurons under certain conditions¹. ESC-based neurogenesis provides the best platform to mimic neurogenesis, thus serving as a useful tool for developmental biology studies and potentially aid in regenerative medicine^2,3. In the past decades, many strategies have been reported for inducing embryonic neurogenesis, such as the transgenic method⁴, using small molecules⁵, using a 3D matrix microenvironment⁶, and the co-culture technique⁷. However, most of these protocols are either condition limited or hard to operate, thus they are not suitable for usage in most laboratories.

To find an easy to operate and low cost method to achieve efficient neural differentiation from mESCs, a combinatorial screening strategy was used here. As described in Figure 1, the whole process of embryonic neurogenesis was divided into 2 phases. Phase I refers to the differentiation process from mESCs into NPCs, and phase II relates to the subsequent differentiation from NPCs into neurons. Based on the principles of easy operation, low cost, easily available materials and high differentiation efficiency, seven protocols in Phase I and three protocols in Phase II were chosen based on the traditional adherent monolayer culture system or embryoid body formation system^8,9. The differentiation efficiency of protocols in both phases was evaluated using cell morphology observation and immunofluorescence assay. Through combining the most efficient protocol of each phase, we established the optimized method for neural differentiation from mESCs.

Protocol

1. Mouse embryonic stem cell culture Prepare 0.1% gelatin coated cell culture dishes or plates. 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. Put the dishes into a 5% CO2 incubator at 37 °C and allow coating for 1 h. 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 …

Representative Results

2-day embryoid body formation + 6-day RA induction works best on directing the differentiation of mESCs into NPCs (Phase I). To determine the optimal protocol that best promote the differentiation of mESCs into NPCs (Phase I), 7 protocols were tested on both A2lox and 129 mESCs (Table 1) and the differentiation status of each group was monitored using light microscope. As shown in Figure 3A, most A2lox and 129 derivatives under "2-day embryoid body formation + 6-day RA i…

Discussion

In the present study, we established a simple and effective method for neuronal differentiation from mESCs, with low cost and easily obtained materials. In this method, 2 days of embryoid body formation followed by 6 days of RA induction can effectively promote the differentiation of mESCs into NPCs (Phase I-protocol 3). For the phase II differentiation, N2B27 medium II (Phase II-protocol 3) most effectively induce the differentiation from NPCs into neurons. To ensure success, more attention should be paid to several cri…

Materials

Anti-Nestin antibody [Rat-401]	Abcam	Ab11306	stored at -80 °C, avoid repeated freezing and thawing
Anti-β-Tubulin III antibody produced in rabbit	Sigma Aldrich	T2200	stored at -80 °C, avoid repeated freezing and thawing
Alexa Fluor 488-Labeled Goat Anti-Mouse IgG	Beyotime	A0428	stored at -20 °C and protect from light
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
Cy3-Labeled Goat Anti-Rabbit IgG	Beyotime	A0516	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
KnockOut Serum Replacement	Gibco	10828028	stored at -20 °C, avoid repeated freezing and thawing
Leukemia Inhibitory Factor human	Sigma	L5283	stored at -20 °C
Mounting Medium With DAPI – Aqueous, Fluoroshield	Abcam	ab104139	stored at 4 °C and protect from light
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
Stem-Cellbanker (DMSO free)	ZENOAQ	stem cellbanker DMSO free	stored at -20 °C, avoid repeated freezing and thawing
Trypsin 0.25% (1X) Solution	HyClone	SH30042.01	stored at 4 °C
Triton X-100	Sigma	T8787
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