Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

Summary

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

Abstract

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

Introduction

Since their first derivation from the inner cell mass of the developing mouse blastocysts^1,2, mouse embryonic stem cells (mESC) have been used as powerful tools to study stem cell self-renewal and differentiation³. Furthermore, studying mESC differentiation leads to tremendous understanding of molecular mechanisms that may improve efficiency and safety in stem cell-based therapy in treating diseases such as neurodegenerative disorders⁴. Compared to animal models, this in vitro system provides many advantages including simplicity in practice and assessment, low cost in maintaining cell lines in contrast to animals, and relative ease in genetic manipulations. However, the efficiency and quality of differentiated cell types are often affected by different lines of mESCs as well as the differentiation methods^5,6. Also, the traditional assays to evaluate differentiation efficiency rely on qualitative examination of selected marker genes which lack robustness and they therefore fail to grasp global changes in gene expression.

Here we aim to use a battery of assays for systematic assessment of the neuronal differentiation. Using both traditional in vitro analyses on selected markers and RNA-seq, we establish a platform for measurement of the differentiation efficiency as well as the transcriptomic changes during this process. Based on a previously established protocol⁷, we generated embryoid bodies (EBs) through the hanging drop technique, followed by induction using supraphysiologic amount of retinoic acid (RA) to generate neural progenitor cells (NPCs), which were subsequently differentiated to neurons with neural induction medium. To examine the efficiency of the differentiation, in addition to traditional RT-qPCR and immunofluorescence (IF) assays, we performed RNA-seq and flow cytometry. These analyses provide comprehensive measurement of the progression of the stage-specific differentiation.

Protocol

1. mESC culture

1. Coat a 10 cm tissue-culture-treated plate with 0.1% gelatin and allow the gelatin to set for at least 15–30 min before aspirating it out.
2. Seed γ-irradiated mouse embryonic fibroblasts (MEFs) one day before culturing the mESCs in the pre-warmed mESC medium (Dulbecco’s modified Eagle medium (DMEM) with 15% fetal bovine serum (FBS), non-essential amino acids, β-mercaptoethanol, L-glutamine, penicillin/streptomycin, sodium pyruvate, LIF, PD0325901 (PD), and Chir99021 (CH)).
3. Allow the γ-irradiated MEFs to settle and attach to the plate surface before culturing E14 cells.
4. Thaw E14 ESCs in 37 °C water bath and quickly transfer the cells in a 15 cm conical tube with warm mESC medium. Pellet the cells at 200 x g for 3 min and remove the supernatant.
5. Resuspend the cells in 10 mL of mESC medium and plate the cells on the culture plate containing the γ-irradiated MEFs seeded earlier. Incubate the cell culture in a 37 °C incubator under 5% CO₂.
6. For culture passaging, aspirate e the medium and wash the plate with sterile 1x PBS. Add enough 0.05% trypsin to cover the plate surface and incubate at 37 °C for 3 min.
7. Neutralize the trypsin with mESC medium and pipette to generate single-cell suspension. Centrifuge the cells at 200 x g for 3 min and remove the supernatant.
8. Count the cells with a hemocytometer or cell counter and seed about 5.0 x 10⁵ cells in a 10 cm culture plate.
9. Resuspend the cells in 10 mL of mESC medium, plate the cells on the gelatin-coated tissue culture plate and incubate cultures as described earlier.
   NOTE: It is recommended that mESCs be passaged every 2 days to prevent the cells from differentiating in their colonies. Phenol red in the medium functions solely as a pH indicator and depending on the cellular density, it can turn yellowish (more acidic), sooner than 2 days. Hence, it may be necessary to change the medium every day. The γ-irradiated MEFs will eventually die off after a couple of passages.

