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

この研究は、中国国立自然科学財団(No. 31501099)と中国湖北省教育部の中高年と若者(No.Q20191104)。そして、武漢科学技術大学のウェンシェン・デン教授が、マウス胚性幹細胞株A2loxを提供してくれたことに感謝します。

1. マウス胚性幹細胞培養
<ol><li>0.1%ゼラチンコーティングされた細胞培養皿またはプレートを準備します。<ol>
<li>滅菌した0.1%のゼラチン(水中0.1%w/v)を60mm細胞培養皿に2mL加えます。細胞培養皿のコーティングを確実にするために、穏やかにロックします。</li>
		<li>37°Cで5%CO2インキュベーターに皿を入れ、1時間コーティングを行います。</li>
		<li>細胞を播種する前に0.1%ゼラチン溶液を...

<ol>
	<li>Vieira, M. S., 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=Neural+stem+cell+differentiation+into+mature+neurons:+mechanisms+of+regulation+and+biotechnological+applications.">Neural stem cell differentiation into mature neurons: mechanisms of regulation and biotechnological applications.</a> Biotechnology Advances. 36 (7), 1946-1970 (2018).</li><li>Yang, J. R., Lin, Y. T., Liao, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+embryonic+stem+cells+on+Parkinson's+disease+therapy.">Application of embryonic stem cells on Parkinson's disease therapy.</a> Genomic Medicine, Biomarkers, and Health Sciences. 3 (1), 17-26 (2011).</li><li>Liu, C., Zhong, Y. W., Apostolou, A., Fang, S. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+differentiation+of+mouse+embryonic+stem+cells+modified+by+the+Neural+Growth+Factor+(NGF)+gene.">Proliferation and differentiation of mouse embryonic stem cells modified by the Neural Growth Factor (NGF) gene.</a> Biology Bulletin. 45 (3), 219-225 (2018).</li><li>Yoshimatsu, S., 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=Evaluating+the+efficacy+of+small+molecules+for+neural+differentiation+of+common+marmoset+ESCs+and+iPSCs.">Evaluating the efficacy of small molecules for neural differentiation of common marmoset ESCs and iPSCs.</a> Neuroscience research. , (2019).</li><li>Kothapalli, C. R., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+matrix+microenvironment+for+targeted+differentiation+of+embryonic+stem+cells+into+neural+and+glial+lineages.">3D matrix microenvironment for targeted differentiation of embryonic stem cells into neural and glial lineages.</a> Biomaterials. 34, 5995-6007 (2013).</li><li>Gazina, E. V., 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=Method+of+derivation+and+differentiation+of+mouse+embryonic+stem+cells+generating+synchronous+neuronal+networks.">Method of derivation and differentiation of mouse embryonic stem cells generating synchronous neuronal networks.</a> Journal of Neuroscience Methods. 293, 53-58 (2018).</li><li>Ohnuki, Y., Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hanging+drop+culture+conditions+on+embryoid+body+formation+and+neuronal+cell+differentiation+using+mouse+embryonic+stem+cells:+Optimization+of+culture+conditions+for+the+formation+of+well-controlled+embryoid+bodies.">Effects of hanging drop culture conditions on embryoid body formation and neuronal cell differentiation using mouse embryonic stem cells: Optimization of culture conditions for the formation of well-controlled embryoid bodies.</a> Journal of Bioscience and Bioengineering. 115 (5), 571-574 (2013).</li><li>Wongpaiboonwattana, W., Stavridis, M. 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>

本研究では、低コストで容易に得られる物質を用いて、mESCからの神経細胞分化の簡便かつ効果的な方法を確立した。この方法では、2日間の胚体形成後に6日間のRA誘導が行われ、MESCsのNPCへの分化を効果的に促進することができる(フェーズI-プロトコル3)。第II相分化のために、N2B27培地II(フェーズII-プロトコル3)は、NPCからニューロンへの分化を最も効果的に誘導する。成功を確実にするため?...

