Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

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.

1. Mouse embryonic stem cell culture

1. Prepare 0.1% gelatin coated cell culture dishes or plates.
   1. 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.
   2. Put the dishes into a 5% CO₂ incubator at 37 °C and allow coating for 1 h.
   3. 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 dishes.
2. Mouse embryonic stem cells (A2lox and 129) culture
   1. Incubate mESCs (A2lox and 129) cells in the 0.1% gelatin coated 60 mm cell culture dishes in mESC growth medium at 37 °C in a 5% CO₂ incubator, respectively. The mESC growth medium consists of 85% knock-out DMEM/F12, 15% Knock-out serum replacement (KSR), 0.1 mM β-mercaptoethanol (2ME), 2 mM GlutaMAX, 1% non-essential amino acid (NEAA), 1% penicillin/streptomycin (P/S), 1000 U/mL leukemia inhibitory factor (LIF), 10 nM CHIR-99021 (GSK-3 inhibitor) and 0.33 nM PD0325901 (MEK inhibitor).
      CAUTION: β-mercaptoethanol is flammable and has inhalation toxicity. Keep away from fire sources and wear a mask to avoid inhalation when use.
   2. Change the mESC growth medium daily for better growth of A2lox and 129.
   3. When the cells reach 80% confluence, remove the medium and add 1 mL of 0.1% trypsin to the dish. Gently rock for 30 s to ensure even cover of trypsin on all cells.
   4. Leave the cells for about 1 min to trypsinize and then remove the trypsin using 1 mL pipette.
   5. Add 2 mL of mESC growth medium to the dish, pipette up and down several times to make a single cell suspension.
   6. Count the density of the cells in the suspension as accurately as possible using hemocytometer.
   7. Divide the cells into 7 groups and induce differentiation using different protocols shown in Table 1.

