Name: the use of sc1 (pluripotin) to support mesc self-renewal in the absence of lif
Uploaded: 2009-11-18T17:30:00
Duration: 5 min 58 s
Description: Watch this Scientific Journal Video about The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF at JoVE.com

作者衷心感谢他们的协助和方向盛鼎博士在斯克里普斯研究所和北京大学的博士洪奎邓小平。

1。 MEF的接驳板的制备<ol><li>前一天培养ES细胞，解冻之一小瓶的照射E14.5 CF - 1 MEF饲养层细胞（见我们的程序就如何解冻ES细胞）。 </li><li>板万细胞密度的MEF饲养层细胞/孔在12孔板。在37 ° C和5％的CO 2孵育过夜的细胞。 MEF细胞介质：DMEM培养基辅以10％的ES - PBS，谷氨酰胺1X和1X非必需氨基酸。 </li></ol> 2。小鼠胚胎干细胞的自我更新维护<ol><li>分离的ES细胞，用胰蛋白酶。种子/以及在先前准备的MEF的生长介质的接驳板（淘汰赛的MEM补充KSR 15％，4毫米L -谷氨酰胺，0.1毫米的非必需氨基酸和1X BME）在50,000个细胞的密度细胞辅以SC1所需浓度（我们使用100纳米，300纳米，和1μM）。我们还添加了一个积极的控制，以及1000μ/ ml的LIF的补充。在37 ° C和5％的CO 2孵育过夜的细胞。 </li><li>培养细胞在37 ° C和5％的CO 2在每个通道的3-4天。刷新媒体每48小时。 </li><li>传代细胞1:10至1:15与胰蛋白酶，以保持作为最初的播种密度相似密度。 5代后，细胞可表达典型的多能性标志物，用免疫细胞化学检查。 </li></ol> 3。多潜能性标志物的免疫细胞化学考试<ol><li>清洗细胞，用PBS轻轻三次。 </li><li>修复细胞在室温下20分钟的固定液500μL。 </li><li>清洗细胞，用PBS轻轻三次。 </li><li>阻止非特异性结合，用500μl堵在室温下一个小时的缓冲。 </li><li>孵育细胞的特定抗体与250μL（我们Nanog的，SOX2，SSEA1和Oct4的使用在以下稀释1:500，1:2000，1:500，而1:500） </li><li>清洗细胞，用PBS轻轻三次。 </li><li>两个小时在室温下，保持避光（我们用1:2000和驴驴的Alexa Fluor ® 488标记的抗兔抗鼠的共轭的Alexa Fluor ® 555 1 250μL二次抗体孵育细胞：2000）。 </li><li>清洗细胞，用PBS轻轻三次。 </li><li>添加的DAPI（终浓度为1微克/毫升）的最后一次洗涤，孵育5分钟，以可视化核。 </li><li>分析荧光显微镜下的细胞。 </li></ol> 4。代表性的成果： 1。形态学结果： LIF或SC1 MEF饲养层细胞的小鼠胚胎干细胞（ESC R1）的生长，维持自我更新，在未分化状态。分化后的第二通道，可以在细胞中观察到无LIF或SC1（阴性对照），5代后，基本上没有无差别的殖民地。 LIF（阳性对照）处理殖民地保持在未分化状态，整个5单元段落。然而，SC1的治疗浓度为300纳米或100纳米的细胞也保持未分化，5代后，细胞治疗1微米SC1的经验丰富的细胞死亡之后的第四代。应用笔记（图2）表1总结了形态学观察。 2。多能性标志物的表达固定维持5代的小鼠胚胎干细胞和免疫细胞化学研究SSEA1，OCT4，Nanog和Sox2基因。标记表达LIF在维持细胞相似。 （图3和4应用笔记） <img src="/files/ftp_upload/1550/1550fig2a.jpg" alt="图1" /> 图1：在LIF或SC1维持细胞的形态比较。在形态上没有差异。 <img src="/files/ftp_upload/1550/1550fig2c.jpg" alt="图2和3" /> 图2和图3：Nanog的，SSEA1，SOX2，和Oct4的SC1或LIF（图4）应用笔记维持ES细胞的染色。保持在SC1的细胞显示出类似的多能性标志物表达LIF在维持细胞。 表1 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> 阴性对照 </td><td> LIF的积极的控制 </td><td> SC1的1微米 </td><td> SC1的300纳米 </td><td> SC1的100纳米 </td></tr><tr><td> 第一通道 </td><td>正常的胚胎干细胞的形态</td><td>正常的胚胎干细胞的形态</td><td>正常的胚胎干细胞的形态</td><td>正常的胚胎干细胞的形态</td><td>正常的胚胎干细胞的形态</td></tr><tr><td> 第二通道 </td><td>一些差异化的形态</td><td>常规e手机信号形态</td><td>正常的胚胎干细胞的形态</td><td>正常的胚胎干细胞的形态</td><td>正常的胚胎干细胞的形态</td></tr><tr><td> 第3代 </td><td>大部分细胞分化</td><td>正常的胚胎干细胞的形态</td><td>正常的胚胎干细胞的形态</td><td>正常的胚胎干细胞的形态</td><td>正常的胚胎干细胞的形态</td></tr><tr><td> 第四通道 </td><td>没有人事编制小组委员会的殖民地</td><td>正常的胚胎干细胞的形态</td><td>观察细胞死亡</td><td>正常的胚胎干细胞的形态</td><td>正常的胚胎干细胞的形态</td></tr><tr><td> 第5代 </td><td>有区别的</td><td>正常的胚胎干细胞的形态</td><td>观察细胞死亡</td><td>正常的胚胎干细胞的形态</td><td>正常的胚胎干细胞的形态</td></tr></tbody></table>