胚性幹細胞(ESC)は多能性であり、神経前駆細胞(NPC)に分化し、その後、特定の条件下でニューロンに分化することができる1。ESCベースの神経新生は、神経新生を模倣するための最良のプラットフォームを提供し、したがって、発生生物学研究のための有用なツールとして機能し、潜在的に再生医療2、3を支援する。過去数十年の間に、トランスジェニック法4のような胚性神経新生を誘導するための多くの戦略が報告されている、小分子5を用いて、3Dマトリックス微小環境6を用いて、および共培養技術7を用いた。しかし、これらのプロトコルのほとんどは、条件が制限されているか、操作が困難であるため、ほとんどの研究室での使用には適していません。
mESCからの効率的な神経分化を達成するために、操作が容易で低コストの方法を見つけるために、ここで組み合わせスクリーニング戦略を使用しました。図1に記載されているように、胚性神経新生の全過程を2相に分けた。フェーズIはmESCからNPCへの分化プロセスを指し、第II相はNPCからニューロンへのその後の分化に関連する。容易な操作、低コスト、容易に利用できる材料および高い分化効率の原則に基づいて、フェーズIの7つのプロトコルおよびフェーズIIの3つのプロトコルは、伝統的な付着単層培養システムまたは胚体形成システム8、9に基づいて選択された。両相におけるプロトコルの分化効率を、細胞形態観察および免疫蛍光アッセイを用いて評価した。各フェーズの最も効率的なプロトコルを組み合わせることで、mESCからの神経分化のための最適化された方法を確立しました。

<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

マウス胚性幹細胞(mESC)の神経分化は、神経新生に関与する重要なメカニズムを解明し、再生医療に役立つ可能性のあるツールです。ここでは、組み合わせスクリーニングの戦略を用いて、 インビトロでのmESCからのニューロン分化のための効率的かつ低コストの方法を確立しました。ここで定義された条件の下で、2日間の胚体形成+6日間のレチノイン酸誘導プロトコルは、ネスチン陽性であるよく積み重ねられ、神経突起のようなA2loxおよび129誘導体の形成によって見られるように、mESCから神経前駆細胞(NPC)への迅速かつ効率的な分化を可能にする。胚体の健全な状態とレチノイン酸(RA)が適用される時点は、RA濃度と同様に、その過程で重要である。NPCからニューロンへのその後の分化において、N2B27培地II(神経基底培地によって補完される)は、神経細胞の長期的な維持および成熟をよりよく支えることができる。提示された方法は、高効率、低コスト、操作が容易であり、神経生物学や発生生物学の研究のための強力なツールとなり得る。

2日間の胚体形成+ 6日間のRA誘導は、MESCのNPCへの分化(フェーズI)を導くのに最適です。MESCsのNPCへの分化を最も促進する最適なプロトコル(フェーズI)を決定するために、A2loxと129 mESCsの両方で7つのプロトコルをテストし、各群の分化状況を光顕微鏡で監視した。 図3Aに示すように、ほとんどのA2loxおよび129誘導体は「2日間の胚体形成+6日間のRA誘導」処置(フェ...

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

ここでは、低コストで使いやすい方法を確立し、胚性幹細胞からニューロンへの迅速かつ効率的な分化を導く方法を確立しました。この方法は、研究室間で普及に適しており、神経学的研究のための有用なツールとなり得る。

