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

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.

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

Name	Company	Catalog Number	Comments
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