Name: directed differentiation of induced pluripotent stem cells towards t lymphocytes
Uploaded: 2012-05-14T13:00:00
Description: Watch this Scientific Journal Video about Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes at JoVE.com

我们感谢提供山中伸弥博士（京都大学）IPS-MEF的NG-20D-17细胞系，支持OT1-2A的达里奥Vignali博士（圣裘德儿童研究医院），博士胡安•pMig II结构卡洛斯·祖尼加Pflucker（免疫学系，多伦多大学）支持OP9-DL1的细胞系，博士，肯特，éVrana（宾夕法尼亚州立大学医学院药理学教研室）为帮助本研究设计。该项目资助下，拨款补助人数从国家癌症研究所，Barsumian信托和黑色素瘤研究基金会（J宋）K18CA151798。

1。细胞培养<ol><li>制备辐照SNL76 / 7（irSNL76 / 7）饲养层细胞培养。 SNL76 / 7细胞一般都保持在10％胎牛血清（FBS），杜尔贝科的改良Eagle培养基（DMEM培养基）媒体。 <ol><li>培养皿或瓶，将涂有0.1％的明胶溶液在37°C，30分钟孵化器前恢复SNL76 / 7细胞从液氮。 </li><li>达到汇合SNL76 / 7细胞时，细胞就会被胰酶消化，离心5分钟在400Ğ和悬浮在新鲜的媒体。 </li><li>悬浮SNL76 / 7细胞，将在5000拉德的剂量...

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<html>为使基于疗法， 在体外产生大量高活性银特定的T细胞在体内重新输液是最佳方法。虽然我们在体外培养的方法，给出了iPS细胞，iPS细胞源性细胞的死亡，尤其是在第四个星期，在四个星期内的大量功能性T细胞上升。我们得出这样的结论：从缺口的生存信号的信号介导的DL1的IL-7和FLt3L不足以维持iPS细胞衍生的祖T细胞的生存以及其他存活的因素可能是合作，以调节这些细胞...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> C57BL/6J小鼠</td><td>杰克逊实验室</td><td> 000664 </td></tr><tr><td> B6.129S7 RAG1 tm1Mom / J的 </td><td>杰克逊实验室</td><td> 002216 </td></tr><tr><td>抗CD3抗体（2C11） </td><td>屋宇署Pharmingen公司</td><td> 553058 </td></tr><tr><td>抗CD28（37.51）抗体</td><td>屋宇署Pharmingen公司</td><td> 553295 </td></tr><tr><td>抗CD3抗体（17A2） </td><td> BioLegend </td><td> 100202 </td></tr><tr><td>抗CD4（GK1.5）抗体</td><td> BioLegend </td><td> 100417 </td></tr><tr><td>抗-CD8（53-6.7）抗体</td><td> BioLegend </td><td> 100714 </td></tr><tr><td>抗CD25抗体（3C7） </td><td> BioLegend </td><td> 101912 </td></tr><tr><td>一个TI-CD44的抗体（1M7） </td><td> BioLegend </td><td> 103012 </td></tr><tr><td>抗-CD117的抗体（2B8） </td><td> BioLegend </td><td> 105812 </td></tr><tr><td>抗TCR-β（H57597）抗体</td><td> BioLegend </td><td> 109220 </td></tr><tr><td>抗IL-2（JES6-5H4）抗体</td><td> BioLegend </td><td> 503810 </td></tr><tr><td>抗γ-干扰素（XMG1.2）抗体</td><td> BioLegend </td><td> 505822 </td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> ABCD1234 </td></tr><tr><td> α-MEM中</td><td> Invitrogen公司</td><td> A10490-01 </td></tr><tr><td>胎牛血清</td><td>胎牛血清</td><td> SH3007.01 </td></tr><tr><td>布雷菲德菌素A </td><td>西格玛</td><td> B7651 </td></tr><tr><td>凝聚胺</td><td>西格玛</td><td> 107689 </td></tr><tr><td> GeneJammer </td><td>综合科学</td><td> 204130 </td></tr><tr><td> RNA试剂盒&lt;/ TD&gt; <td> Qiagen公司</td><td> 74104 </td></tr><tr><td> DNA试剂盒</td><td> Qiagen公司</td><td> 69504 </td></tr><tr><td> CD8分离试剂盒</td><td>美天旎生物技术</td><td> 130-095-236 </td></tr><tr><td> ACK裂解液</td><td>龙沙</td><td> 10-548E </td></tr><tr><td> mFlt-3L </td><td> PeproTech公司</td><td> 250-31L </td></tr><tr><td> MIL-7 </td><td> PeproTech公司</td><td> 217-17 </td></tr><tr><td>明胶</td><td>西格玛</td><td> G9391 </td></tr><tr><td> FITC标记的抗卵抗体</td><td>罗克兰Immunochemicals </td><td> 200-4233 </td></tr><tr><td>通透性缓冲区</td><td> Biolegend </td><td> 421002 </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A7906 </td></tr><tr><td>甲醛</td><td>西格玛</td><td> F8775 </td></tr><tr><td> 0.4微米过滤器</td><td> Millipore公司</td><td></td></tr><tr><td&gt; Moflo细胞分选仪</td><td>岳Cytomation </td><td></td></tr><tr><td>刀魂流式细胞仪</td><td>屋宇署</td><td></td></tr><tr><td> LSR的第二流式细胞仪</td><td>屋宇署</td><td></td></tr><tr><td>鼠标限位</td><td>布伦特里科学</td><td></td></tr></tbody></table>

