Name: efficient differentiation of mouse embryonic stem cells into motor neurons
Uploaded: 2012-06-09T14:00:00
Description: Watch this Scientific Journal Video about Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons at JoVE.com

这手稿是致力于到文澜王博士的记忆2011年05月26日，通过了。我们感谢道格拉斯A·克尔博士慷慨提供的HBG3 MES在这项研究中使用的细胞。这项工作是由内穆尔，赠款（2 RR016472-10）根据国家研究资源中心（NCRR），和1 COBRE补助金从NCRR（5 P20的RR020173-05），以支持中心的INBRE方案儿童，美国特拉华州威尔明顿，在阿尔弗雷德一，杜邦医院的儿科研究。

1。步骤1：小鼠胚胎干细胞（MES）文化（定时：3天） 1。电镀主小鼠胚胎成纤维细胞（PMEF） <ol><li>大衣4个100毫米组织培养皿中，用明胶PMEF粘附。加入8毫升0.1％的明胶溶液（干细胞），每道菜，并在室温下30分钟的孵育。 </li><li>去除多余的明胶从菜肴。不要冲洗碗碟。 </li><li>稀包含约一小瓶丝裂霉素C灭活PMEF（Millipore公司）。 5×10 6细胞（配方见表1），?...

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	<li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110, 385-397 (2002).</li><li>Miles, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+motoneurons+derived+from+mouse+embryonic+stem+cells.">Functional properties of motoneurons derived from mouse embryonic stem cells.</a> J. Neurosci. 24, 7848-7858 (2004).</li><li>Wichterle, H., Peljto, M. <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+to+spinal+motor+neurons.">Differentiation of mouse embryonic stem cells to spinal motor neurons.</a> Curr. Protoc. Stem Cell. Biol. Chapter 1, Unit 1H.1.1-Unit 1H.1.9 (2008).</li><li>Kiris, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-derived+motoneurons+provide+a+highly+sensitive+cell+culture+model+for+botulinum+neurotoxin+studies,+with+implications+for+high-throughput+drug+discovery.">Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery.</a> Stem Cell Res. 6, 195-205 (2011).</li><li>Wu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+assessment+of+a+cell+model+of+spinal+muscular+atrophy.">Proteomic assessment of a cell model of spinal muscular atrophy.</a> BMC Neurosci. 12, 25 (2011).</li><li>McMahon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin-mediated+antagonism+of+BMP+signaling+is+required+for+growth+and+patterning+of+the+neural+tube+and+somite.">Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite.</a> Genes Dev. 12, 1438-1452 (1998).</li><li>Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+posterior+neural+tissue+is+induced+by+FGF+in+the+chick+embryo.">Early posterior neural tissue is induced by FGF in the chick embryo.</a> Development. 125, 473-484 (1998).</li><li>Launay, C., Fromentoux, V., Shi, D. L., Boucaut, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+truncated+FGF+receptor+blocks+neural+induction+by+endogenous+Xenopus+inducers.">A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers.</a> Development. 122, 869-880 (1996).</li><li>Sinha, S., Chen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purmorphamine+activates+the+Hedgehog+pathway+by+targeting+Smoothened.">Purmorphamine activates the Hedgehog pathway by targeting Smoothened.</a> Nat. Chem. Biol. 2, 29-30 (2006).</li><li>Chen, J. K., Taipale, J., Young, K. E., Maiti, T., Beachy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+modulation+of+Smoothened+activity.">Small molecule modulation of Smoothened activity.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 14071-14076 (2002).</li><li>Lee, S. M., Danielian, P. S., Fritzsch, B., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+FGF8+signaling+from+the+midbrain-hindbrain+junction+regulates+growth+and+polarity+in+the+developing+midbrain.">Evidence that FGF8 signaling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain.</a> Development. 124, 959-969 (1997).</li></ol>