2. Differentiation from mESCs to NPCs (Phase I)

1. Prepare 0.1% gelatin coated cell culture plates or coverslips.
   1. Before use, prepare 0.1% gelatin coated 6-well plates or coverslips as in step 1.1.
2. Phase I differentiation using protocol 1 (Table 1)
   1. Seed about 2 x 10⁴ mESCs in 2 mL of basal differentiation medium I per well in the 0.1% gelatin-coated 6-well plates. Check the cell density under a microscope.
   2. Incubate the cells at 37 °C in a 5% CO₂ incubator for 6 h to allow for attachment.
   3. After attachment, take out of the cells from incubator, and wash the cells twice with 2 mL of PBS.
   4. Add 2 mL of basal differentiation medium I (Table 1) to each well and put the cells back into the incubator.
   5. Leave the cells for differentiation for 8 days. Replace the basal differentiation medium I every 2 days.
3. Phase I differentiation using protocol 2 (Table 1)
   1. Add 1.5 x 10⁶ mESCs into a nonadhesive bacterial dish in 10 mL of basal differentiation medium I to allow for embryoid body formation at 37 °C in a 5% CO₂ incubator.
   2. After 2 days, transfer cell aggregates into 15 mL centrifuge tubes and let them settle by gravity.
   3. Remove the supernatant and add 10 mL of fresh basal differentiation medium I to resuspend the embryoid bodies. Replant them into a new nonadhesive bacterial dish and allow differentiation for another 2 days.
   4. Check the formation of embryoid bodies under the microscope (Figure 2A).
   5. Collect embryoid bodies as described in steps 2.3.2-2.3.3. Seed about 50 embryoid bodies in 2 mL of basal differentiation medium I per well onto 0.1% gelatin-coated 6-well plates.
   6. Prepare 1 mM all-trans RA stock (in DMSO) and store away from light in a -80 °C freezer after sub-packaging.
      NOTE: RA is unstable, and attention should be paid to keeping it away from light and reducing air contact during preparation of RA stock.
   7. For RA induction, add 2 µL of RA stock into each well to make a final concentration of 1 µM.
   8. Place the plate into the 5% CO₂ incubator at 37 °C and differentiate for another 4 days.
   9. Change the entire 2 mL of basal differentiation medium I (with 1 µM RA) every 2 days.
4. Phase I differentiation using protocol 3 (Table 1)
   1. Plant 1.5 x 10⁶ mESCs into a nonadhesive bacterial dish in 10 mL of basal differentiation medium I. Leave for 2 days for embryoid body formation at 37 °C in a 5% CO₂ incubator.
   2. Check the formation of embryoid bodies under a microscope (Figure 2B).
   3. Transfer cell aggregates into 15 mL centrifuge tubes and let them settle by gravity.
   4. Remove the supernatant carefully and add 10 mL of fresh basal differentiation medium I to resuspend them.
   5. Seed about 50 embryoid bodies into 2 mL of basal differentiation medium I per well onto 0.1% gelatin-coated 6-well plates.
   6. For RA induction, add 2 µL of RA stock into each well to make a final concentration of 1 µM.
   7. Place the plate into the 5% CO₂ incubator at 37 °C and differentiate for another 6 days. Change the entire basal differentiation medium I (with 1 µM RA) every 2 days.
5. Phase I differentiation using protocol 4 (Table 1)
   1. Seed about 2 x 10⁴ mESCs within 2 mL of basal differentiation medium I per well onto the 0.1% gelatin-coated 6-well plates. Place the plate into the 5% CO₂ incubator at 37 °C to allow for attachment for 6 h.
   2. After attachment, wash the cells twice with 2 mL of PBS. Add 2 mL of basal differentiation medium I to each well and allow for differentiation for 4 days in the 5% CO₂ incubator at 37 °C.
   3. For RA induction, add 2 µL of all-trans RA stock into each well (the working concentration is 1 µM) to induce differentiation for another 4 days.
   4. In the whole process, replace the entire medium every 2 days.
6. Phase I differentiation using protocol 5 (Table 1)
   1. Seed about 2 x 10⁴ mESCs within 2 mL of basal differentiation medium I per well onto the 0.1% gelatin-coated 6-well plates. Place the plate into the 5% CO₂ incubator at 37 °C to allow for attachment for 6 h.
   2. Wash the cells twice with 2 mL of PBS. Add 2 mL of basal differentiation medium I to each well and allow for differentiation for 2 days in the 5% CO₂ incubator at 37 °C.
   3. For the subsequent RA induction, add 2 µL of RA stock into each well to make a final concentration of 1 µM. Place the plate into the 5% CO₂ incubator at 37 °C to induce differentiation for another 6 days.
   4. In the whole process, replace the entire medium every 2 days.
7. Phase I differentiation using protocol 6 (Table 1)
   1. Plant 1.5 x 10⁶ mESCs into a nonadhesive bacterial dish in 10 mL of N2B27 medium II (Table 1) to allow for embryoid bodies formation.
   2. On the 2^nd day, collect the cell aggregates as described in steps 2.3.2-2.3.3 and resuspend the embryoid bodies using 10 mL of fresh N2B27 medium II.
   3. Replant them into a new nonadhesive bacterial dish and allow differentiation for another 2 days in the 5% CO₂ incubator at 37 °C. Check the formation of embryoid bodies under microscope.
   4. On the 4^th day, collect embryoid bodies. Seed about 50 embryoid bodies per well onto 0.1% gelatin-coated 6-well plates with 2 mL of N2B27 medium II.
   5. Add 2 µL of all-trans RA stock into each well and induce differentiation for another 4 days. Replace the entire medium (N2B27 medium II with 1 µM RA) every two days.
8. Phase I differentiation using protocol 7 (Table 1)
   1. Seed about 2 x 10⁴ mESCs within 2 mL of basal differentiation medium I per well onto the 0.1% gelatin-coated 6-well plates. Place the plate into the 5% CO₂ incubator at 37 °C to allow for attachment for 6 h.
   2. Wash the cells twice with PBS. Then, add 2 mL of N2B27 medium II to each well and allow for differentiation for 8 days at 37 °C in a 5% CO₂ incubator.
   3. Change the entire N2B27 medium II every 2 days.