インビトロにおけるマウス胚性幹細胞からの神経細胞分化

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 assays is excellent model for illustrating the key mechanisms involved in neurogenesis. We demonstrate an optimized method for embryoid neurogenesis using a company toro screening strategy. This technique has the advantages of high efficiency, low cost, and easy operation, and is suitable for popularization among laboratory.It is a powerful tool for neuroscience research. To perform differentiation using protocol one, seed 20, 000 mouse embryoid stem cells in two milliliters of basal differentiation medium into each well of a 0.1%gelatin-coated six-well plate. After cell attachment, wash the cells with two milliliters of PBS and add two milliliters of basal differentiation medium to each well, then put the cells back in the incubator.Allow the cells to differentiate for eight days, replacing the medium every two days. To perform protocol two, add 1.5 million mouse embryoid stem cells into a non-adhesive bacterial dish in 10 milliliters of basal differentiation medium and incubate the plate at 37 degrees Celsius and 5%carbon dioxide to allow for embryoid body formation. After two days transfer the cell aggregates into 15 milliliter centrifuge tubes and let them settle by gravity.Remove the supernatant and add 10 milliliters of fresh basal differentiation medium to re-suspend the embryoid bodies. Then replant them into a new non-adhesive dish and incubate them for another two days. Collect and seed about 50 embryoid bodies in two milliliters of basal differentiation medium per well onto a gelatin-coated six well plate.For retinoic acid induction, add two microliters of RA stock into each well to a final concentration of one micromolar. Return the plate to the incubator and differentiate the cells for another four days. To perform differentiation using protocol three, repeat protocol two but allow only two days for embryonic body formation and six days for RA induction.Quality control of embryoid bodies formation should be carried out using microscopy. Only those with bright cores can differentiate successfully in the subsequent process. For protocol four, seed 20, 000 mouse embryonic stem cells per well into the gelatin coated plates and follow protocol one.After allowing the cells to differentiate for four days add two microliters of all trans RA stock into each well and incubate the plate for another four days. To perform protocol five, repeat protocol four but begin RA induction after only two days of differentiation. For protocol six, plant 1.5 million stem cells into a non-adhesive bacterial dish in 10 milliliters of N2B27 medium two and incubate the plate at 37 degrees Celsius and 5%carbon dioxide to allow for embryoid body formation.After two days, collect cell aggregates as previously described and re-suspend the embryoid bodies with 10 milliliters of fresh N2B27 medium two. Replant them into a new non-adhesive bacterial dish and allow differentiation for another two days. On the fourth day, collect and seed about 50 embryoid bodies per well onto gelatin-coated six well plates with two milliliters of N2B27 medium two.Add two microliters of all trans RA stock into each well and induce differentiation for another four days. To perform protocol seven, seed about 20, 000 cells in two milliliters of basal differentiation medium per well onto the gelatin-coated plates. After cell attachment and two washes with PBS add two milliliters of N2B7 medium two to each well and allow the cells to differentiate for eight days at 37 degrees Celsius and 5%carbon dioxide.After performing phase one differentiation using protocol three, seed about 500, 000 mouse embryonic stem cell derivatives in two milliliters of basal differentiation medium per well onto the 0.1%gelatin-coated plates. Randomly divide the derivatives into three groups in order to test three phase two differentiation protocols. Incubate the plate for six hours, then wash the cells twice with two milliliters of PBS and add two milliliters of basal differentiation medium N2B27 medium one or N2B27 medium two to each well, depending on the protocol.Place the blades into the incubator and allow the cells to differentiate for another 10 days, changing the corresponding medium every two days. Seven protocols were tested to determine the optimal protocol for the differentiation of mouse embryonic stem cells in general precursor cells or NPCs. The protocols were tested on both A2lox locks and 129 cells and the differentiation status of each group was monitored using a light microscope.Most A2lox and 129 derivatives showed well-stacked and neurite-like morphologies under protocol three indicating the formation of NPCs. However, cells differentiated using protocol two appeared apoptotic which may be due to the lack of nutrients within the embryoid bodies. To further confirm the formation of NPCs nestin-positive cells were detected using an immunofluorescence assay.Protocol three resulted in the highest percentage of nestin-positive cells reaching up to 78 and 69%in A2lox and 129 derivatives respectively. Three protocols were tested for the differentiation of NPCs into neurons. It was determined that protocol three most effectively induces the differentiation.Most A2lox and 129 derivatives in phase two protocol three had prolonged neuron-like structures with clear neurites and cell body extensions. Immunofluorescence assays further confirmed the generation of neurons with the percentage of beta tubulin three positive cells up to 68 and 59%in A2lox and 129 derivatives respectively using protocol three. When attempting this procedure, it is important to ensure that mouse assays are healthy and non-differentiated before differentiation.In addition, strict quality control should be carried out after formation in bore out bodies and before phase two differentiation. This technique provides a standard model for researchers of neurobiology and developmental biology and is expected to be a powerful tool for illustrating the key mechanisms involved in neurogenesis.

Research

JoVE Journal

Neuroscience

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

小分子の誘導と多能性ヒト胚性幹細胞からヒト神経前駆細胞と神経細胞の効率的な導出

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

神経科学

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

胚腹側中脳の器官型スライス培養：ドーパミン作動性ニューロンの発達を研究するための体制 in vitroで</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

胎児マウス神経管から神経堤細胞の単離および培養

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

ヒト多能性幹細胞からドーパミン作動性ニューロンの分化を監督

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

マウス胚性幹細胞の皮質介在ニューロン前駆細胞への分化

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

Isolation and Culture of Embryonic Mouse Neural Stem Cells

マウス萌芽期の神経幹細胞の分離と培養

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 ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

生体内ニューロン回路接続を模倣するためのインビトロウェッジスライスの準備

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

線維芽細胞からニューロン前駆細胞への直接変換とアストロサイトへの分化を用いた神経疾患のインビトロモデリング

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

In vivo Calcium Imaging in Mouse Inferior Olive

In vivo Calcium Imaging in Mouse Inferior Olive

インビボ マウス劣オリーブにおけるカルシウムイメージング

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

ヒトミクログリア様細胞:人工多能性幹細胞からの分化とヒトシナプトソームを用いた in vitro 生細胞貪食アッセイ

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...