Name: neuronal differentiation from mouse embryonic stem cells in vitro
Uploaded: 2020-06-02T14:00:00
Description: Watch this Scientific Journal Video about Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro at JoVE.com

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 ...

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+of+human+embryonic+stem+cells+as+an+in+vitro+tool+for+the+study+of+the+expression+patterns+of+the+neuronal+cytoskeleton+during+neurogenesis.">Neural differentiation of human embryonic stem cells as an in vitro tool for the study of the expression patterns of the neuronal cytoskeleton during neurogenesis.</a> Biochemical and Biophysical Research Communications. 439 (1), 154-159 (2013).</li><li>Khramtsova, E. A., Mezhevikina, L. M., Fesenko, E. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+of+mouse+embryonic+stem+cells+in+serum-free+monolayer+culture.">Neural differentiation of mouse embryonic stem cells in serum-free monolayer culture.</a> Journal of Visualized experiments. (99), e52823 (2015).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+neuronal+differentiation+of+embryonic+stem+cells+in+vitro.">An optimized method for neuronal differentiation of embryonic stem cells in vitro.</a> Journal of Neuroscience Methods. 330 (15), 108486 (2020).</li><li>Zhou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+optimal+suspension+culture+time+for+the+osteoblastic+differentiation+of+human+induced+pluripotent+stem+cells+using+the+embryoid+body+method.">Investigation of the optimal suspension culture time for the osteoblastic differentiation of human induced pluripotent stem cells using the embryoid body method.</a> Biochemical and Biophysical Research Communications. 515 (4), 586-592 (2019).</li><li>Lu, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-trans+retinoic+acid+promotes+neural+lineage+entry+by+pluripotent+embryonic+stem+cells+via+multiple+pathways.">All-trans retinoic acid promotes neural lineage entry by pluripotent embryonic stem cells via multiple pathways.</a> BMC Cell Biology. 10, 57 (2009).</li><li>Kanungo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+signaling+in+P19+stem+cell+differentiation.">Retinoic acid signaling in P19 stem cell differentiation.</a> Anticancer Agents in Medicinal Chemistry. 17 (9), 1184-1198 (2017).</li><li>Rochette-Egly, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+signaling+and+mouse+embryonic+stem+cell+differentiation:+Cross+talk+between+genomic+and+non-genomic+effects+of+RA.">Retinoic acid signaling and mouse embryonic stem cell differentiation: Cross talk between genomic and non-genomic effects of RA.</a> Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1851 (1), 66-75 (2015).</li><li>Wang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+maintains+self-renewal+of+murine+embryonic+stem+cells+via+a+feedback+mechanism.">Retinoic acid maintains self-renewal of murine embryonic stem cells via a feedback mechanism.</a> Differentiation. 76 (9), 931-945 (2008).</li><li>Wu, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid-induced+upregulation+of+miR-219+promotes+the+differentiation+of+embryonic+stem+cells+into+neural+cells.">Retinoic acid-induced upregulation of miR-219 promotes the differentiation of embryonic stem cells into neural cells.</a> Cell Death Disease. 8, 2953 (2017).</li><li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+regulates+germ+cell+differentiation+in+mouse+embryonic+stem+cells+through+a+Smad-dependent+pathway.">Retinoic acid regulates germ cell differentiation in mouse embryonic stem cells through a Smad-dependent pathway.</a> Biochemical and Biophysical Research Communications. 418 (3), 571-577 (2012).</li><li>Nakayama, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+for+neuronal+induction+of+the+P19+embryonic+carcinoma+cell+line.">A rapid and efficient method for neuronal induction of the P19 embryonic carcinoma cell line.</a> Journal of Neuroscience Methods. 227, 100-106 (2014).</li><li>Bibel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mouse+embryonic+stem+cells+into+a+defined+neuronal+lineage.">Differentiation of mouse embryonic stem cells into a defined neuronal lineage.</a> Nature neuroscience. 7 (9), 1003-1009 (2004).</li><li>Coyle, D. E., Li, J., Baccei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+Differentiation+of+Retinoic+Acid-Induced+Human+Pluripotent+Embryonic+Carcinoma+Stem+Cell+Neurons.">Regional Differentiation of Retinoic Acid-Induced Human Pluripotent Embryonic Carcinoma Stem Cell Neurons.</a> PLoS ONE. 6 (1), 16174 (2011).</li><li>Okada, Y., Shimazaki, T., Sobue, G., Okano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic-acid-concentration-dependent+acquisition+of+neural+cell+identity+during+in+vitro+differentiation+of+mouse+embryonic+stem+cells.">Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells.</a> Developmental Biology. 275, 124-142 (2004).</li></ol>

In the present study, we established a simple and effective method for neuronal differentiation from mESCs, with low cost and easily obtained materials. In this method, 2 days of embryoid body formation followed by 6 days of RA induction can effectively promote the differentiation of mESCs into NPCs (Phase I-protocol 3). For the phase II differentiation, N2B27 medium II (Phase II-protocol 3) most effectively induce the differentiation from NPCs into neurons. To ensure success, more attention should be paid to several cri...

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 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin III antibody produced in rabbit</td>
			<td>Sigma Aldrich</td>
			<td>T2200</td>
			<td>stored at -80 &#176;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 &#176;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 &#176;C, and protect from light</td>
		</tr>
		<tr>
			<td>CHIR-99021 (CT99021)</td>
			<td>Selleck</td>
			<td>S1263</td>
			<td>stored at -20 &#176;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 &#176;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 &#176;C</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td>stored at -20 &#176;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 &#176;C</td>
		</tr>
		<tr>
			<td>Immunol Staining Primary Antibody dilution Buffer</td>
			<td>Beyotime</td>
			<td>P0103</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12</td>
			<td>Gibco</td>
			<td>12660012</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>stored at -20 &#176;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 &#176;C</td>
		</tr>
		<tr>
			<td>Mounting Medium With DAPI - Aqueous, Fluoroshield</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td>stored at 4 &#176;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 &#176;C</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>stored at -20 &#176;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 &#176;C</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>stored at 4 &#176;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 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin Solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>PD0325901(Mirdametinib)</td>
			<td>Selleck</td>
			<td>S1036</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>stored at -80 &#176;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 &#176;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 &#176;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 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Beyotime</td>
			<td>P0098</td>
			<td>stored at -20 &#176;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 i...

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 ...

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