2. EB, NPC, and neuron differentiation

1. Perform culture passaging protocol mentioned earlier and count the cells (steps 1.7–1.10).
2. Hanging drop method (day 0)
   1. For a 10 cm cell culture plate, count roughly 2.5 x 10⁴ cells where 5.0 x 10² cells will be suspended in 20 µL differentiation medium (DMEM with 15% FBS, non-essential amino acids, β-mercaptoethanol, L-glutamine, penicillin/streptomycin, and sodium pyruvate). Roughly fifty 20 µL droplets containing the cells can be plated on one 10 cm plate.
   2. Aliquot the appropriate number of cells, and then centrifuge the cells at 200 x g for 3 min and remove the supernatant.
   3. Resuspend the cells in the appropriate volume of differentiation medium for a cell density of 5.0 x 10² cells per 20 µL (e.g., 2.5 x 10⁴ cells in 1 mL of differentiation medium).
   4. Using a micropipette or a repeater pipette, place 20 µL droplets of the cell suspension onto the lid of the tissue culture plate. Make sure that the droplets are not too close to one another to prevent them from merging.
      NOTE: The droplets can be plated on either a tissue-culture-treated attachment plate or suspension plate as they will be placed on the lid and not the plate itself. For a more feasible and sterile approach, plate the droplets on an attachment tissue-culture plate and transfer them to a suspension plate as described below.
   5. Fill up the plate with 5–10 mL of 1x PBS and carefully put the lid back on the plate. Incubate the culture in the 37 °C incubator.
      NOTE: PBS is added to the culture plate to prevent the droplets from drying up.
3. On day 2, use a micropipette to collect the droplets from the lid and place them in a 10 cm cell culture suspension plate filled with 10 mL of differentiation medium. Incubate the culture on an orbital shaker shaking at low speed in the incubator.
4. On day 4, to harvest the EBs, collect the cells, centrifuge at 200 x g for 3 min, and remove the supernatant.
   NOTE: The EBs can also be washed with 1x PBS as per the requirements of subsequent experiments.
5. To continue the procedure and induce the EB differentiation into neural progenitor cells (NPCs), prepare the differentiation medium with 5 µM retinoic acid (RA).
6. Remove the old medium by pelleting the EBs at 100 x g for 3 min or allow the EBs to settle before aspirating out the old medium. Add 10 mL of the differentiation medium containing 5 µM RA to the culture plate.
7. On day 6, replace at least half of the medium with fresh medium containing 5 µM RA by tilting the plate and pipetting out the medium as described above.
   NOTE: It is recommended that at least half of the medium be replaced with fresh RA-containing medium on days 5 and 7. Take note of the phenol red indicator; if it turns yellowish, it is best to replace all of the medium.
8. On day 8, harvest the NPCs by collecting the cells, centrifuging at 200 x g for 3 min, and removing the supernatant.
   NOTE: The NPCs can also be washed with 1x PBS according to the needs of subsequent experiments. If needed, NPCs can be frozen down and thawed again for later culture and analysis. If the NPCs are to be cultured, accutase can also be used as an alternative to trypsin.
9. To continue the procedure and to differentiate NPCs into neurons, collect NPCs in a 15 mL conical tube by centrifugation, dissociate them with trypsin and incubate them at 37 °C for 3 min. Pipette the NPCs to ensure that all NPC aggregates are dissociated and neutralize the trypsin with the medium.
10. Filter the cells with 40 μm nylon cell strainer and count the cells before plating them at a density of 1.5 x 10⁵/cm² in N2 medium (DMEM/F12 medium + 3 mg/mL glucose + 3 mg/mL lipid-rich bovine serum albumin (LBSA) + 1:100 N2 supplement + 10 ng/mL bFGF + 50 U/mL pen/strep + 1 mM L-glutamine) on a tissue-culture-treated plate for subsequent PCR and western blot experiments; or on a tissue culture chamber for immunofluorescence experiments.
11. On day 9, replace the old medium with a fresh N2 medium.
12. On day 10, switch the N2 medium with N2/B27 medium (50% DMEM/F12 and 50% neural basal, 3 mg/mL LBA, 1:200 N2 supplement, 1:100 B27 supplement, 50 U/mL pen/strep, and 1 mM L-glutamine).
13. On days 11–12, harvest the neurons as follows. Wash the cells with 1x PBS, add trypsin, and incubate the culture in the 37 °C incubator for 3 min before neutralizing the trypsin with medium and centrifuge at 200 x g for 3 min.

