This work was supported by a grant from the NIH (1R35GM133496-01) to Z. Gao. We would like to thank Dr. Ryan Hobbs for the assistance in sectioning. We thank&#160;Penn State College of Medicine&#39;s core facilities, including the Genome Sciences and Bioinformatics, the Advanced Light&#160;Microscopy Imaging, and the Flow Cytometry. We also thank Dr.&#160;Yuka Imamura&#160;for the assistance in RNA-seq analysis.&#160;

1. mESC culture
<ol>
	<li>Coat a 10 cm tissue-culture-treated plate with 0.1% gelatin and allow the gelatin to set for at least 15&#8211;30 min before aspirating it out.</li>
	<li>Seed &#947;-irradiated mouse embryonic fibroblasts (MEFs) one day before culturing the mESCs in the pre-warmed mESC medium (Dulbecco&#8217;s modified Eagle medium (DMEM) with 15% fetal bovine serum (FBS), non-essential amino acids, &#946;-mercaptoethanol, L-glutamine, penicillin/streptomycin, sodium pyruvate, LIF, PD0325901 (PD), and Chir99021 (CH)).</li>
	<li>Allow the &#947;-irradiated MEFs to settle and attach to the plate surface before culturing E14 cells.</li>
	<li>Thaw E14 ESCs in 37 &#176;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.</li>
	<li>Resuspend the cells in 10 mL of mESC medium and plate the cells on the culture plate containing the &#947;-irradiated MEFs seeded earlier. Incubate the cell culture in a 37 &#176;C incubator under 5% CO2.</li>
	<li>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 &#176;C for 3 min.</li>
	<li>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.</li>
	<li>Count the cells with a hemocytometer or cell counter and seed about 5.0 x 105 cells in a 10 cm culture plate.</li>
	<li>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 &#947;-irradiated MEFs will eventually die off after a couple of passages.</li>
</ol>
2. EB, NPC, and neuron differentiation
<ol>
	<li>Perform culture passaging protocol mentioned earlier and count the cells (steps 1.7&#8211;1.10).</li>
	<li>Hanging drop method (day 0)
	<ol>
		<li>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 &#181;L differentiation medium (DMEM with 15% FBS, non-essential amino acids, &#946;-mercaptoethanol, L-glutamine, penicillin/streptomycin, and sodium pyruvate). Roughly fifty 20 &#181;L droplets containing the cells can be plated on one 10 cm plate.</li>
		<li>Aliquot the appropriate number of cells, and then centrifuge the cells at 200 x g for 3 min and remove the supernatant.</li>
		<li>Resuspend the cells in the appropriate volume of differentiation medium for a cell density of 5.0 x 102 cells per 20 &#181;L (e.g., 2.5 x 104 cells in 1 mL of differentiation medium).</li>
		<li>Using a micropipette or a repeater pipette, place 20 &#181;L 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.</li>
		<li>Fill up the plate with 5&#8211;10 mL of 1x PBS and carefully put the lid back on the plate. Incubate the culture in the 37 &#176;C incubator. 
		NOTE: PBS is added to the culture plate to prevent the droplets from drying up.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>To continue the procedure and induce the EB differentiation into neural progenitor cells (NPCs), prepare the differentiation medium with 5 &#181;M retinoic acid (RA).</li>
	<li>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 &#181;M RA to the culture plate.</li>
	<li>On day 6, replace at least half of the medium with fresh medium containing 5 &#181;M 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.</li>
	<li>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.</li>
	<li>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 &#176;C for 3 min. Pipette the NPCs to ensure that all NPC aggregates are dissociated and neutralize the trypsin with the medium.</li>
	<li>Filter the cells with 40 &#956;m 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.</li>
	<li>On day 9, replace the old medium with a fresh N2 medium.</li>
	<li>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).</li>
	<li>On days 11&#8211;12, harvest the neurons as follows. Wash the cells with 1x PBS, add trypsin, and incubate the culture in the 37 &#176;C incubator for 3 min before neutralizing the trypsin with medium and centrifuge at 200 x g for 3 min.</li>
</ol>
3. Characterization of mESCs and differentiated cells
<ol>
