我们感谢三宅义子，早矢香Kanaami，奇哈纳藤田，美穗山口，夏子Henmi，和丽大野耐一从庆应义塾大学医学院提供技术援助。这部分工作是由R＆D Systems的支持计划，以加速实际使用的健康研究成果，以及高速公路项目为再生医学的实现提供资金。

1.准备激活人类T细胞<ol><li>收集肝素化的全血从供体通过静脉穿刺10毫升<ol><li>获得捐赠者的知情同意提前静脉穿刺。静脉穿刺必须由合格的，经过培训的人员来完成。 </li><li>处理的血液样本，用无菌设备及以下适当的生物安全准则。 </li></ol></li><li>稀释肝素化的全血用10ml D-PBS中的10毫升（ - ）。 </li><li>将15个毫升聚蔗糖溶液（菲可 - 帕克溢价）到一个新的50毫升锥形管。 </li><li>层在步骤1.2制得到在步骤1.3中所述的Ficoll溶液20毫升血液。使用电吸移管，让血液溶液制备步骤1.2运行慢慢沿管的内壁上。要小心，不要混用血液和聚蔗糖的解决方案。 </li><li>离心机在400×g下在室温30分钟。将离心减速速度“慢”。 注意：离心后，为白色薄层浮现的上部乳白色血浆层和下清晰的Ficoll层之间。这白色的薄薄的一层含有单核细胞。 </li><li>的单核细胞层转移到一个新的50毫升锥形管用1毫升吸移管。要小心，不要包括聚蔗糖的解决方案。 </li><li>添加D-PBS（ - ）与单核细胞，以10毫升的总体积，以及离心机在200×g下5分钟，在室温。 </li><li>弃去上清液。加入10 mL D-PBS的（ - ）对单核细胞，并在离心分离机200×g下5分钟，在室温。 </li><li>弃去上清液，加入1 ml的KBM502培养基向收集的单核细胞，并使用血细胞计数器计数细胞。超过5×10 6单核细胞可以从肝素化的全血与适当的分离用Ficoll 10 ml的获得。 </li><li>制备的抗人CD3抗体溶液以10微克/毫升在D-PBS的浓度（ - ）。抗人CD3抗体溶液添加至6孔板，浸泡冲浪每个王牌孔，孵育菜，在37℃在一个5％CO 2培养箱中至少30分钟。 </li><li>除去从各抗人CD3抗体溶液以及与用D-PBS洗一次（ - ）以接种细胞之前。 </li><li>分别以2.5×10 5个细胞/ cm 2的对抗人CD3抗体包被6孔板中KBM502介质的密度制备步骤1.9 ​​那种子的单核细胞。在5％-CO 2培养箱中3-7天在37℃的无介质改变，直到T细胞达到汇合。 T细胞在此期间都暂停，细胞粘附。 </li></ol> 2.感染人体T细胞SeV载体<ol><li>经过3-7天的培养，收集活化的T细胞，吹打现有的网上平台。它们转移到15毫升锥形管中并离心，在200×g下5分钟，在室温。 </li><li>去除上清，加入1毫升新鲜KBM502网上平台。计数细胞，和板1。5×10 6个细胞（在2ml新鲜培养基KBM502）插入抗CD3抗体包被的6孔板的每个孔中。 </li><li>冰上解冻OCT3 / 4-SeV载体/TSΔF，​​SOX2-的SeV /TSΔF，​​KLF4-的SeV /TSΔF，​​和c-MYC（HNL）-sev /TS15ΔF的SeV的解决方案。 </li><li>添加包含OCT3 / 4-SeV载体/TSΔF，​​SOX2-的SeV /TSΔF，​​KLF4-的SeV /TSΔF，​​和c-MYC（HNL）SeV的溶液-sev /TS15ΔF单独的井，每于感染复数（MOI） 10.