mES细胞的质量是最关键的参数为运动神经元的高效发电。 mES细胞必须培养，对PMEF防止自发分化。此外LIF的帮助mES细胞保持在未分化状态。由于MES细胞分裂每12至15小时，培养基变得枯竭迅速，必须每天更换。 高效活化Shh的途径是一个关键的参数，需要引起运动神经元的规范。 Shh蛋白（研发体系），Shh信号激动剂，如HH激动剂HhAg1.3（Curis公司），purmorphamine（Calbiochem公司），?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 终浓度 </td></tr><tr><td> PMEF </td><td> Millipore公司</td><td> PMEF - H </td><td>不适用</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> PMEF中等 </td><td></td><td></td><td></td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 11965-118 </td><td>不适用</td></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 16140-071 </td><td> 10％ </td></tr><tr><td> L-谷氨酰胺</td><td> Invitrogen公司</td><td> 25030-081 </td><td> 1％ </td></tr><tr><td>青霉素/ </td><td> Invitrogen公司</td><td> 15240-063 </td><td> 1％ </td></tr><tr><td></td><td></TD&gt; <td></td><td></td></tr><tr><td> MES中等 </td><td></td><td></td><td></td></tr><tr><td> DMEM培养液</td><td>干细胞技术</td><td> 36250 </td><td>不适用</td></tr><tr><td> ES细胞合格的胎牛血清</td><td> Invitrogen公司</td><td> 16141-079 </td><td> 15％ </td></tr><tr><td> GlutaMax我</td><td> Invitrogen公司</td><td> 35050-061 </td><td> 1％ </td></tr><tr><td>非必需氨基酸</td><td> Invitrogen公司</td><td> 11140-050 </td><td> 1％ </td></tr><tr><td>核苷</td><td> Millipore公司</td><td> ES-008 - D </td><td> 1％ </td></tr><tr><td> β-巯基乙醇</td><td> Millipore公司</td><td> ES-007-E的</td><td> 0.1毫米</td></tr><tr><td>青霉素/ </td><td> Millipore公司</td><td> TMS-AB2型 - C </td><td> 1％ </td></tr><tr><td> LIF的</td><td> Millipore公司</td><td&gt; LIF2010 </td><td> 10纳克/毫升</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 神经分化培养基 </td><td></td><td></td><td></td></tr><tr><td> DMEM培养液</td><td>干细胞技术</td><td> 36250 </td><td>不适用</td></tr><tr><td> ES的邪教胎牛血清</td><td>干细胞技术</td><td> 06905 </td><td> 15％ </td></tr><tr><td>非必需氨基酸</td><td> Invitrogen公司</td><td> 11140-050 </td><td> 1％ </td></tr><tr><td>单硫代甘油</td><td> Sigma-Aldrich公司</td><td>的M-6145 </td><td> 1MM </td></tr><tr><td> Noggin与</td><td> Invitrogen公司</td><td> PHC1506 </td><td> 50毫微克/毫升</td></tr><tr><td> FGF-8 </td><td> Invitrogen公司</td><td> PHG0274 </td><td> 20毫微克/毫升</td></tr><tr><td>碱性成纤维细胞生长因子</td><td> Invitrogen公司</td><td> PHG0024 </td><td> 20毫微克/毫升</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 国防部介质（分化） </td><td></td><td></td><td></td></tr><tr><td> ES邪教的基础培养基- </td><td>干细胞技术</td><td> 5801 </td><td>不适用</td></tr><tr><td> Knockout血清替代品</td><td> Invitrogen公司</td><td> 10828-028 </td><td> 10％ </td></tr><tr><td>的N-2的补充</td><td> Invitrogen公司</td><td> 17502-048 </td><td> 1％ </td></tr><tr><td>其补编-B的</td><td>干细胞技术</td><td> 07155 </td><td> 1％ </td></tr><tr><td>抗坏血酸</td><td>干细胞技术</td><td> 07157 </td><td> 1％ </td></tr><tr><td>青霉素/ </td><td> Millipore公司</td><td> TMS-AB2型 - C </td><td> 1％ </td></tr><tr><td> GlutaMax我</td><td>Invitrogen公司</td><td> 35050-061 </td><td> 1％ </td></tr><tr><td> D-葡萄糖</td><td> Sigma-Aldrich公司</td><td> G - 8270 </td><td> D 2 H 2 O 0.15％ </td></tr><tr><td>纤维连接蛋白</td><td>干细胞技术</td><td> 07159 </td><td> 5微克/毫升</td></tr><tr><td>肝素</td><td> Sigma-Aldrich公司</td><td> H3149 </td><td> 20 D 2微克/毫升氢 Ø </td></tr><tr><td> β-巯基乙醇</td><td> Millipore公司</td><td> ES-007-E的</td><td> 0.1毫米</td></tr><tr><td>全反式维甲酸</td><td> Sigma-Aldrich公司</td><td>的R-2625 </td><td> 1 mM </td></tr><tr><td>凹陷</td><td>电解二氧化锰化学品</td><td> 566660 </td><td> 1 mM </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 国防部介质（运动神经元的文化） </td><td></td><td></td><td></td></tr><tr><td> ES邪教的基础培养基- </td><td>干细胞技术</td><td> 5801 </td><td>不适用</td></tr><tr><td> Knockout血清替代品</td><td> Invitrogen公司</td><td> 10828-028 </td><td> 10％ </td></tr><tr><td>的N-2的补充</td><td> Invitrogen公司</td><td> 17502-048 </td><td> 1％ </td></tr><tr><td>其补编-B的</td><td>干细胞技术</td><td> 07155 </td><td> 1％ </td></tr><tr><td>抗坏血酸</td><td>干细胞技术</td><td> 07157 </td><td> 1％ </td></tr><tr><td>青霉素/ </td><td> Millipore公司</td><td> TMS-AB2型 - C </td><td> 1％ </td></tr><tr><td> GlutaMax我</td><td> Invitrogen公司</td><td> 35050-061 </td><td> 1％ </td></tr><tr><td> D-葡萄糖</td><td> Sigma-Aldrich公司</td><td> G - 8270 </td><td> D 2 H 2 O 0.15％ </td></tr><tr><td>纤维连接蛋白</td><td> StemCELL技术</td><td> 07159 </td><td> 5微克/毫升</td></tr><tr><td>肝素</td><td> Sigma-Aldrich公司</td><td> H3149 </td><td> 20 D 2微克/毫升氢 Ø </td></tr><tr><td> β-巯基乙醇</td><td> Millipore公司</td><td> ES-007-E的</td><td> 0.1毫米</td></tr><tr><td>脑源性神经营养因子</td><td> R＆D系统</td><td> 248-BD-005/CF </td><td> 10纳克/毫升</td></tr><tr><td>睫状神经营养因子</td><td> R＆D系统</td><td> 257-NY-010/CF </td><td> 10纳克/毫升</td></tr><tr><td> GDNF的</td><td> R＆D系统</td><td> 212-GD-010/CF </td><td> 10纳克/毫升</td></tr><tr><td> NT-3 </td><td> R＆D系统</td><td> 267-N3-005/CF </td><td> 10纳克/毫升</td></tr></tbody></table> ，NA =不适用。 表1。MES细胞培养或分化PMEF和媒体。 <table border="1"><tbody><tr><td> &lt;强&gt;试剂名称</td><td> 公司 </td><td> 目录编号 </td><td> 终浓度 </td></tr><tr><td> 0.1％的明胶</td><td>干细胞技术</td><td> 07903 </td><td>不适用</td></tr><tr><td>聚-DL-鸟氨酸</td><td> Sigma-Aldrich公司</td><td> P0421 </td><td>在D 0.1毫克/毫升2 H 2 O </td></tr><tr><td>鼠标粘连</td><td> Millipore公司</td><td> CC095 </td><td> 2微克/毫升的PBS </td></tr><tr><td> 0.25％胰蛋白酶/ EDTA </td><td>干细胞技术</td><td> 07901 </td><td>不适用</td></tr><tr><td> accumax </td><td> Millipore公司</td><td> SCR006 </td><td>不适用</td></tr></tbody></table> ，NA =不适用。 表2。用于涂料和分离试剂。