	<li>Alkaline phosphatase (AP) assay 
	<ol>
		<li>Use a kit to assess alkaline phosphatase activity (see the Table of Materials).</li>
		<li>Remove the medium from the culture plate and wash the ESCs with 1x PBS.</li>
		<li>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&#8211;5 min. 
		NOTE: Over-incubation in the fix solution can compromise the AP activity.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Remove the 1x PBS and add the AP solution prepared earlier.</li>
		<li>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.</li>
		<li>Monitor the reaction and remove the reaction solution when the solution turns bright to avoid non-specific staining.</li>
		<li>Wash the ESCs twice with 1x PBS.</li>
		<li>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 &#176;C refrigerator.</li>
	</ol>
	</li>
	<li>RT-qPCR 
	<ol>
		<li>Collect cells at various stages by following steps 1.6&#8211;1.7 and 2.4 for ESCs and EBs and NPCs, respectively.</li>
		<li>Isolate RNA using RNA, DNA, and protein extraction solution (see Table of Materials).</li>
		<li>Generate cDNA with a reverse transcriptase kit (see the Table of Materials) and follow the manufacturer&#8217;s manual.</li>
	</ol>
	</li>
	<li>Fixation and embedding 
	<ol>
		<li>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.</li>
		<li>Remove the PFA and wash the sample with 1x PBS for 5 min.</li>
		<li>Place the sample in a serial dilution of 1x PBS, 10%-, 20%-, and 30%-sucrose solutions at 25&#8210;28 &#176;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 &#176;C before continuing with the embedding step.</li>
		<li>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.</li>
		<li>Carefully add optimum cutting temperature (OCT) solution to the mold without resuspending the samples and remove excess bubbles with a pipette.</li>
		<li>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.</li>
		<li>Quickly freeze the sample by placing the mold in liquid nitrogen or on dry ice. 
		NOTE: Samples can be stored in a -70 &#176;C freezer before continuing to the next step.</li>
	</ol>
	</li>
	<li>Cryosectioning 
	<ol>
		<li>Set the cryostat to cool down to -20 to -18 &#176;C before transferring the sample to the instrument.</li>
		<li>Detach the frozen OCT block from the mold and secure it on the holder with a little OCT solution placed on&#160;the surface of the holder.</li>
		<li>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.</li>
		<li>Carefully section 10 &#956;m of the block and pay close attention to the slices that contain the sample.</li>
		<li>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 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Immunofluorescence (IF) 
	<ol>
		<li>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.</li>
		<li>Incubate the samples in primary antibody diluted in 5% normal donkey serum/0.05% Triton X-100 in 1x PBS overnight at 4 &#176;C.</li>
		<li>Wash samples in 1x PBS/0.1% Triton X-100 thrice for 5 min each wash.</li>
		<li>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.</li>
		<li>Wash samples in 1x PBS/0.1% Triton X-100 thrice for 5 min each wash then incubate the samples in 1 &#956;g/mL DAPI.</li>
		<li>Mount the samples with a coverslip and some mounting medium and allow it to dry.</li>
		<li>Observe the samples under a fluorescence microscope.</li>
	</ol>
	</li>
	<li>RNA-seq analysis 
	<ol>
		<li>Collect the cells at various stages and perform RNA extraction (see step 3.2).</li>
		<li>Prepare the cDNA libraries, perform deep sequencing and data analysis according to the protocol described in Wang et al.8.</li>
		<li>Perform the Gene Ontology (GO) analysis using the R package, clusterProfiler.</li>
	</ol>
	</li>
	<li>Flow cytometry 
	<ol>
		<li>Collect ESCs by following the steps 1.6&#8211;1.7 and resuspend the cells in medium. Collect the EBs and NPCs by following step 2.4 and resuspend the cells in medium.</li>
		<li>Filter the cell suspension using the 40 &#956;m nylon cell strainer into a new 15 mL conical tube.</li>
		<li>Measure the GFP signal of the samples using the flow cytometer (performed by the institution&#8217;s core facility).</li>
	</ol>
	</li>
</ol>

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Authors declare that there are no competing financial interests.