孵育在37℃下在5％-CO 2培养箱中另外24小时。 </li></ol> 3.从人类T细胞SeV载体<ol><li> 24小时感染后，收集感染的细胞用吸管。如果有粘附细胞，他们可以很容易被拆卸吹打他们反复向上和向下。它们转移到15毫升锥形管中，并离心分离，在200×g下5分钟，在室温。 </li><li>删除包含SeV载体上清液，加2ml新鲜KBM502介质。 Replate细胞EAC小时井。在5％-CO 2培养箱在37℃下另外24小时。 </li></ol>在基底膜层4.重新设定种子细胞<ol><li>准备通过解冻基质凝胶基底膜基质溶液（请参阅材料清单用于本研究中使用的特定的品牌），在4℃和在10毫升的DMEM / F12培养基中溶解0.2毫升 注：基质胶（请参阅材料清单用于本研究中使用的特定的品牌）需要解冻O / N在4℃下被完全解冻。保持它在冰上使用前至。 </li><li>在RT种子细胞之前，每盘盘5毫升基底膜基质溶液为100毫米的菜肴，并留下了至少30分钟。都需要一个实验的至少两个100-mm培养皿中。 </li><li>收集的SeV感染的细胞通过移液，将它们在室温下转移到15毫升锥形管中，并离心分离，在200×g下5分钟。 </li><li>去除上清，加入1毫升新鲜KBM502网上平台。伯爵CE数量LLS使用血细胞计数器，删除在步骤4.2和板制备100-mm培养皿中的基底膜基质溶液1×10 5和1×10 6个细胞（在10ml新鲜mTeSR培养基）上的100毫米基底膜基质涂覆菜肴。最后，使用板，其菌落容易上手。 </li><li>孵育在5％-CO 2培养箱O / N的菜在37℃。 </li><li>介质更改至10ml充实mTeSR培养基隔日1次。 注意：在感染后约20-30天，菌落可以非常类似于胚胎干细胞（ESC）将出现。 </li></ol> 5.展开T细胞来源的iPS细胞<ol><li>准备通过解冻基质凝胶基底膜基质溶液（请参阅材料清单用于本研究中使用的特定的品牌），在4℃和在10毫升的DMEM / F12培养基中溶解0.2毫升</li><li>板1 ml的在6孔板每孔基底膜基质溶液中，并在室温离开在采摘菌落前至少30分钟。 </li><li>填的96孔平板与20μlmTeSR培养基中每孔。 </li><li>选择像用20微升移液管的体视显微镜下，胚胎干细胞的殖民地。 注意：胚胎干细胞和iPS细胞的集落是圆形和扁平，如图1B所示 。 </li><li>传输所选的菌落放入96孔板在步骤5.3充满mTeSR培养基。 </li><li>加入200μlmTeSR培养基到每个孔中，并仔细吸取上下打破菌落成小团块用200μl移液管的立体显微镜下。取下充分的准备基底膜基质解决方案5.2，并把每个菌落到基底膜基质包被6孔板2毫升mTeSR培养基中的一个孔，每孔。 </li><li>介质更改〜2毫升充实mTeSR培养基隔日1次。 </li></ol> 6.保持T细胞来源的iPS细胞 T细胞来源的iPS细胞可以保持并用作为人类的iPSC和人ESC相同的技术存储。一旦IPSC的殖民地长大，重复同样的程序扩展成更大的菜肴。 <ol><li>更改mTeSR培养基每1-2天。 </li><li>当菌落变得汇合每5-7天，通过用解离溶液用于人类的iPSC和根据制造商的说明人ESC细胞。 </li></ol>