3. Characterization of mESCs and differentiated cells

1. Alkaline phosphatase (AP) assay
   1. Use a kit to assess alkaline phosphatase activity (see the Table of Materials).
   2. Remove the medium from the culture plate and wash the ESCs with 1x PBS.
   3. Add 1 mL of the fix solution (consists of formaldehyde and methanol) provided with the kit to the plate and incubate it at room temperature for 2–5 min.
      NOTE: Over-incubation in the fix solution can compromise the AP activity.
   4. Remove the fix solution, wash the ESCs with 1x PBS and leave some amount of PBS in the plate.
      NOTE: Keep the ESCs moist in PBS to not compromise the AP activity.
   5. Prepare the AP solution by mixing the A, B, and C substrate solutions at 1:1:1 ratio. Mix A and B solutions first and incubate the mixture at room temperature for 2 min before adding the C solution.
   6. Remove the 1x PBS and add the AP solution prepared earlier.
   7. Incubate the ESCs for about 15 min in the dark by wrapping the culture plate with aluminum foil or performing step 3.5 in a dark room.
   8. Monitor the reaction and remove the reaction solution when the solution turns bright to avoid non-specific staining.
   9. Wash the ESCs twice with 1x PBS.
   10. Prevent the sample from drying by covering the ESCs with 1x PBS or mounting medium.
       NOTE: A red or purple stain will appear for AP expression. The plate can be stored in a 4 °C refrigerator.
2. RT-qPCR
   1. Collect cells at various stages by following steps 1.6–1.7 and 2.4 for ESCs and EBs and NPCs, respectively.
   2. Isolate RNA using RNA, DNA, and protein extraction solution (see Table of Materials).
   3. Generate cDNA with a reverse transcriptase kit (see the Table of Materials) and follow the manufacturer’s manual.
3. Fixation and embedding
   1. Harvest EBs and NPCs as described above (step 2.6) and fix them with 4% paraformaldehyde (PFA) solution in 1x PBS for 30 min at room temperature.
   2. Remove the PFA and wash the sample with 1x PBS for 5 min.
   3. Place the sample in a serial dilution of 1x PBS, 10%-, 20%-, and 30%-sucrose solutions at 25‒28 °C where the sample is transferred to the next solution after 30 min of incubation.
      NOTE: The sample can be stored in the 30% sucrose solution at 4 °C before continuing with the embedding step.
   4. Wet the pipette tip with sucrose solution before placing the sample (without stacking them) at the center of the cryo-mold and pipette out the excess liquid.
      NOTE: Filter paper can also be used to remove the excess solution. Wetting the pipette tip with sucrose solution is important to prevent the EBs and NPCs from sticking to the walls of the tip.
   5. Carefully add optimum cutting temperature (OCT) solution to the mold without resuspending the samples and remove excess bubbles with a pipette.
   6. Place the mold with the sample on a laboratory mixer at low speed to mildly agitate the sample for 15 min. This helps to settle the EBs and NPCs to the bottom if they are resuspended in the OCT solution.
   7. Quickly freeze the sample by placing the mold in liquid nitrogen or on dry ice.
      NOTE: Samples can be stored in a -70 °C freezer before continuing to the next step.
4. Cryosectioning
   1. Set the cryostat to cool down to -20 to -18 °C before transferring the sample to the instrument.
   2. Detach the frozen OCT block from the mold and secure it on the holder with a little OCT solution placed on the surface of the holder.
   3. Align the OCT block so that the EBs and NPCs are closest to the blade to ensure that the sample is not lost during sectioning.
   4. Carefully section 10 μm of the block and pay close attention to the slices that contain the sample.
   5. Quickly place the OCT slice containing the sample onto the tissue-embedding glass slide and allow the OCT slices to air-dry for 1 h at room temperature.
      NOTE: The samples can be stored at -70 °C for later use.
5. Immunofluorescence (IF)
   1. Block OCT sections or culture chambers containing neurons in 10% normal donkey serum/0.1% Triton X-100 in 1x PBS for 1 h at room temperature.
   2. Incubate the samples in primary antibody diluted in 5% normal donkey serum/0.05% Triton X-100 in 1x PBS overnight at 4 °C.
   3. Wash samples in 1x PBS/0.1% Triton X-100 thrice for 5 min each wash.
   4. Incubate the samples with secondary antibody diluted in 5% normal donkey serum/0.05% Triton X-100 in 1x PBS for 1 h at room temperature.
   5. Wash samples in 1x PBS/0.1% Triton X-100 thrice for 5 min each wash then incubate the samples in 1 μg/mL DAPI.
   6. Mount the samples with a coverslip and some mounting medium and allow it to dry.
   7. Observe the samples under a fluorescence microscope.
6. RNA-seq analysis
   1. Collect the cells at various stages and perform RNA extraction (see step 3.2).
   2. Prepare the cDNA libraries, perform deep sequencing and data analysis according to the protocol described in Wang et al.⁸.
   3. Perform the Gene Ontology (GO) analysis using the R package, clusterProfiler.
7. Flow cytometry
   1. Collect ESCs by following the steps 1.6–1.7 and resuspend the cells in medium. Collect the EBs and NPCs by following step 2.4 and resuspend the cells in medium.
   2. Filter the cell suspension using the 40 μm nylon cell strainer into a new 15 mL conical tube.
   3. Measure the GFP signal of the samples using the flow cytometer (performed by the institution’s core facility).

Results

As a representation of our method, we performed an EB, NPC, and neuron differentiation experiment on E14 cells. E14 cells were cultured on γ-irradiated MEFs (Figure 1A) until the γ-irradiated MEF population diluted out. We confirmed the pluripotency of the E14 cells by performing Alkaline Phosphatase (AP) staining (Figure 1B) and later RT-qPCR (see below) for Nanog and Oct4 markers. The γ-irradiated MEF-free E14 cells were then i...