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 purposes7,10,11. 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 ESCs. Furthermore, our approaches have proved to be useful tools to evaluate the impact of specific genes or pathways on neuronal differentiation in vitro8.
With our methods, pluripotent un-committed ESCs treated with retinoic acid (RA) commit to the neural lineage with a high efficiency and are further induced to generate neurons7. To improve the successful differentiation of ES cells into neural cells and reduce the heterogeneity, it is important to keep the ES cells in an undifferentiated state12. Non-proliferative MEFs, treated with &#947;-irradiation or mitomycin C, function to maintain the pluripotency of the ESCs and provide a scaffold for their growth13,14. To obtain consistent results, we start each differentiation experiment with mESCs cultured on &#947;-irradiated MEFs. After a few passages, the &#947;-irradiated MEFs die out and the culture eventually becomes homogenous for mESC cells. Alternatively, the mESCs can be pre-plated on gelatin for about 45 min before &#947;-irradiated MEFs are seeded to better remove them on the next passage. Leukemia inhibitory factor (LIF) has long been used to maintain the pluripotency state of cultured mouse ES cells by activating the JAK/STAT pathway15,16,17. More recently, PD0325901 (PD, a MEK inhibitor) and CHIR99021 (CH, a GSK inhibitor) were found to provide additional pluripotency maintenance of the ES cells3,18. In our protocol, we culture mESCs with these inhibitors together with LIF to maintain high pluripotency of mESCs.
Another critical factor to achieve successful differentiation is the quality of EBs. We perform the differentiation of E14 cells by the hanging drop method, which has been applied by other investigators5,19,20. With this method, single ES cells are allowed to suspend in the differentiation medium droplet for 2 days where they spontaneously aggregate and form EBs. The resulting EBs are typically more well-defined in terms of their morphology (Figure 2B) compared to the method of suspending isolated mESCs in medium, which results in EB sizes in a much wider range in our experience (data not shown). To prevent EBs from attaching to the plates, it is important to do a low speed rotation starting from day 3 continuing through the differentiation process. The EBs are induced to differentiate into NPCs by treating them with RA. The resulting NPCs from RA treatment at EB day 4 are typically heterogenous for neural cells such as oligodendrocytes and astrocytes21. Using RT-qPCR or immunofluorescence, the NPC population can be probed for neuron and other neural lineage markers such as Gfap for astrocytes and Olig2 for oligodendrocytes. To further induce the differentiation of NPCs into neurons, the NPCs are cultured in optimal neuron medium where the most important components are the N2 and B27 supplements. N2 supplement mainly functions to help the NPCs to commit to the neuronal lineage while the B27 supplement functions to maintain the longevity of the neurons.
The samples can be collected across the differentiation period (e.g., ESC stage, EB day 2, EB day 4, NPC day 6, NPC day 8, neurons day 10, and neurons day 12) to track the differentiation process by performing RT-qPCR for pluripotency and ectoderm markers. Comparing the pluripotency markers such as Oct4, Nanog, Sox2, Klf4, and Myc between the different cell stages will verify the pluripotency of the mESCs (Figure 3B,F). To investigate the efficiency of the neural differentiation, markers for the mesoderm layer such as Hand1, Snai1, and Tbxt; and endoderm layer such as Eomes and Gata4 can also be probed for (data not shown). Further verification can be performed with immunofluorescence (IF) probing for NPC or neuronal markers (Figure 3A). However, these methods are not quantitative and biased toward the selected markers. To overcome these limitations, we incorporate flow cytometry and RNA-seq analyses (Figure 3C&#8210;F). The cell line used in the flow cytometry experiment is a Sox1-GFP E14 cell line, which was used specifically in this experiment to assess the quality of the NPC differentiation procedure. Sox1 is one of the earliest specific neuronal marker during neuroectoderm development22 hence making it an excellent marker for NPC lineage. Sox1 can be probed for using RT-qPCR or Western blot to evaluate the NPC population. These analyses are particularly beneficial to investigate the differentiation defect caused by gene manipulation or chemical treatment.
It is important to note that there are a few limitations to our protocol presented here. First of all, we are only presenting the comprehensive analysis for one wild-type mESC cell line. Other ESC lines originating from mice or humans might require changes and further optimization in the protocol to ensure successful and efficient neuron differentiation. Secondly, we present an in vitro neuron differentiation method, which naturally bears its own set of limitations. As mentioned before, EBs are treated with a supraphysiological level of RA to drive them towards the NPC lineage. The resulting NPCs are then placed in neuron-optimum media to mimic the physiological conditions and encourage neuron lineage commitment, growth, and longevity. Here, N2 and B27 supplements are used to culture neurons but other supplements are also available such as NS2123 for similar purposes, which may alter the success and efficiency of neuron differentiation. These conditions are synthetically reconstituted in the cell culture assays, which may not fully represent physiological conditions. The quality of the EBs, NPCs, and neurons highly depend on the starting mESCs. mESCs that have been passaged for too many times and kept in culture for more than 1 week typically start to lose pluripotency and may not successfully undergo differentiation. Thus, maintaining the mESCs in an optimal condition is key in ensuring that they can effectively differentiate into EBs, NPCs, and neurons. Other neuron culture methods such as 3D models have also been proposed to better mimic physiological conditions24,25,26 sometimes at the expense of throughput and feasibility27,28. We believe our protocols are useful to characterize these 3D culture models.

