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.
1. Mouse embryonic stem cell culture
2. Differentiation from mESCs to NPCs (Phase I)
3. Cell morphology observation
4. Immunofluorescence staining
5. Differentiation from NPCs to neurons (Phase II)
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 induction" treatment (Phase I-protocol 3) showed well-stacked and neurite-like morphologies, which indicating the formation of NPCs. However, cells with "4-day embryoid body formation + 4-day RA induction" treatment (Phase I-protocol 2) showed poor and apoptotic status, which may be due to the lack of nutrient within embryoid bodies. Monolayer culture combined with RA induction (Phase I-protocol 4 and 5) could also direct the differentiation of mESCs, while the proportion of neurite-like cells was not as much as that in Phase I-protocol 3. Meanwhile, most A2lox and 129 derivatives in Phase I-protocol 6 and 7 showed smaller cell bodies and tended to undergo apoptosis, suggesting that N2B27 medium II could not support embryonic neurogenesis effectively.
To further confirm the formation of NPCs, the percentage of Nestin+ cells (marker for NPCs) in each group were detected using an immunofluorescence assay. In Figure 3B, the percentage of Nestin+ cells in Phase I-protocol 3 were the highest and reached up to 77.67 ± 4.33% and 69.33 ± 2.33% in A2lox and 129 derivatives, respectively. Collectively, Phase I-protocol 3 works best on directing the differentiation of mESCs into NPCs.
N2B27 medium II can most effectively induce the differentiation from NPCs into neurons (Phase II). Three protocols in phase II differentiation were examined. As shown in Figure 4A, morphological observation showed that most A2lox and 129 derivatives in phase II-protocol 3 (differentiation with N2B27 medium II) appeared the most prolonged neuron-like structures with clear neurites and cell body extensions by Day 18, indicating the efficient occurrence of neurogenesis. Immunofluorescence assays further confirmed the generation of neurons, with the percentage of β-Tubulin III+ cells up to 67.75 ± 4.01% and 58.73 ± 7.25%, respectively, in A2lox and 129 derivatives on D18 (Figure 4B).
To make it clearer, a schematic diagram of the optimized method for embryonic neurogenesis is shown in Figure 5. Briefly, 1.5 x 106 mESCs are seeded into a nonadhesive bacterial dish in 10 mL of basal differentiation medium I and allow for embryoid body formation for 2 days. Then, embryoid bodies are collected and planted into the 0.1% gelatin-coated 6-well plates with the concentration of 50 embryoid bodies per well. Meanwhile, RA (1 µM) is added for another 6 days. From Day 8 to Day 18, RA is removed, and N2B27 medium II is applied to direct the subsequent differentiation from NPCs to neurons. With such a combined method, robust neurons can be formed on Day 18.
Figure 1: Diagram of the embryonic neurogenesis process. This figure has been modified from Li et al.10. Please click here to view a larger version of this figure.
Figure 2: The morphology of the embryoid bodies. (A) Embryoid bodies cultured for 4 days. (B) Embryoid bodies cultured for 2 days. Please click here to view a larger version of this figure.
Figure 3: Efficiency comparison of the 7 protocols on phase I differentiation using A2lox and 129 mESCs. (A) Morphological analysis of A2lox and 129 mESCs derivatives on Day 8. Upper panel: A2lox derivatives; Lower panel: 129 derivatives. (B) Immunofluorescence detection for the formation of NPCs (Nestin+, green). The nuclei were labeled blue with DAPI. Upper panel: A2lox derivatives on D8; Lower panel: 129 derivatives on D8. Percentages of Nestin+ cells of each group were shown by histogram. Each column represents the mean±SEM of three independent experiments. *, p≤0.05; **, p≤0.01. This figure has been modified from Li et al.10. Please click here to view a larger version of this figure.
Figure 4: Efficiency comparison of the 3 protocols on phase II differentiation. (A) Morphological analysis of A2lox and 129 mESCs derivatives on Day 18. Upper panel: A2lox derivatives; Lower panel: 129 derivatives. (B) Immunofluorescence detection for the formation of neurons (β-Tubulin III+, red). The nuclei were labeled blue with DAPI. Upper panel: A2lox derivatives on D18; Lower panel: 129 derivatives on D18. Percentages of β-Tubulin III+ cells of each group were shown by histogram. Each column represents the mean ± SEM of three independent experiments. *, p≤0.05; **, p≤0.01. This figure has been modified from Li et al.10. Please click here to view a larger version of this figure.
Figure 5: Brief model of the optimized method for neuronal differentiation from mESCs in vitro This figure has been modified from Liet al.10. Please click here to view a larger version of this figure.