directed differentiation of induced pluripotent stem cells towards t lymphocytes

抗原特异性CD8 +细胞毒性T淋巴细胞（CTL）的收养细胞转移（ACT）是一个很有前途的治疗各种恶性肿瘤的1。淋巴细胞可以识别肿瘤抗原与T细胞受体（TCR），释放细胞毒素以及细胞因子的相互作用，以杀死肿瘤细胞的恶性细胞。据了解，少分化和中央内存（称为高活性 ）CTL的是为使基于免疫的最佳人口，因为这些淋巴细胞具有较高的增殖潜能，是不易，更分化的细胞凋亡，并有更高的响应能力2-7稳态细胞因子。然而，由于在这种淋巴细胞从患者获得大量的困难，是一个迫切需要找到一种新的方法产生高活性的银特有的成功为基础的ACT疗法的淋巴细胞。 自我再生的干的TCR转导细胞免疫重建具有治疗疾病的治疗8-10潜力。然而，从病人身上获取胚胎干细胞（ESCs）的方法是不可行的。利用造血干细胞（造血干细胞用于治疗目的）虽然已被广泛应用在临床上11-13减少，造血干细胞的分化和增殖能力，造血干细胞难以在体外细胞培养14-16扩大。 iPS细胞技术和基因传递在体外系统的发展能够产生iPS细胞从患者没有任何的手术方法。此外，胚胎干细胞一样，iPS细胞具有无限期的体外增殖能力，已被证明可以分化成造血干细胞。因此，iPS细胞具有更大的潜力，使用胚胎干细胞或造血干细胞相比，在ACT - 基于免疫。 在这里，我们提出了T淋巴细胞的生成方法iPS细胞在体外 cytes，并在促进癌细胞的免疫监视iPS细胞的抗原特异性CTL的体内编程。推动iPS细胞在体外与Notch配体刺激T细胞的分化和TCR基因转导到抗原特异性T细胞分化的iPS细胞在体内 ，从而阻止肿瘤的生长结果。因此，我们证明iPS细胞的抗原特异性T细胞的分化。我们的研究提供了一个潜在的更有效的方法使基于疗法产生抗原特异性CTL和促进疾病的治疗策略的发展。

Watch this Scientific Journal Video about Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes at JoVE.com

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

诱导多能干细胞（iPS）细胞，T淋巴细胞的生成，给出了一个使用基于T细胞免疫治疗的胚胎干细胞的替代方法。该方法表明，利用任<em&gt;在体外</em&gt;或<em&gt;在体内</em&gt;感应系统，iPS细胞能够分化成传统和抗原特异性T淋巴细胞。

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

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