This work was supported by the National Natural Science Foundation of China (No. 31501099) and&#160;the&#8194;Middle-aged and&#8194;Young&#8194;of&#8194;the&#8194;Education&#8194;Department of&#8194;Hubei&#8194;Province, China (No. Q20191104). And, we thank Professor Wensheng Deng at Wuhan University of Science and Technology for providing the mouse embryonic stem cell lines A2lox.

1. Mouse embryonic stem cell culture
<ol>
	<li>Prepare 0.1% gelatin coated cell culture dishes or plates.
	<ol>
		<li>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.</li>
		<li>Put the dishes into a 5% CO2 incubator at 37 &#176;C and allow coating for 1 h.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Mouse embryonic stem cells (A2lox and 129) culture
	<ol>
		<li>Incubate mESCs (A2lox and 129) cells in the 0.1% gelatin coated 60 mm cell culture dishes in mESC growth medium at 37 &#176;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 &#946;-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: &#946;-mercaptoethanol is flammable and has inhalation toxicity. Keep away from fire sources and wear a mask to avoid inhalation when use.</li>
		<li>Change the mESC growth medium daily for better growth of A2lox and 129.</li>
		<li>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.</li>
		<li>Leave the cells for about 1 min to trypsinize and then remove the trypsin using 1 mL pipette.</li>
		<li>Add 2 mL of mESC growth medium to the dish, pipette up and down several times to make a single cell suspension.</li>
		<li>Count the density of the cells in the suspension as accurately as possible using hemocytometer.</li>
		<li>Divide the cells into 7 groups and induce differentiation using different protocols shown in Table 1.</li>
	</ol>
	</li>
</ol>
2. Differentiation from mESCs to NPCs (Phase I)
<ol>
	<li>Prepare 0.1% gelatin coated cell culture plates or coverslips.
	<ol>
		<li>Before use, prepare 0.1% gelatin coated 6-well plates or coverslips as in step 1.1.</li>
	</ol>
	</li>
	<li>Phase I differentiation using protocol 1 (Table 1)
	<ol>
		<li>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.</li>
		<li>Incubate the cells at 37 &#176;C in a 5% CO2 incubator for 6 h to allow for attachment.</li>
		<li>After attachment, take out of the cells from incubator, and wash the cells twice with 2 mL of PBS.</li>
		<li>Add 2 mL of basal differentiation medium I (Table 1) to each well and put the cells back into the incubator.</li>
		<li>Leave the cells for differentiation for 8 days. Replace the basal differentiation medium I every 2 days.</li>
	</ol>
	</li>
	<li>Phase I differentiation using protocol 2 (Table 1)
	<ol>
		<li>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 &#176;C in a 5% CO2 incubator.</li>
		<li>After 2 days, transfer cell aggregates into 15 mL centrifuge tubes and let them settle by gravity.</li>
		<li>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.</li>
		<li>Check the formation of embryoid bodies under the microscope (Figure 2A).</li>
		<li>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.</li>
		<li>Prepare 1 mM all-trans RA stock (in DMSO) and store away from light in a -80 &#176;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.</li>
		<li>For RA induction, add 2 &#181;L of RA stock into each well to make a final concentration of 1 &#181;M.</li>
		<li>Place the plate into the 5% CO2 incubator at 37 &#176;C and differentiate for another 4 days.</li>
		<li>Change the entire 2 mL of basal differentiation medium I (with 1 &#181;M RA) every 2 days.</li>
	</ol>
	</li>
	<li>Phase I differentiation using protocol 3 (Table 1)
	<ol>
		<li>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 &#176;C in a 5% CO2 incubator.</li>
		<li>Check the formation of embryoid bodies under a microscope (Figure 2B).</li>
		<li>Transfer cell aggregates into 15 mL centrifuge tubes and let them settle by gravity.</li>
		<li>Remove the supernatant carefully and add 10 mL of fresh basal differentiation medium I to resuspend them.</li>
		<li>Seed about 50 embryoid bodies into 2 mL of basal differentiation medium I per well onto 0.1% gelatin-coated 6-well plates.</li>
		<li>For RA induction, add 2 &#181;L of RA stock into each well to make a final concentration of 1 &#181;M.</li>
		<li>Place the plate into the 5% CO2 incubator at 37 &#176;C and differentiate for another 6 days. Change the entire basal differentiation medium I (with 1 &#181;M RA) every 2 days.</li>
	</ol>
	</li>
	<li>Phase I differentiation using protocol 4 (Table 1)
	<ol>
		<li>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 &#176;C to allow for attachment for 6 h.</li>
		<li>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 &#176;C.</li>
		<li>For RA induction, add 2 &#181;L of all-trans RA stock into each well (the working concentration is 1 &#181;M) to induce differentiation for another 4 days.</li>