Since their first derivation from the inner cell mass of the developing mouse blastocysts1,2, 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 methods5,6. 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.

<table><tbody><tr><td>0.05% Trypsin + 0.53mM EDTA 1X</td><td>Corning</td><td>25-052-CV</td><td></td></tr><tr><td>0.1% Gelatin</td><td>Sigma</td><td>G1890-100G</td><td>Prepared in de-ionized water</td></tr><tr><td>16% Paraformaldehyde</td><td>Thermo Scientific</td><td>28908</td><td>Diluted in 1X PBS</td></tr><tr><td>40-&amp;mu;m cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>Albumax</td><td>Thermo Fisher Scientific</td><td>11020021</td><td></td></tr><tr><td>AlexaFluor 488 goat anti-mouse IgG (H+L)</td><td>Invitrogen</td><td>A11001</td><td>Antibody was diluted at 1:500 for IF</td></tr><tr><td>Alkaline Phosphatase Staining Kit II</td><td>Stemgent</td><td>00-0055</td><td></td></tr><tr><td>AzuraQuant Green Fast qPCR Mix LoRox</td><td>Azura Genomics</td><td>AZ-2105</td><td></td></tr><tr><td>B27 supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>BD FACSCanto</td><td>BD</td><td>657338</td><td></td></tr><tr><td>bFGF</td><td>Sigma</td><td>11123149001</td><td></td></tr><tr><td>BioAnalyzer High Sensitivity DNA Kit</td><td>Agilent</td><td>5067-4626</td><td></td></tr><tr><td>Chir99021</td><td>Cayman Chemicals</td><td>13122</td><td></td></tr><tr><td>Chloroform</td><td>C298-500</td><td>Fisher Chemical</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>R37606</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-017-CM</td><td></td></tr><tr><td>DMEM/F12 medium</td><td>Thermo Fisher Scientific</td><td>11320033</td><td></td></tr><tr><td>EB buffer</td><td>Qiagen</td><td>19086</td><td></td></tr><tr><td>Ethanol</td><td>111000200</td><td>Pharmco</td><td>Diluted in de-ionized water</td></tr><tr><td>Fetal bovine serum</td><td>Atlanta Biologicals</td><td>S10250</td><td></td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>HiSeq 2500 Sequencing System</td><td>Illumina</td><td>SY-401-2501</td><td></td></tr><tr><td>Isopropanol</td><td>BDH1133-4LG</td><td>BDH VWR Analytical</td><td>Diluted in de-ionized water</td></tr><tr><td>L-glutamine</td><td>Thermo Fisher Scientific</td><td>25030024</td><td></td></tr><tr><td>LIF</td><td>N/A</td><td>N/A</td><td>Collected from MEF supernatant</td></tr><tr><td>m18srRNA primers</td><td>IDTDNA</td><td>N/A</td><td>5&#039;-GCAATTATTCCCCATGAACG-3&#039;&lt;br/&gt; 5&#039;-GGCCTCACTAAACCATCCAA-3&#039;</td></tr><tr><td>MEM Non-essential amino acids</td><td>Corning</td><td>25-025-Cl</td><td></td></tr><tr><td>mNanog primers</td><td>IDTDNA</td><td>N/A</td><td>5&#039;-AGGCTTTGGAGACAGTGAGGTG-3&#039;&lt;br/&gt; 5&#039;-TGGGTAAGGGTGTTCAAGCACT-3&#039;</td></tr><tr><td>mNes