Differentiation Phase I (8d) | ||
Protocols | Media | |
protocol 1 | Differentiation naturally: With basal differentiation medium I only | Basal differentiation medium I: DMEM/F12 +15%FBS + 1%NEAA+0.1mM 2ME+ 1%P/S |
protocol 2 | 4-day Embryoid Bodies formation + 4-day RA induction | |
protocol 3 | 2-day Embryoid Bodies formation + 6-day RA induction | |
protocol 4 | Monolayer culture combined with RA induction: 4d (-RA) 4d (+RA) | |
protocol 5 | Monolayer culture combined with RA induction: 2d (-RA) 6d(+RA) | |
protocol 6 | Embryoid Bodies formation (4 d) and differentiation induced with N2B27 medium II | N2B27 medium II: 49% DMEM/F12+ 1% N2 + 48% Neurobasal medium + 2% B27 +1%GlutaMAX+ 0.1mM 2ME |
protocol 7 | Monolayer culture with N2B27 medium II |
Table 1: Details of the 7 protocols used in phase I differentiation. This table has been modified from Li et al.10.
Differentiation Phase II (10d) | ||
Protocols | Media | |
protocol 1 | Differentiation naturally: With basal differentiation medium I only | Basal differentiation medium I: DMEM/F12 +15%FBS +1%NEAA +0.1mM 2ME+ 1%P/S |
protocol 2 | Differentiation with N2B27 medium I | N2B27 medium I: DMEM/F12 + 1%N2 + 2%B27 + 1%GlutaMAX +0.1mM 2ME |
protocol 3 | Differentiation with N2B27 medium II | N2B27 medium II: 49% DMEM/F12+ 1% N2 + 48% Neurobasal medium + 2% B27 +1%GlutaMAX+ 0.1mM 2ME |
Table 2: Details of the 3 protocols used in phase II differentiation. This table has been modified from Li et al.10.
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 critical steps.
Firstly, the healthy state of embryoid bodies is the key for the whole differentiation process. Three-dimensional embryoid body formation is usually used to direct the differentiation of ESCs8. In this study, we investigated the proper suspension culture time of embryoid bodies. As shown in Figure 2, round embryoid bodies with bright cores were formed after suspension culture for 2 days in this condition. However, when cultured for 4 days, many embryoid bodies adhere to each other, and the cores become dark, indicating the apoptosis of cells in the cores. The subsequent differentiation further confirmed the worse effect of prolonged embryoid body formation. In some reported studies, suspension culture of embryoid bodies could last for as long as 10 days, using medium with lower FBS concentration or without FBS11. The reduced time for embryoid body formation in the study may be due to the higher FBS concentration (15%) used here, and it has been proven that 15% FBS can better promote the formation and differentiation of embryoid bodies.
Secondly, the timepoint at which RA is applied and the RA working concentration are critical for cell fate determination of mESCs. RA, a derivative of vitamin A, is one of the most important morphogens with pleiotropic actions12. RA can regulate multiple signal pathways and affect cell fate determination of ESCs13,14. Reports showed that short-term treatment of mESCs with RA during the early differentiation stage prevented spontaneous differentiation and maintain self-renewal capacity of mESCs15. Others suggested that RA could regulate both germ cell differentiation and neural differentiation from ESCs, which are timepoint dependent16,17,18. In the condition presented here, RA added on the 2nd day after embryoid body formation is appropriate for directing the differentiation into NPCs. Meanwhile, the working concentration of RA is also critical. Low RA concentrations (~10 nM) may induce the differentiation of mESCs into endoderm-like cells, whereas high RA concentrations (1-5 µM) are more likely to induce differentiation into NPCs13,14,15,16,17,18,19. Due to the use of RA, one would expect a caudalization effect; the differentiation into fore brain neurons would be rarely seen and yielding neurons of hindbrain and spinal cord fates would occur20,21. Furthermore, RA is an easily available and low cost agent, and the use of this protocol can save research funds for most laboratory.
Thirdly, in condition presented here, the NPCs generated after phase I differentiation can be stored and passaged in proper conditions. Cryopreservation with high cell density (>2 x 106) using Stem-Cellbanker can effectively reduce the cell damage caused by freezing to get a high recovery rate. Meanwhile, NPCs generated in the study can be passaged using N2B27 medium (49% DMEM/F12+ 1% N2 + 48% Neurobasal medium + 2% B27) with a cell density more than 5 x 105/cm2. Under low cell density (less than 0.5 x 105/cm2), NPCs tend to differentiate. Such cell cryopreservation and recovery can bring great convenience to the research.
Moreover, Neurobasal medium is essential for the phase II differentiation. Neurobasal medium is designed specifically for long-term maintenance and maturation of neuronal cell. As listed in N2B27 medium II (Table 2), the addition of Neurobasal medium could better support the differentiation from NPCs into neurons.
Collectively, we reported an efficient and low-cost method for neuronal differentiation from mESCs in vitro, using the strategies of combinatorial screening. The established method is very easy to implement and is suitable for use by most laboratories. Such an optimized method can be a powerful tool for neurobiology and developmental biology research.
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 |