		<li>In the whole process, replace the entire medium every 2 days.</li>
	</ol>
	</li>
	<li>Phase I differentiation using protocol 5 (Table 1)
	<ol>
		<li>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 &#176;C to allow for attachment for 6 h.</li>
		<li>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 &#176;C.</li>
		<li>For the subsequent RA induction, add 2 &#181;L of RA stock into each well to make a final concentration of 1 &#181;M. Place the plate into the 5% CO2 incubator at 37 &#176;C to induce differentiation for another 6 days.</li>
		<li>In the whole process, replace the entire medium every 2 days.</li>
	</ol>
	</li>
	<li>Phase I differentiation using protocol 6 (Table 1)
	<ol>
		<li>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.</li>
		<li>On the 2nd 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.</li>
		<li>Replant them into a new nonadhesive bacterial dish and allow differentiation for another 2 days in the 5% CO2 incubator at 37 &#176;C. Check the formation of embryoid bodies under microscope.</li>
		<li>On the 4th 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.</li>
		<li>Add 2 &#181;L of all-trans RA stock into each well and induce differentiation for another 4 days. Replace the entire medium (N2B27 medium II with 1 &#181;M RA) every two days.</li>
	</ol>
	</li>
	<li>Phase I differentiation using protocol 7 (Table 1)
	<ol>
		<li>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 &#176;C to allow for attachment for 6 h.</li>
		<li>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 &#176;C in a 5% CO2 incubator.</li>
		<li>Change the entire N2B27 medium II every 2 days.</li>
	</ol>
	</li>
</ol>
3. Cell morphology observation
<ol>
	<li>Check the differentiation status of the above-mentioned 7 groups daily under an inverted phase contrast light microscope.</li>
	<li>Randomly select at least 12 fields and take photos to record the morphological changes of each group on D8.</li>
</ol>
4. Immunofluorescence staining
<ol>
	<li>Sample preparation: Seed mESCs on 0.1% gelatin-coated coverslips and allow for differentiation for 8 days using the protocols mentioned in step 2.</li>
	<li>Rinse: On the 8th 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.</li>
	<li>Fixation: Add 1 mL of 4% paraformaldehyde to each sample and fix the cells for 20 min at room temperature (RT).</li>
	<li>Rinse: After fixation, gently rinse the cells with 1 mL of PBS 3 times, for 5 min each.</li>
	<li>Permeabilization: Add 1 mL of 0.2% TritonX-100 in PBS to each sample and leave for 8 min at RT.</li>
	<li>Rinse: After permeabilization, gently rinse the cells with 1 mL of PBS 3 times, for 5 min each.</li>
	<li>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.</li>
	<li>Incubation with primary antibody
	<ol>
		<li>Dilute the anti-Nestin antibody at a ratio of 1:100 using 5% goat serum in PBS.</li>
		<li>Apply 500 &#181;L of diluted antibody to different samples and incubate overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Rinse: Remove the antibody and rinse the samples gently with 1 mL of PBS 3 times for 8 min each.</li>
	<li>Incubation with secondary antibody
	<ol>
		<li>Dilute the Alexa Fluor 488-labeled goat anti-mouse IgG at a ratio of 1:500 using 5% goat serum in PBS.</li>
		<li>Apply 500 &#181;L 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.</li>
	</ol>
	</li>
	<li>Rinse: Remove the secondary antibody and rinse the samples gently with 1 mL of PBS 3 times for 8 min each.</li>
	<li>Nuclear staining and mounting
	<ol>
		<li>Place one drop of DAPI mounting medium onto the clean microslide.</li>
		<li>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.</li>
		<li>Remove excess DAPI mounting medium with absorbent paper.</li>
	</ol>
	</li>
	<li>Fluorescence microscopy observation
	<ol>
		<li>Place the specimens under the fluorescence microscopy and detect the signal for DAPI and Alexa Fluor 488 using proper filters.</li>
		<li>Evenly and randomly pick 10-15 different visual fields for each sample and record the images with a CCD camera.</li>
	</ol>
	</li>
</ol>
5. Differentiation from NPCs to neurons (Phase II)
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Place the plate into the 5% CO2 incubator at 37 &#176;C to allow for attachment for 6 h. Wash them twice with 2 mL of PBS.</li>
	<li>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.</li>
	<li>Place the plates into the incubator and allow to differentiate for another 10 days. Change the corresponding medium every 2 days.</li>
	<li>Check the differentiation status and record the morphological changes as mentioned in step 3.</li>
	<li>On Day 18, evaluate the generation of neurons (&#946;-Tubulin III positive) and determine the differentiation efficiency of the 3 protocols using in step 4.</li>
</ol>

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The authors have nothing to disclose.