primers</td><td>IDTDNA</td><td>N/A</td><td>5&#039;-AGTGCCCAGTTCTAGTGGTGTCC-3&#039;&lt;br/&gt; 5&#039;-CCTCTAAAATAGAGTGGTGAGGGTTG-3&#039;</td></tr><tr><td>mNeuroD1 primers</td><td>IDTDNA</td><td>N/A</td><td>5&#039;-CGAGTCATGAGTGCCCAGCTTA-3&#039;&lt;br/&gt; 5&#039;-CCGGGAATAGTGAAACTGACGTG-3&#039;</td></tr><tr><td>mOct4 primers</td><td>IDTDNA</td><td>N/A</td><td>5&#039;-AGATCACTCACATCGCCAATCA-3&#039;&lt;br/&gt; 5&#039;-CGCCGGTTACAGAACCATACTC-3&#039;</td></tr><tr><td>mPax6 primers</td><td>IDTDNA</td><td>N/A</td><td>5&#039;-CTTGGGAAATCCGAGACAGA-3&#039;&lt;br/&gt; 5&#039;-CTAGCCAGGTTGCGAAGAAC-3&#039;</td></tr><tr><td>N2 supplement</td><td>Thermo Fisher Scientific</td><td>17502048</td><td></td></tr><tr><td>Nestin primary antibody</td><td>Millipore</td><td>MAB5326</td><td>Antibody was diluted at 1:200 for IF</td></tr><tr><td>Neural basal</td><td>Thermo Fisher Scientific</td><td>21103049</td><td></td></tr><tr><td>Neurofilament primary antibody</td><td>DSHB</td><td>2H3</td><td></td></tr><tr><td>NEXTflex Illumina Rapid Directional RNA-Seq Library Prep Kit</td><td>BioO Scientific</td><td>NOVA-5138-07</td><td></td></tr><tr><td>PD0325901</td><td>Cayman Chemicals</td><td>13034</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>N/A</td><td>N/A</td><td>Prepared in de-ionized water</td></tr><tr><td>- Potassium chloride</td><td>P217-500G</td><td>VWR</td><td></td></tr><tr><td>- Potassium phosphate monobasic anhydrous</td><td>0781-500G</td><td>VWR</td><td></td></tr><tr><td>- Sodium chloride</td><td>BP358-10</td><td>Fisher Bioreagents</td><td></td></tr><tr><td>- Sodium phosphate, dibasic, heptahydrate</td><td>SX0715-1</td><td>Milipore</td><td></td></tr><tr><td>Random hexamer primer</td><td>Thermo Scientific</td><td>SO142</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma</td><td>R2625</td><td>Prepared in DMSO</td></tr><tr><td>Sodium pyruvate</td><td>Corning</td><td>25-000-Cl</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>84097</td><td>Diluted in 1X PBS</td></tr><tr><td>SuperScript III Reverse Transcriptase</td><td>Invitrogen</td><td>18064022</td><td></td></tr><tr><td>Tissue-Tek O.C.T. compound</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>TriPure Isolation Reagent</td><td>Sigma-Aldrich</td><td>11667165001</td><td></td></tr><tr><td>TruSeq Rapid</td><td>Illumina</td><td>20020616</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Fisher BioReagents</td><td>BP176-100</td><td></td></tr></tbody></table>

differentiation and characterization of neural progenitors and neurons from mouse embryonic stem cells

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&#160;reverse transcription&#160;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.