3. Cell morphology observation

1. Check the differentiation status of the above-mentioned 7 groups daily under an inverted phase contrast light microscope.
2. Randomly select at least 12 fields and take photos to record the morphological changes of each group on D8.

4. Immunofluorescence staining

1. Sample preparation: Seed mESCs on 0.1% gelatin-coated coverslips and allow for differentiation for 8 days using the protocols mentioned in step 2.
2. Rinse: On the 8^th day, take the samples out from the incubator and remove the differentiation medium by aspiration. Gently rinse the cells once with 1 mL of PBS for 5 min.
3. Fixation: Add 1 mL of 4% paraformaldehyde to each sample and fix the cells for 20 min at room temperature (RT).
4. Rinse: After fixation, gently rinse the cells with 1 mL of PBS 3 times, for 5 min each.
5. Permeabilization: Add 1 mL of 0.2% TritonX-100 in PBS to each sample and leave for 8 min at RT.
6. Rinse: After permeabilization, gently rinse the cells with 1 mL of PBS 3 times, for 5 min each.
7. Blocking: Add 1 mL of 10% goat serum in PBS to each sample and incubate at RT for 1 h to block any non-specific interactions.
8. Incubation with primary antibody
   1. Dilute the anti-Nestin antibody at a ratio of 1:100 using 5% goat serum in PBS.
   2. Apply 500 µL of diluted antibody to different samples and incubate overnight at 4 °C.
9. Rinse: Remove the antibody and rinse the samples gently with 1 mL of PBS 3 times for 8 min each.
10. Incubation with secondary antibody
    1. Dilute the Alexa Fluor 488-labeled goat anti-mouse IgG at a ratio of 1:500 using 5% goat serum in PBS.
    2. Apply 500 µL of diluted antibody to different samples and incubate in dark for 2 h at RT.
       NOTE: After applying fluorescent secondary antibody, perform all the subsequent steps in the dark to prevent fluorescence quenching.
11. Rinse: Remove the secondary antibody and rinse the samples gently with 1 mL of PBS 3 times for 8 min each.
12. Nuclear staining and mounting
    1. Place one drop of DAPI mounting medium onto the clean microslide.
    2. Carefully take out of the samples from the plates and place the sample on top of the DAPI mounting medium with the cell face down. Leave in the dark for 5 min at RT.
    3. Remove excess DAPI mounting medium with absorbent paper.
13. Fluorescence microscopy observation
    1. Place the specimens under the fluorescence microscopy and detect the signal for DAPI and Alexa Fluor 488 using proper filters.
    2. Evenly and randomly pick 10-15 different visual fields for each sample and record the images with a CCD camera.

5. Differentiation from NPCs to neurons (Phase II)

1. Prepare mESC derivatives under Phase I differentiation using protocol 3 (8 days, Table 1) as detailed in step 2.4, which has the highest differentiation efficiency (See Figure 3).
   NOTE: After phase I differentiation, quality control should be carried out using cell morphology observation and immunofluorescence assay mentioned above, to ensure a healthy and high-yield NPCs.
2. Seed about 5 x 10⁵ 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.
3. 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.
4. Add 2 mL of basal differentiation medium I, N2B27 medium I and N2B27 medium II (Table 2), respectively, to each well of the above groups.
5. Place the plates into the incubator and allow to differentiate for another 10 days. Change the corresponding medium every 2 days.
6. Check the differentiation status and record the morphological changes as mentioned in step 3.
7. On Day 18, evaluate the generation of neurons (β-Tubulin III positive) and determine the differentiation efficiency of the 3 protocols using in step 4.

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.

Table 1: Details of the 7 protocols used in phase I differentiation. This table has been modified from Li et al.¹⁰.

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 ESCs^13,14. 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 dependent^16,17,18. In the condition presented here, RA added on the 2^nd 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 NPCs^{13,14,15,16,17,18,19}. 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 occur^20,21. 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.