In the present study, we established a simple and effective method for neuronal differentiation from mESCs, with low cost and easily obtained materials. In this method, 2 days of embryoid body formation followed by 6 days of RA induction can effectively promote the differentiation of mESCs into NPCs (Phase I-protocol 3). For the phase II differentiation, N2B27 medium II (Phase II-protocol 3) most effectively induce the differentiation from NPCs into neurons. To ensure success, more attention should be paid to several critical steps.
Firstly, the healthy state of embryoid bodies is the key for the whole differentiation process. Three-dimensional embryoid body formation is usually used to direct the differentiation of ESCs8. In this study, we investigated the proper suspension culture time of embryoid bodies. As shown in Figure 2, round embryoid bodies with bright cores were formed after suspension culture for 2 days in this condition. However, when cultured for 4 days, many embryoid bodies adhere to each other, and the cores become dark, indicating the apoptosis of cells in the cores. The subsequent differentiation further confirmed the worse effect of prolonged embryoid body formation. In some reported studies, suspension culture of embryoid bodies could last for as long as 10 days, using medium with lower FBS concentration or without FBS11. The reduced time for embryoid body formation in the study may be due to the higher FBS concentration (15%) used here, and it has been proven that 15% FBS can better promote the formation and differentiation of embryoid bodies.
Secondly, the timepoint at which RA is applied and the RA working concentration are critical for cell fate determination of mESCs. RA, a derivative of vitamin A, is one of the most important morphogens with pleiotropic actions12. RA can regulate multiple signal pathways and affect cell fate determination of ESCs13,14. Reports showed that short-term treatment of mESCs with RA during the early differentiation stage prevented spontaneous differentiation and maintain self-renewal capacity of mESCs15. Others suggested that RA could regulate both germ cell differentiation and neural differentiation from ESCs, which are timepoint dependent16,17,18. In the condition presented here, RA added on the 2nd day after embryoid body formation is appropriate for directing the differentiation into NPCs. Meanwhile, the working concentration of RA is also critical. Low RA concentrations (~10 nM) may induce the differentiation of mESCs into endoderm-like cells, whereas high RA concentrations (1-5 &#181;M) are more likely to induce differentiation into NPCs13,14,15,16,17,18,19. Due to the use of RA, one would expect a caudalization effect; the differentiation into fore brain neurons would be rarely seen and yielding neurons of hindbrain and spinal cord fates would occur20,21. Furthermore, RA is an easily available and low cost agent, and the use of this protocol can save research funds for most laboratory.
Thirdly, in condition presented here, the NPCs generated after phase I differentiation can be stored and passaged in proper conditions. Cryopreservation with high cell density (&#62;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.

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&#160;developmental biology studies and potentially aid in regenerative medicine2,3. In the past decades, many strategies have been reported for inducing embryonic neurogenesis, such as the transgenic method4, using small molecules5, using a 3D matrix microenvironment6, and the co-culture technique7. However, most of these protocols are either condition limited or hard to operate, thus they are not suitable for usage in most laboratories.
To find an easy to operate and low cost method to achieve efficient neural differentiation from mESCs, a combinatorial screening strategy was used here. As described in Figure 1, the whole process of embryonic neurogenesis was divided into 2 phases. Phase I refers to the differentiation process from mESCs into NPCs, and phase II relates to the subsequent differentiation from NPCs into neurons. Based on the principles of easy operation, low cost, easily available materials and high differentiation efficiency, seven protocols in Phase I and three protocols in Phase II were chosen based on the traditional adherent monolayer culture system or embryoid body formation system8,9. The differentiation efficiency of protocols in both phases was evaluated using cell morphology observation and immunofluorescence assay. Through combining the most efficient protocol of each phase, we established the optimized method for neural differentiation from mESCs.