As a representation of our method, we performed an EB, NPC, and neuron differentiation experiment on E14 cells. E14 cells were cultured on &#947;-irradiated MEFs (Figure 1A) until the &#947;-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 &#947;-irradiated MEF-free E14 cells were then induced for differentiation using the protocol outlined in Figure 2A. Briefly, differentiation media droplets of 20 &#181;L containing 500 cells were seeded on the lid of the culture plate (see protocol section 2 for details). The EBs formed were then collected and placed in suspension in fresh differentiation media. From day 4 to day 8 of differentiation, 5 &#956;M RA was added to the culture plates to induce NPCs. Differentiated EBs showed round shape and their size continued to increase during differentiation (Figure 2B). At day 8, the NPCs were harvested and trypsinized, and then the resulting single cell suspension was plated in a tissue culture chamber in DMEM/F12 medium with N2 supplement and later in B27 supplement. By day 10, NPCs differentiating into neurons appear to have elongated-cell shape (Figure 2B).
To further evaluate our differentiation experiment, we performed immunofluorescence (IF) experiments on E14 NPCs at day 8 and E14 neurons at day 12. We observed positive staining for nestin in NPCs and neurofilament (NF) signal for neurons (Figure 3A). Alternatively, RT-qPCR and RNA-seq confirmed the induction of NPC marker genes and loss of pluripotency genes in NPCs (Figure 3B,D,F). As a quantitative method to test the success of ESC differentiation, we differentiated a mouse ESC line expressing a Sox1 promoter-driven GFP reporter9, followed by flow cytometry analysis on ESCs and NPCs. We found that 58.7% of total cells at the NPC stage are GFP-positive while the GFP signal is 0.0% at the ESC stage (Figure 3C). To profile the transcriptomic changes during differentiation, RNA-seq experiments for E14 ESCs, EB day 3, and NPC day 8 were performed and revealed gene clusters associated with the respective stages (Figure 3D). The genes in the RNA-seq heat-map were sorted based on their expression levels to identify differentially expressed genes in the different stages during differentiation. Gene Ontology (GO) analysis for the four gene clusters showed that these clusters correspond to distinct cellular functions or pathways indicating that the three cell stages of mESC neuronal differentiation each have a group of genes that are highly expressed in their respective stage but not others (Figure 3E). For example, genes in Cluster 3 are highly expressed in E14 NPCs compared to other stages and correspond to pathways related to neuronal development. Clusters 1, 2, and 4 do not contain highly expressed genes related to any germ layer lineage specifications but they are related to cellular growth and proliferation. Thus, the RNA-seq and accompanying GO analysis showed that the E14 cells have differentiated into the neuronal lineage by day 8 of differentiation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61446/61446fig1.jpg" /> 
Figure 1: E14 ESCs in culture. (A) The light microscope images show E14 cells (black arrow) growing in colonies atop the &#947;-irradiated MEFs. E14 colonies continue to proliferate as seen in the colony size difference between day 1 and day 3 cultures. (B) Confirmation of the pluripotency of E14 ESCs by the alkaline phosphatase (AP) stain. Purple arrows indicate the mESCs that were positive for AP stain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61446/61446fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61446/61446fig2.jpg" /> 
Figure 2: E14 differentiation into EBs, NPCs, and neurons. (A) The schematic summarizes the major steps for differentiating E14 cells into EBs, NPCs, and neurons. (B) E14 ESCs cultured in medium without LIF and 2i in a suspension plate form individual spheres of EBs visible at day 2 where they continue to grow and expand in size in subsequent days. RA is added at day 4 of differentiation to induce the differentiation into NPCs. After 4 days of induction, these NPCs are plated for differentiation into neurons, which are shown in the bottom panel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61446/61446fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61446/61446fig3.jpg" /> 
Figure 3: Characterization of differentiated cells. (A) Immunofluorescence images in the top panel show NPC-containing EBs at day 8 probed for nestin (green) and nuclei (DAPI, blue). The bottom panel shows immunofluorescence images for neurons at day 12 probed for neurofilament (green). The red box in the merged images are zoomed in 3x for better view. (B) RT-qPCR analysis showing the pluripotency markers (Nanog and Oct4) and NPC markers (Pax6, NeuroD1, and Nes) of E14 ESCs and NPCs. Error bars are mean &#177; SD. (C) E14 cells expressing a Sox1 promoter-driven GFP reporter were differentiated into NPCs. Both the ESCs and NPCs at day 8 were quantified for positive GFP fluorescent signal with flow cytometry. (D) The heatmap shows the z-scores for the determined FPKM of the genes expressed in the ESC, EB, and NPC stages. Four distinct gene clusters were identified signifying groups of genes that are differentially expressed in either the ESC, EB, or NPC stage. (E) GO analysis was performed using the R package, clusterProfiler, on the four clusters identified in the RNA-seq. (F) The graph shows the FPKM values for three other pluripotency markers, Sox2, Klf4, and Myc as well as neural markers, NeuN, Map2, and Tubb3 for E14 cells at the ESC, EB day 3, and NPC day 8 stages. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61446/61446fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells at JoVE.com