<ol>
	<li>Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L., Gearing, D. P., Wagner, E. F., Metcalf, D., Nicola, N. A., Gough, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+leukaemia+inhibitory+factor+maintains+the+developmental+potential+of+embryonic+stem+cells.">Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.</a> Nature. 336, 684-687 (1988).</li><li>Ying, Q. L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ground+state+of+embryonic+stem+cell+self-renewal.">The ground state of embryonic stem cell self-renewal.</a> Nature. 453, 519-523 (2008).</li><li>Chen, S., Do, J. T., Zhang, Q., Yao, S., Yan, F., Peters, E. C., Scholer, H. R., Schultz, P. G., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+of+embryonic+stem+cells+by+a+small+molecule.">Self-renewal of embryonic stem cells by a small molecule.</a> Proc. Natl. Acat. Sci. USA. 103, 17266-17271 (2006).</li></ol>

The authors of this article are employed by Stemgent that produces reagents and instruments used in this article.

可用于小分子SC1在小鼠胚胎干细胞的未分化状态的维持与自我更新。然而，在使用一个未经考验的细胞株，它应该被滴定，以确定最佳浓度。例如，我们测试了三种浓度的小鼠胚胎干细胞株ESC R1的。 100 nm和300 nm的浓度能够维持小鼠胚胎干细胞，然而，1微米的浓度是有毒的。 SC1也可用于馈线条件下。

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the use of sc1 (pluripotin) to support mesc self-renewal in the absence of lif

小鼠胚胎干（ES细胞）是传统的培养与白血病抑制因子（LIF），以维持自我更新。<sup&gt; 1</sup然而，LIF的是昂贵的，活化的LIF/JAK/STAT3途径也不是绝对需要保持自我更新的状态。<sup&gt; 2</sup星际1的小分子可能是一种经济的替代LIF的。通过双抑制的Ras GAP和ERK1 SC1功能。<sup&gt; 3</sup&gt;插图，其作用机制，使得一个有用的工具，究其根本的自我更新的分子机制。在这里，我们展示了在SC1的存在为培养小鼠胚胎干细胞的过程，并表明他们是在LIF的情况下能够保持自我更新。与SC1的培养细胞中LIF维持细胞相比，表现出类似的形态。展出后，五个通道典型的小鼠胚胎干形态。典型的多能性标志物的表达（OCT4，SOX2，NANOG，和SSEA1）观察SC1的存在，在五年后的段落。此外，SC1引起的小鼠胚胎干细胞没有明显的毒性。

Watch this Scientific Journal Video about The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF at JoVE.com

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

通过双抑制的Ras GAP和ERK1 SC1功能。我们SC1的功能，支持在没有LIF的小鼠ES细胞的自我更新和测试表明，SC1的是能够保持自我更新的小鼠胚胎干细胞培养。

SC1（Pluripotin）使用，以支持在没有LIF的自我更新MESC

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

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The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

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