<table><tbody><tr><td>Anti-Nestin antibody [Rat-401]</td><td>Abcam</td><td>Ab11306</td><td>stored at -80 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>Anti-&amp;beta;-Tubulin III antibody produced in rabbit</td><td>Sigma Aldrich</td><td>T2200</td><td>stored at -80 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>Alexa Fluor 488-Labeled Goat Anti-Mouse IgG</td><td>Beyotime</td><td>A0428</td><td>stored at -20 &amp;deg;C and protect from light</td></tr><tr><td>B-27 Supplement (50X), serum free</td><td>Gibco</td><td>17504044</td><td>stored at -20 &amp;deg;C, and protect from light</td></tr><tr><td>CHIR-99021 (CT99021)</td><td>Selleck</td><td>S1263</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Coverslips</td><td>NEST</td><td>801007</td><td></td></tr><tr><td>Cy3-Labeled Goat Anti-Rabbit IgG</td><td>Beyotime</td><td>A0516</td><td>stored at -20 &amp;deg;C and protect from light</td></tr><tr><td>DME/F-12 1:1 (1x)</td><td>HyClone</td><td>SH30023.01B</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Fetal bovine serum</td><td>HyClone</td><td>SH30084.03</td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>Fluorescence microscopy</td><td>Olympus</td><td>CKX53</td><td></td></tr><tr><td>Gelatin</td><td>Gibco</td><td>CM0635B</td><td>stored at room temperature</td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050061</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Immunol Staining Primary Antibody dilution Buffer</td><td>Beyotime</td><td>P0103</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>KnockOut DMEM/F-12</td><td>Gibco</td><td>12660012</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>KnockOut Serum Replacement</td><td>Gibco</td><td>10828028</td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>Leukemia Inhibitory Factor human</td><td>Sigma</td><td>L5283</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Mounting Medium With DAPI - Aqueous, Fluoroshield</td><td>Abcam</td><td>ab104139</td><td>stored at 4 &amp;deg;C and protect from light</td></tr><tr><td>MEM Non-essential amino acids solution</td><td>Gibco</td><td>11140076</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>N-2 Supplement (100X)</td><td>Gibco</td><td>17502048</td><td>stored at -20 &amp;deg;C and protect from light</td></tr><tr><td>Normal goat serum</td><td>Jackson</td><td>005-000-121</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Neurobasal Medium</td><td>Gibco</td><td>21103049</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Nonadhesive bacterial dish</td><td>Corning</td><td>3262</td><td></td></tr><tr><td>Phosphate Buffered Saline (1X)</td><td>HyClone</td><td>SH30256.01B</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Penicillin/ Streptomycin Solution</td><td>HyClone</td><td>SV30010</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>PD0325901(Mirdametinib)</td><td>Selleck</td><td>S1036</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Retinoic acid</td><td>Sigma</td><td>R2625</td><td>stored at -80 &amp;deg;C and protect from light</td></tr><tr><td>Strain 129 Mouse Embryonic Stem Cells</td><td>Cyagen</td><td>MUAES-01001</td><td>Maintained in feeder-free culture system</td></tr><tr><td>Stem-Cellbanker (DMSO free)</td><td>ZENOAQ</td><td>stem cellbanker DMSO free</td><td>stored at -20 &amp;deg;C, avoid repeated freezing and thawing</td></tr><tr><td>Trypsin 0.25% (1X) Solution</td><td>HyClone</td><td>SH30042.01</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco</td><td>21985023</td><td>stored at 4 &amp;deg;C and protect from light</td></tr><tr><td>4% paraformaldehyde</td><td>Beyotime</td><td>P0098</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>6 - well plate</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>60 mm cell culture dish</td><td>Corning</td><td>430166</td><td></td></tr><tr><td>15 ml centrifuge tube</td><td>NUNC</td><td>339650</td><td></td></tr></tbody></table>

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.