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

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.

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

differentiation-characterization-neural-progenitors-neurons-from

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

4.8K Views.  Penn State Hershey Cancer Institute. 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.

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

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Neuroscience

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Biology

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

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.

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 ...

Differentiating Human Embryonic Stem Cells into Neural Progenitors

Source: Jeon, K. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60571">Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells.</a> J. Vis. Exp.&#160;(2020)
This video demonstrates the differentiation of human embryonic stem cells, or hESCs, directly into neural progenitor cells without using feeder cells or any intermediate steps. The selective inhibition of bone morphogenetic protein or BMP and transforming growth factor-beta, or TGF-&#946;, signaling pathways leads to hESCs differentiation into neural progenitor cells, or NPCs.

1. Preparation of human embryonic stem cells (hESC)-qualified Basement Membrane Matrix-coated Plates

	Slowly thaw the hESC-qualified basement membrane ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Source: Hanafiah, A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61446">Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells.</a> J. Vis. Exp. (2020).
The video demonstrates the differentiation of mouse embryonic stem cells (mESCs) to embryoid bodies (EBs) by the hanging drop method. Upon suspending the mESCs in hanging drops and incubating, the cells aggregate to form EBs, which are further differentiated into neuronal progenitor cells (NPCs) using a differentiation medium supplemented with retinoic acid (RA).

1. mESC culture

	Coat a 10 cm tissue-culture-treated plate with 0.1% gelatin and allow the gelatin to set for at least 15&#8211;30 min before aspirating ...

Primary Neuronal Culture

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

Source: Mao, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
This video demonstrates a method for the in vitro generation of neural progenitor cells (NPCs) from mouse embryonic stem cells (mESCs). The mESCs are cultured in suspension to form embryoid bodies (EBs), which are then guided to differentiate into NPCs via retinoic acid treatment in an adherent culture.

1. Mouse embryonic stem cell culture

	Prepare 0.1% gelatin coated cell culture dishes or plates.
	
		Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in ...

Differentiation of Neural Progenitor Cells into Neurons

Source: Mao, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
The video showcased a cell culture method for differentiating neural progenitor cells into neurons. By incubating cells in a nutrient-rich medium supplemented with growth factors, the progenitor cells matured into neurons with developed axons and dendrites. A stabilizing agent ensured cell viability and protected against oxidative damage throughout the differentiation process.