Discussion

The method for neural differentiation of mouse embryonic stem cells has been established for decades and researchers have continued to modify the previous protocols or create new ones for various purposes^7,10,11. We utilized a series of assays to comprehensively analyze the efficiency and progress of the differentiation stages of mESCs to neurons, which may be used in analysis of other lineage differentiation of mouse or human E...

Materials

Name	Company	Catalog Number	Comments
0.05% Trypsin + 0.53mM EDTA 1X	Corning	25-052-CV
0.1% Gelatin	Sigma	G1890-100G	Prepared in de-ionized water
16% Paraformaldehyde	Thermo Scientific	28908	Diluted in 1X PBS
40-μm cell strainer	Falcon	352340
Albumax	Thermo Fisher Scientific	11020021
AlexaFluor 488 goat anti-mouse IgG (H+L)	Invitrogen	A11001	Antibody was diluted at 1:500 for IF
Alkaline Phosphatase Staining Kit II	Stemgent	00-0055
AzuraQuant Green Fast qPCR Mix LoRox	Azura Genomics	AZ-2105
B27 supplement	Thermo Fisher Scientific	17504044
BD FACSCanto	BD	657338
bFGF	Sigma	11123149001
BioAnalyzer High Sensitivity DNA Kit	Agilent	5067-4626
Chir99021	Cayman Chemicals	13122
Chloroform	C298-500	Fisher Chemical
DAPI	Invitrogen	R37606
DMEM	Corning	10-017-CM
DMEM/F12 medium	Thermo Fisher Scientific	11320033
EB buffer	Qiagen	19086
Ethanol	111000200	Pharmco	Diluted in de-ionized water
Fetal bovine serum	Atlanta Biologicals	S10250
Fisherbrand Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15
HiSeq 2500 Sequencing System	Illumina	SY-401-2501
Isopropanol	BDH1133-4LG	BDH VWR Analytical	Diluted in de-ionized water
L-glutamine	Thermo Fisher Scientific	25030024
LIF	N/A	N/A	Collected from MEF supernatant
m18srRNA primers	IDTDNA	N/A	5'-GCAATTATTCCCCATGAACG-3' 5'-GGCCTCACTAAACCATCCAA-3'
MEM Non-essential amino acids	Corning	25-025-Cl
mNanog primers	IDTDNA	N/A	5'-AGGCTTTGGAGACAGTGAGGTG-3' 5'-TGGGTAAGGGTGTTCAAGCACT-3'
mNes primers	IDTDNA	N/A	5'-AGTGCCCAGTTCTAGTGGTGTCC-3' 5'-CCTCTAAAATAGAGTGGTGAGGGTTG-3'
mNeuroD1 primers	IDTDNA	N/A	5'-CGAGTCATGAGTGCCCAGCTTA-3' 5'-CCGGGAATAGTGAAACTGACGTG-3'
mOct4 primers	IDTDNA	N/A	5'-AGATCACTCACATCGCCAATCA-3' 5'-CGCCGGTTACAGAACCATACTC-3'
mPax6 primers	IDTDNA	N/A	5'-CTTGGGAAATCCGAGACAGA-3' 5'-CTAGCCAGGTTGCGAAGAAC-3'
N2 supplement	Thermo Fisher Scientific	17502048
Nestin primary antibody	Millipore	MAB5326	Antibody was diluted at 1:200 for IF
Neural basal	Thermo Fisher Scientific	21103049
Neurofilament primary antibody	DSHB	2H3
NEXTflex Illumina Rapid Directional RNA-Seq Library Prep Kit	BioO Scientific	NOVA-5138-07
PD0325901	Cayman Chemicals	13034
Penicillin/streptomycin	Corning	30-002-Cl
Phosphate-buffered saline (PBS)	N/A	N/A	Prepared in de-ionized water
- Potassium chloride	P217-500G	VWR
- Potassium phosphate monobasic anhydrous	0781-500G	VWR
- Sodium chloride	BP358-10	Fisher Bioreagents
- Sodium phosphate, dibasic, heptahydrate	SX0715-1	Milipore
Random hexamer primer	Thermo Scientific	SO142
Retinoic acid	Sigma	R2625	Prepared in DMSO
Sodium pyruvate	Corning	25-000-Cl
Sucrose	Sigma	84097	Diluted in 1X PBS
SuperScript III Reverse Transcriptase	Invitrogen	18064022
Tissue-Tek O.C.T. compound	Sakura	4583
TriPure Isolation Reagent	Sigma-Aldrich	11667165001
TruSeq Rapid	Illumina	20020616
β-mercaptoethanol	Fisher BioReagents	BP176-100