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 &#34;2-day embryoid body formation + 6-day RA induction&#34; treatment (Phase I-protocol 3) showed well-stacked and neurite-like morphologies, which indicating the formation of NPCs. However, cells with &#34;4-day embryoid body formation + 4-day RA induction&#34; 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 &#177; 4.33% and 69.33 &#177; 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 &#946;-Tubulin III+ cells up to 67.75 &#177; 4.01% and 58.73 &#177; 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 &#181;M) 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61190/61190fig01.jpg" /> 
Figure 1: Diagram of the embryonic neurogenesis process.&#160;This figure has been modified from Li et al.10. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61190/61190fig01large.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/61190/61190fig02.jpg" /> 
Figure 2: The morphology of the embryoid bodies.&#160;(A) Embryoid bodies cultured for 4 days. (B) Embryoid bodies cultured for 2 days. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61190/61190fig02large.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/61190/61190fig03.jpg" /> 
Figure 3: Efficiency comparison of the 7 protocols on phase I differentiation using A2lox and 129 mESCs.&#160;(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&#177;SEM of three independent experiments. *, p&#8804;0.05; **, p&#8804;0.01. This figure has been modified from Li et al.10. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61190/61190fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61190/61190fig04.jpg" /> 
Figure 4: Efficiency comparison of the 3 protocols on phase II differentiation.&#160;(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 (&#946;-Tubulin III+, red). The nuclei were labeled blue with DAPI. Upper panel: A2lox derivatives on D18; Lower panel: 129 derivatives on D18. Percentages of &#946;-Tubulin III+ cells of each group were shown by histogram. Each column represents the mean &#177; SEM of three independent experiments. *, p&#8804;0.05; **, p&#8804;0.01. This figure has been modified from Li et al.10. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61190/61190fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61190/61190fig05.jpg" /> 
Figure 5: Brief model of the optimized method for neuronal differentiation from mESCs in vitro&#160;This figure has been modified from Liet al.10. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61190/61190fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Differentiation Phase I (8d)</td>
		</tr>
		<tr>
			<td></td>
			<td>Protocols</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>protocol 1</td>
			<td>Differentiation naturally: With basal differentiation medium I only</td>
			<td rowspan="5">Basal differentiation medium I: 
			DMEM/F12 +15%FBS + 1%NEAA+0.1mM 2ME+ 1%P/S</td>
		</tr>
		<tr>
			<td>protocol 2</td>
			<td>4-day Embryoid Bodies formation + 4-day RA induction</td>
		</tr>
		<tr>
			<td>protocol 3</td>
			<td>2-day Embryoid Bodies formation + 6-day RA induction</td>
		</tr>
		<tr>
			<td>protocol 4</td>
			<td>Monolayer culture combined with RA induction: 4d (-RA) 4d (+RA)</td>
		</tr>
		<tr>
			<td>protocol 5</td>
			<td>Monolayer culture combined with RA induction: 2d (-RA) 6d(+RA)</td>
		</tr>
		<tr>
			<td>protocol 6</td>
			<td>Embryoid Bodies formation (4 d) and differentiation induced with N2B27 medium II</td>
			<td rowspan="2">N2B27 medium II: 49% DMEM/F12+ 1% N2 + 48% Neurobasal medium + 2% B27 +1%GlutaMAX+ 0.1mM 2ME</td>
		</tr>
		<tr>
			<td>protocol 7</td>
			<td>Monolayer culture with N2B27 medium II</td>
		</tr>
	</tbody>
</table>
Table 1: Details of the 7 protocols used in phase I differentiation. This table has been modified from Li et al.10.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Differentiation Phase II (10d)</td>
		</tr>
		<tr>
			<td></td>
			<td>Protocols</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>protocol 1</td>
			<td>Differentiation naturally: With basal differentiation medium I only</td>
			<td>Basal differentiation medium I: DMEM/F12 +15%FBS +1%NEAA +0.1mM 2ME+ 1%P/S</td>
		</tr>
		<tr>
			<td>protocol 2</td>
			<td>Differentiation with N2B27 medium I</td>
			<td>N2B27 medium I: DMEM/F12 + 1%N2 + 2%B27 + 1%GlutaMAX +0.1mM 2ME</td>
		</tr>
		<tr>
			<td>protocol 3</td>
			<td>Differentiation with N2B27 medium II</td>
			<td>N2B27 medium II: 49% DMEM/F12+ 1% N2 + 48% Neurobasal medium + 2% B27 +1%GlutaMAX+ 0.1mM 2ME</td>
		</tr>
	</tbody>
</table>
Table 2: Details of the 3 protocols used in phase II differentiation. This table has been modified from Li et al.10.

Watch this Scientific Journal Video about Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro at JoVE.com

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

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 ...

neuronal-differentiation-from-mouse-embryonic-stem-cells-in-vitro

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

8.8K Views.  Wuhan Center for Disease Control and Prevention. 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.

Video: Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

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 ...

Developmental Biology

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 ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

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.

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 ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

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 ...

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 ...

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

Source: Mao, X., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/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 ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Differentiation of Neural Progenitor Cells into Neurons

Source: Mao, X., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/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.