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.
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.
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 medicine2,3. 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 system8,9. 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.
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…
The authors have nothing to disclose.
This work was supported by the National Natural Science Foundation of China (No. 31501099) and the Middle-aged and Young of the Education Department of Hubei Province, China (No. Q20191104). And, we thank Professor Wensheng Deng at Wuhan University of Science and Technology for providing the mouse embryonic stem cell lines A2lox.
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 |