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作者有没有竞争或利益冲突的披露。

We describe a protocol for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors. For clinical applications of iPSCs, it is important to have a protocol for stably generating iPSCs and a less-invasive method for cell sampling. Although generating iPSCs with the combination of matrigel and mTeSR medium showed lower reprogramming efficiency than that with knockout serum replacement (KSR) medium and feeder-cells, this combination achieves stable generation of iPSCs from donors16. Stable iPSC generation and less-invasive cell sampling has the advantage of being able to increase the number of donors for iPSC generation. 
SeV vectors, a minus-strand RNA virus, is not integrated into the host genome. Therefore the risks of tumorigenesis can be avoided at the step of reprogramming factor induction20-24. Additionally, the feeder-free conditions, which use a combination of defined culture medium and matrigel instead of a feeder layer, make it possible to minimize the potential risks of exposure to unknown exogenous factors. The fusion of these techniques provides a less invasive and safer iPSC technology for regenerative medicine16.
Important steps in this protocol are the step of activating human T cells (Step 1) and infecting them with SeV vectors (Step 2). When no ESC-like colony is obtained in the culture dish at an optimal time after SeV infection, the following should be considered. First, the confluency of the mononuclear cells before T cell activation may not be appropriate because optimal activation of T cells is critical for SeV infection25,26. Too high a density of mononuclear cells leads to cell death, interfering with the proper activation of T cells. Too low a density of mononuclear cells also disturbs the proper activation and proliferation of T cells. Therefore, the confluency of mononuclear cells should be checked and adjusted accordingly. Second, the dosage of SeV vectors may not be sufficient. The induction efficiency of iPSC colonies depends on the dosage of the virus6. If no ESC-like colony is observed after SeV infection, the option of increasing the virus dosage up to an MOI of 15-20 should be considered. If signs of iPSC colony differentiation are observed, the frequency at which medium is changed may be increased up to every other day, or to every day when colonies are larger. 
The limitation in this protocol consists of this method not being virus free. Although SeV for cell reprogramming is commercially available with ease, users need to prepare equipment according to the appropriate biosafety level. As another method for generating integration-free iPSCs, episomal vectors have been used until now27-29. Although episomal vectors can be used with equipment with a lower level of biosafety, episomal vectors might insert into the host genome at extremely low rates. Therefore, additional checks are required to confirm the disappearance of transgenes, as when using SeV.
There is another limitation in that this technique is not absolutely free from animal-derived products. Some substrates, such as matrigel, anti-CD3 mAb, dissociation solution and SeV solution are derived from animal products and are therefore associated with a risk of transferring xenogeneic pathogens. However, the reduction of animal-derived substrates in the culture system is meaningful for the clinical application of iPSC technology due to the lower risk.

iPS细胞引起了极大关注，因为细胞在再生医学1-3一个突破性的来源。迄今为止，用于产生iPSCs的多样的方法已被报道4,5。其中，从人T细胞产生iPSCs的已经因为细胞采样6-8的较少创伤的方法的特别的兴趣。此外，从iPSCs的T细胞衍生的含有T细胞受体（TCR）基因重排，因而抗原特异性T细胞9,10的来源。因此，产生的T细胞衍生的iPS细胞安全是正在进行再生医学有用。 此方法是基于减少不可预知的致病性的风险的概念。为iPS细胞的临床应用安全是很重要的，以减少病原体11暴露的风险。先前在多能干细胞的许多培养系统，胎牛血清和饲养细胞已被用来作为基本试剂第 12条。然而，去除这两个从培养系统试剂是优选的iPSC产生，以减少不可预知的致病性的危险。 此外，此方法具有避免从患者和饲养细胞的制备费力侵袭性细胞采样的优点。因为T细胞衍生的iPSCs已经被成功地用于在疾病研究13,14，这种方法也适用和有益的，从患者产生疾病特异性的iPSC。 间T细胞重新编程的方法，使用仙台病毒（SeV载体）载体作为基因载体是能够产生iPSCs的高效率7,16的方法。此外，由于SeV载体是一个单链RNA病毒，并且不需要的DNA相对于复制，其在的iPSC代使用避免断宿主基因组17-19。因此，我们已经建立的方案，用于从人外周血T细胞的iPSC在无血清的FRee值，并使用基质胶，mTeSR培养基的组合无饲养条件下，和SeV载体。