efficient differentiation of mouse embryonic stem cells into motor neurons

功能运动神经元分化成胚胎干细胞（ES细胞）的直接代表一个有前途的资源，研究疾病机理，筛选新的药物化合物，并制定，如脊肌萎缩症（SMA）和肌萎缩性侧索硬化症的运动神经元疾病的新疗法（ ALS）的。目前许多协议使用维甲酸（RA）和刺猬（SHH）的组合分化成运动神经元1-4小鼠胚胎干细胞（MES）。然而，mES细胞分化成运动神经元的效率只达到中等成功。我们已经开发了一个两步分化协议5，显着提高目前建立的协议相比，分化效率。第一步是Noggin与成纤维细胞生长因子（FGFs）的加入，以加强neuralization过程。 Noggin与骨形态发生蛋白（BMP）受体拮抗剂和被牵连在神经诱导1ccording神经发生和结果形成前神经图案6默认模型。 FGF信号协同诱导神经组织的形成，促进后神经身份7-9 Noggin的行为。在这一步，mES细胞被引两天，以促进对神经谱系分化与Noggin的，碱性成纤维细胞生长因子，FGF-8。第二步是诱发运动神经元的规范。 Noggin与/ FGFs暴露mES细胞培养与RA和Shh的激动剂，理顺激动剂（凹陷），再过5天，以促进运动神经元生成。为了监测乱七八糟的分化成运动神经元，我们使用胚胎干细胞系，从转基因小鼠表达EGFP派生控制下运动神经元特异性启动子HB9 1。使用这个强大的协议，我们实现了51±0.8％的分化效率（N = 3，P &lt;0.01，学生t-检验）5。从免疫的结果染色表明，绿色荧光蛋白+细胞表达运动神经元的特异性标志物，胰岛-1和胆碱乙酰转移酶（CHAT）。我们两步分化协议mES细胞分化成脊髓运动神经元提供了一条有效途径。

Watch this Scientific Journal Video about Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons at JoVE.com

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

我们开发了一个新的协议，以提高效率，在体外分化成运动神经元的小鼠胚胎干细胞。分化的胚胎干细胞获得运动神经元功能的神经元和运动神经元采用免疫组化技术标记表达证明。

高效的小鼠胚胎干细胞分化成运动神经元

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

Research

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Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

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小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

人类多能干细胞的神经元在一个Multititre的板格式的快速和高效地生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

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新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

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Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...