<table border="0">
	<tbody>
		<tr>
			<td>Ficoll-Paque PREMIUM</td>
			<td>GE Healthcare</td>
			<td>17-5442-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified NA/LE mouse anti-human CD3</td>
			<td>BD Pharmingen</td>
			<td>555336</td>
			<td></td>
		</tr>
		<tr>
			<td>KBM502 medium</td>
			<td>KOHJIN BIO</td>
			<td>16025020</td>
			<td>Warm in 37 &#8451; water bath before use</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V solution</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Matrigel Matrix Growth Factor Reduced</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td>Thaw completely at 4&#8451; overnight and dilute it 50 times with Dulbecco&#39;s Modified Eagle&#39;s Medium before coating culture dishes</td>
		</tr>
		<tr>
			<td>mTeSR1 medium kit</td>
			<td>STEM CELL</td>
			<td>5850</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr>
			<td>Dissociation Solution</td>
			<td>ReproCELL</td>
			<td>RCHETP002</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS(&#8211;)</td>
			<td>Wako</td>
			<td>045-29795</td>
			<td></td>
		</tr>
		<tr>
			<td>SeV Vector kit CytoTune-iPS ver.1.0</td>
			<td>DNAVEC</td>
			<td>DV-0303c</td>
			<td>Thaw on ice before use</td>
		</tr>
		<tr>
			<td>100-mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353078</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>227261</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from human peripheral t cells using sendai virus in feeder-free conditions

最近，iPS细胞已经引起关注，因为细胞再生疗法的新来源。尽管用于产生iPSCs的初始方法依赖于通过侵入性活组织检查和转基因的逆转录病毒基因组的插入得到的皮肤成纤维细胞，已经出现了许多努力来避免这些缺点。人外周血T细胞是唯一的小区源用于产生iPSCs的。从T细胞衍生的iPSC含有T细胞受体（TCR）基因的重排，并且抗原特异性T细胞的来源。此外，在基因组中的T细胞受体的重排有标记单个细胞系和移植的和供体细胞区分的可能性。为iPS细胞的安全的临床应用中，它以最小化暴露新产生iPSCs的有害试剂的风险是重要的。虽然胎牛血清和饲养细胞已为多能干细胞培养必需的，最好是从培养系统中删除他们减少风险不可预知的致病性。为了解决这个问题，我们已经建立了一个协议，用于使用仙台病毒，以减少暴露的iPSC未定义的病原体的风险产生于人类外周T细胞的iPS细胞。虽然处理仙台病毒要求设备具备相应的生物安全水平，仙台病毒感染活化的T细胞无基因组插入，但效率很高。在这个协议中，我们展示了使用活化的T细胞培养和仙台病毒的组合从人外周血T细胞在无饲养条件iPS细胞的产生。

<html>使用此协议，用户能够生成从人外周血T细胞的iPSC稳定。 IPSC的一代，从T细胞的SeV和基底膜呈约0.002％ -细胞重编程的几个段落后，均未检出几个捐助情况和SeV载体的效率为0.005％，16 图1A显示源细胞的协议产生吨示意图iPS细胞使用基质胶和mTeSR中等无饲养条件。感染的SeV后约20至30天，iPSC的菌落通过菌落ESC样形态（图1B）识别。免疫荧光染色显示的典型的多能细胞标记物的表达（NANOG，OCT3 / 4，SSEA4，TRA-1-60，和TRA-1-81）中的T细胞来源的iPS细胞无饲养条件（图1C）下产生的。 “WIDTH =”700“/&gt; 图1（A）：无饲养条件本研究下的协议重新编程性T细胞的示意图。 （B）：在无饲养条件下采血后27天一个典型的ESC样IPSC的殖民地。 （℃）：ALP染色和免疫荧光染色为多能性和表面标记（NANOG，OCT3 / 4，SSEA4，TRA-1-60，和TRA-1-81）中的T细胞来源的iPS细胞无饲养条件下产生的。 <a href="https://www.jove.com/files/ftp_upload/53225/53225fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions at JoVE.com

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

从人外周T细胞无饲养条件下，使用仙台病毒诱导多能干细胞的生成

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

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11.9K 次观看.  Keio University School of Medicine. This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.